CN116411070A - Leukemia related markers and uses thereof - Google Patents

Abstract

The invention relates to a leukemia stem cell specific expression biomarker Allergin-1 and application thereof. In particular to a diagnosis product, a therapeutic drug or composition of leukemia, especially Acute Myeloid Leukemia (AML), application of a detection reagent of a detection marker Allergin-1 in preparation of products for diagnosis, disease course monitoring and prognosis judgment of leukemia, and application of a therapeutic agent/regulator in preparation of drugs for leukemia treatment.

Description

Translated fromChinese

白血病相关标志物以及应用Leukemia-related markers and their applications

技术领域technical field

本发明涉及白血病干细胞特异性表达生物标志物Allergin-1及其应用。具体而言，涉及白血病尤其是急性髓系白血病(AML)的诊断产品、治疗药物或组合物、检测标志物Allergin-1的检测试剂在制备用于白血病的诊断、病程监控和预后判断的产品中的应用、试剂在制备用于白血病治疗的药物中的用途。The invention relates to the specific expression biomarker Allergin-1 of leukemia stem cells and its application. Specifically, it relates to leukemia, especially acute myeloid leukemia (AML), diagnostic products, therapeutic drugs or compositions, and detection reagents for the detection marker Allergin-1 in the preparation of products for the diagnosis, course monitoring, and prognosis of leukemia. The application of the reagent and the use of the reagent in the preparation of medicines for the treatment of leukemia.

另一方面，本发明涉及通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来鉴别白血病干细胞(LSCs)、诊断、病程监控和预后判断白血病(尤其是AML)或用于确定发生白血病(尤其是AML)的风险或用于确定严重的白血病(尤其是AML)的方法，其中Allergin-1在LSCs中特异性异常高表达。本发明还提供了通过抑制/拮抗Allergin-1表达/活性而治疗受试者白血病(尤其是AML)的方法。此外，提供了包含用于白血病干细胞特异性表达生物标志物的检测工具、特别是抗体的试剂盒以及阵列。In another aspect, the present invention relates to the identification of leukemia stem cells (LSCs), diagnosis, course monitoring and prognosis of leukemia (especially AML) or a method for determining the risk of developing leukemia (especially AML) or for determining severe leukemia (especially AML), wherein Allergin-1 is abnormally highly expressed specifically in LSCs. The present invention also provides a method for treating leukemia (especially AML) in a subject by inhibiting/antagonizing the expression/activity of Allergin-1. Furthermore, kits and arrays comprising detection means, in particular antibodies, for the specific expression of biomarkers by leukemia stem cells are provided.

背景技术Background technique

急性髓系白血病(Acute myeloid leukemia,AML)是由造血干细胞(Hematopoietic stem cells,HSCs)分化受阻断并无限增殖而引起的恶性疾病，其病情凶险、易复发、致死率高，发病率逐年上升(Dores GM,Devesa SS,Curtis RE,Linet MS,Morton LM:Acute leukemia incidence and patient survival among children andadults in the United States,2001-2007.Blood 2012,119(1):34-43)。随着白血病发病率的增高以及全球性的人口老龄化，AML在恶性肿瘤中所占比例越来越大，成为人类健康的重大威胁。在AML的基础和临床研究方面，虽然国内外学者做了大量工作并取得了进展，但AML患者依然存在复发率高、5年以上长期生存率低等诸多问题，尤其是年龄>60岁的患者中约60％-80％复发(Dombret H,Gardin C:An update of current treatments for adultacute myeloid leukemia.Blood 2016,127(1):53-61；Short NJ,Konopleva M,Kadia TM,Borthakur G,Ravandi F,DiNardo CD,Daver N:Advances in the Treatment of AcuteMyeloid Leukemia:New Drugs and New Challenges.Cancer Discov 2020,10(4):506-525)。近年来，新兴的细胞疗法与免疫疗法在淋系白血病的治疗中取得了较好的效果，但在AML中实施起来却困难重重，因为靶向治疗AML对作用靶点的选择有更高的要求。随着肿瘤发生分子机制研究的不断深入，分子靶向治疗成为攻克肿瘤的突破口，因此进一步探索治疗AML的有效分子靶点成为现今AML研究的重要科学问题(Sami SA,Darwish NHE,BarileANM,Mousa SA:Current and Future Molecular Targets for Acute Myeloid LeukemiaTherapy.Curr Treat Option On 2020,21(1))。Acute myeloid leukemia (AML) is a malignant disease caused by blocked differentiation and unlimited proliferation of hematopoietic stem cells (HSCs). (Dores GM, Devesa SS, Curtis RE, Linet MS, Morton LM: Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood 2012, 119(1): 34-43). With the increasing incidence of leukemia and the aging of the global population, AML accounts for an increasing proportion of malignant tumors and has become a major threat to human health. In terms of basic and clinical research on AML, although scholars at home and abroad have done a lot of work and made progress, there are still many problems in AML patients, such as high recurrence rate and low long-term survival rate of more than 5 years, especially for patients aged > 60 years old. About 60%-80% of patients relapse (Dombret H, Gardin C: An update of current treatments for adultacute myeloid leukemia. Blood 2016,127(1):53-61; Short NJ, Konopleva M, Kadia TM, Borthakur G, Ravandi F, DiNardo CD, Daver N: Advances in the Treatment of Acute Myeloid Leukemia: New Drugs and New Challenges. Cancer Discov 2020,10(4):506-525). In recent years, emerging cell therapy and immunotherapy have achieved good results in the treatment of lymphoid leukemia, but it is difficult to implement in AML, because targeted therapy for AML has higher requirements for the selection of targets . With the continuous deepening of research on the molecular mechanism of tumorigenesis, molecular targeted therapy has become a breakthrough to overcome tumors, so further exploration of effective molecular targets for the treatment of AML has become an important scientific issue in current AML research (Sami SA, Darwish NHE, Barile ANM, Mousa SA : Current and Future Molecular Targets for Acute Myeloid Leukemia Therapy. Curr Treat Option On 2020, 21(1)).

研究表明，白血病干细胞(Leukemia stem cells,LSCs)在AML发生、发展和复发中起着关键作用(Ishikawa F,Yoshida S,Saito Y,Hijikata A,Kitamura H,Tanaka S,Nakamura R,Tanaka T,Tomiyama H,Saito N et al:Chemotherapy-resistant human AMLstem cells home to and engraft within the bone-marrow endosteal region.NatBiotechnol 2007,25(11):1315-1321；Saito Y,Yuki H,Kuratani M,Hashizume Y,TakagiS,Honma T,Tanaka A,Shirouzu M,Mikuni J,Handa N et al:A Pyrrolo-PyrimidineDerivative Targets Human Primary AML Stem Cells in Vivo.Sci Transl Med 2013,5(181)；Pabst C,Krosl J,Fares I,Boucher G,Ruel R,Marinier A,Lemieux S,Hebert J,Sauvageau G:Identification of small molecules that support human leukemiastem cell activity ex vivo.Nat Methods 2014,11(4):436-442)。1994年，Lapido等率先从AML患者骨髓细胞中分离出CD34⁺CD38^-LSCs亚群，并证实此亚群具有HSCs样强大的自我更新和增殖能力(Lapidot T,Sirard C,Vormoor J,Murdoch B,Hoang T,CacerescortesJ,Minden M,Paterson B,Caligiuri MA,Dick JE:A Cell Initiating Human AcuteMyeloid-Leukemia after Transplantation into Scid Mice.Nature 1994,367(6464):645-648)。该亚群96％以上的细胞处于G0期，能逃逸化学药物的杀伤作用，导致耐药和易复发(Ishikawa F,Yoshida S,Saito Y,Hijikata A,Kitamura H,Tanaka S,Nakamura R,Tanaka T,Tomiyama H,Saito N et al:Chemotherapy-resistant human AML stem cellshome to and engraft within the bone-marrow endosteal region.Nat Biotechnol2007,25(11):1315-1321)。2014年，John E.Dick等发现25％的AML病人具有DNMT3a基因突变，而该基因突变导致pre-LSCs形成，其功能与正常HSCs相似，但却异常生长(Shlush LI,Zandi S,Mitchell A,Chen WC,Brandwein JM,Gupta V,Kennedy JA,Schimmer AD,SchuhAC,Yee KW et al:Identification of pre-leukaemic haematopoietic stem cells inacute leukaemia.Nature 2014,508(7496):420-420)。这些存在于骨髓的pre-LSCs对化疗等疗法不敏感，可能最终获得其他一些改变而导致AML的复发。上述研究表明，LSCs是AML发病、发展和复发的主要原因。因此，探索和发现有效抑制LSCs活性的新分子靶点从而开发相应的治疗药物，有望从根本上达到控制白血病的最终目的。Studies have shown that leukemia stem cells (Leukemia stem cells, LSCs) play a key role in the occurrence, development and recurrence of AML (Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, Nakamura R, Tanaka T, Tomiyama H, Saito N et al: Chemotherapy-resistant human AMLstem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007, 25(11): 1315-1321; Saito Y, Yuki H, Kuratani M, Hashizume Y, Takagi S ,Honma T,Tanaka A,Shirouzu M,Mikuni J,Handa N et al:A Pyrrolo-PyrimidineDerivative Targets Human Primary AML Stem Cells in Vivo.Sci Transl Med 2013,5(181);Pabst C,Krosl J,Fares I, Boucher G, Ruel R, Marinier A, Lemieux S, Hebert J, Sauvageau G: Identification of small molecules that support human leukemiastem cell activity ex vivo. Nat Methods 2014,11(4):436-442). In 1994, Lapido et al. first isolated the CD34⁺ CD38^- LSCs subpopulation from the bone marrow cells of AML patients, and confirmed that this subpopulation has HSCs-like strong self-renewal and proliferation capabilities (Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Cacerescortes J, Minden M, Paterson B, Caligiuri MA, Dick JE: A Cell Initiating Human Acute Myeloid-Leukemia after Transplantation into Scid Mice. Nature 1994, 367(6464):645-648). More than 96% of the cells in this subpopulation are in the G0 phase, which can escape the killing effect of chemical drugs, resulting in drug resistance and easy relapse (Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, Nakamura R, Tanaka T , Tomiyama H, Saito N et al: Chemotherapy-resistant human AML stem cellshome to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007, 25(11): 1315-1321). In 2014, John E. Dick et al. found that 25% of AML patients had DNMT3a gene mutations, which led to the formation of pre-LSCs, which functioned similarly to normal HSCs but grew abnormally (Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V, Kennedy JA, Schimmer AD, Schuh AC, Yee KW et al: Identification of pre-leukaemic haematopoietic stem cells inacute leukaemia. Nature 2014, 508(7496):420-420). These pre-LSCs present in the bone marrow are insensitive to therapies such as chemotherapy and may eventually acquire other changes that lead to AML recurrence. The above studies show that LSCs are the main cause of AML pathogenesis, development and recurrence. Therefore, exploring and discovering new molecular targets that effectively inhibit the activity of LSCs to develop corresponding therapeutic drugs is expected to fundamentally achieve the ultimate goal of controlling leukemia.

大量研究表明，细胞表面分子在LSCs的活性和AML的发生、发展中扮演着重要的角色(Zheng JK,Umikawa M,Cui CH,Li JY,Chen XL,Zhang CZ,Huynh H,Kang XL,SilvanyR,Wan X et al:Inhibitory receptors bind ANGPTLs and support blood stem cellsand leukaemia development.Nature 2012,488(7413):684-684；Majeti R,Chao MP,Alizadeh AA,Pang WW,Jaiswal S,Gibbs KD,van Rooijen N,Weissman IL:CD47 Is anAdverse Prognostic Factor and Therapeutic Antibody Target on Human AcuteMyeloid Leukemia Stem Cells.Cell 2009,138(2):286-299；van Rhenen A,van DongenGAMS,Kelder A,Rombouts EJ,Feller N,Moshaver B,Stigter-van WalsumM,Zweegman S,Ossenkoppele GJ,Schuurhuis GJ:The novel AML stem cell-associated antigen CLL-1aids in discrimination between normal and leukemic stem cells.Blood 2007,110(7):2659-2666；Kikushige Y,Shima T,Takayanagi S,Urata S,Miyamoto T,Iwasaki H,Takenaka K,Teshima T,Tanaka T,Inagaki Y et al:TIM-3Is a Promising Target toSelectively Kill Acute Myeloid Leukemia Stem Cells.Cell Stem Cell 2010,7(6):708-717；Kang XL,Lu ZG,Cui CH,Deng M,Fan YQ,Dong BJ,Han X,Xie FC,Tyner JW,Coligan JE et al:The ITIM-containing receptor LAIR1 is essential for acutemyeloid leukaemia development.Nat Cell Biol 2015,17(5):665-U286)，而在LSCs中特异性表达的细胞表面分子可以作为靶向治疗AML的靶点。以细胞表面分子作为靶点的AML靶向治疗研究具有十分重要的现实临床意义：一方面，利用LSCs中特异性表达的细胞表面分子可以进行AML的病程监控和预后判断；另一方面，以LSCs中特异性表达的细胞表面分子作为靶点可以开发更加有效、甚至靶向性治愈AML的新治疗方法。近年来，随着人们对LSCs研究深入，发现CD33(Hauswirth AW,Florian S,Printz D,Sotlar K,Krauth MT,Fritsch G,Schernthaner GH,Wacheck V,Selzer E,Sperr WR et al:Expression of the targetreceptor CD33 in CD34(+)/CD38(-)/CD123(+)AML stem cells.Eur J Clin Invest2007,37(1):73-82)、CD47(Majeti R,Chao MP,Alizadeh AA,Pang WW,Jaiswal S,GibbsKD,van Rooijen N,Weissman IL:CD47 Is an Adverse Prognostic Factor andTherapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells.Cell2009,138(2):286-299)、CD96(Hosen N,Park CY,Tatsumi N,Oji Y,Sugiyama H,Gramatzki M,Krensky AM,Weissman IL:CD96 is a leukemic stem cell-specificmarker in human acute myeloid leukemia.P Natl Acad Sci USA 2007,104(26):11008-11013)、CD123(Florian S,Sonneck K,Hauswirth AW,Krauth MT,SchernthanerGH,Sperr WR,Valent P:Detection of molecular targets on the surface of CD34+/CD38-stem cells in various myeloid malignancies.Leukemia Lymphoma 2006,47(2):207-222；Hwang K,Park CJ,Jang S,Chi HS,KimDY,Lee JH,Lee JH,Lee KH,Im HJ,SeoJJ:Flow cytometric quantification and immunophenotyping of leukemic stemcells in acute myeloid leukemia.Ann Hematol 2012,91(10):1541-1546；Abdel-WahabO,Mullally A,Hedvat C,Garcia-Manero G,Patel J,Wadleigh M,Malinge S,Yao JJ,Kilpivaara O,Bhat R et al:Genetic characterization of TET1,TET2,and TET3alterations in myeloid malignancies.Blood 2009,114(1):144-147)、TIM-3(Kikushige Y,Shima T,Takayanagi S,Urata S,Miyamoto T,Iwasaki H,Takenaka K,Teshima T,Tanaka T,Inagaki Y et al:TIM-3Is a Promising Target to SelectivelyKill Acute Myeloid Leukemia Stem Cells.Cell Stem Cell 2010,7(6):708-717)和CLL-1(van Rhenen A,van Dongen GAMS,Kelder A,Rombouts EJ,Feller N,Moshaver B,Stigter-van Walsum M,Zweegman S,Ossenkoppele GJ,Schuurhuis GJ:The novel AMLstem cell-associated antigen CLL-1aids in discrimination between normal andleukemic stem cells.Blood 2007,110(7):2659-2666)等细胞表面分子的差异表达可以区分LSCs与HSCs。然而，上述细胞表面分子在LSCs中或弱表达或部分表达，或者在HSCs中也部分表达，使其作为治疗靶点存在一定局限性。因此，需要进一步探索更加有效的细胞表面分子作为新的靶点分子。A large number of studies have shown that cell surface molecules play an important role in the activity of LSCs and the occurrence and development of AML (Zheng JK, Umikawa M, Cui CH, Li JY, Chen XL, Zhang CZ, Huynh H, Kang XL, SilvanyR, Wan X et al: Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature 2012, 488(7413):684-684; Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, van Rooijen N , Weissman IL: CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells. Cell 2009, 138(2): 286-299; van Rhenen A, van DongenGAMS, Kelder A, Rombouts EJ, Feller N, Moshaver B ,Stigter-van WalsumM,Zweegman S,Ossenkoppele GJ,Schuurhuis GJ:The novel AML stem cell-associated antigen CLL-1aids in discrimination between normal and leukemic stem cells.Blood 2007,110(7):2659-2666; , Shima T, Takayanagi S, Urata S, Miyamoto T, Iwasaki H, Takenaka K, Teshima T, Tanaka T, Inagaki Y et al: TIM-3 Is a Promising Target to Selectively Kill Acute Myeloid Leukemia Stem Cells. Cell Stem Cell 2010, 7( 6):708-717; Kang XL, Lu ZG, Cui CH, Deng M, Fan YQ, Dong BJ, Han X, Xie FC, Tyner JW, Coligan JE et al: The ITIM-containing receptor LAIR1 is essential for acutemyeloid leukaemia development.Nat Cell Biol 2015,17(5):665-U286), and cell surface molecules specifically expressed in LSCs can be used as targets for targeted therapy of AML. The study of AML targeted therapy using cell surface molecules as targets has very important practical and clinical significance: on the one hand, the use of cell surface molecules specifically expressed in LSCs can be used to monitor the course of AML and judge the prognosis; on the other hand, using LSCs Cell surface molecules specifically expressed in cells can be used as targets to develop new treatments that are more effective and even targeted to cure AML. In recent years, with the in-depth study of LSCs, it was found that CD33 (Hauswirth AW, Florian S, Printz D, Sotlar K, Krauth MT, Fritsch G, Schernthaner GH, Wacheck V, Selzer E, Sperr WR et al: Expression of the target receptor CD33 in CD34(+)/CD38(-)/CD123(+) AML stem cells. Eur J Clin Invest2007,37(1):73-82), CD47 (Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, GibbsKD, van Rooijen N, Weissman IL: CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells. Cell2009,138(2):286-299), CD96 (Hosen N, Park CY, Tatsumi N , Oji Y, Sugiyama H, Gramatzki M, Krensky AM, Weissman IL: CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia.P Natl Acad Sci USA 2007,104(26):11008-11013), CD123(Florian S, Sonneck K, Hauswirth AW, Krauth MT, SchernthanerGH, Sperr WR, Valent P: Detection of molecular targets on the surface of CD34+/CD38-stem cells in various myeloid malignancies. Leukemia Lymphoma 2006,47(2):207-222 ; Hwang K, Park CJ, Jang S, Chi HS, KimDY, Lee JH, Lee JH, Lee KH, Im HJ, SeoJJ: Flow cytometric quantification and immunophenotyping of leukemic stemcells in acute myeloid leukemia. Ann Hematol 2012, 91(10) :1541-1546; Abdel-Wahab O, Mullally A, Hedvat C, Garcia-Manero G, Patel J, Wadleigh M, Malinge S, Yao JJ, Kilpivaara O, Bhat R et al: Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood 2009, 114(1):144-147), TIM-3 (Kikushige Y, Shima T, Takayanagi S, Urata S, Miyamoto T, Iwasaki H, Takenaka K, Teshima T, Tanaka T, Inagaki Y et al al:TIM-3Is a Promising Target to SelectivelyKill Acute Myeloid Leukemia Stem Cells.Cell Stem Cell 2010,7(6):708-717) and CLL-1(van Rhenen A,van Dongen GAMS,Kelder A,Rombouts EJ,Feller N, Moshaver B, Stigter-van Walsum M, Zweegman S, Ossenkoppele GJ, Schuurhuis GJ: The novel AMLstem cell-associated antigen CLL-1aids in discrimination between normal and leukemic stem cells. Blood 2007,110(7):2659-2666) Differential expression of such cell surface molecules can distinguish LSCs from HSCs. However, the above cell surface molecules are either weakly or partially expressed in LSCs, or also partially expressed in HSCs, which limits their use as therapeutic targets. Therefore, it is necessary to further explore more effective cell surface molecules as new target molecules.

Allergin-1，别称MILR1，是一种属于免疫球蛋白样受体超家族的免疫抑制性受体，在人和小鼠主要表达于巨噬细胞、中性粒细胞、肥大细胞等髓系细胞中。Allergin-1参与体内多种病理生理过程(Hitomi K,Tahara-Hanaoka S,Someya S,Fujiki A,Tada H,Sugiyama T,Shibayama S,Shibuya K,Shibuya A:An immunoglobulin-like receptor,Allergin-1,inhibits immunoglobulin E-mediated immediate hypersensitivityreactions.Nat Immunol.2010,11(7):601-607)，然而，Allergin-1在LSCs和AML细胞中的表达特性，Allergin-1在AML发生、发展中的生物学作用，以及以Allergin-1作为生物标志物或分子靶点探讨AML的病程监控、预后判断、靶向治疗和免疫治疗可行性的研究，尚未见报道。Allergin-1, also known as MILR1, is an immunosuppressive receptor belonging to the immunoglobulin-like receptor superfamily. It is mainly expressed in myeloid cells such as macrophages, neutrophils, and mast cells in humans and mice. . Allergin-1 is involved in various pathophysiological processes in vivo (Hitomi K, Tahara-Hanaoka S, Someya S, Fujiki A, Tada H, Sugiyama T, Shibayama S, Shibuya K, Shibuya A: An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions.Nat Immunol.2010,11(7):601-607), however, the expression characteristics of Allergin-1 in LSCs and AML cells, and the biology of Allergin-1 in the occurrence and development of AML The role of Allergin-1 as a biomarker or molecular target to explore the feasibility of AML disease course monitoring, prognosis judgment, targeted therapy and immunotherapy has not been reported yet.

因此，急需深入研究Allergin-1作为新的生物标志物对于白血病的生物学效应，以获得用于高特异性诊断白血病的方法和体系以及相关的靶向治疗和免疫治疗的靶点、方法和药物。Therefore, there is an urgent need to study the biological effects of Allergin-1 as a new biomarker on leukemia in order to obtain methods and systems for highly specific diagnosis of leukemia, as well as targets, methods and drugs for related targeted therapy and immunotherapy .

发明内容Contents of the invention

针对以上技术问题，我们发现：Allergin-1在正常HSCs中不表达，而在LSCs和AML单核细胞中特异性异常高表达；在M4和M5亚型AML患者中Allergin-1的表达水平与患者的生存率呈负相关，尤其在生存率极低的M5亚型中特异性异常高表达；Allergin-1⁺LSCs自我更新能力显著高于Allergin-1^-LSCs；Allergin-1⁺AML细胞晚期髓细胞晚期分化标志CD11b的表达水平显著底于Allergin-1^-AML细胞；IFN-γ促进AML细胞的增殖及Allergin-1的表达水平；敲降Allergin-1显著抑制AML细胞增殖能力、显著促进AML细胞CD11b的表达水平、显著抑制AML细胞的体内定植(或体内生长)；在体外，激发Allergin-1抗体介导的效应细胞对AML细胞的细胞毒作用；在体内，Allergin-1抗体可介导效应细胞消除AML的根源性LSCs和AML单核细胞；Allergin-1^-AML细胞促进正常人外周血单核细胞来源CD3^-CD56⁺NK细胞的CD25(活化NK细胞标志)的表达水平，并促进CD3^-CD56⁺NK细胞的细胞毒作用。基于此，Allergin-1作为生物标志物用于LSCs的鉴别、白血病(尤其是AML)的诊断、病程监控、预后判断，是用于白血病尤其是AML靶向治疗和/或免疫治疗的靶标。In response to the above technical problems, we found that: Allergin-1 is not expressed in normal HSCs, but is abnormally highly expressed in LSCs and AML monocytes; the expression level of Allergin-1 in M4 and M5 subtype AML patients is similar to that of patients The survival rate of AML is negatively correlated, especially in the M5 subtype with extremely low survival rate; the self-renewal ability of Allergin-1⁺ LSCs is significantly higher than that of Allergin-1^- LSCs; Allergin-1⁺ AML cells are late myeloid cells The expression level of late differentiation marker CD11b was significantly lower than that of Allergin-1^- AML cells; IFN-γ promoted the proliferation of AML cells and the expression level of Allergin-1; knocking down Allergin-1 significantly inhibited the proliferation of AML cells and significantly promoted the proliferation of AML cells CD11b The expression level of AML cells significantly inhibits the colonization (or growth) of AML cells in vivo; in vitro, stimulates the cytotoxic effect of Allergin-1 antibody-mediated effector cells on AML cells; in vivo, Allergin-1 antibody can mediate effector cells Eliminates the root-causing LSCs of AML and AML monocytes; Allergin-1^- AML cells promote the expression level of CD25 (a marker of activated NK cells) in normal human peripheral blood mononuclear cell-derived CD3^- CD56⁺ NK cells, and promote the expression level of CD3^- CD56⁺ Cytotoxicity of NK cells. Based on this, Allergin-1 is used as a biomarker for the identification of LSCs, the diagnosis of leukemia (especially AML), the monitoring of disease course, and the judgment of prognosis, and it is a target for targeted therapy and/or immunotherapy of leukemia, especially AML.

在本发明的一个方面，本发明涉及通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来鉴别LSCs的方法，其中Allergin-1在LSCs中特异性异常高表达。In one aspect of the invention, the invention relates to a method for identifying LSCs by detecting the expression or activity of the biomarker Allergin-1 in a subject or a sample from a subject, wherein Allergin-1 is specifically abnormal in LSCs High expression.

在本发明的一个方面，本发明涉及通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来诊断、病程监控和预后判断白血病(尤其是AML)或用于确定发生白血病(尤其是AML)的风险或用于确定白血病(尤其是AML)严重程度的的方法，其中在M4和M5亚型AML患者中Allergin-1的表达水平与患者的生存率呈负相关，在LSCs和AML单核细胞中Allergin-1异常高表达。在一些实施方案中，Allergin-1在AML患者中高表达，尤其在M5亚型中特异性异常高表达。In one aspect of the present invention, the present invention relates to the diagnosis, course monitoring and prognosis of leukemia (especially AML) by detecting the expression or activity of the biomarker Allergin-1 in a subject or a sample from a subject or using A method for determining the risk of developing leukemia (especially AML) or for determining the severity of leukemia (especially AML), wherein the expression level of Allergin-1 in M4 and M5 subtype AML patients is negatively correlated with the patient's survival rate Related, Allergin-1 is abnormally high expressed in LSCs and AML monocytes. In some embodiments, Allergin-1 is highly expressed in AML patients, especially abnormally highly expressed in the M5 subtype.

在本发明的一个方面，本发明涉及一种鉴别LSCs的方法，所述方法包括通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来鉴别LSCs；In one aspect of the present invention, the present invention relates to a method of identifying LSCs, said method comprising identifying LSCs by detecting the expression or activity of the biomarker Allergin-1 in a subject or a sample from a subject;

任选地，通过检测所述样本中造血干细胞中的所述生物标志物Allergin-1的表达或活性；Optionally, by detecting the expression or activity of the biomarker Allergin-1 in hematopoietic stem cells in the sample;

任选地，Allergin-1在造血干细胞中的表达水平或活性显著高于正常造血干细胞中Allergin-1的表达水平或活性，是造血干细胞为LSCs的指示。Optionally, the expression level or activity of Allergin-1 in hematopoietic stem cells is significantly higher than the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the hematopoietic stem cells are LSCs.

在上述方法中，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的至少约3倍以上，是造血干细胞为LSCs的指示，优选的，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的约3倍-约100倍，更优选地，为约10-90倍、为约20-80倍、为约30-70倍，例如40倍、45倍、50倍、55倍、60倍。In the above method, the expression level or activity of Allergin-1 in hematopoietic stem cells is at least about 3 times the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the hematopoietic stem cells are LSCs, preferably, The expression level or activity of Allergin-1 in hematopoietic stem cells is about 3 times to about 100 times that of Allergin-1 in normal hematopoietic stem cells, more preferably about 10-90 times, about 20 times -80 times, about 30-70 times, such as 40 times, 45 times, 50 times, 55 times, 60 times.

在本发明的一个方面，本发明涉及检测样本中Allergin-1表达水平或活性的试剂在制备鉴别LSCs的产品中的应用。In one aspect of the present invention, the present invention relates to the application of a reagent for detecting the expression level or activity of Allergin-1 in a sample in the preparation of a product for identifying LSCs.

在本发明的一个方面，本发明涉及检测样本中Allergin-1表达水平或活性的试剂在制备用于白血病(尤其是AML)的诊断、病程监控和预后判断的产品中的应用。In one aspect of the present invention, the present invention relates to the application of a reagent for detecting the expression level or activity of Allergin-1 in a sample in the preparation of products for diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML).

优选的，所述检测样本中Allergin-1表达水平或活性的试剂与一种或多种其他试剂组合。Preferably, the reagent for detecting the expression level or activity of Allergin-1 in a sample is combined with one or more other reagents.

更优选的，所述一种或多种其他试剂为IFN-γ。More preferably, the one or more other agents are IFN-γ.

在本发明的一个方面，本发明涉及用于检测Allergin-1和至少一种生物标志物的组合的试剂在制备用于鉴别LSCs的产品中的应用。在一些实施方案中，所述Allergin-1和至少一种生物标志物的组合为Allergin-1⁺CD34⁺CD38^-。In one aspect of the invention, the invention relates to the use of a reagent for detecting a combination of Allergin-1 and at least one biomarker for the manufacture of a product for the identification of LSCs. In some embodiments, the combination of Allergin-1 and at least one biomarker is Allergin-1⁺ CD34⁺ CD38⁻ .

在本发明的一个方面，本发明涉及用于检测Allergin-1和至少一种生物标志物的组合的试剂在制备用于白血病(尤其是AML)的诊断、病程监控和预后判断的产品中的应用。在一些实施方案中，所述Allergin-1和至少一种生物标志物的组合为Allergin-1⁺CD34⁺CD38^-。In one aspect of the present invention, the present invention relates to the application of the reagent for detecting the combination of Allergin-1 and at least one biomarker in the preparation of products for the diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML) . In some embodiments, the combination of Allergin-1 and at least one biomarker is Allergin-1⁺ CD34⁺ CD38⁻ .

优选的，上文所述的试剂包括但不限于用于RT-PCR、实时定量PCR、免疫检测(例如ELISA、RIA、多重免疫实验、免疫荧光实验、蛋白印记、或斑点印记实验)、原位杂交或芯片技术检测Allergin-1表达水平用的试剂。更优选的，所述试剂包括针对Allergin-1基因的引物/探针、分子信标或针对Allergin-1蛋白的抗体和/或配体、以及小分子化合物。Preferably, the reagents described above include but are not limited to RT-PCR, real-time quantitative PCR, immunoassay (such as ELISA, RIA, multiplex immunoassay, immunofluorescence assay, Western blot, or dot blot assay), in situ Reagents for detecting the expression level of Allergin-1 by hybridization or chip technology. More preferably, the reagents include primers/probes against Allergin-1 gene, molecular beacons or antibodies and/or ligands against Allergin-1 protein, and small molecule compounds.

优选的，上文所述的产品包括但不限于：芯片、试剂、试纸、制剂、试剂盒或高通量筛选平台。Preferably, the above-mentioned products include but are not limited to: chips, reagents, test strips, preparations, kits or high-throughput screening platforms.

在本发明的一个方面，本发明涉及Allergin-1表达或活性的调节剂(抑制剂/拮抗剂)在制备用于治疗受试者的白血病(尤其是AML)的产品中的应用。在一些实施方案中，所述治疗为分子免疫治疗。In one aspect of the present invention, the present invention relates to the use of a modulator (inhibitor/antagonist) of Allergin-1 expression or activity in the manufacture of a product for treating leukemia (especially AML) in a subject. In some embodiments, the therapy is molecular immunotherapy.

在本发明的一个方面，本发明提供了了一种治疗白血病(尤其是AML)的药物或组合物，所述药物或组合物包括针对Allergin-1分子具备引起免疫效应细胞对AML细胞毒性反应的、和/或Allergin-1功能性表达的抑制剂/拮抗剂，和/或与所述抑制剂配伍的其他药类以及药学上可接受的载体和/或辅料。In one aspect of the present invention, the present invention provides a medicine or composition for the treatment of leukemia (especially AML), said medicine or composition comprising allergin-1 molecules capable of causing immune effector cells to AML cytotoxic response , and/or an inhibitor/antagonist of Allergin-1 functional expression, and/or other drugs compatible with the inhibitor, as well as pharmaceutically acceptable carriers and/or adjuvants.

上文所述的适合的抑制剂和/或拮抗剂是指任何可降低编码Allergin-1的核酸的表达、降低Allergin-1蛋白质水平，或抑制Allergin-1的活性的物质。例如，降低Allergin-1蛋白的活性、降低Allergin-1基因或蛋白的稳定性、下调Allergin-1的表达、减少Allergin-1蛋白有效作用时间、或抑制Allergin-1的转录和翻译的物质。Suitable inhibitors and/or antagonists mentioned above refer to any substance that can reduce the expression of nucleic acid encoding Allergin-1, reduce the protein level of Allergin-1, or inhibit the activity of Allergin-1. For example, substances that reduce the activity of Allergin-1 protein, reduce the stability of Allergin-1 gene or protein, down-regulate the expression of Allergin-1, reduce the effective time of Allergin-1 protein, or inhibit the transcription and translation of Allergin-1.

优选的，上文所述的抑制剂/或拮抗剂包括但不限于：核酸抑制物、蛋白抑制剂、蛋白水解酶、蛋白结合分子；及其组合。其中核酸抑制物选自：以Allergin-1或其转录本为靶序列、且能够抑制Allergin-1基因表达或基因转录的干扰分子，包括：shRNA(小发夹RNA)、小干扰RNA(siRNA)、dsRNA、微小RNA，或能表达或形成所述shRNA、小干扰RNA、dsRNA、微小RNA的构建物。蛋白结合分子选自：与Allergin-1蛋白特异性结合的物质，如能够抑制Allergin-1蛋白活性的抗体或配体；Allergin-1配体结合位点的竞争剂，包括Allergin-1受体与其配体结合片段、可溶性截短的Allergin-1受体、可溶性Allergin-1受体融合蛋白，例如包含IgG免疫球蛋白的Fc部分的Allergin-1融合蛋白、配体融合蛋白；拟肽；肽抑制剂；小分子化合物；及其组合。Preferably, the aforementioned inhibitors/antagonists include, but are not limited to: nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein-binding molecules; and combinations thereof. Wherein the nucleic acid inhibitor is selected from: interfering molecules that take Allergin-1 or its transcript as the target sequence and can inhibit Allergin-1 gene expression or gene transcription, including: shRNA (small hairpin RNA), small interfering RNA (siRNA) , dsRNA, microRNA, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microRNA. The protein-binding molecules are selected from: substances that specifically bind to the Allergin-1 protein, such as antibodies or ligands capable of inhibiting the activity of the Allergin-1 protein; competitors of the Allergin-1 ligand binding site, including Allergin-1 receptors and their Ligand-binding fragment, soluble truncated Allergin-1 receptor, soluble Allergin-1 receptor fusion protein, e.g., Allergin-1 fusion protein comprising the Fc portion of an IgG immunoglobulin, ligand fusion protein; peptidomimetic; peptide inhibitory agents; small molecule compounds; and combinations thereof.

更优选的，所述抑制剂/或拮抗剂为shRNA；尤其优选的，所述shRNA序列具有SEQID NO：12、13、14、15、16或17所示的序列。More preferably, the inhibitor/antagonist is shRNA; especially preferably, the shRNA sequence has the sequence shown in SEQ ID NO: 12, 13, 14, 15, 16 or 17.

更优选的，所述抑制剂/或拮抗剂为特异性与Allergin-1结合的抗体；优选的，例如，所述特异性抗体包括单克隆抗体、多克隆抗体、中和抗体、抗原结合片段、偶联Allergin-1抗体。More preferably, the inhibitor/or antagonist is an antibody that specifically binds to Allergin-1; preferably, for example, the specific antibody includes a monoclonal antibody, a polyclonal antibody, a neutralizing antibody, an antigen-binding fragment, Conjugated Allergin-1 antibody.

在本发明的一个方面，本发明提供了一种白血病(尤其是AML)的诊断、病程监控和预后判断的产品，所述产品包括检测Allergin-1表达水平的芯片、制剂或检测试剂盒。进一步，所述试剂盒包括至少一对特异性扩增Allergin-1基因的引物；优选的，所述引物具有SEQ ID NO：18、19、20、21、22或23所示的序列。In one aspect of the present invention, the present invention provides a product for the diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML), the product includes a chip, a preparation or a detection kit for detecting the expression level of Allergin-1. Further, the kit includes at least one pair of primers for specifically amplifying the Allergin-1 gene; preferably, the primers have the sequence shown in SEQ ID NO: 18, 19, 20, 21, 22 or 23.

在本发明的一个方面，本发明提供了一种生物标志物组合，其包括Allergin-1、CD34和CD38。In one aspect of the present invention, the present invention provides a biomarker combination comprising Allergin-1, CD34 and CD38.

在本发明的一个方面，本发明提供了生物标志物组合Allergin-1、CD34和CD38在鉴定LSC中应用。根据本发明的具体实施例，所述生物标志物中Allergin-1阳性、CD34阳性和CD38阴性是LSCs的指示。In one aspect of the present invention, the present invention provides the use of the biomarker combination Allergin-1, CD34 and CD38 in identifying LSC. According to a specific embodiment of the present invention, among the biomarkers, Allergin-1 positive, CD34 positive and CD38 negative are indicators of LSCs.

本发明的一个方面，提供了具备引起免疫效应细胞对AML细胞毒性反应的、或对AML细胞功能性表达降低的抑制剂/拮抗剂如Allergin-1单克隆抗体、Allergin-1中和抗体、抗肿瘤药偶联Allergin-1抗体、抗原结合片段，小分子化合物。One aspect of the present invention provides an inhibitor/antagonist capable of causing the cytotoxic response of immune effector cells to AML cells, or reducing the functional expression of AML cells, such as Allergin-1 monoclonal antibody, Allergin-1 neutralizing antibody, anti- Oncology drug conjugated Allergin-1 antibody, antigen-binding fragment, small molecule compound.

在本发明的一个方面，本发明提供了一种提高NK细胞活性或免疫治疗效果的方法，向受试者给予有效量的Allergin-1表达或活性的抑制剂或拮抗剂。In one aspect of the present invention, the present invention provides a method for improving the activity of NK cells or the effect of immunotherapy, by administering an effective amount of an inhibitor or antagonist of Allergin-1 expression or activity to a subject.

在本发明的一个方面，本发明提供了生物标志物Allergin-1在筛选治疗白血病(尤其是AML)的候选药物的方法。优选的，所述方法包括：用待筛选物质处理表达或含有Allergin-1基因的体系；和检测所述体系中Allergin-1基因的表达水平；其中，若所述待筛选的物质可以降低Allergin-1基因的表达水平，则表明该待筛选物质是治疗白血病(尤其是AML)的候选药物。更优选的，上文所述的体系包括(但不限于)：细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系；所述候选药物包括(但不限于)：针对Allergin-1基因或其上游或下游基因设计的干扰分子、核酸抑制物、小分子化合物。In one aspect of the present invention, the present invention provides a method for screening a candidate drug for the treatment of leukemia (especially AML) using the biomarker Allergin-1. Preferably, the method includes: treating the system expressing or containing the Allergin-1 gene with the substance to be screened; and detecting the expression level of the Allergin-1 gene in the system; wherein, if the substance to be screened can reduce the Allergin-1 gene expression level; 1 gene expression level, it indicates that the substance to be screened is a candidate drug for treating leukemia (especially AML). More preferably, the systems described above include (but are not limited to): cell systems, subcellular systems, solution systems, tissue systems, organ systems or animal systems; the candidate drugs include (but are not limited to): targeting Allergin- 1 Interfering molecules, nucleic acid inhibitors, and small molecular compounds designed for genes or their upstream or downstream genes.

附图说明Description of drawings

图1是检测Allergin-1在AML患者中的表达情况图，其中：Figure 1 is a diagram showing the detection of the expression of Allergin-1 in AML patients, wherein:

图1A表明Allergin-1的表达水平与AML患者的生存率呈负相关；Figure 1A shows that the expression level of Allergin-1 is negatively correlated with the survival rate of AML patients;

图1B表明Allergin-1在M5亚型AML患者中异常高表达；Figure 1B shows that Allergin-1 is abnormally highly expressed in patients with M5 subtype AML;

图2是检测Allergin-1在健康脐带血细胞(HSCs)和原发AML患者的LSCs中的表达情况图，其中：Figure 2 is a graph showing the detection of Allergin-1 expression in healthy umbilical cord blood cells (HSCs) and LSCs of patients with primary AML, wherein:

图2A和2C表明Allergin-1在健康人脐带血CD34⁺CD38^-，CD34⁺CD38⁺和CD34^-细胞中的表达；Figure 2A and 2C show the expression of Allergin-1 in healthy human cord blood CD34⁺ CD38^- , CD34⁺ CD38⁺ and CD34^- cells;

图2B和2D表明Allergin-1在原发AML患者骨髓CD34⁺CD38^-，CD34⁺CD38⁺和CD34^-细胞中的表达；Figure 2B and 2D show the expression of Allergin-1 in the bone marrow CD34⁺ CD38^- , CD34⁺ CD38⁺ and CD34^- cells of patients with primary AML;

图2E表明Allergin-1在健康人和原发AML患者单核细胞中的表达；Figure 2E shows the expression of Allergin-1 in monocytes of healthy people and patients with primary AML;

图3是检测Allergin-1阳性的LSCs的自我更新能力的情况图；Fig. 3 is a situation diagram of detecting the self-renewal ability of Allergin-1 positive LSCs;

图4是检测Allergin-1的表达与AML细胞的增殖和分化相关性的情况图，其中：Figure 4 is a situation diagram of detecting the correlation between the expression of Allergin-1 and the proliferation and differentiation of AML cells, wherein:

图4A和图4B表明Allergin-1阳性AML细胞的增殖能力显著高于Allergin-1阴性AML细胞；Figure 4A and Figure 4B show that the proliferation ability of Allergin-1 positive AML cells is significantly higher than that of Allergin-1 negative AML cells;

图4C和图4D表明Allergin-1阳性AML细胞的CD11b表达水平显著低于Allergin-1阴性AML细胞；Figure 4C and Figure 4D show that the CD11b expression level of Allergin-1 positive AML cells is significantly lower than that of Allergin-1 negative AML cells;

图5是检测shRNA对Allergin-1表达的干扰的效果图，其中：Figure 5 is an effect diagram of detecting the interference of shRNA on the expression of Allergin-1, wherein:

图5A和图5B通过Western blot法检测Thp-1(A)和U937(B)细胞敲降效率；Figure 5A and Figure 5B detect the knockdown efficiency of Thp-1 (A) and U937 (B) cells by Western blot method;

图5C和图5D表明敲降Allergin-1抑制Thp-1(C)和U937(D)细胞的增殖；Figure 5C and Figure 5D show that knocking down Allergin-1 inhibits the proliferation of Thp-1 (C) and U937 (D) cells;

图5E和图5F表明敲降Allergin-1抑制Thp-1(E)和U937(F)细胞的CD11b表达水平；Figure 5E and Figure 5F show that knocking down Allergin-1 inhibits the CD11b expression levels of Thp-1 (E) and U937 (F) cells;

图5G和图5H通过瑞氏-吉姆萨染色检测敲降Allergin-1对Thp-1(G)和U937(H)细胞形态的影响；Figure 5G and Figure 5H detected the effect of knocking down Allergin-1 on the morphology of Thp-1(G) and U937(H) cells by Wright-Giemsa staining;

图6是检测IFN-γ对AML细胞的Allergin-1表达水平和增殖的影响的情况图，其中：Figure 6 is a situation diagram of detecting the effect of IFN-γ on the expression level of Allergin-1 and the proliferation of AML cells, wherein:

图6A和图6B表明IFN-γ促进AML细胞的增殖(图6A)和Allergin-1的表达水平(图6B)；Figure 6A and Figure 6B show that IFN-γ promotes the proliferation of AML cells (Figure 6A) and the expression level of Allergin-1 (Figure 6B);

图6C表明IFN-γ预处理的Allergin-1⁺AML细胞的增殖显著高于对照组Allergin-1^-AML细胞；Figure 6C shows that the proliferation of Allergin-1⁺ AML cells pretreated with IFN-γ was significantly higher than that of control Allergin-1^- AML cells;

图7是测定敲降Allergin-1抑制AML细胞在小鼠体内的定植的情况图，其中：Figure 7 is a diagram of the determination of knockdown of Allergin-1 inhibiting the colonization of AML cells in mice, wherein:

图7A表明Allergin-1敲降的Thp-1细胞移植的小鼠肝脏及脾脏的重量显著小于对照组；Figure 7A shows that the weight of the liver and spleen of mice transplanted with Allergin-1 knockdown Thp-1 cells was significantly smaller than that of the control group;

图7B表明Allergin-1敲降的Thp-1细胞在小鼠肝脏和脾脏细胞中的定植能力显著低于对照组细胞；Figure 7B shows that the colonization ability of Allergin-1 knockdown Thp-1 cells in mouse liver and spleen cells is significantly lower than that of control cells;

图8是检测Allergin-1抗体依赖效应细胞对AML细胞的细胞毒作用(ADCC)的情况图，其中：Figure 8 is a diagram of the detection of the cytotoxicity (ADCC) of Allergin-1 antibody-dependent effector cells on AML cells, wherein:

左图通过流式细胞仪检测Allergin-1在Thp-1细胞的表达；右图表明Allergin-1抗体介导的外周血单核细胞(PBMCs)对Thp-1细胞的细胞毒作用显著高于对照组IgG1抗体；The left figure detects the expression of Allergin-1 in Thp-1 cells by flow cytometry; the right figure shows that the cytotoxicity of peripheral blood mononuclear cells (PBMCs) to Thp-1 cells mediated by Allergin-1 antibody is significantly higher than that of the control Group IgG1 antibody;

图9是检测Allergin-1抗体在NOD/SCID小鼠体内消除原发AML患者骨髓细胞的效果图，其中，Figure 9 is a diagram showing the effect of detecting Allergin-1 antibody in NOD/SCID mice to eliminate the bone marrow cells of patients with primary AML, wherein,

图9A是Allergin-1抗体处理原发AML患者骨髓细胞移植的人鼠异种移植模型小鼠示意图；Figure 9A is a schematic diagram of Allergin-1 antibody treatment of human-mouse xenograft model mice transplanted with bone marrow cells from patients with primary AML;

图9B表明Allergin-1抗体处理组小鼠肝脏细胞中人CD45⁺细胞所占比例显著少于对照组IgG1抗体处理组；Figure 9B shows that the proportion of human CD45⁺ cells in the mouse liver cells of the Allergin-1 antibody treatment group was significantly less than that of the IgG1 antibody treatment group in the control group;

图9C-图9D表明Allergin-1抗体处理组小鼠骨髓细胞中：人CD45⁺细胞(图D左上)，人CD45⁺细胞中CD34⁺细胞(图C上，图D右上)，人CD45⁺细胞中的CD34⁺CD38^-细胞(图C下，图D左下)和人CD45⁺细胞中Allergin-1⁺细胞(图C中，图D右下)所占比率显著少于对照组IgG1抗体处理组；Figure 9C-Figure 9D shows that in bone marrow cells of mice treated with Allergin-1 antibody: human CD45⁺ cells (upper left in Figure D), CD34⁺ cells in human CD45⁺ cells (upper Figure C, upper right in Figure D), human CD45⁺ cells The proportion of CD34⁺ CD38^- cells in human CD34 + CD38 - cells (bottom of Figure C, lower left of Figure D) and Allergin-1⁺ cells in human CD45⁺ cells (middle of Figure C, lower right of Figure D) was significantly less than that of the IgG1 antibody treatment group in the control group;

图10是检测Allergin-1抗体对健康人脐带血CD34阳性细胞在NOD/SCID小鼠体内造血重建能力的影响图，其中：Figure 10 is a graph showing the influence of Allergin-1 antibody on the hematopoietic reconstitution ability of healthy human umbilical cord blood CD34 positive cells in NOD/SCID mice, wherein:

图10A是Allergin-1抗体处理CD34⁺健康人脐带血细胞移植的人鼠异种移植模型小鼠示意图；Figure 10A is a schematic diagram of human-mouse xenograft model mice treated with Allergin-1 antibody for transplantation of CD34⁺ healthy human umbilical cord blood cells;

图10B-图10D表明，与对照组IgG1抗体处理组相比Allergin-1抗体处理组小鼠骨髓细胞中：人CD45⁺细胞(图10B)，人CD45⁺细胞中CD19⁺或CD33⁺细胞(图10C)所占比例无显著差异；Allergin-1抗体处理组小鼠骨髓的人CD45⁺细胞中Allergin-1⁺CD33⁺细胞所占比率明显减少于对照组IgG1抗体处理组(图10D)；Figure 10B-Figure 10D shows that, compared with the control group IgG1 antibody treatment group, in the mouse bone marrow cells of the Allergin-1 antibody treatment group: human CD45⁺ cells (Figure 10B), CD19⁺ or CD33⁺ cells in human CD45⁺ cells (Figure 1 10C) There was no significant difference in the proportion; the proportion of Allergin-1⁺ CD33⁺ cells in the human CD45⁺ cells in the mouse bone marrow of the Allergin-1 antibody treatment group was significantly lower than that of the IgG1 antibody treatment group in the control group (Fig. 10D);

图11是检测Allergin-1抑制健康人外周血单核细胞来源NK细胞的活性及细胞毒作用的效果图；Figure 11 is a diagram showing the effect of Allergin-1 inhibiting the activity and cytotoxicity of NK cells derived from peripheral blood mononuclear cells of healthy people;

图11A-D表明，与Allergin-1^-Thp-1细胞共培养的CD3^-CD56⁺NK细胞的CD25的表达水平(图11A-C)及细胞毒作用(图11D)显著高于与Allergin-1⁺Thp-1细胞共培养的CD3^-CD56⁺NK细胞；Figure 11A-D shows that the CD25 expression level (Figure 11A-C) and cytotoxicity (Figure 11D) of CD3^- CD56⁺ NK cells co-cultured with Allergin-1^- Thp-1 cells were significantly higher than those with Allergin-1 CD3^- CD56⁺ NK cells co-cultured with⁺ Thp-1 cells;

图12为Allergin-1基因的序列；Figure 12 is the sequence of the Allergin-1 gene;

图13是本发明实施例所使用的原发AML患者骨髓样本信息；Figure 13 is the bone marrow sample information of primary AML patients used in the embodiment of the present invention;

图14是以Allergin-1基因为靶点的shRNA序列；Figure 14 is the shRNA sequence targeting the Allergin-1 gene;

图15是扩增Allergin-1基因的引物序列。Figure 15 is the sequence of primers for amplifying Allergin-1 gene.

具体实施方式Detailed ways

定义definition

除非另有说明，本文使用的所有技术和科学术语具有与本发明所属领域的技术人员通常理解相同的含义，但如有冲突，则以本说明书中的定义为准。Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in case of conflict, the definitions in this specification shall prevail.

如说明书和权利要求书中所用，单数形式“一”、“一个”和“该(所述)”包括复数形式，除非上下文另有明确说明。As used in the specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

如无特殊说明，本说明书中的百分比(％)均为重量百分比(重量％)。Unless otherwise specified, the percentages (%) in this specification are all percentages by weight (% by weight).

在说明书和权利要求书中使用的涉及组分量、工艺条件等的所有数值或表述在所有情形中均应理解被“约”修饰。术语“约”当指数量或数值范围时，意思是所指数量或者数值范围是试验变异性内(或统计学实验误差内)的近似值，因此该数量或者数值范围可以在所述数量或数值范围的例如+5之间变化。All numbers or expressions referring to amounts of components, process conditions, etc. used in the specification and claims are to be understood as modified by "about" in all instances. The term "about" when referring to an amount or a numerical range means that the indicated amount or numerical range is an approximation within experimental variability (or within statistical experimental error) such that the amount or numerical range may be within the stated amount or numerical range Varies between eg+5 .

涉及相同组分或性质的所有范围均包括端点，该端点可独立地组合。由于这些范围是连续的，因此它们包括在最小值与最大值之间的每一数值。还应理解的是，本申请引用的任何数值范围预期包括该范围内的所有子范围。All ranges referring to the same component or property are inclusive of endpoints, which are independently combinable. Since these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all subranges within that range.

当本发明针对物理性质例如分子量或者针对化学性质以范围定义时，应包括范围的所有组合和亚组合以及其内的具体实施方式。术语“包含”(以及相关术语例如“含有”或“含”或“具有”或“包括”)包括这样一些实施方式，该实施方式为例如，物质、组合物、方法或过程等的任何组合，其“由所描述的特征组成”或者“基本上由所描述的特征组成”。When the invention is defined in ranges for a physical property, such as molecular weight, or for a chemical property, all combinations and subcombinations of ranges and embodiments therein are intended to be included. The term "comprising" (and related terms such as "comprising" or "containing" or "having" or "comprising") includes those embodiments which are, for example, any combination of substances, compositions, methods or processes, etc., It "consists of" or "consists essentially of" the described features.

本说明书和权利要求中使用的“和/或”，应当理解为相关联的组分“二者择一或二者”，即组分在一些情况中联合存在而在另一些情况中分开存在。多个用“和/或”列出的组分应当以同样的方式理解，即“一种或多种”相关联的组分。除了“和/或”从句具体确定的组分，其它组分可任选地存在，无论与那些具体确定的组分相关还是不相关。因此，作为非限制性实例，提及“A和/或B”，当用于连接开放式结尾的文字如“包括”，在一个实施方案中，可仅指A(任选地包括除B外的组分)；在另一实施方案，可仅指B(任选地包括除A外的组分)；在再一实施方案中，指A和B(任选的包括其它组分)等。"And/or" used in the specification and claims should be understood as "either or both" of the associated components, that is, the components exist jointly in some cases and exist separately in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, ie, "one or more" of the associated elements. Other components may optionally be present other than the components specifically identified by the "and/or" clause, whether related or unrelated to those components specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with an open-ended word such as "comprising", may, in one embodiment, refer to only A (optionally including other than B) In another embodiment, it may refer to only B (optionally including components other than A); in yet another embodiment, it may refer to A and B (optionally including other components) and so on.

应当理解，除非明确地相反指示，否则在本文要求保护的包括多于一步或一个行为的任何方法中，该方法的步骤和行为的顺序不必限制于所叙及的方法的步骤和行为的顺序。It should be understood that in any method claimed herein that includes more than one step or act, the order of the method steps and acts is not necessarily limited to the order of the method steps and acts recited, unless expressly indicated to the contrary.

本发明使用的缩写具有在化学、生物学和药学领域的通常含义。Abbreviations used herein have their usual meanings in the fields of chemistry, biology and pharmacy.

术语“有效量”或“治疗有效量”是指足以提供希望的生物结果的试剂的量。该结果可为疾病的征兆、症状或原因的减少和/或减轻，或任何其它希望的生物系统的变化。例如，治疗用途的“有效量”是指包含作为本发明活性成分的化合物的临床上显著减少疾病所需要的组合物的量。在任何个案中，适当的“有效”量可由本领域普通技术人员使用常规实验来测定。因此，表达方式“有效量”通常是指活性物质具有治疗效果时的量。The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to provide a desired biological result. The result may be reduction and/or alleviation of the signs, symptoms or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use refers to the amount of a composition comprising a compound of the present invention as an active ingredient required for a clinically significant reduction in disease. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Thus, the expression "effective amount" usually refers to the amount of active substance which has a therapeutic effect.

本申请使用的术语“治疗(treat)”或“处置(treatment)”与术语“预防(prevent)”同义，意在表示推迟疾病发展、防止疾病发展和/或降低将会发展或预期会发展的所述症状的严重性。因此，这些术语包括改善已有的疾病症状、预防另外的症状、改善或预防症状的潜在的代谢原因、抑制障碍或疾病，例如，阻止障碍或疾病的发展、减轻障碍或疾病、使障碍或疾病退行、减轻由疾病或障碍导致的病症，或使疾病或障碍的症状停止。As used herein, the terms "treat" or "treatment" are synonymous with the term "prevent" and are intended to mean delaying disease development, preventing disease development and/or reducing the severity of the symptoms. Accordingly, these terms include ameliorating existing disease symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, alleviating the disorder or disease, rendering the disorder or disease Regression, alleviation of a condition caused by a disease or disorder, or cessation of symptoms of a disease or disorder.

“疾病”、“障碍”和“病症”在本文中可互换地使用。"Disease", "disorder" and "condition" are used interchangeably herein.

除非另作说明，否则，本文使用的术语“治疗”包括受试者患有具体疾病、障碍或病症时所发生的作用，它降低疾病、障碍或病症的严重程度，或延迟或减缓疾病、障碍或病症的发展(“治疗性治疗”)，还包括受试者开始患有具体疾病、障碍或病症之前发生的作用(“预防性治疗”)。As used herein, unless otherwise specified, the term "treating" includes an effect on a subject suffering from a particular disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a disease, disorder or condition ("therapeutic treatment") and also includes effects that occur before a subject begins to suffer from a particular disease, disorder or condition ("prophylactic treatment").

术语“药用的”、“药理上可接受的”或“药学上可接受的”在本文中定义为那些在合理医学判断范围内适合接触个体(例如哺乳动物或人)的组织而不会产生过量毒性、刺激过敏性反应和其他并发症问题且具有与之相称的合理的效益/风险比的化合物、材料、试剂、组合物和/或剂型。The terms "pharmaceutically acceptable", "pharmacologically acceptable" or "pharmaceutically acceptable" are defined herein as those which, within the scope of sound medical judgment, are suitable for contacting the tissues of an individual (e.g., a mammal or a human) without producing Compounds, materials, agents, compositions and/or dosage forms that have a reasonable benefit/risk ratio commensurate with excessive toxicity, irritation of allergic reactions and other complications.

“施用”或“给药”在其应用于动物、人类、实验受试者、细胞、组织、器官或生物流体时是指外源性药物、治疗剂、诊断剂或组合物与受试者、细胞、组织、器官或生物流体的接触。"Administering" or "administering" when it is applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid refers to the interaction of an exogenous drug, therapeutic, diagnostic or composition with a subject, Contact of cells, tissues, organs or biological fluids.

本文中所使用的术语“共同施用”或“组合施用”定义为涵盖向单个患者施用所选治疗剂，且意指包括药物不一定通过相同途径施用或同时施用的治疗方案。The term "co-administration" or "administration in combination" as used herein is defined to encompass the administration of selected therapeutic agents to a single patient and is meant to include treatment regimens in which the drugs are not necessarily administered by the same route or at the same time.

本申请使用的术语“受试者”包括哺乳动物和非哺乳动物。哺乳动物的实例包括但不限于哺乳动物纲的任何成员：人、非人灵长类如黑猩猩及其它猿类和猴类；农场动物如牛、马、绵羊、山羊、猪；家养动物如兔、狗和猫；实验室动物，包括啮齿类动物如大鼠、小鼠和豚鼠等。非哺乳动物的实例包括但不限于鸟、鱼等。在本发明一个实施方案中，哺乳动物为人。The term "subject" as used herein includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, Dogs and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the invention the mammal is a human.

优选的，所述“受试者”包括人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人)和/或非人的动物，例如，哺乳动物，例如，灵长类(例如，食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人动物。本文可互换使用术语“人”、“患者”和“受试者”。Preferably, the "subject" includes a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, adolescent) or an adult subject (e.g., a young adult, middle-aged adults or older adults) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, Rodents, cats and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein tester".

术语“Allergin-1”是一种属于免疫球蛋白样受体超家族的免疫抑制性受体，又称为MILR1，其与程序性死亡分子1(PD-1)同属于免疫检查点分子。Allergin-1与PD-1有许多相似的特征，如表达模式、免疫受体酪氨酸抑制基序(ITIM)模式、可与酪氨酸磷酸酶SHP-1结合和可通过磷酸化及去磷酸化作用来调节细胞的信号传导等。SHP-1及其下游信号通路的活化对肿瘤的发展和维持肿瘤干细胞的特性具有重要作用。The term "Allergin-1" is an immunosuppressive receptor belonging to the immunoglobulin-like receptor superfamily, also known as MILR1, which belongs to the same immune checkpoint molecule as programmed death molecule 1 (PD-1). Allergin-1 shares many similar features with PD-1, such as expression pattern, immunoreceptor tyrosine inhibitory motif (ITIM) pattern, binding to tyrosine phosphatase SHP-1, and phosphorylation and dephosphorylation to regulate cellular signal transduction, etc. The activation of SHP-1 and its downstream signaling pathways play an important role in the development of tumors and the maintenance of the characteristics of cancer stem cells.

MILR1是位于人17号染色体上。本发明中的MILR1包括野生型、突变型或其片段。代表性的MILR1基因序列如目前国际公共核酸数据库GeneBank中MILR1基因(NC_000017.11)所示。MILR1基因序列也可如下文SEQ ID NO:1、2、3、4、5、6、7、8、9、10、或11所示。MILR1 is located onhuman chromosome 17. MILR1 in the present invention includes wild type, mutant type or fragments thereof. The representative MILR1 gene sequence is shown in the MILR1 gene (NC_000017.11) in the current international public nucleic acid database GeneBank. The MILR1 gene sequence can also be as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 below.

>XR_002957990.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X4]>XR_002957990.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X4]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGGGCCAGTTGCAATCCCCAAGATCCAGCAAGACTGCCTCGCCTTTCCACCCGACACCTGTTTTGAAGCATGAAATCGTGAAGCATACCGTGAAGAAGGTTCTCCCGTAGTTTCCCAGAAGTTTTTAATACGTTCTCAAGAAATGCTACTA(SEQ ID NO：1)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAA ATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTG CTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAA ACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCGT GATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGGGCCAGTTGCAATCCCCAAGATCCAGCAAGACTGCCTCGCCTTTCCACCCGACACCTGTTTTGAAGCATGAAATCGTGAAGCATACCGTGAA GAAGGTTCTCCCGTAGTTTCCCCAGAAGTTTTTAATACGTTTCCAAGAAATGCTACTA (SEQ ID NO: 1)

>XR_002957989.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X2]>XR_002957989.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X2]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGATGTGGCCAGTGACAACCCTCCTCAAGCCAGCATCTGTGACCTTTTTATATGACCCATTAGTCTTCAAGCAATTCTTTGCTTTTTAGCACCATAAAGTGTTTCAAACTCACCTTGTACTTTCCCTAACCCCAGACCCGGAATCAGCCATTTCTCCCAGTACTGGTTCATCTTAGAAACCAAGATCTGGAGGCCGGGCGCAGTGGC(SEQ ID NO：2)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAA ATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTG CTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAA ACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCGT GATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGATGTGGCCAGTGACAACCCTCCTCAAGCCAGCATCTGTGACCTTTTTATATGACCCATTAGTCTTCAAGCAATTCTTTGCTTTTAGCACCATAAA GTGTTTCAAACTCACCTTGTACTTTTCCCTAACCCCAGACCCGGAATCAGCCATTTCTCCCAGTACTGGTTCATCTTAGAAACCAAGATCTGGAGGCCGGGCGCAGTGGC (SEQ ID NO: 2)

>XM_024450706.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X3]>XM_024450706.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X3]

CACAGAGGAAGACAGATTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGGTAGGTAGCAAAAGCTGAAGCTGTGACAATGGTCAAGGGTCAAACTCTACATCAGCCAGGAATGCTAGGTGAGGGCTTTGAATTTTACCTTACACAGAAAGGCTAATTAACAAACACATCTGAGCCCAACAAATGTTTTTACCAAAGTAACTTTGTAATCTAGAACAATGAAAATTGTTACAAAGAGTTTTTGTATTTGTATAATATATTAATAGCTACATACATCAAATATAATTTCTTGTATGTCAGAAGAATTACCACTGACACATTAAGACAATTTATGTATTAACTATTAAATTAAAGGTAATTATTGCAGTACCTTTCTCCACCACTGGAGTCGATGACGTAACCAGAAATCAAGCCACTGGTTTGAAGTTCTCGGAGGAGTAAACCATACTAACGAAGCTTCAGTCTTCTCACCAA(SEQ ID NO：3)CACAGAGGAAGACAGATTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAA AGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCATGA ATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCTCAGTCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTAT GACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTA CTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCAAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAG AGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTGAGTACTAAAACTTGATCATCAATTACAGAGCCATTTTGACTAATATGGAAAGTTCATGGAGGTAGGTAGCAAAAGCTGAAGCTGTGACAATGGTCAAAGGGTCAAACTCTACATCAGCCAGGAATGCTAGGTGAGGGCT TTGAATTTTACCTTACACAGAAAGGCTAATTAACAAACACATCTGAGCCCAACAAATGTTTTTACCAAAGTAACTTTGTAATCTAGAACAATGAAAATTGTTACAAGAGTTTTTGTATTTGTATAATATATTAATAGCTACATACATCAAATATAATTTCTTGTATGTCAGAAGAATTACCACTGACACATTAAAGACAATTTATGTATTAACTATTAAAATTAAAGGTAATTATTGCA GTACCTTTCTCCACCACTGGAGTCGATGACGTAACCAGAAATCAAGCCACTGGTTTGAAGTTCTCGGAGGAGTAAACCATACTAACGAAGCTTCAGTCTTCTCACCAA (SEQ ID NO: 3)

>XM_024450708.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X6]>XM_024450708.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X6]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGGCCAGTTGCAATCCCCAAGATCCAGCAAGACTGCCTCGCCTTTCCACCCGACACCTGTTTTGAAGCATGAAATCGTGAAGCATACCGTGAAGAAGGTTCTCCCGTAGTTTCCCAGAAGTTTTTAATACGTTCTCAAGAAATGCTACTA(SEQID NO：4)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAA ATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTG CTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAA ACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCGT GATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGGCCAGTTGCAATCCCCAAGATCCAGCAAGACTGCCTCGCCTTTCCACCCGACACCTGTTTTGAAGCATGAAATCGTGAAGCATACCGTGAAGAAGGTTCTCCCGTAGTTTCCAGAAGTTTTTAATACGTTTCCAAGAAATGCTACTA(SEQ ID NO: 4)

>XM_024450709.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X7]>XM_024450709.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X7]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTAGAGACGGGGTTTTGC(SEQ ID NO：5)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAA ATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTG CTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAA ACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCGT GATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTAGAGACGGGGTTTTGC (SEQ ID NO: 5)

>XM_024450707.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X5]>XM_024450707.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X5]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTAGAGACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTTCAACTCCTGACCTCAAGTGATCCGCCCACCTCGGACTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCTCGCCTGGCCCTCCATTTCCTGATCTAGTCTTATATCCACGCTCACCACCTCAGCACGCTCAGACCCACGCTGCTGTGGGCTCCTCTGGCTCCTGGAAGAGTGCGTCCGCAGATGCTGCAGTCTTTGTGTGGCTCAGCAATTGCCACTCACATCAGGAACTGCCTTTACCCTGTCAGGCTCTACTGAGACCCGACCCTGGTTATTAAGCTATAGGGGAGACAAGGATGGATCTTAAAGAAGACAAGCAAAATGTAGTGAAGCAAATAGAATGGTGGTTCCTGGGGGATGGGGGCAGGGGACAACGAGGAGTGACTGGCTAACAGATACAGCGTTTCAGTTTGGAAAGACAAAAAAGTTCTGGAAAAAAGATGGAAGGTGGTGATGGTTGCACAATAATATGAGTTTTGTTGTTGTTGTTTTTTGAG(SEQ ID NO：6)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAA ATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTG CTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAA ACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCGT GATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGTAGAGACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTTCAACTCCCTGACCTCAAGTGATCCGCCCACCTCGGACTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCTCGCCTGGCCCTCCATTTCCTGATCTAGTCTTATATCCACG CTCACCACCTCAGCACGCTCAGACCCACGCTGCTGTGGGCTCCTCTGGCTCCTGGAAGAGTGCGTCCGCAGATGCTGCAGTCTTTGTGTGGCTCAGCAATTGCCACTCACATCAGGAACTGCCTTTACCCTGTCAGGCTCTACTGAGACCCGACCCTGGTTATTAAGCTATAGGGGAGACAAGGATGGATCTTAAAGAAGACAAGCAAAATGTAGTGAAGCAAA TAGAATGGTGGTTCCTGGGGGATGGGGGCAGGGGACAACGAGGAGTGACTGGCTAACAGATACAGCGTTTCAGTTTGGAAAGACAAAAAAGTTCTGGAAAAAAGATGGAAGGTGGTGATGGTTGCACAATAATATGAGTTTTGTTGTTGTTGTTTTTTGAG (SEQ ID NO: 6)

>XM_017024487.1MILR1[生物体＝智人][基因ID＝284021][转录物＝X8]>XM_017024487.1 MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = X8]

TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGTAAGATGCTTTTTATGAAGCTGATTTCCATGAACAAAAAGCAAACTTGAGGCTGAGGCAGGTGGATTACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGCAAAAACCCCAT(SEQ ID NO：7)TTGGTCGAACAAACCAGTATTATGCAAACCTCATCCAAACCCTCTGATTTCCTTAACTTGGCTAAGAAAAAGAGGAAGTTCTCCGAGTTACTCACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATT GTGAGGCAATGAAAACAAATGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCGT GCTGAACATTATGGTCATTCAAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGC AACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTT GAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGTAAG ATGCTTTTTATGAAGCTGATTTCCATGAACAAAAAGCAAACTTGAGGCTGAGGCAGGTGGATTACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGCAAAAACCCCAT (SEQ ID NO: 7)

>NM_001369493.1MILR1[生物体＝智人][基因ID＝284021][转录物＝4]>NM_001369493.1MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = 4]

ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGATTTAAAGGGGAGACCTGGCCAGAGGAATCATCAACAGTGGATTTGTGATGACTGGAGTTCATTTCCTTCTTTCTCAACTGTCCTGGAAGGAAATGGACTAGTGCCTTACCTCTGACTCTTCCGCAGCACACACCCACCACCAGAGACAGTAACTGAGATCTGCAGTAGCACAGAAACTTACAGGTTAGAAAAATTAGACACCTAAGGATTTCATGACTCCAGACCTGTTTCCTTAGCATAATACCACACCAGTAGGAGAAATACAACTTACGAAATTTCTTTGTAAATAAATTCTATAACATTTTGGGTAAAAATTTATCATAAAAAAATTATTAAAAGCAATTGGGTTGGAAGGAAGTTCTGTCAAAATACAATCCAGGTGTGCAGTTCTCTGATAAATTTAGCAGTGGTTACTACTGAGGCAGTCTCAAAAAAAAGTGTAAAAGGACTTTAAGGGGTGATGAAAATTTTCTACATTGCCTTTGTGGAGATGGTTATACATTTGTCAATACTCATCAATTTGTACATTTAAAAGAGGTAAATTTATTGTATGTAAATTGTATCTTAATAAAACTGATTAAAACAGACATACAAACCCCA(SEQ ID NO：8)ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTT GTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATT ATGGTCATTCAAAACAGAAACAGACCGACATATAACATTACATTGCCTCCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCA CCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCA AAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGGATTTAAAGGGGAG ACCTGGCCAGAGGAATCATCAACAGTGGATTTGTGATGATGACTGGAGTTCATTTCCTTCTTTCTCAACTGTCCTGGAAGGAAATGGACTAGTGCCTTACCTCTGACTCTTCCGCAGCACACCACCCACCAGAGACAGTAACTGAGACAGTAACTGAGATCTGCAGTAGCACAGAAACTTACAGGTTAGAAAAAATTAGACACCTAAGGATTTCATGACTCCAGACCTGTTTCCTTAGCATAAT ACCACACCAGTAGGAGAAATACAACTTACGAAATTTCTTTGTAAATAAAATTCTATAACATTTTGGGTAAAAAATTTATTCATAAAAAAATTATTAAAAGCAATTGGGTTGGAAGGAAGTTCTGTCAAAATACAATCCAGGTGTGCAGTTCTCTGATAAATTTAGCAGTGGTTACTACTGAGGCAGTCTCAAAAAAAGTGTAAAAGGACTTTAAGGGGTGATGAAAAT TTTTCTACATTGCCTTTGTGGAGATGGTTATACATTTGTCAATACTCATCAATTTGTACATTTAAAAGAGGTAAATTATTGTATGTAAATTGTATCTTAATAAAACTGATTAAAACAGACATACAAACCCCA (SEQ ID NO: 8)

>NM_001085423.2MILR1[生物体＝智人][基因ID＝284021][转录物＝L]>NM_001085423.2MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = L]

ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCACTTCAATCTCCATCTTCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAACTACAAGCCTGAGCCACCGTGCCCGGCCCTGAATCGCTTTAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA(SEQ ID NO：9)ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTT GTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGACCCGGTGACTTCCCCAGTGCTGAACATT ATGGTCATTCAAAACAGAAACAGACCGACATATAACATTACATTGCCTCCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCA CCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCA AAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCAAGCCCAAGATGAGGCCAAACACTCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGT TGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCACTTCAATCTCCATCTTCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCA AAGTGCTGGAACTACAAGCCTGAGCCACCGTGCCCGGCCCTGAATCGCTTTAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA (SEQ ID NO: 9)

>NM_001291316.2MILR1[生物体＝智人][基因ID＝284021][转录物＝S1]>NM_001291316.2MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = S1]

ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTTGTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCACTTCAATCTCCATCTTCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAACTACAAGCCTGAGCCACCGTGCCCGGCCCTGAATCGCTTTAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA(SEQ ID NO：10)ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGAATTCCCTTCTCCATGTTTGGACTCAAAAGACTAAGGTGGTTATGAAGGGTCAAAATGTATCTATGTTTT GTTCCCATAAGAACAAATCACTGCAGATCACCTATTCATTGTTTCGACGTAAGACACACCTGGGAACCCAGGATGGAAAAGGTGAACCTGCGATTTTTAACCTAAGCATCACAGAAGCCCATGAATCAGGCCCCTACAAATGCAAAGCCCAAGTTACCAGCTGTTCAAAATACAGTCGTGACTTCAGCTTCACGATTGTCGGCGGAGACAGCTGTCCTTTCTGTCTGAAG CTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAG CCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCACTTCAATCTCCATCT TCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAACTACAAAGCCTGAGCCACCGTGCCCGGCCCTGAATCGCTT TAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA (SEQ ID NO: 10)

>NM_001291317.2MILR1[生物体＝智人][基因ID＝284021][转录物＝S2]>NM_001291317.2MILR1 [Organism = Homo sapiens] [Gene ID = 284021] [Transcript = S2]

ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAAACAGAAACAGACCGACATATAACATTACATTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTCCTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCCGTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCACTTCAATCTCCATCTTCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAACTACAAGCCTGAGCCACCGTGCCCGGCCCTGAATCGCTTTAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA(SEQ ID NO：11)ACCACTGTGGTTCTACTATGCCTTCTGACCCCGTCTTGGACTTCAACTGGGAGAATGTGGAGCCATTTGAACAGGCTCCTCTTCTGGAGCATATTTCTTCTGTCACTTGTAGAAAAGCTGTATTGGATTGTGAGGCAATGAAAACAAATGACCCGGTGACTTCCCCAGTGCTGAACATTATGGTCATTCAACAGAAACAGACCGACATATAACATTACA TTGCCTCTCAGTCAATGGCTCGCTGCCCATCAATTACACTTTCTTTGAAAACCATGTTGCCATATCACCAGCTATTTCCAAGTATGACAGGGAGCCTGCTGAATTTAACTTAACCAAGAAGAATCCTGGAGAAGAGGAAGAGTATAGGTGTGAAGCTAAAAACAGATTGCCTAACTATGCAACATACAGTCACCCCTGTCACCATGCCCTCAACAGGCGGAGACAGCTGTC CTTTCTGTCTGAAGCTACTACTTCCAGGGTTATTACTGTTGCTGGTGGTGATAATCCTAATTCTGGCTTTTTGGGTACTGCCCCAAATACAAAACAAGAAAAGCTATGAGAAATAATGTGCCCAGGGACCGTGGAGACACAGCCATGGAAGTTGGAATCTATGCAAATATCCTTGAAAAACAAGCAAAGGAGGAATCTGTGCCAGAAGTGGGATCCAGGCC GTGTGTTTCCACAGCCCAAGATGAGGCCAAACACTCCCAGGAGCTACAGTATGCCACCCCCGTGTTCCAGGAGGTGGCACCAAGAGAGCAAGAAGCCTGTGATTCTTATAAATCTGGATATGTCTATTCTGAACTCAACTTCTGAAATTTACAGAAACAAACTACATCTCAGGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTTCAGTAGCGCGATCTTGGCTCAC TTCAATCTCCATCTTCCCAGTTCAAGCGATTCTCATGCCTCGACCTCCCGAGTAGCTGGGATTACAGGTGCCCGCTACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACTATGGTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGATGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAACTACAAGCCTGAGCCACCGTGCCCGG CCCTGAATCGCTTTAGTAAATAAAGGGTCTCCAAGAATAAATTCATCCGAACATGCA (SEQ ID NO: 11)

术语“AML”亚型：1976年，法，美，英(FAB)白血病分类协作组提出的急性白血病分型标准已作为该病诊断分型的基本方法，以后又作了多次补充和修改，AML的FAB分型是目前最常用的类型。AML可分成8种类型，分别为M0(急性髓系白血病微分化型)，M1(急性粒细胞白血病未分化型)，M2(急性粒细胞性白血病部分分化型)，M3(急性早幼粒细胞白血病)，M4(急性粒单核细胞白血病)，M5(急性单核细胞白血病)，M6(红白血病)，M7(急性巨核细胞白血病)。The term "AML" subtype: In 1976, the acute leukemia classification standard proposed by the French, American and British (FAB) Leukemia Classification Collaborative Group has been used as the basic method for the diagnosis and classification of the disease, and has been supplemented and modified many times since then. The FAB type of AML is currently the most commonly used type. AML can be divided into 8 types, namely M0 (micro-differentiated acute myeloid leukemia), M1 (undifferentiated acute myeloid leukemia), M2 (partially differentiated acute myeloid leukemia), and M3 (acute promyelocytic leukemia). leukemia), M4 (acute myelomonocytic leukemia), M5 (acute monocytic leukemia), M6 (erythroleukemia), M7 (acute megakaryoblastic leukemia).

AML单核细胞：AML患者骨髓液中分离的单核细胞(Mononuclear cells)为AML单核细胞。例如，可利用Ficoll-Paque分离液，行骨髓穿刺术抽取AML患者骨髓液进行分离单核细胞。AML mononuclear cells: the mononuclear cells (Mononuclear cells) isolated from the bone marrow fluid of AML patients are AML mononuclear cells. For example, Ficoll-Paque separation fluid can be used to extract the bone marrow fluid of AML patients through bone marrow aspiration to isolate mononuclear cells.

正常单核细胞：正常健康机体血液中的单核细胞为正常单核细胞。例如，可利用Ficoll-Paque分离液在足月健康新生儿脐带血中分离获得。Normal monocytes: The monocytes in the blood of a normal healthy body are normal monocytes. For example, it can be isolated from cord blood of full-term healthy newborns using Ficoll-Paque separation fluid.

白血病干细胞(Leukemia stem cells，LSCs)又称为白血病起始细胞(Leukemiainitiating cells，LICs)是一群具有自我更新能力，并能产生异质性白血病细胞群体的白血病细胞。1994年，Tsvee Lapidot等率先从AML患者骨髓细胞中分离出CD34⁺CD38^-LICs亚群，并发现该亚群具有HSCs样强大的自我更新能力，并可在免疫缺陷小鼠体内重建白血病。2007年，Fumihiko Ishikawa等发现LSCs能逃逸化学药物的杀伤作用，导致耐药和易复发。Leukemia stem cells (Leukemia stem cells, LSCs), also known as leukemia initiating cells (Leukemia initiating cells, LICs), are a group of leukemia cells that have self-renewal ability and can generate heterogeneous leukemia cell populations. In 1994, Tsvee Lapidot et al took the lead in isolating CD34⁺ CD38^- LICs subpopulation from bone marrow cells of AML patients, and found that this subpopulation had HSCs-like strong self-renewal ability and could reconstitute leukemia in immunodeficient mice. In 2007, Fumihiko Ishikawa found that LSCs can escape the killing effect of chemical drugs, leading to drug resistance and easy recurrence.

术语“抑制剂”是指能够抑制或下调物质表达或活性的调节剂。例如，可为能够特异性敲降或沉默Allergin-1的siRNA或能够特异性结合和抑制Allergin-1活性的抗体。The term "inhibitor" refers to a modulator capable of inhibiting or down-regulating the expression or activity of a substance. For example, it may be an siRNA capable of specifically knocking down or silencing Allergin-1 or an antibody capable of specifically binding and inhibiting the activity of Allergin-1.

术语“拮抗剂”是指能够拮抗Allergin-1活性，降低或抑制Allergin-1与Allergin-1配体相互作用的调节剂。例如，可为Allergin-1配体的竞争结合剂。The term "antagonist" refers to a modulator capable of antagonizing the activity of Allergin-1, reducing or inhibiting the interaction between Allergin-1 and Allergin-1 ligands. For example, it may be a competitive binder for Allergin-1 ligand.

“抗体”是能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合至靶标诸如碳水化合物、多核苷酸、脂质、多肽的免疫球蛋白分子。如本文中所使用，该术语不仅涵盖完整多克隆或单克隆抗体，而且涵盖其片段(诸如Fab、Fab’、F(ab’)2、Fv)、单链(scFv)和结构域抗体(包括例如鲨鱼和骆驼抗体)以及包含抗体的融合蛋白，和包含抗原识别位点的免疫球蛋白分子的任何其他修饰的构型。抗体包括任何类别的抗体，诸如IgG、IgA或IgM(或其亚类)，且该抗体不必为任何特定类别。根据其重链的恒定区的抗体氨基酸序列，免疫球蛋白可被归为不同类别。存在五种主要类别的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，且这些中的几种可进一步分成亚类(同种型)，例如IgGl、IgG2、IgG3、IgG4、IgAl和IgA2。对应于不同种类的免疫球蛋白的重链恒定区分别称为α、δ、ε、γ和μ。不同类别的免疫球蛋白的亚单位结构和三维构型是众所周知的。An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term covers not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) and domain antibodies (including such as shark and camel antibodies), as well as fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

如本文中所使用，术语“抗体”包括完整抗体和任何抗原结合片段(即“抗原结合部分”)或其单链。“抗体”是指包含至少两条重链(H)和两条轻链(L)并通过二硫键相互连接的，或其抗原结合部分的蛋白质。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。重链恒定区由三个结构域，CH1、CH2和CH3组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区的。轻链恒定区由一个结构域CL组成。VH和VL区可以进一步细分成高变区，称为互补决定区(CDR)，与更保守的称为构架区(FR)的区域散布。每个VH和VL由三个CDR和四个FR组成，从氨基末端到羧基末端以下面的顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包含与抗原相互作用的结合结构域。As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof. "Antibody" refers to a protein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

术语“抗体”，在本申请中所用的是指免疫球蛋白或其片段或它们的衍生物，并且包括其包含的抗原结合位点的任何多肽，而不管其是否是在体外或体内产生。该术语包括，但不限于，多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化、单链的、嵌合的、合成的、重组的、杂合的、突变的、嫁接的抗体。术语“抗体”还包括抗体片段例如Fab、F(ab')2、FV、scFv、Fd、dAb和其它保留抗原结合功能的抗体片段，即，能够与PD-1的特异性结合。通常情况下，这样的片段将包括抗原结合片段。The term "antibody", as used in this application, refers to an immunoglobulin or fragment or derivative thereof, and includes any polypeptide comprising an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutated, grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function, ie, are capable of specific binding to PD-1. Typically, such fragments will include antigen binding fragments.

如本文所使用的术语抗体的“抗原结合片段”或“抗原结合部分”是指保留与给定抗原特异性结合的能力的完整抗体的一个或多个片段。抗体的抗原结合功能可由完整抗体的片段执行。涵盖在术语抗体的“抗原结合片段”内的结合片段的实例包括Fab；Fab’；F(ab’)2；由VH结构域和CH1结构域组成的Fd片段；由抗体的单臂的VL结构域和VH结构域组成的Fv片段；单结构域抗体(dAb)片段(Ward等人，Nature 341:544-546,1989)和分离的互补决定区(CDR)。术语“抗原结合片段”、“抗原结合结构域”和“结合片段”是指一种抗体分子，其包含负责具体的抗体和抗原之间的结合的氨基酸。例如，其中的抗原是大的，抗原结合片段只结合抗原的一部分。即抗原分子中负责与抗原结合片段特异性相互作用的部分被称为“表位”或“抗原决定簇”。The term "antigen-binding fragment" or "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an intact antibody that retain the ability to specifically bind a given antigen. The antigen binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab; Fab'; F(ab')2; the Fd fragment consisting of a VH domain and a CH1 domain; the VL structure consisting of a single arm of an antibody domains and VH domains; single domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989) and isolated complementarity determining regions (CDRs). The terms "antigen-binding fragment", "antigen-binding domain" and "binding fragment" refer to an antibody molecule comprising the amino acids responsible for the binding between a particular antibody and antigen. For example, where the antigen is large, the antigen-binding fragment only binds a portion of the antigen. That is, the part of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called "epitope" or "antigenic determinant".

抗原结合片段通常包括抗体轻链可变区(VL)和抗体重链可变区(VH)，然而，它不一定必须包括两者。例如，一个所谓的Fd抗体片段仅由VH结构域组成，但仍保留了完整抗体的一些抗原结合功能。An antigen-binding fragment typically includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to include both. For example, a so-called Fd antibody fragment consists of only the VH domain but still retains some of the antigen-binding functions of the intact antibody.

术语“同源性”是指当两个多肽序列最佳比对时在两个序列之间的序列相似性。当两个比较的序列的两者中的位置由相同氨基酸单体亚单位占据时，例如如果两个不同Ab的轻链CDR中的位置由丙氨酸占据时，贝两个Ab在该位置处同源。同源性百分比是由两个序列共有的同源性位置的数目除以所比较的位置的总数目X 100。例如，如果当序列最佳比对时，两个序列中的10个位置中的8个位置匹配或同源，则该两个序列是80％同源的。通常，在两个序列被比对以产生最大同源性百分比时进行比较。例如，比较可通过BLAST算法进行，其中该算法的参数被选择以在各参考序列的完整长度上的各序列之间产生最大匹配。The term "homology" refers to the sequence similarity between two polypeptide sequences when they are optimally aligned. When a position in both of the two compared sequences is occupied by the same amino acid monomer subunit, for example if a position in the light chain CDR of two different Abs is occupied by alanine, then both Abs are at that position homologous. The percent homology is the number of homologous positions shared by the two sequences divided by the total number of positions comparedX 100. For example, two sequences are 80% homologous if 8 out of 10 positions in two sequences match or are homologous when the sequences are optimally aligned. Generally, comparisons are made when two sequences are aligned to yield the greatest percent homology. For example, a comparison can be performed by the BLAST algorithm, the parameters of which are chosen to yield the largest match between the sequences over the full length of each reference sequence.

在本发明中，术语“芯片”、“微阵列”、“阵列”可以等同替代，包括但不限于：DNA微阵列(例如，cDNA微阵列和寡核苷酸微阵列)、蛋白质微阵列、组织微阵列、转染或细胞微阵列、化学化合物微阵列和抗体微阵列。通常称为基因芯片、DNA芯片或生物芯片的DNA微阵列是微观DNA点的集合，这些点连接到固体表面(例如，玻璃、塑料或硅芯片)上，形成用于对数千种基因同时进行表达谱分析或表达水平监测的阵列。固定的DNA片段称为探针，其数千个可用于单个DNA微阵列中。微阵列可用于通过比较疾病和正常细胞中的基因表达而识别疾病基因或转录本(例如，ncRNA)。微阵列可使用多种技术加以制造，包括但不限于：用细尖针印刷到载玻片上、使用预制的掩模进行光刻、使用动态微镜器件进行光刻、喷墨印刷或微电极阵列上的电化学方法。In the present invention, the terms "chip", "microarray", and "array" can be substituted equivalently, including but not limited to: DNA microarrays (for example, cDNA microarrays and oligonucleotide microarrays), protein microarrays, tissue Microarrays, transfection or cellular microarrays, chemical compound microarrays, and antibody microarrays. DNA microarrays, often called gene chips, DNA chips, or biochips, are collections of microscopic DNA spots attached to a solid surface (for example, a glass, plastic, or silicon chip) that form a pattern for simultaneous sequencing of thousands of genes. Arrays for expression profiling or expression level monitoring. The immobilized DNA fragments are called probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify disease genes or transcripts (eg, ncRNAs) by comparing gene expression in disease and normal cells. Microarrays can be fabricated using a variety of techniques including, but not limited to: printing onto glass slides with fine-tipped needles, photolithography using prefabricated masks, photolithography using dynamic micromirror devices, inkjet printing, or microelectrode arrays Electrochemical method on.

如在本文中使用的，术语“生物标志物”是指能够特异性标记细胞或指示疾病状态的物质。在旨在特异性标记细胞的本发明的上下文中，生物标志物组合Allergin-1、CD34和CD38可作为LSC特异性标记。在旨在诊断白血病的本发明的上下文中，“生物标志物”是指指示诊断、病程监控和预后判断白血病(尤其是AML)的物质。“生物标志物”包括多肽和糖蛋白，与正常的健康受试者相比，Allergin-1在AML患者中特异性异常高表达。As used herein, the term "biomarker" refers to a substance capable of specifically marking cells or indicating a disease state. In the context of the present invention aimed at specifically marking cells, the biomarker combination Allergin-1, CD34 and CD38 may serve as LSC specific markers. In the context of the present invention aimed at diagnosing leukemia, "biomarker" refers to a substance indicative of the diagnosis, monitoring of the disease course and prognosis of leukemia, especially AML. "Biomarkers" include peptides and glycoproteins. Compared with normal healthy subjects, Allergin-1 is specifically and abnormally highly expressed in AML patients.

在本发明中，术语“样本”、“样品”或“生物样品”可以等同替代，包括但不限于：多种从个体得到的样品类型，并且可用于诊断或监测测试。生物流体样品涵盖了血液、脑脊液(CSF)、尿和其它生物来源的液体样品。例如，本发明所述的生物样品可为血液。如果需要，样品可预先进行处理，例如浓缩或分离。In the present invention, the terms "specimen", "sample" or "biological sample" may be substituted equivalently, including but not limited to: a variety of sample types obtained from individuals and used in diagnostic or monitoring tests. Biological fluid samples encompass blood, cerebrospinal fluid (CSF), urine and other liquid samples of biological origin. For example, the biological sample described in the present invention can be blood. Samples can be pretreated, eg concentrated or separated, if desired.

应当理解本文采用的术语用于描述具体实施方案的目的，而不意在进行限制。此外，在本发明的实践或试验中可以使用与本文描述的那些类似或等价的任何方法、装置和材料，下文描述了优选的方法、装置和材料。It is to be understood that terminology employed herein is for the purpose of describing particular embodiments and is not intended to be limiting. In addition, any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials being described below.

疾病的诊断、病程监控和/或预后判断的方法、应用和产品Methods, applications and products for disease diagnosis, disease course monitoring and/or prognosis judgment

方法和/或应用method and/or application

如前所述，本发明提供了通过检测或定量Allergin-1表达或活性来诊断、监测和评价白血病诸如AML的方法。在本发明的一个方面，本发明涉及通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来鉴别LSCs的方法，其中Allergin-1在LSCs中特异性异常高表达。As previously stated, the present invention provides methods for diagnosing, monitoring and evaluating leukemias such as AML by detecting or quantifying Allergin-1 expression or activity. In one aspect of the invention, the invention relates to a method for identifying LSCs by detecting the expression or activity of the biomarker Allergin-1 in a subject or a sample from a subject, wherein Allergin-1 is specifically abnormal in LSCs High expression.

在本发明的一个方面，本发明涉及通过检测受试者或来自受试者的样本中的生物标志物Allergin-1的表达或活性来诊断、病程监控和预后判断白血病(尤其是AML)或用于确定发生白血病(尤其是AML)的风险或用于确定白血病(尤其是AML)严重程度的方法，其中Allergin-1在AML患者中特异性异常高表达。在一些实施例中，所述受试者造血干细胞中Allergin-1的的表达水平或活性显著高于正常造血干细胞中Allergin-1的表达水平或活性，是受试者患有白血病的指示或是受试者处于白血病疾病状态的指示或是受试者处于白血病疾病高发风险的指示或是受试者患有严重白血病的指示。在一些实施方案中，Allergin-1在AML患者中高表达，尤其在M4或M5亚型中异常高表达。In one aspect of the present invention, the present invention relates to the diagnosis, course monitoring and prognosis of leukemia (especially AML) by detecting the expression or activity of the biomarker Allergin-1 in a subject or a sample from a subject or using A method for determining the risk of developing leukemia (especially AML) or for determining the severity of leukemia (especially AML), wherein Allergin-1 is specifically abnormally highly expressed in AML patients. In some embodiments, the expression level or activity of Allergin-1 in the subject's hematopoietic stem cells is significantly higher than the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the subject has leukemia or An indication that the subject is in a leukemic disease state or an indication that the subject is at high risk for a leukemic disease or an indication that the subject has severe leukemia. In some embodiments, Allergin-1 is overexpressed in AML patients, especially abnormally overexpressed in M4 or M5 subtypes.

在本发明的一个方面，本发明涉及检测样本中Allergin-1表达水平的试剂在制备鉴别LSCs的产品中的应用。优选的，所述检测样本中Allergin-1表达水平或活性的试剂与一种或多种其他试剂组合；更优选的，所述一种或多种其他试剂为IFN-γ。In one aspect of the present invention, the present invention relates to the application of a reagent for detecting the expression level of Allergin-1 in a sample in the preparation of a product for identifying LSCs. Preferably, the reagent for detecting the expression level or activity of Allergin-1 in a sample is combined with one or more other reagents; more preferably, the one or more other reagents are IFN-γ.

在本发明的一个方面，本发明涉及检测样本中Allergin-1表达水平的试剂在制备用于白血病(尤其是AML)的诊断、病程监控和预后判断的产品中的应用。优选的，所述检测样本中Allergin-1表达水平或活性的试剂与一种或多种其他试剂组合；更优选的，所述一种或多种其他试剂为IFN-γ。In one aspect of the present invention, the present invention relates to the application of a reagent for detecting the expression level of Allergin-1 in a sample in the preparation of products for diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML). Preferably, the reagent for detecting the expression level or activity of Allergin-1 in a sample is combined with one or more other reagents; more preferably, the one or more other reagents are IFN-γ.

在本发明的一个方面，本发明涉及用于检测Allergin-1和至少一种生物标志物的组合的试剂在制备用于鉴别LSCs的产品中的应用。在一些实施方案中，所述Allergin-1和至少一种生物标志物的组合为Allergin-1⁺CD34⁺CD38^-。In one aspect of the invention, the invention relates to the use of a reagent for detecting a combination of Allergin-1 and at least one biomarker for the manufacture of a product for the identification of LSCs. In some embodiments, the combination of Allergin-1 and at least one biomarker is Allergin-1⁺ CD34⁺ CD38⁻ .

在本发明的一个方面，本发明涉及用于检测Allergin-1和至少一种生物标志物的组合的试剂在在制备用于白血病(尤其是AML)的诊断、病程监控和预后判断的产品中的应用。在一些实施方案中，所述Allergin-1和至少一种生物标志物的组合为Allergin-1⁺CD34⁺CD38^-。In one aspect of the present invention, the present invention relates to the use of reagents for detecting the combination of Allergin-1 and at least one biomarker in the preparation of products for the diagnosis, course monitoring and prognosis of leukemia (especially AML) application. In some embodiments, the combination of Allergin-1 and at least one biomarker is Allergin-1⁺ CD34⁺ CD38⁻ .

在上述方法或应用的实施方案中，术语检测、确定或诊断包括物理地测定、物理地确定或物理地诊断白血病诸如AML的生物标志物的水平或量的步骤。也就是说，该方法包括以下步骤：利用对于测定水平或量有用的已知方法测定Allergin-1的生物标志物的水平或量，和使用所述测定的水平或量相应地确定或诊断。在一个优选实施方式中，生物标志物以其肽或蛋白的形式加以确定。然而，也可以在核酸水平，例如基于生物样品中的mRNA水平或量测定Allergin-1生物标志物的水平或量。In embodiments of the methods or uses described above, the terms detecting, determining or diagnosing include the step of physically determining, physically determining or physically diagnosing the level or amount of a biomarker for leukemia, such as AML. That is, the method comprises the steps of determining the level or amount of a biomarker of Allergin-1 using known methods useful for determining the level or amount, and correspondingly determining or diagnosing using said determined level or amount. In a preferred embodiment, the biomarkers are determined in their peptide or protein form. However, the level or amount of the Allergin-1 biomarker can also be determined at the nucleic acid level, for example based on the level or amount of mRNA in a biological sample.

在上述方法或应用的实施方案中，对受试者的疾病的进展可以通过测量对应于诸如Allergin-1等一个或多个生物标志物的mRNA，或所编码的蛋白质表达水平或其活性来检测。Allergin-1的mRNA或蛋白质表达水平可以在体内检测，或在取自例如，血液、骨髓样品中检测。对应于基因的mRNA和/或蛋白质的表达水平可以通过如上所述的标准方法检测。通过将受试者的把标蛋白质或RNA水平与该受试者靶标蛋白质或RNA基线水平比较，可以监测受试者的疾病状态(例如，疾病的改善、加剧或复发)。例如，第一时间检测的受试者的Allergin-1表达水平可以与后来的第二时间受试者的Allergin-1表达水平比较。Allergin-1mRNA或蛋白质的表达水平随时间的升高是白血病疾病进展的指征。Allergin-1mRNA或蛋白质的表达水平随时间的降低是白血病疾病减轻的指征。In embodiments of the above methods or uses, the progression of the disease in the subject can be detected by measuring the mRNA corresponding to one or more biomarkers, such as Allergin-1, or the expression level of the encoded protein or its activity . Allergin-1 mRNA or protein expression levels can be detected in vivo, or in samples taken from, eg, blood, bone marrow. Expression levels of mRNA and/or protein corresponding to a gene can be detected by standard methods as described above. By comparing the subject's target protein or RNA level to the subject's baseline level of the target protein or RNA, the subject's disease state (eg, improvement, exacerbation, or relapse of the disease) can be monitored. For example, the expression level of Allergin-1 detected in a subject at a first time can be compared to the expression level of Allergin-1 in a subject at a later time at a second time. An increase in the expression level of Allergin-1 mRNA or protein over time is an indicator of leukemia disease progression. A reduction in the expression level of Allergin-1 mRNA or protein over time is indicative of remission of leukemia disease.

在上述方法或应用的实施方案中，受试者中例如Allergin-1蛋白质或RNA的水平也可用于监测治疗效果。通常，获得受试者目的蛋白质或RNA的基线水平(例如治疗前的)，并且与在治疗后或治疗期间的多个时间点(例如治疗后1天或更多天、数周或数月)测得的目的蛋白质或RNA的水平相比较。比较的结果可表明过去治疗的效果，并可由此对未来的治疗作出相应的修改。In embodiments of the above methods or uses, levels of eg Allergin-1 protein or RNA in a subject may also be used to monitor the effect of the treatment. Typically, a baseline level of the subject's protein or RNA of interest (e.g., before treatment) is obtained and compared to various time points after or during treatment (e.g., 1 or more days, weeks or months after treatment) The measured target protein or RNA levels are compared. The results of the comparison can indicate the effectiveness of past treatments, and future treatments can be modified accordingly.

根据本发明，本文中公开的方法分别涉及体外和/或体内方法。优选地，所述方法是基于获自个体的和体外提供的样品的体外方法。According to the present invention, the methods disclosed herein relate to in vitro and/or in vivo methods, respectively. Preferably, the method is an in vitro method based on a sample obtained from an individual and provided in vitro.

目的基因转录物可以用多种本领域已知的技术检测。一些有用的核酸检测系统包括准备纯化的样品的核酸组分，以及对样品进行直接检测，或扩增过程之后进行检测，例如检测骨髓或血液组织样本中的Allergin-1mRNA。可以通过例如聚合酶链式反应(PCR)、反转录酶(RT)和偶联的RT-PCR进行扩增。核酸的检测可以通过例如用与目的核酸杂交的探针探测纯化的核酸组分来达成，许多情况下还会涉及扩增。Northern印迹、点印迹、微阵列、定量PCR和定量RT-PCR都是用于检测样本中核酸的众所周知的方法。核酸扩增也可以通过连接酶链反应、链置换扩增、自主序列复制或基于核酸序列的扩增进行。核酸也可以通过测序检测，测序可以采用对目的核酸特异的引物(例如Allergin-1cDNA序列)或针对接到目的核酸的接头序列的引物。随机选择的mRNA或cDNA序列的测序可以提供对生物标志物的相对表达量的表征，这通过所有测序的含有对应于该生物标志物的核酸序列(例如Allergin-1的cDNA或mRNA序列)的转录物的百分比来表征。或者，核酸可以原位检测而不经提取或纯化，例如通过杂交来检测。Gene transcripts of interest can be detected using a variety of techniques known in the art. Some useful nucleic acid detection systems include preparation of the nucleic acid fraction of a sample for purification, and detection of the sample directly, or after an amplification process, eg detection of Allergin-1 mRNA in bone marrow or blood tissue samples. Amplification can be performed by, for example, polymerase chain reaction (PCR), reverse transcriptase (RT), and coupled RT-PCR. Detection of nucleic acids can be achieved, for example, by probing purified nucleic acid fractions with probes that hybridize to the nucleic acid of interest, and in many cases will involve amplification. Northern blots, dot blots, microarrays, quantitative PCR and quantitative RT-PCR are all well known methods for detecting nucleic acids in samples. Nucleic acid amplification can also be performed by ligase chain reaction, strand displacement amplification, autonomous sequence replication, or nucleic acid sequence-based amplification. Nucleic acid can also be detected by sequencing, which can use primers specific to the target nucleic acid (such as Allergin-1 cDNA sequence) or primers directed at the linker sequence connected to the target nucleic acid. Sequencing of randomly selected mRNA or cDNA sequences can provide a characterization of the relative expression level of a biomarker by all sequenced transcripts containing a nucleic acid sequence corresponding to that biomarker (e.g., the cDNA or mRNA sequence of Allergin-1). Characterized by the percentage of the substance. Alternatively, nucleic acids can be detected in situ without extraction or purification, for example by hybridization.

在上述方法或应用的实施方案中，通过已知方法进行根据本发明的生物标志物的水平或量的测定。例如，在蛋白水平确定水平或量的情况下，进行免疫试验，例如ELISA、RIA、放射免疫扩散、蛋白印迹、奥克特洛尼免疫扩散、火箭免疫电泳、免疫组化染色、免疫沉淀试验、补体结合试验、FACS和蛋白质芯片试验以及免疫荧光试验、多重免疫试验、线性试验或斑点印迹试验。In embodiments of the methods or uses described above, the determination of the level or amount of a biomarker according to the invention is performed by known methods. For example, where the level or amount of the protein level is determined, immunoassays such as ELISA, RIA, radioimmunodiffusion, Western blot, octroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, Complement fixation assays, FACS and protein microarray assays, and immunofluorescence assays, multiplex immunoassays, linearity assays, or dot blot assays.

根据本发明的方法或应用，在一个优选实施方式中，进行ELISA。用于测定生物标志物的水平或量的测定工具的典型实例包括蛋白特异性抗体。特定的分子抗体在本领域是已知的或者可基于本领域描述的方法容易地制备。测定蛋白水平意味着通过使用与蛋白特异结合的测定工具如抗体测定蛋白的量。所述测定方法可限定为上述方法中的任一种。通常，所述方法包括形成抗原抗体复合物和通过已知方法确定该复合物。According to the method or application of the present invention, in a preferred embodiment, ELISA is performed. Typical examples of assay means for determining the level or amount of a biomarker include protein-specific antibodies. Specific molecular antibodies are known in the art or can be readily prepared based on methods described in the art. Determining protein levels means determining the amount of protein by using an assay tool that specifically binds to the protein, such as an antibody. The assay method may be limited to any one of the above-mentioned methods. Generally, the methods involve forming an antigen-antibody complex and identifying the complex by known methods.

通过测定生物标志物蛋白的测定标签或表达标签的信号大小，可以确定抗原-抗体复合物形成的量。也就是说，抗体可贴上本领域已知的检测标签，所述检测标签包括但不限于酶、荧光标志物、配体、发光体、微粒子和氧化还原分子。可用作检测标签的酶的实例包括但不限于D-葡萄糖苷酶、脲酶、过氧化物酶、碱性磷酸酶、乙酰胆碱酯酶、葡萄糖氧化酶、己糖激酶、GDP酶、RNA酶、萤光素酶、磷酸果糖激酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和磷酸烯醇丙酮酸脱羧酶。荧光标志物的实例包括但不限于异硫氰酸荧光素、罗丹明、藻红蛋白、藻蓝蛋白、别藻蓝蛋白和荧光胺。配体的实例包括但不限于生物素、亲和素以及生物素和亲和素的衍生物。The amount of antigen-antibody complex formation can be determined by measuring the signal magnitude of the assay tag or the expressed tag of the biomarker protein. That is, antibodies can be labeled with detection labels known in the art, including but not limited to enzymes, fluorescent markers, ligands, luminophores, microparticles, and redox molecules. Examples of enzymes that can be used as detection labels include, but are not limited to, D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, GDPase, RNase, fluorescent Luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, and phosphoenolpyruvate decarboxylase. Examples of fluorescent markers include, but are not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorescamine. Examples of ligands include, but are not limited to, biotin, avidin, and derivatives of biotin and avidin.

在上述方法或应用的实施方案中，例如，靶蛋白可以用一个或多个抗体免疫检测。在免疫检测中，可以直接或间接标记对生物标志物有特异结合亲和性的抗体，或与该抗体结合的第二抗体。抗体不需要是完整的：抗体可变结构域或其人工模拟物(如单链抗体)即可。合适的标记物包括但不限于，放射性核素(例如，1251、1311、35S、3H、32P、33P或14C)、荧光基团(例如，荧光素、FITC、多曱藻(黄)素叶绿素蛋白、罗丹明或PE)、发光基团、吸收确定波长光的化合物或酶(如碱性磷酸酶或辣根过氧化物酶)。可以通过与生物素结合然后用采用上述分子标记的亲和素或链霉素检测而间接标记抗体。根据标记的性质检测或定量标记物的方法是本领域已知的。检测器的例子包括但不限于X光片、辐射计数器、闪烁计数器、分光光度计、色度计、荧光计、光度计和光密度计。可以使用本领域熟悉的这些方法的组合(包括“多层”检测)来提高检测的灵敏度。In embodiments of the methods or uses described above, for example, the target protein can be immunodetected with one or more antibodies. In immunoassays, an antibody with specific binding affinity for a biomarker, or a secondary antibody that binds to the antibody, can be labeled directly or indirectly. Antibodies need not be complete: antibody variable domains or their artificial mimics (such as single chain antibodies) are sufficient. Suitable labels include, but are not limited to, radionuclides (e.g., 1251, 1311, 35S, 3H, 32P, 33P, or 14C), fluorophores (e.g., fluorescein, FITC, dinoflagellate chlorophyll protein , rhodamine or PE), luminescent groups, compounds or enzymes that absorb light of a defined wavelength (such as alkaline phosphatase or horseradish peroxidase). Antibodies can be labeled indirectly by conjugation with biotin followed by detection with avidin or streptomycin using molecular labels as described above. Methods for detecting or quantifying markers depending on the nature of the marker are known in the art. Examples of detectors include, but are not limited to, radiographs, radiation counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, photometers, and densitometers. Combinations of these methods familiar in the art, including "multilayer" detection, can be used to increase the sensitivity of detection.

在上述方法或应用的实施方案中，例如，检测目标蛋白质的免疫学检验可以以各种已知方式进行，包括夹心法、竞争法(竞争RIA)，或桥免疫测定。检测目标蛋白质的方法一般包括将生物学样本与结合该蛋白质的抗体接触，并检测该蛋白质与该抗体的结合。例如，可以通过本领域已知的各种方法中的任何方法将对Allergin-1有特异性结合亲和力的抗体固定在固体基质上，然后将该抗体暴露给该生物学样本。In embodiments of the methods or uses described above, for example, immunological assays to detect the protein of interest can be performed in a variety of known ways, including sandwich, competition (competition RIA), or bridge immunoassays. Methods for detecting a target protein generally include contacting a biological sample with an antibody that binds the protein, and detecting the binding of the protein to the antibody. For example, an antibody having specific binding affinity for Allergin-1 can be immobilized on a solid substrate by any of a variety of methods known in the art and then exposed to the biological sample.

在其他实施方案中，使用捕获抗体被固定于固体基质的“夹心”检测法来检测目的蛋白质的水平。该固体基质可以与生物学样本接触，从而使得该样本中的任何目的蛋白质能够结合到该固定化的抗体上。可以利用对目的蛋白质有特异性结合亲和性的“检测”抗体来测定结合到上述捕获抗体的目的蛋白质的水平，方法如上所述。应了解，在这些夹心检验法中，捕获抗体不应该结合到与检测抗体所结合的抗原表位相同的抗原表位上(或在使用多克隆抗体的情况下，捕获抗体结合的抗原表位的范围不该与检测抗体所结合的抗原表位的范围相同)。因此，如果一种单克隆抗体用作捕获抗体，检测抗体可以是另一种单克隆抗体，它结合的抗原表位或者与该捕获单克隆抗体结合的抗原表位在物理上完全分隔，或者仅仅与其部分重叠；检测抗体也可以是多克隆抗体，其结合到非捕获单克隆抗体结合的抗原表位上或结合到除捕获单克隆抗体结合的抗原表位以外的抗原表位上。如果多克隆抗体作为捕获抗体，检测抗体可以是单克隆抗体，它结合的抗原表位或者与该捕获多克隆抗体结合的任何抗原表位在物理上完全分隔，或者与其部分重叠；检测抗体或也可以是多克隆抗体，其结合到非捕获多克隆抗体结合的抗原表位上，或结合到除捕获多克隆抗体结合的抗原表位以外的抗原表位上。夹心检测可以作为夹心ELISA法、夹心蛋白质印迹检测或夹心免疫磁性检测法来实施。In other embodiments, the level of the protein of interest is detected using a "sandwich" assay in which the capture antibody is immobilized to a solid substrate. The solid substrate can be contacted with a biological sample, thereby enabling any protein of interest in the sample to bind to the immobilized antibody. A "detection" antibody having specific binding affinity for the protein of interest can be used to determine the level of protein of interest bound to the capture antibody described above, as described above. It will be appreciated that in these sandwich assays, the capture antibody should not bind to the same epitope (or in the case of polyclonal antibodies, a fraction of the epitope to which the capture antibody binds) as the detection antibody. The range should not be the same as the range of the epitope bound by the detection antibody). Thus, if one mAb is used as the capture antibody, the detection antibody can be another mAb that binds an epitope that is either physically completely separated from the epitope that the capture mAb binds, or simply It partially overlaps; the detection antibody can also be a polyclonal antibody that binds to the epitope bound by the non-capture monoclonal antibody or binds to an epitope other than the epitope bound by the capture monoclonal antibody. If a polyclonal antibody is used as the capture antibody, the detection antibody may be a monoclonal antibody that binds to an epitope that is physically completely separated from, or partially overlaps with, any epitope bound by the capture polyclonal antibody; the detection antibody or It may be a polyclonal antibody that binds to an epitope to which the non-capture polyclonal antibody binds, or to an epitope other than the epitope to which the capture polyclonal antibody binds. Sandwich assays can be performed as sandwich ELISA assays, sandwich Western blot assays, or sandwich immunomagnetic assays.

适用的抗体(如捕获抗体)可以结合的固体基质包括但不限于，微孔板、管、如尼龙膜或硝酸纤维素膜等的膜、和珠子或颗粒(如，琼脂糖、纤维素、玻璃、聚苯乙烯，聚丙烯酰胺、磁性的、或可磁化珠或颗粒)。当使用自动免疫检测系统时，特别适用磁性颗粒或可磁化颗粒。Solid matrices to which suitable antibodies (e.g., capture antibodies) can be bound include, but are not limited to, microwell plates, tubes, membranes such as nylon or nitrocellulose membranes, and beads or particles (e.g., agarose, cellulose, glass , polystyrene, polyacrylamide, magnetic, or magnetizable beads or particles). Magnetic or magnetisable particles are particularly suitable when using automated immunoassay systems.

检测目标多肽的其他技术包括质谱-分光光度技术，例如电喷射离子化(ESI)和基质辅助激光解吸电离(MALDI)。Other techniques for detecting target polypeptides include mass spectrophotometric techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).

在上述方法或应用的实施方案中，优选的，上文所述的试剂包括但不限于：适于通过RT-PCR、实时定量PCR、免疫检测(例如ELISA、RIA、多重免疫实验、免疫荧光实验、蛋白印记、或斑点印记实验)、原位杂交或芯片技术检测Allergin-1表达水平用的试剂。更优选的，所述试剂包括针对Allergin-1基因的引物/探针、分子信标、RNAi、或针对Allergin-1蛋白的抗体和/或配体、锌指、以及小分子化合物。In the embodiments of the above methods or applications, preferably, the reagents described above include but are not limited to: suitable for RT-PCR, real-time quantitative PCR, immunoassay (such as ELISA, RIA, multiplex immunoassay, immunofluorescence assay) , western blotting, or dot blot), in situ hybridization or chip technology to detect the expression level of Allergin-1 reagents. More preferably, the reagents include primers/probes for Allergin-1 gene, molecular beacons, RNAi, or antibodies and/or ligands for Allergin-1 protein, zinc fingers, and small molecule compounds.

在上述方法或应用的实施方案中，优选的，上文所述的产品包括但不限于：芯片、试剂、试纸、制剂、试剂盒或高通量筛选平台。In the embodiment of the above method or application, preferably, the above-mentioned products include but are not limited to: chips, reagents, test strips, preparations, kits or high-throughput screening platforms.

在上述方法或应用的实施方案中，优选的，在一个实施方式中，根据本发明的方法或应用包括以下步骤：In the embodiment of the above method or application, preferably, in one embodiment, the method or application according to the present invention comprises the following steps:

a)收集受试者生物样品，优选骨髓细胞；a) collecting a subject's biological sample, preferably bone marrow cells;

b)检测在上述生物样品中CD34⁺CD38^-细胞中Allergin-1的表达水平；b) detecting the expression level of Allergin-1 in CD34⁺ CD38^- cells in the above biological sample;

c)将所述生物样品中测定的Allergin-1生物标志物的水平或量与生物标志物的参比水平或量进行比较。c) comparing the level or amount of the Allergin-1 biomarker determined in said biological sample to a reference level or amount of the biomarker.

优选的，所述参比标签是：1)从未患有白血病例如AML的群体获得的平均标签；和/或2)来自包括白血病例如AML患者的个体的组的平均或中值水平。Preferably, the reference signature is: 1) an average signature obtained from a population without leukemia, eg AML; and/or 2) an average or median level from a group comprising individuals with leukemia, eg AML.

应理解，采用上述技术中的一种或多种可以容易地检测根据本发明的任何目的基因转录物或目的蛋白质的表达。It will be appreciated that expression of any gene transcript of interest or protein of interest according to the present invention can readily be detected using one or more of the techniques described above.

诊断或病程监控产品Diagnostic or disease process monitoring products

在本发明的一个方面，本发明提供了一种白血病(尤其是AML)的诊断、病程监控和预后判断的产品，所述产品包括检测Allergin-1表达水平的芯片、制剂或检测试剂盒。In one aspect of the present invention, the present invention provides a product for the diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML), the product includes a chip, a preparation or a detection kit for detecting the expression level of Allergin-1.

优选的，本发明提供了一种白血病(尤其是AML)的诊断、病程监控和预后判断的检测试剂盒。进一步，所述检测试剂盒包括至少一对特异性扩增Allergin-1基因的引物；优选的，所述引物具有表3所示的SEQ ID NO：18、19、20、21、22或23的序列。Preferably, the present invention provides a detection kit for diagnosis, disease course monitoring and prognosis judgment of leukemia (especially AML). Further, the detection kit includes at least one pair of primers for specifically amplifying the Allergin-1 gene; preferably, the primers have SEQ ID NO: 18, 19, 20, 21, 22 or 23 shown in Table 3 sequence.

表3：Allergin-1Q-PCR引物(5`→3`)Table 3: Primers for Allergin-1Q-PCR (5`→3`)

优选的，用于检测生物标志物的本发明的检测试剂盒包含对以上蛋白有特异性的抗体，并且可还包含与用于与底物的显色反应的标签例如酶偶联的第二抗体；诱发与标签的显色反应的显色底物溶液；洗液；和酶反应停止液。其可还包含合适的微孔板、标准溶液和方案。对上文和下文描述的生物标志物有特异性的(第一)抗体可自身代替与标签偶联的第二抗体而与标签偶联。Preferably, the detection kit of the present invention for detecting biomarkers comprises antibodies specific to the above proteins, and may further comprise a second antibody coupled to a label for color reaction with the substrate, such as an enzyme ; a chromogenic substrate solution to induce a chromogenic reaction with the label; a washing solution; and an enzyme reaction stop solution. It may also contain suitable microplates, standard solutions and protocols. The (primary) antibody specific for the biomarkers described above and below may itself be coupled to the tag instead of a second antibody coupled to the tag.

用于检测生物标志物的本发明的检测试剂盒可通过经抗原-抗体结合反应定性地或定量地分析抗原而诊断白血病，并且抗原-抗体结合反应可通过常规方法如ELISA(酶联免疫吸附试验)或夹心试验进行测定。例如，用于检测如上文或下文所述的检测生物标志物的检测试剂盒可以设置为以下方式：通过使用表面涂布有分析物和对照的96孔微量滴定板进行用于与重组单克隆抗体蛋白反应的ELISA。作为抗原-抗体结合反应的接受器，可以使用聚乙烯树脂或聚苯乙烯树脂，硝基纤维素膜，或载玻片。进行显色反应的常规标签如HRP(辣根过氧化物酶)，碱性磷酸酶，胶体金，荧光标签，如荧光素，FITC(聚L-赖氨酸-异硫氰酸荧光素)或RITC(异氰酸罗丹明-B)或染料可优选用作第二抗体偶联物的标签或者用作第一抗体偶联物的标签。也考虑确定生物标记物的量的其它方法，特别是RIA(放射免疫检测)，聚丙烯酰胺凝胶上的蛋白印迹，免疫印迹和免疫组化染色。如本领域众所周知的，针对考虑用于生物标志物的定量确定的特定方法调整检测试剂盒。The detection kit of the present invention for detecting biomarkers can diagnose leukemia by qualitatively or quantitatively analyzing the antigen through the antigen-antibody binding reaction, and the antigen-antibody binding reaction can be detected by a conventional method such as ELISA (enzyme-linked immunosorbent assay). ) or sandwich test. For example, an assay kit for detecting a biomarker as described above or below can be configured in such a way that it can be used for the detection of a recombinant monoclonal antibody by using a 96-well microtiter plate coated with an analyte and a control surface. ELISA of protein reaction. As a receptor for antigen-antibody binding reaction, polyethylene resin or polystyrene resin, nitrocellulose membrane, or slide glass can be used. Conventional labels for chromogenic reactions such as HRP (horseradish peroxidase), alkaline phosphatase, colloidal gold, fluorescent labels such as fluorescein, FITC (poly-L-lysine-fluorescein isothiocyanate) or RITC (rhodamine-B isocyanate) or a dye can preferably be used as a label for the second antibody conjugate or as a label for the primary antibody conjugate. Other methods of quantifying biomarkers are also contemplated, in particular RIA (radioimmunoassay), Western blotting on polyacrylamide gels, immunoblotting and immunohistochemical staining. As is well known in the art, the assay kit is tailored to the particular method contemplated for the quantitative determination of the biomarkers.

如上指出的，在蛋白水平确定生物标志物的水平或量的情况下，试剂例如是抗体。可选地，在核酸水平检测水平或量的情况下，试剂可以是核酸分子。As noted above, where the level or amount of a biomarker is determined at the protein level, the reagent is, for example, an antibody. Alternatively, in the case of nucleic acid level detection levels or amounts, the reagent may be a nucleic acid molecule.

优选的，在上述产品的实施方案中，本发明中的检测试剂盒可用于检测Allergin-1的表达，优选的，所述的试剂盒包括检测有效量的检测Allergin-1基因的试剂，选自下组的一种或多种物质：容器、使用说明书、阳性对照物、阴性对照物、缓冲剂、助剂或溶剂。例如用于混悬或固定细胞的溶液，可检测的标签或标记，使核酸易于杂交的溶液，用于裂解细胞的溶液，或用于核酸纯化的溶液。Preferably, in the embodiment of the above product, the detection kit of the present invention can be used to detect the expression of Allergin-1, preferably, the kit includes a detection effective amount of reagents for detecting the Allergin-1 gene, selected from One or more substances of the group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent. Examples include solutions for suspending or fixing cells, detectable labels or labels, solutions for facilitating hybridization of nucleic acids, solutions for lysing cells, or solutions for nucleic acid purification.

更优选的，本发明的试剂盒中还可附有试剂盒的使用说明书，其中记载了如何采用试剂盒进行检测，和如何利用检测结果对白血病发展进行判断、对治疗方案进行选择。More preferably, the kit of the present invention can also be accompanied with an instruction manual of the kit, which records how to use the kit for detection, and how to use the detection results to judge the development of leukemia and select a treatment plan.

在上述产品的实施方案中，采用本发明的试剂盒，可通过选自下组的各种方法(包括但不限于)检测Allergin-1：实时定量反转录PCR、生物芯片检测法、DNA印迹法、或RNA印迹法或原位杂交法。本领域普通技术人员可根据实际条件和需要对检测方式进行调整和改变。In the embodiment of the above product, using the kit of the present invention, Allergin-1 can be detected by various methods selected from the group (including but not limited to): real-time quantitative reverse transcription PCR, biochip detection method, Southern blotting method, or Northern blot or in situ hybridization. Those skilled in the art can adjust and change the detection method according to actual conditions and needs.

在本发明的一个方面，用于检测生物标志物的本发明的阵列可以是微量滴定板。In one aspect of the invention, an array of the invention for detecting biomarkers may be a microtiter plate.

此外，本发明涉及诊断白血病或确定发生白血病的风险的计算机实施的方法，所述方法包括以下步骤：Furthermore, the present invention relates to a computer-implemented method of diagnosing leukemia or determining the risk of developing leukemia, said method comprising the steps of:

a)获得如本文中限定的白血病生物标志物，即Allergin-1在受试者和任选对照的生物样品中的测定水平或量的数据；a) obtaining data on the measured level or amount of a leukemia biomarker as defined herein, namely Allergin-1, in a biological sample of the subject and optionally a control;

b)通过将所述样品中的生物标志物的水平或量与从数据库获得的界限值或作为界限对照的测定水平或测定量获得的界限值进行比较而对步骤a)的数据进行运算；b) calculating the data of step a) by comparing the level or amount of the biomarker in said sample with a cut-off value obtained from a database or a measured level or measured amount as a cut-off control;

c)分析和鉴定其水平或量升高或降低至界限水平之上或之下的生物标志物。c) Analyzing and identifying biomarkers whose levels or amounts have increased or decreased above or below a cut-off level.

任选地，所述方法还包括在输出单元将每一种生物标志物的结果或仅仅阳性或阴性诊断或风险评估的结果以例如有色和突出显示的形式展示呈现的步骤。例如，通过主成分分析进行分析。所述分析可伴有误差分析。Optionally, the method further comprises the step of displaying the results of each biomarker or only positive or negative diagnostic or risk assessment results in eg colored and highlighted form in the output unit. For example, analysis by principal component analysis. The analysis may be accompanied by an error analysis.

本发明还涉及计算机介质或计算机程序产品，其具有用于执行根据本发明的方法的步骤的计算机可执行的指令。The invention also relates to a computer medium or a computer program product having computer-executable instructions for carrying out the steps of the method according to the invention.

在一些实施方式中，测定值和参比值的比较包括计算测定值和参比值之间的倍数差异，从而相应地鉴定测定值是否在界限值之上或之下。In some embodiments, the comparison of the measured value and the reference value comprises calculating the fold difference between the measured value and the reference value, thereby identifying whether the measured value is above or below a cutoff value, accordingly.

治疗和/或缓解疾病的方法和/或应用Method and/or use for treating and/or alleviating disease

本发明提供了在受试者中治疗和/或缓解白血病(例如，AML)疾病的方法或应用。具体而言通过调节Allergin-1在LSCs和AML细胞中的表达来影响上述细胞的增殖、分化和体内定植(或体内生长)，从而应用于白血病(例如，AML)疾病的治疗和/或缓解。在本发明的一个方面，本发明涉及试剂在制备用于治疗受试者的白血病(尤其是AML)的产品中的应用。优选的，所述试剂为Allergin-1表达或活性的的调节剂(例如抑制剂/拮抗剂)。优选的，所述产品为药物或药物组合。The present invention provides methods or uses for treating and/or ameliorating leukemia (eg, AML) disease in a subject. Specifically, by regulating the expression of Allergin-1 in LSCs and AML cells to affect the proliferation, differentiation and in vivo colonization (or in vivo growth) of the above cells, so as to be applied to the treatment and/or alleviation of leukemia (for example, AML) diseases. In one aspect of the invention, the invention relates to the use of an agent for the manufacture of a product for the treatment of leukemia, especially AML, in a subject. Preferably, the agent is a modulator (eg inhibitor/antagonist) of Allergin-1 expression or activity. Preferably, the product is a drug or a drug combination.

在上述方法或应用的实施方案中，调节Allergin-1的表达或活性可通过Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)来达成，例如降低编码Allergin-1的核酸的表达、降低Allergin-1蛋白质水平，或抑制Allergin-1的活性。例如，降低Allergin-1蛋白的活性、降低Allergin-1基因或蛋白的稳定性、下调Allergin-1的表达、减少Allergin-1蛋白有效作用时间、或抑制Allergin-1的转录和翻译的物质。In embodiments of the above-mentioned methods or uses, regulating the expression or activity of Allergin-1 can be achieved by a modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity, such as reducing the expression of a nucleic acid encoding Allergin-1 , reducing the protein level of Allergin-1, or inhibiting the activity of Allergin-1. For example, substances that reduce the activity of Allergin-1 protein, reduce the stability of Allergin-1 gene or protein, down-regulate the expression of Allergin-1, reduce the effective time of Allergin-1 protein, or inhibit the transcription and translation of Allergin-1.

优选的，上文所述的调节剂(例如抑制剂/拮抗剂)包括但不限于：核酸抑制物、蛋白抑制剂、蛋白水解酶、蛋白结合分子；及其组合。其中核酸抑制物选自：以Allergin-1或其转录本为靶序列、且能够抑制Allergin-1基因表达或基因转录的干扰分子，包括：shRNA(小发夹RNA)、小干扰RNA(siRNA)、dsRNA、微小RNA，或能表达或形成所述shRNA、小干扰RNA、dsRNA、微小RNA的构建物。蛋白结合分子选自：与Allergin-1蛋白特异性结合的物质，如能够抑制Allergin-1蛋白活性的抗体或配体；Allergin-1配体结合位点的竞争剂，包括Allergin-1受体与其配体结合片段、可溶性截短的Allergin-1受体、可溶性Allergin-1受体融合蛋白，例如包含IgG免疫球蛋白的Fc部分的Allergin-1融合蛋白、配体融合蛋白；拟肽；肽抑制剂；小分子化合物；及其组合。Preferably, the aforementioned modulators (eg inhibitors/antagonists) include, but are not limited to: nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein-binding molecules; and combinations thereof. Wherein the nucleic acid inhibitor is selected from: interfering molecules that take Allergin-1 or its transcript as the target sequence and can inhibit Allergin-1 gene expression or gene transcription, including: shRNA (small hairpin RNA), small interfering RNA (siRNA) , dsRNA, microRNA, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microRNA. The protein-binding molecules are selected from: substances that specifically bind to the Allergin-1 protein, such as antibodies or ligands capable of inhibiting the activity of the Allergin-1 protein; competitors of the Allergin-1 ligand binding site, including Allergin-1 receptors and their Ligand-binding fragment, soluble truncated Allergin-1 receptor, soluble Allergin-1 receptor fusion protein, e.g., Allergin-1 fusion protein comprising the Fc portion of an IgG immunoglobulin, ligand fusion protein; peptidomimetic; peptide inhibitory agents; small molecule compounds; and combinations thereof.

更优选的，本发明还涉及使用RNA干扰(RNAi)抑制Allergin-1的表达。任何类型的RNA干扰序列都可用于本发明。其非限制性的例子包括短干扰RNA(siRNA)分子或短发夹RNA(shRNA)。可用多种算法进行RNAi序列设计。更优选的，所述调节剂(例如抑制剂/拮抗剂)为shRNA；尤其优选的，所述shRNA序列如表2中的SEQ ID NO：12、13、14、15、16或17所示的序列。More preferably, the present invention also relates to using RNA interference (RNAi) to inhibit the expression of Allergin-1. Any type of RNA interference sequence can be used in the present invention. Non-limiting examples thereof include short interfering RNA (siRNA) molecules or short hairpin RNA (shRNA). RNAi sequence design can be performed using a variety of algorithms. More preferably, the modulator (such as an inhibitor/antagonist) is shRNA; especially preferably, the shRNA sequence is as shown in SEQ ID NO: 12, 13, 14, 15, 16 or 17 in Table 2 sequence.

表2：shRNA序列Table 2: shRNA sequences

此外，目的序列可以选择为具有与其他变种或基因序列同源性低的序列。可以通过在表达目的基因产物的细胞中引入或表达该RNAi序列来对RNAi分子的效果进行评估。目的基因产物的mRNA或蛋白质水平的实质改变是RNAi分子在抑制该基因表达中的有效性的指征。在细胞中表达RNAi分子的方法为本领域所公知，其中包括，例如，慢病毒载体。In addition, the target sequence can be selected as a sequence with low homology to other variants or gene sequences. The effect of RNAi molecules can be assessed by introducing or expressing the RNAi sequence in cells expressing the gene product of interest. A substantial change in the mRNA or protein levels of a gene product of interest is indicative of the effectiveness of the RNAi molecule in inhibiting expression of that gene. Methods for expressing RNAi molecules in cells are known in the art and include, for example, lentiviral vectors.

更优选的，所述调节剂(例如抑制剂/拮抗剂)为配体结合竞争剂。Allergin-1信号也可以通过给予结合Allergin-1配体的竞争剂来抑制。这可以例如通过给予Allergin-1细胞外结构域的可溶性片段来达成，其可选择地偶联到载体蛋白(如本领域已知的IgG免疫球蛋白)上。例如，已经公开了给予用Allergin-1片段和人IgGl的Fc部分形成的Allergin-1-Fc融合蛋白。融合蛋白的IgG Fc部分可来自任何IgG亚类(如IgGl，IgG2，IgG3和IgG4)。重要的是，对人的治疗并不一定需要给予野生型的人Allergin-1片段：也可以使用来自其他哺乳动物的其他Allergin-1片段，而且其中可以引入一个或多个氨基酸取代，只要该片段还保有与内源性人Allergin-1竞争与配体结合的能力即可。More preferably, the modulator (eg inhibitor/antagonist) is a ligand binding competitor. Allergin-1 signaling can also be inhibited by administering a competitor that binds Allergin-1 ligand. This can be achieved, for example, by administering a soluble fragment of the extracellular domain of Allergin-1, optionally coupled to a carrier protein such as an IgG immunoglobulin as known in the art. For example, administration of an Allergin-1-Fc fusion protein formed with an Allergin-1 fragment and the Fc portion of human IgG1 has been disclosed. The IgG Fc portion of the fusion protein can be from any IgG subclass (eg IgGl, IgG2, IgG3 and IgG4). Importantly, treatment in humans does not necessarily require the administration of wild-type human Allergin-1 fragments: other Allergin-1 fragments from other mammals can also be used, and one or more amino acid substitutions can be introduced therein, as long as the fragment It only needs to retain the ability to compete with endogenous human Allergin-1 for ligand binding.

在上述方法或应用的实施方案中，治疗白血病例如AML的其他手段包括给予结合剂，如蛋白质，肽和/或抗体或其部分(例如，Fab,F(ab’)2，Fv或单链Fv片段)，这种结合剂与治疗靶点相互作用，例如结合和/或中和治疗靶点。对白血病例如AML患者给予抗Allergin-1结合剂，例如抗Allergin-1抗体，可以通过抑制和/或拮抗Allergin-1而减轻疾病的症状。该抗体可以是分离的抗体。在一个实施方案中，该抗体是拮抗性抗体。在另一实施方案中，该抗体是中和抗体。在进一步的实施方案中，抗体调节、降低和/或抑制一个或多个Allergin-1相关的活性，包括但不限于调节、降低和/或抑制Allergin-1与Allergin-1配体相互作用；调节、降低和/或抑制Allergin-1介导的信号转导；和调节、降低和/或抑制Allergin-1激活的基因的表达。本发明的抗Allergin-1抗体可以包括，例如，特异性结合Allergin-1的抗体和/或结合Allergin-1受体的膜结合形式而不激活Allergin-1受体的抗体。本发明的抗Allergin-1抗体也可包括来自任何物种的单结构域抗体。也可以使用替代的结合结构域多肽来抑制和/或拮抗Allergin-1活性或Allergin-1信号转导。In embodiments of the above methods or uses, other means of treating leukemia such as AML include administration of binding agents, such as proteins, peptides and/or antibodies or portions thereof (e.g., Fab, F(ab')2, Fv or single chain Fv Fragments), such binding agents interact with, e.g., bind and/or neutralize, a therapeutic target. Administration of anti-Allergin-1 binding agents, such as anti-Allergin-1 antibodies, to patients with leukemia such as AML can alleviate the symptoms of the disease by inhibiting and/or antagonizing Allergin-1. The antibody can be an isolated antibody. In one embodiment, the antibody is an antagonist antibody. In another embodiment, the antibody is a neutralizing antibody. In a further embodiment, the antibody modulates, reduces and/or inhibits one or more Allergin-1-related activities, including but not limited to modulating, reducing and/or inhibiting the interaction of Allergin-1 with an Allergin-1 ligand; modulating , reducing and/or inhibiting Allergin-1 mediated signal transduction; and regulating, reducing and/or inhibiting the expression of Allergin-1 activated genes. Anti-Allergin-1 antibodies of the invention can include, for example, antibodies that specifically bind Allergin-1 and/or antibodies that bind a membrane-bound form of the Allergin-1 receptor without activating the Allergin-1 receptor. Anti-Allergin-1 antibodies of the invention may also include single domain antibodies from any species. Alternative binding domain polypeptides can also be used to inhibit and/or antagonize Allergin-1 activity or Allergin-1 signaling.

更优选的，所述调节剂(例如抑制剂/拮抗剂)为特异性与Allergin-1结合的抗体；优选的，例如，所述特异性抗体包括单克隆抗体、多克隆抗体、中和抗体、抗原结合片段、偶联Allergin-1抗体。More preferably, the modulator (such as an inhibitor/antagonist) is an antibody that specifically binds to Allergin-1; preferably, for example, the specific antibody includes a monoclonal antibody, a polyclonal antibody, a neutralizing antibody, Antigen-binding fragment, conjugated to Allergin-1 antibody.

在上述方法或应用的实施方案中，优选的，所述治疗为免疫治疗。In the embodiments of the above methods or uses, preferably, the treatment is immunotherapy.

在上述方法或应用的实施方案中，优选的，所述治疗为基因治疗。例如，可直接将Allergin-1的调节剂(例如抑制剂/拮抗剂)通过诸如注射等方法给药于受试者；或者，可通过一定的途径将携带Allergin-1的调节剂(例如抑制剂/拮抗剂)的表达单位(比如表达载体或病毒等，或siRNA或shRNA)递送到靶点上，并使之表达活性的Allergin-1调节剂(例如抑制剂/拮抗剂)，具体情况需视所述的抑制剂的类型而定，这些均是本领域技术人员所熟知的。In the embodiments of the above methods or uses, preferably, the therapy is gene therapy. For example, the modulator (such as inhibitor/antagonist) of Allergin-1 can be directly administered to the subject through methods such as injection; /antagonist) expression unit (such as expression vector or virus, etc., or siRNA or shRNA) delivered to the target, and make it express active Allergin-1 modulator (such as inhibitor/antagonist), depending on the specific situation It depends on the type of inhibitor, which are well known to those skilled in the art.

在上述应用的实施方案中，用于治疗白血病的方法还包括向所述受试者施用治疗有效量的Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)与一种或多种治疗剂的组合。In the embodiment of the above-mentioned application, the method for treating leukemia further comprises administering to the subject a therapeutically effective amount of a modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity together with one or more Combination of therapeutic agents.

优选的，所述一种或多种治疗剂为IFN-γ。Preferably, the one or more therapeutic agents are IFN-γ.

在本发明的一个方面，本发明涉及调节剂在制备药物、制剂或治疗试剂盒中的应用，所述药物、制剂或治疗试剂盒用于调节Allergin-1在LSCs和AML细胞中的表达来影响上述细胞的增殖、分化和体内定植(或体内生长)，从而应用于白血病(例如，AML)疾病的治疗和/或缓解。In one aspect of the present invention, the present invention relates to the use of modulators in the preparation of medicaments, preparations or therapeutic kits for regulating the expression of Allergin-1 in LSCs and AML cells to affect Proliferation, differentiation and in vivo colonization (or in vivo growth) of the above cells are applied to the treatment and/or alleviation of leukemia (eg, AML) diseases.

优选的，在上述应用的实施方案中，所述药物包含上文所述的Allergin-1表达或活性的的调节剂(例如抑制剂/拮抗剂)或其药学上可接受衍生物或功能类似物和药学上可接受的辅料、赋形剂、载体、溶媒或它们的组合。Preferably, in the embodiment of the above-mentioned application, the drug comprises the above-mentioned regulator of Allergin-1 expression or activity (such as an inhibitor/antagonist) or a pharmaceutically acceptable derivative or functional analog thereof And pharmaceutically acceptable adjuvant, excipient, carrier, vehicle or their combination.

也应认识到，本发明公开的上述调节剂(例如抑制剂/拮抗剂)或功能类似物可以以游离形式存在用于治疗，或者如果适当可以以其药学上可接受的衍生物的形式存在。It will also be appreciated that the aforementioned modulators (eg inhibitors/antagonists) or functional analogs disclosed herein may exist in free form for use in therapy or, if appropriate, in the form of pharmaceutically acceptable derivatives thereof.

药学上可接受衍生物的一些非限制性的实施方案包括药学上可接受的前药，盐，酯，这些酯的盐，或者对有需要的患者给药时能直接或间接提供本发明所述上述激动剂、促进剂、拮抗剂或抑制剂或功能类似物或其代谢产物或残留物的任何另外的加合物或衍生物。Some non-limiting embodiments of pharmaceutically acceptable derivatives include pharmaceutically acceptable prodrugs, salts, esters, salts of these esters, or the ability to directly or indirectly provide the present invention when administered to a patient in need thereof. Any additional adducts or derivatives of the aforementioned agonists, enhancers, antagonists or inhibitors or functional analogs or metabolites or residues thereof.

可以施用有效量的上文中所述的调节剂(例如抑制剂/拮抗剂)以预防或治疗疾病。上文中所述的调节剂(例如抑制剂/拮抗剂)，例如抗Allergin-1抗体或其抗原结合片段适当剂量可以根据待治疗的疾病类型，调节剂(例如抑制剂/拮抗剂)的类型，疾病的严重程度和病程，治疗的严重程度、个体的临床症状，个体的临床病史和对治疗的反应来确定。在一些实施方案中，用上文中所述的调节剂(例如抑制剂/拮抗剂)与其他药物的联合治疗是协同的，因此相对于单独的调节剂(例如抑制剂/拮抗剂)的有效剂量而言，降低了组合中的有效剂量。An effective amount of a modulator (eg, inhibitor/antagonist) described above can be administered to prevent or treat a disease. The modulator (such as inhibitor/antagonist) mentioned above, such as anti-Allergin-1 antibody or its antigen-binding fragment, the appropriate dosage can be according to the type of disease to be treated, the type of modulator (such as inhibitor/antagonist), The severity and course of the disease, the severity of the treatment, the individual's clinical symptoms, the individual's clinical history and response to treatment are determined. In some embodiments, combination therapy with a modulator (e.g., inhibitor/antagonist) described above with other agents is synergistic, and thus an effective dose relative to the modulator (e.g., inhibitor/antagonist) alone In terms of , the effective dose in the combination is reduced.

一般而言，本文提供给人的调节剂(例如抑制剂/拮抗剂)，例如Allergin-1抗体或其抗原结合片段的治疗有效量为约0.0001mg/kg至约100mg/kg(例如，约0.01mg/kg、约0.1mg/kg、约0.5mg/kg、约1mg/kg)的患者体重，无论是一次还是多次给药。在一些实施方案中，所使用的抗体或其抗原结合片段是约0.01mg/kg、约0.015mg/kg、约0.1mg/kg、约0.15mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约2mg/kg、约5mg/kg、约10mg/kg、约15mg/kg、约20mg/kg、25mg/kg、约30mg/kg、约35mg/kg、约40mg/kg、约45mg/kg、约50mg/kg、约55mg/kg、约60mg/kg、约65mg/kg、约70mg/kg、约75mg/kg、约80mg/kg、约85mg/kg、约90mg/kg、约95mg/kg或约100mg/kg。在一些实施方案中，以约50mg/kg或更少、10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少、或小于或等于0.1mg/kg的剂量施用抗体或其抗原结合片段。在一些实施方案中，所使用的抗体或其抗原结合片段是约0.01mg/kg-0.2mg/kg。Generally, a therapeutically effective amount of a modulator (e.g., an inhibitor/antagonist), such as an Allergin-1 antibody or antigen-binding fragment thereof, provided herein to a human is from about 0.0001 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg), regardless of single or multiple doses. In some embodiments, the antibody or antigen-binding fragment thereof used is about 0.01 mg/kg, about 0.015 mg/kg, about 0.1 mg/kg, about 0.15 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 2mg/kg, about 5mg /kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg. In some embodiments, at about 50 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or less than or equal to 0.1 mg The antibody or antigen-binding fragment thereof was administered at a dose of 1/kg. In some embodiments, the antibody or antigen-binding fragment thereof used is about 0.01 mg/kg-0.2 mg/kg.

本发明公开的上述调节剂(例如抑制剂/拮抗剂)或其功能类似物可制备并包装为散装形式，其中可提取安全有效量，然后以粉末或糖浆形式给予患者。或者，本发明公开的上述调节剂(例如抑制剂/拮抗剂)可制备并包装为单位剂型，其中每个物理上离散的单位含有安全有效量。当以单位剂型制备时，本发明公开的上述调节剂(例如抑制剂/拮抗剂)例如，Allergin-1抗体或其抗原结合片段以约1mg至约100mg的单位剂量，优选以约5mg、约10mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、50mg的单位剂量施用。The above modulators (such as inhibitors/antagonists) disclosed in the present invention or their functional analogues can be prepared and packaged in bulk form, from which a safe and effective amount can be extracted, and then administered to patients in the form of powder or syrup. Alternatively, the aforementioned modulators (eg, inhibitors/antagonists) disclosed herein may be prepared and packaged in unit dosage form, wherein each physically discrete unit contains a safe and effective amount. When prepared in unit dosage form, the above modulators (such as inhibitors/antagonists) disclosed in the present invention, for example, Allergin-1 antibody or antigen-binding fragment thereof, are in a unit dosage of about 1 mg to about 100 mg, preferably about 5 mg, about 10 mg , 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg in unit dose administration.

在一些实施方案中，施用剂量可以在治疗过程中改变。例如，在一些实施方案中，初始施用剂量可以高于随后的施用剂量。在一些实施方案中，取决于受试者的反应，给药剂量可以在治疗过程中变化。In some embodiments, the dosage administered may vary during the course of treatment. For example, in some embodiments, the initial dose administered may be higher than subsequent doses administered. In some embodiments, the dosage administered may vary during the course of treatment, depending on the subject's response.

上述调节剂(例如抑制剂/拮抗剂)或者一种多种其他药物/治疗剂可以多次间隔施用特定剂量，例如每天一次，每天两次或更多次；每周一次，每周两次或三次或更多次；每月一次，两次或更多次；每周一次，每两周一次，每三周一次，每月一次或每两个月一次，或者更少。在一些实施方案中，施用的剂量可以随治疗过程而变化。例如，在某些实施方案中，施用的初始剂量可以高于随后的剂量。在某些实施方案中，根据治疗对象的反应，在治疗过程中调节给药剂量。可以调整剂量方案以达到最佳应答(例如治疗应答)。例如，可以在一段时间内施用单剂量或多次分剂量施用。The aforementioned modulators (e.g. inhibitors/antagonists) or one or more of the other drugs/therapeutics may be administered in a given dose at multiple intervals, e.g. once a day, twice a day or more; once a week, twice a week or Three or more times; once a month, two or more times; once a week, once every two weeks, once every three weeks, once a month or once every two months, or less often. In some embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial dose administered may be higher than subsequent doses. In certain embodiments, the dosage administered is adjusted during the course of treatment based on the response of the treated subject. Dosage regimens may be adjusted to achieve an optimal response (eg, a therapeutic response). For example, a single dose or multiple divided doses may be administered over a period of time.

本发明公开的上述“药学上可接受的辅料、赋形剂、载体、溶媒”意指与给药剂型或药物组合物一致性相关的药学上可接受的材料，混合物或溶媒。每种辅料在混合时必须与药物组合物的其它成分相容，以避免对患者给药时会大大降低本发明公开的上述调节剂(例如抑制剂/拮抗剂)的功效的相互作用和会导致不是药学上可接受的药物组合物的相互作用。此外，每种辅料必须是药学上可接受的，例如，具有足够高的纯度。The above-mentioned "pharmaceutically acceptable adjuvant, excipient, carrier, vehicle" disclosed in the present invention means a pharmaceutically acceptable material, mixture or vehicle related to the consistency of the dosage form or pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when mixed in order to avoid interactions that would substantially reduce the efficacy of the aforementioned modulators (e.g. inhibitors/antagonists) disclosed in the present invention when administered to a patient and would lead to Not a pharmaceutically acceptable drug composition interaction. Furthermore, each excipient must be pharmaceutically acceptable, eg, of sufficiently high purity.

合适的药学上可接受的“药学上可接受的辅料、赋形剂、载体、溶媒”会依所选具体剂型而不同。此外，可根据它们在组合物中的特定功能来选择药学上可接受的辅料。例如，可选择能有助于生产均一剂型的某些药学上可接受的辅料。可选择能有助于生产稳定剂型的某些药学上可接受的辅料。可选择对患者给药时有助于携带或运输本发明公开化合物从身体的一个器官或部分到身体的另一个器官或部分的某些药学上可接受的辅料。可选择增强患者依从性的某些药学上可接受的辅料。Suitable pharmaceutically acceptable "pharmaceutically acceptable excipients, excipients, carriers, and vehicles" will vary depending on the selected specific dosage form. Furthermore, pharmaceutically acceptable excipients can be selected according to their specific function in the composition. For example, certain pharmaceutically acceptable excipients can be selected to aid in the production of uniform dosage forms. Certain pharmaceutically acceptable excipients can be selected to aid in the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be selected to aid in the carrying or transport of the disclosed compounds from one organ or part of the body to another organ or part of the body when administered to a patient. Certain pharmaceutically acceptable excipients can be selected to enhance patient compliance.

用于本文公开的“药学上可接受的辅料、赋形剂、载体、溶媒”可包括例如药学上可接受的液体、凝胶或固体载体、水性介质、非水性介质、抗微生物材料、渗透材料、缓冲剂、抗氧化剂、麻醉剂、悬浮/分散剂、螯合剂、稀释剂、佐剂或无毒辅助物质，本领域已知的其他组分或上述的各种组合。"Pharmaceutically acceptable excipients, excipients, carriers, vehicles" used in the disclosure herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial materials, osmotic materials , buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants or non-toxic auxiliary substances, other components known in the art or various combinations of the above.

合适的组分可以包括例如抗氧化剂、填充剂、粘合剂、崩解剂、缓冲剂、防腐剂、润滑剂、增香剂、增稠剂、着色剂、乳化剂或稳定剂，例如糖和环糊精。合适的抗氧化剂可以包括例如甲硫氨酸、抗坏血酸、EDTA、硫代硫酸钠、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙醇酸、脱水山梨醇、丁基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colourants, emulsifiers or stabilizers, such as sugars and Cyclodextrin. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, sorbitan, butyl anise ether, butylated hydroxytoluene and/or propyl gallate.

此外，药学上可接受的赋形剂或载体可包括例如水性媒介物，例如氯化钠注射剂、林格氏注射剂、等渗右旋糖注射剂、无菌水注射剂或右旋糖和乳酸林格氏剂注射剂，非水媒介物例如固定油。植物来源、棉籽油、玉米油、芝麻油或花生油、抑菌或抑菌浓度的抗菌剂、等渗剂(例如氯化钠或葡萄糖)、缓冲剂(例如磷酸盐或柠檬酸盐缓冲剂)、抗氧化剂(例如硫酸氢钠)、局部麻醉剂作为盐酸普鲁卡因、悬浮剂和分散剂，例如羧甲基纤维素钠，羟丙基甲基纤维素或聚乙烯吡咯烷酮、乳化剂，例如聚山梨酯80(TWEEN-80)，螯合剂或螯合剂，例如EDTA(乙二胺四乙酸)或EGTA(乙二醇四乙酸)、乙醇、聚乙二醇、丙二醇、钠氢氧化物、盐酸、柠檬酸或乳酸。可用作载体的抗微生物剂可以在多剂量容器中添加到药物组合物中，该容器包括苯酚或甲酚、汞、苯甲醇、氯丁醇、对羟基苯甲酸甲酯和丙基对羟基苯甲酸酯、硫柳汞、苯扎氯铵和苄索氯铵。合适的赋形剂可包括例如水、盐水、右旋糖、甘油或乙醇。合适的无毒辅助物质可以包括，例如，润湿剂或乳化剂、pH缓冲剂、稳定剂、溶解度增强剂、或诸如乙酸钠、脱水山梨糖醇单月桂酸酯、三乙醇胺油酸酯或环糊精的试剂。In addition, pharmaceutically acceptable excipients or carriers may include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's For injection, non-aqueous vehicles such as fixed oils. Vegetable sources, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial agents at bacteriostatic or bacteriostatic concentrations, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antibacterial Oxidizing agents such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), chelating agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents useful as carriers can be added to pharmaceutical compositions in multi-dose containers that include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben, and propylparaben Formate, Thimerosal, Benzalkonium Chloride, and Benzethonium Chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclic Dextrin reagent.

优选的，合适的药学上可接受的辅料包括以下类型的辅料：稀释剂、填充剂、粘合剂、崩解剂、润滑剂、助流剂、造粒剂、包衣剂、润湿剂、溶剂、共溶剂、助悬剂、乳化剂、甜味剂、矫味剂、掩味剂、着色剂、防结块剂、保湿剂、螯合剂、塑化剂、增粘剂、抗氧化剂、防腐剂、稳定剂、表面活性剂和缓冲剂。技术人员可认识到，某些药学上可接受的辅料可提供不止一种功能，并提供可供选择的功能，这取决于制剂中存在多少该辅料和制剂中存在哪些其他辅料。Preferably, suitable pharmaceutically acceptable auxiliary materials include the following types of auxiliary materials: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, Solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste-masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, tackifiers, antioxidants, preservatives agents, stabilizers, surfactants and buffers. The skilled artisan will recognize that certain pharmaceutically acceptable excipients can serve more than one function, and serve alternative functions, depending on how much of that excipient is present in the formulation and which other excipients are present in the formulation.

本发明公开的上述调节剂(例如抑制剂/拮抗剂)或其功能类似物通常被配制成适合于通过所需途径对患者给药的剂型。例如，剂型包括那些适合于以下给药途径的剂型：(1)口服给药，例如片剂、胶囊剂、囊片剂、丸剂、含片剂、粉剂、糖浆剂、酏剂、混悬剂、溶液剂、乳剂、颗粒剂和扁囊剂；(2)胃肠外给药，例如无菌溶液剂、混悬剂和冻干粉末剂；(3)透皮给药，例如透皮贴片剂；(4)直肠给药，例如栓剂；(5)吸入，例如气雾剂、溶液剂和干粉剂；和(6)局部给药，例如乳膏剂、油膏剂、洗剂、溶液剂、糊剂、喷雾剂、泡沫剂和凝胶剂。The aforementioned modulators (eg, inhibitors/antagonists) or functional analogs thereof disclosed herein are generally formulated into dosage forms suitable for administration to a patient by the desired route. For example, dosage forms include those suitable for the following routes of administration: (1) oral administration, such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, Solutions, emulsions, granules, and cachets; (2) parenteral administration, such as sterile solutions, suspensions, and lyophilized powders; (3) transdermal administration, such as transdermal patches (4) rectal administration, such as suppository; (5) inhalation, such as aerosol, solution and dry powder; and (6) topical administration, such as cream, ointment, lotion, solution, paste , sprays, foams and gels.

在一些实施方案中，上述调节剂(例如抑制剂/拮抗剂)或其功能类似物例如Allergin-1抗体或其抗原结合片段通过植入、通过吸入、鞘内、心室内或鼻内静脉内、肌内、皮下、局部、口服、经皮、腹膜内、眶内、口服给药，优选静脉内给药。In some embodiments, the aforementioned modulators (e.g., inhibitors/antagonists) or functional analogs thereof, such as Allergin-1 antibodies or antigen-binding fragments thereof, are administered by implantation, by inhalation, intrathecally, intraventricularly or intranasally, intravenously, Intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, oral administration, preferably intravenous administration.

在一些实施方案中、上述方法或应用可以进一步包括另外的疗法。附加疗法可以是放射疗法、手术、化学疗法、基因疗法、DNA疗法、病毒疗法、RNA疗法、免疫疗法、骨髓移植、纳米疗法、单克隆抗体疗法或前述的组合。附加疗法可以采取辅助或新辅助疗法的形式。在一些实施方案中，另外的疗法是小分子酶抑制剂或抗转移剂的施用。在一些实施方案中，另外的疗法是施用副作用限制剂(例如，旨在减轻治疗副作用的发生和/或严重性的试剂，例如抗恶心剂等)。在一些实施方案中，另外的疗法是放射疗法。在一些实施方案中，该另外的疗法是手术。在一些实施例中，附加疗法是放射疗法和手术的组合。在一些实施方案中，另外的疗法是伽马辐射。In some embodiments, the methods or uses described above may further comprise additional therapies. Additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. Add-on therapy can take the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, the additional therapy is the administration of side effect limiting agents (eg, agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma radiation.

本发明的治疗方法或应用可以在手术之前或之后用于去除肿瘤，并且可以在放射疗法之前，之中或之后使用。在一些实施方案中，将本发明治疗方法或应用施用于先前未曾用生物治疗剂或化学治疗剂治疗过的患者。在其他实施方案中，将本发明治疗方法或应用施用于在用生物治疗剂或化学治疗剂(即经历过治疗)的先前治疗后未能实现持续应答的患者。The treatment methods or uses of the invention may be used to remove tumors before or after surgery, and may be used before, during or after radiation therapy. In some embodiments, the treatment methods or uses of the invention are administered to patients who have not previously been treated with a biotherapeutic or chemotherapeutic agent. In other embodiments, the treatment methods or uses of the invention are administered to patients who have failed to achieve a sustained response following prior treatment with a biotherapeutic or chemotherapeutic agent (ie, treatment-experienced).

药物、药物组合和/或药物筛选方法Drugs, drug combinations and/or drug screening methods

在本发明的一个方面，本发明提供了一种治疗白血病的药物、组合物或治疗试剂盒，其中，药物、组合物或治疗试剂盒包括Allergin-1功能性表达的调节剂(例如抑制剂/拮抗剂)，和/或与所述调节剂(例如抑制剂/拮抗剂)配伍的其他类药物以及药学上可接受的辅料、赋形剂、载体、溶媒或它们的组合。In one aspect of the present invention, the present invention provides a medicine, composition or treatment kit for treating leukemia, wherein, the medicine, composition or treatment kit includes a regulator of Allergin-1 functional expression (such as an inhibitor/ antagonist), and/or other drugs compatible with the modulator (eg inhibitor/antagonist) and pharmaceutically acceptable adjuvant, excipient, carrier, vehicle or their combination.

优选的，在上述药物或组合物实施方案中，所述调节剂(例如抑制剂/拮抗剂)如上文“治疗和/或缓解疾病的方法和/或应用”部分中所限定。Preferably, in the above drug or composition embodiment, the modulator (eg inhibitor/antagonist) is as defined above in the section "Methods and/or Applications for Treating and/or Alleviating Diseases".

优选的，在上述药物或组合物实施方案中，所述药学上可接受的辅料、赋形剂、载体、溶媒或它们的组合如上文“治疗和/或缓解疾病的方法和/或应用”部分中所限定。Preferably, in the above drug or composition embodiment, the pharmaceutically acceptable adjuvant, excipient, carrier, vehicle or their combination is as described in the above "methods and/or applications for treating and/or alleviating diseases" limited in.

优选的，所述其他类药物为IFN-γ。Preferably, the other class of drugs is IFN-γ.

优选的，本发明的药物或组合物还可以与其他治疗白血病的药物联用，其他治疗性药物可以与主要的活性成分同时给药，甚至在同一组合物中同时给药。还可以以单独的组合物或与主要的活性成分不同的剂量形式单独给予其它治疗性药物。Preferably, the drug or composition of the present invention can also be used in combination with other drugs for treating leukemia, and other therapeutic drugs can be administered simultaneously with the main active ingredient, even in the same composition. The other therapeutic agent may also be administered alone, in a separate composition or dosage form different from the main active ingredient.

在本发明的一个方面，本发明提供了生物标志物Allergin-1在筛选治疗白血病(尤其是AML)的候选药物的方法。优选的，所述方法包括：用待筛选物质处理表达或含有Allergin-1基因的体系；和检测所述体系中Allergin-1基因的表达水平；其中，若所述待筛选的物质可以降低Allergin-1基因的表达水平，则表明该待筛选物质是治疗白血病(尤其是AML)的候选药物。更优选的，上文所述的体系包括(但不限于)：细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系；所述候选药物包括(但不限于)：针对Allergin-1基因或其上游或下游基因设计的干扰分子、核酸抑制物、小分子化合物。In one aspect of the present invention, the present invention provides a method for screening a candidate drug for the treatment of leukemia (especially AML) using the biomarker Allergin-1. Preferably, the method includes: treating the system expressing or containing the Allergin-1 gene with the substance to be screened; and detecting the expression level of the Allergin-1 gene in the system; wherein, if the substance to be screened can reduce the Allergin-1 gene expression level; 1 gene expression level, it indicates that the substance to be screened is a candidate drug for treating leukemia (especially AML). More preferably, the systems described above include (but are not limited to): cell systems, subcellular systems, solution systems, tissue systems, organ systems or animal systems; the candidate drugs include (but are not limited to): targeting Allergin- 1 Interfering molecules, nucleic acid inhibitors, and small molecular compounds designed for genes or their upstream or downstream genes.

本领域技术人员通过上下文说明以及通过实施例可明了本发明的其他目的。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。在以下实施例中的定量试验中，均设置三次重复实验，结果取平均值。Other objects of the present invention can be understood by those skilled in the art from the context description and the examples. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. In the quantitative experiments in the following examples, three repeated experiments were set up, and the results were averaged.

在以下实施例中，抗人Allergin-1抗体(克隆#767727，小鼠IgG1)购自美国R&DSystems公司。使用噬菌体展示技术制备了针对人Allergin-1L(Allergin-1长形式，GenBank：人ALLERGIN-1L，AB542950)的全人Allergin-1抗体(人IgG1)。In the following examples, anti-human Allergin-1 antibody (clone #767727, mouse IgG1) was purchased from R&D Systems, USA. A fully human Allergin-1 antibody (human IgG1) against human Allergin-1L (Allergin-1 long form, GenBank: human ALLERGIN-1L, AB542950) was prepared using phage display technology.

实施例1Example 1

TARGET(Therapeutically Available Research to Generate EffectiveTreatments)数据库分析TARGET (Therapeutically Available Research to Generate Effective Treatments) database analysis

实验方法:experimental method:

基于TARGET数据库(https://ocg.cancer.gov/programs/target/,accessedDecember 9,2019)中的AML患者RNA Sequencing数据(FPKM)分析Allergin-1表达水平与AML预后的相关性及Allergin-1在AML患者各亚型中的表达。依据Allergin-1表达值的中位数将87位M4/M5亚型AML患者分为高表达组与低表达组，进行不同组AML患者的Kaplan-Meier生存分析。Based on the RNA Sequencing data (FPKM) of AML patients in the TARGET database (https://ocg.cancer.gov/programs/target/, accessed December 9, 2019), the correlation between the expression level of Allergin-1 and the prognosis of AML and the correlation between the expression level of Allergin-1 and the prognosis of AML were analyzed. Expression in each subtype of AML patients. According to the median expression value of Allergin-1, 87 patients with M4/M5 subtype AML were divided into high expression group and low expression group, and the Kaplan-Meier survival analysis of different groups of AML patients was performed.

实验结果：Experimental results:

通过TARGET AML数据库进行分析细胞表面生物标志物在AML患者中的表达情况，发现在M4/M5亚型AML患者中Allergin-1表达与AML患者生存率之间存在明显的相关性，预后差的患者中Allergin-1的表达显著升高(图1A)，且在M5亚型AML患者中其表达高于其他亚型患者(图1B)。其中，图1B中，Allergin-1在M5亚型AML患者中高表达，M0-M5，*p＝0.028；M1-M5，****p<0.0001；M2-M5，****p<0.0001；M4-M5，****p<0.0001；M5-M6，*p＝0.0274；M5-M7，***p＝0.0005；M5-NOS，**p＝0.0045；M5-Unknown，**p＝0.0092。Error bars,s.e.m。The expression of cell surface biomarkers in AML patients was analyzed through the TARGET AML database, and it was found that there was a significant correlation between the expression of Allergin-1 and the survival rate of AML patients in M4/M5 subtype AML patients, and patients with poor prognosis The expression of Allergin-1 in M5 subtype AML patients was significantly increased (Fig. 1A), and its expression was higher than that of other subtypes in patients with AML (Fig. 1B). Among them, in Figure 1B, Allergin-1 is highly expressed in M5 subtype AML patients, M0-M5, *p=0.028; M1-M5, ****p<0.0001; M2-M5, ****p<0.0001 ;M4-M5, ****p<0.0001; M5-M6, *p=0.0274; M5-M7, ***p=0.0005; M5-NOS, **p=0.0045; M5-Unknown, **p = 0.0092. Error bars, s.e.m.

结果提示Allergin-1可应用于AML病程监控和预后判断。The results suggest that Allergin-1 can be applied to the course monitoring and prognosis judgment of AML.

实施例2Example 2

检测HSCs、LSCs、患者原代AML细胞和AML细胞系细胞的Allergin-1表达以及检测LSCs自我更新Detection of Allergin-1 expression in HSCs, LSCs, patient primary AML cells and AML cell lines, and detection of self-renewal of LSCs

实验方法:experimental method:

原发AML患者骨髓样本(详见表1)和足月健康新生儿脐带血由相关医院提供，所有样本，经伦理委员会的许可，将相关事项告知志愿者并知情同意后纳入实验。Bone marrow samples from patients with primary AML (see Table 1 for details) and umbilical cord blood from full-term healthy newborns were provided by relevant hospitals. All samples were included in the experiment after being approved by the ethics committee, and the volunteers were informed of the relevant matters and gave informed consent.

表1:本发明所使用的8例AML患者样本Table 1: 8 routine AML patient samples used in the present invention

HL-60、Thp-1、MV4-11和U937均购于ATCC。通过Ficoll-Paque细胞密度梯度分离液(Pharmacia Biotech,Uppsala,Sweden)分离脐带血和AML患者骨髓的单个核细胞。流式细胞仪检测Allergin-1在富集正常HSCs或LSCs的CD34⁺CD38^-脐带血和原代AML患者骨髓细胞中的表达水平。将人AML细胞系细胞常规培养于DMEM完全培养基(含10％FBS、100U/ml青霉素和100U/ml链霉素)，置于37℃、5％CO₂及饱和湿度的培养箱中培养，每3-4天传代1次。流式细胞仪检测上述AML细胞系细胞中Allergin-1的表达水平。原代AML患者骨髓细胞中分选出3000个CD34⁺CD38^-Allergin-1⁺和CD34⁺CD38^-Allergin-1^-细胞与人造血集落甲基纤维素培养基(M3534,Stem Cell Technologies，Vancouver，BC，Canada)充分混合后加入到35mm细胞培养皿中，置37℃、5％CO₂及饱和湿度的培养箱中培养。6天后通过倒置显微镜计数其中>50个细胞集落形成单位。检测上述细胞的自我更新能力。使用的抗体分别是HumanAllergin-1antibody，PE或FITC-conjugated anti-human CD34(Cone#8G12，BectonDickinson Company，BD Biosciences)和PE-Cyanine7-conjugated anti-human CD38(Clone#HIT2，eBioscience)。HL-60, Thp-1, MV4-11 and U937 were purchased from ATCC. Mononuclear cells from umbilical cord blood and bone marrow of AML patients were separated by Ficoll-Paque cell density gradient separation medium (Pharmacia Biotech, Uppsala, Sweden). Flow cytometry was used to detect the expression level of Allergin-1 in CD34⁺ CD38^- umbilical cord blood enriched with normal HSCs or LSCs and bone marrow cells of primary AML patients. Human AML cell line cells were routinely cultured in DMEM complete medium (containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin), placed in an incubator at 37°C, 5% CO₂ and saturated humidity, Passage once every 3-4 days. Flow cytometry was used to detect the expression levels of Allergin-1 in the above-mentioned AML cell lines. 3000 CD34⁺ CD38^- Allergin-1⁺ and CD34⁺ CD38^- Allergin-1^- cells were sorted from primary AML patient bone marrow cells and artificial hematopoietic colony methylcellulose medium (M3534, Stem Cell Technologies, Vancouver, BC , Canada) were thoroughly mixed and added to a 35mm cell culture dish, and cultured in an incubator at 37°C, 5% CO₂ and saturated humidity. After 6 days, >50 colony-forming units were counted by an inverted microscope. The self-renewal ability of the above cells was detected. The antibodies used were HumanAllergin-1antibody, PE or FITC-conjugated anti-human CD34 (Cone#8G12, BectonDickinson Company, BD Biosciences) and PE-Cyanine7-conjugated anti-human CD38 (Clone#HIT2, eBioscience).

实验结果：Experimental results:

CD34⁺CD38^-细胞中富集HSCs或LSCs，因此本发明收集健康人脐带血和原发AML患者骨髓细胞(表1)，检测在上述CD34⁺CD38^-细胞中Allergin-1的表达水平。发现Allergin-1在健康人脐带血CD34⁺CD38^-细胞中不表达(图2A和2B)，而在原发AML患者骨髓CD34⁺CD38^-细胞中却异常显著高表达(图2C和2D)，提示Allergin-1可应用于正常HSCs与LSCs的鉴别。CD34⁺ CD38^- cells are enriched in HSCs or LSCs, so the present invention collects healthy human umbilical cord blood and primary AML patient bone marrow cells (Table 1), and detects the expression level of Allergin-1 in the above CD34⁺ CD38^- cells. It was found that Allergin-1 was not expressed in CD34⁺ CD38^- cells in cord blood of healthy people (Figure 2A and 2B), but it was abnormally and significantly highly expressed in bone marrow CD34⁺ CD38^- cells of patients with primary AML (Figure 2C and 2D), suggesting Allergin-1 can be used to distinguish normal HSCs from LSCs.

根据图2C和2D可知，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的约3倍-约100倍，平均为约57倍。According to Figures 2C and 2D, the expression level or activity of Allergin-1 in hematopoietic stem cells is about 3 times to about 100 times that of Allergin-1 in normal hematopoietic stem cells, with an average of about 57 times.

从原发AML患者骨髓细胞中分选出CD34⁺CD38^-Allergin-1⁺和CD34⁺CD38^-Allergin-1^-细胞进行CFU形成实验。发现上述CD34⁺CD38^-Allergin-1⁺细胞的CFU形成能力显著高于CD34⁺CD38^-Allergin-1^-细胞(图3，(n＝3)，*p<0.05)，提示CD34⁺CD38^-Allergin-1⁺细胞的自我更新能力显著高于CD34⁺CD38^-Allergin-1^-细胞。CD34⁺ CD38^- Allergin-1⁺ and CD34⁺ CD38^- Allergin-1^- cells were sorted from the bone marrow cells of patients with primary AML for CFU formation experiments. It was found that the above-mentioned CD34⁺ CD38^- Allergin-1⁺ cells had significantly higher CFU formation ability than CD34⁺ CD38^- Allergin-1^- cells (Fig. 3, (n=3), *p<0.05), suggesting that CD34⁺ CD38^- Allergin- The self-renewal ability of¹⁺ cells was significantly higher than that of CD34^+CD38- Allergin-^1- cells.

实施例3Example 3

检测Allergin-1对AML细胞增殖和分化的影响Detection of the effect of Allergin-1 on the proliferation and differentiation of AML cells

实验方法:experimental method:

构建Allergin-1敲降质粒和转染AML细胞：合成3对靶向Allergin-1基因的特异性干扰序列(详见表14)及1对非特异性干扰序列，转染Thp-1和U937细胞。病毒颗粒标记绿色荧光蛋白(GFP)。转染72小时后，用流式细胞仪分选GFP⁺细胞，使用抗Allergin-1抗体(ab177744，abcam)通过Western blot实验验证Thp-1和U937细胞的Allergin-1的敲降效率。Construction of Allergin-1 knockdown plasmid and transfection of AML cells: Synthesize 3 pairs of specific interference sequences targeting Allergin-1 gene (see Table 14 for details) and 1 pair of non-specific interference sequences, and transfect Thp-1 and U937 cells. Virus particles are labeled with green fluorescent protein (GFP). After 72 hours of transfection, GFP⁺ cells were sorted by flow cytometry, and the knockdown efficiency of Allergin-1 in Thp-1 and U937 cells was verified by Western blot experiment using anti-Allergin-1 antibody (ab177744, abcam).

绘制细胞生长曲线和检测细胞分化：转染shRNA 72h后从上述细胞中分选GFP阳性Thp-1和U937细胞置于96孔U型板中(Corning Incorporated Costar 3799，Corning，NY，USA)，每孔容量为0.2ml，含细胞3000个，连续培养6天或12天，每48或72h收集细胞计数并分析Allergin-1对Thp-1和U937细胞增殖的影响。流式细胞仪检测Thp-1(12天)和U937细胞(培养6天)中CD11b(Clone#M1/70，BioLegend)表达水平来探讨Allergin-1对Thp-1和U937分化的影响。Drawing cell growth curves and detecting cell differentiation: 72 hours after transfection of shRNA, GFP-positive Thp-1 and U937 cells were sorted from the above cells and placed in 96-well U-shaped plates (Corning Incorporated Costar 3799, Corning, NY, USA). The well volume is 0.2ml, containing 3000 cells, cultured continuously for 6 days or 12 days, collecting cell counts every 48 or 72 hours and analyzing the effect of Allergin-1 on the proliferation of Thp-1 and U937 cells. The expression levels of CD11b (Clone#M1/70, BioLegend) in Thp-1 (12 days) and U937 cells (cultured 6 days) were detected by flow cytometry to explore the effect of Allergin-1 on the differentiation of Thp-1 and U937.

实验结果：Experimental results:

检测Allergin-1⁺或Allergin-1^-AML细胞系Thp-1和U937细胞的增殖和分化指标。发现Allergin-1⁺AML细胞的增殖能力显著高于对照组Allergin-1^-细胞，(n＝3)，*p<0.05(图4A和4B)，而Allergin-1⁺AML细胞中髓细胞晚期分化标志CD11b的表达水平显著低于对照组Allergin-1^-细胞，(n＝3)，*p<0.05(图4C和4D)。Proliferation and differentiation indicators of Allergin-1⁺ or Allergin-^1- AML cell lines Thp-1 and U937 cells were detected. It was found that the proliferation ability of Allergin-1⁺ AML cells was significantly higher than that of control Allergin-1^- cells, (n=3), *p<0.05 (Figure 4A and 4B), while Allergin-1⁺ AML cells in late myeloid differentiation The expression level of the marker CD11b was significantly lower than that of the control Allergin-^1- cells, (n=3), *p<0.05 (Fig. 4C and 4D).

检测靶向人Allergin-1基因特异性干扰序列或非特异性干扰序列对AML细胞增殖、分化和体内定植的影响。发现Allergin-1敲降组与对照组相比显著抑制AML细胞的增殖，同时显著促进AML细胞CD11b的表达水平，(n＝3)，*p<0.05(图5A-5H)。To detect the effects of targeting human Allergin-1 gene-specific interfering sequences or non-specific interfering sequences on the proliferation, differentiation and in vivo colonization of AML cells. It was found that the Allergin-1 knockdown group significantly inhibited the proliferation of AML cells and significantly promoted the expression level of CD11b in AML cells compared with the control group (n=3), *p<0.05 (Figure 5A-5H).

实施例4Example 4

检测IFN-γ对AML细胞的Allergin-1表达和增殖的影响Detection of the effect of IFN-γ on the expression and proliferation of Allergin-1 in AML cells

实验方法:experimental method:

HL-60、Thp-1、MV4-11和U937细胞(3×10⁴个)在DMEM完全培养基或含30ng/mlrecombinant human IFN-γ(Catalog#300-02，Peprotech)的DMEM完全培养基中培养2天后，通过流式细胞仪和细胞计数检测IFN-γ对Allergin-1表达及AML细胞增殖的影响。进一步，HL-60、Thp-1、MV4-11和U937细胞在含IFN-γ(30ng/ml)的DMEM完全培养基中培养2天后，流式细胞仪分选1×10³个Allergin-1阳性或Allergin-1阴性细胞，继续在DMEM完全培养基中培养6天，通过细胞计数检测IFN-γ预处理细胞对AML增殖的影响。HL-60, Thp-1, MV4-11 and U937 cells (3×10⁴ ) in DMEM complete medium or DMEM complete medium containing 30ng/ml recombinant human IFN-γ (Catalog#300-02, Peprotech) After 2 days of culture, the effect of IFN-γ on the expression of Allergin-1 and the proliferation of AML cells was detected by flow cytometry and cell counting. Further, after HL-60, Thp-1, MV4-11 and U937 cells were cultured in DMEM complete medium containing IFN-γ (30ng/ml) for 2 days, 1×¹⁰³ Allergin-1 cells were sorted by flow cytometry Positive or Allergin-1-negative cells were continued to be cultured in DMEM complete medium for 6 days, and the effect of IFN-γ pretreatment cells on AML proliferation was detected by cell counting.

实验结果：Experimental results:

检测IFN-γ对AML细胞增殖和Allergin-1表达的影响。发现IFN-γ促进AML细胞的增殖(图6A)和Allergin-1的表达水平(图6B)，(n＝3)，*p<0.05，且IFN-γ预处理AML细胞后Allergin-1⁺细胞的增殖能力显著高于对照组Allergin-1^-细胞(图6C)，(n＝3)，*p<0.05。The effect of IFN-γ on the proliferation of AML cells and the expression of Allergin-1 was detected. It was found that IFN-γ promoted the proliferation of AML cells (Fig. 6A) and the expression level of Allergin-1 (Fig. 6B), (n=3), *p<0.05, and after IFN-γ pretreated AML cells, Allergin-1⁺ cells The proliferative ability of the Allergin-^1- cells was significantly higher than that of the control group (Fig. 6C), (n=3), *p<0.05.

实施例5Example 5

检测Allergin-1对AML细胞体内定植的影响Detection of the effect of Allergin-1 on the colonization of AML cells in vivo

实验方法:experimental method:

shRNA预处理细胞进行异种移植实验：GFP阳性Allergin-1敲降的Thp-1细胞经眼窝静脉丛移植至NPG小鼠(北京维通利华实验动物技术有限公司，Beijing Vital RiverLaboratory Animal Technology Co.,Ltd.)，设立实验组和阴性对照组，每组6只。实验组接种Allergin-1shRNA预处理的GFP阳性Thp-1细胞，1×10⁶/只；阴性对照组接种非特异性干扰序列预处理的GFP阳性Thp-1细胞，1×10⁶/只。移植12周后，流式细胞仪检测小鼠脾脏和肝脏细胞中GFP阳性细胞所占比例来评价移植细胞的定植能力。shRNA pretreated cells for xenograft experiments: GFP-positive Allergin-1 knockdown Thp-1 cells were transplanted into NPG mice via the orbital venous plexus (Beijing Vital RiverLaboratory Animal Technology Co., Ltd., Beijing Vital RiverLaboratory Animal Technology Co., Ltd.), set up an experimental group and a negative control group, with 6 rats in each group. The experimental group was inoculated with GFP-positive Thp-1 cells pretreated with Allergin-1 shRNA at 1×10⁶ per mouse; the negative control group was inoculated with GFP-positive Thp-1 cells pretreated with non-specific interference sequences at 1×10⁶ per mouse. Twelve weeks after transplantation, the proportion of GFP-positive cells in spleen and liver cells of mice was detected by flow cytometry to evaluate the colonization ability of transplanted cells.

实验结果：Experimental results:

进一步发现移植Allergin-1敲降Thp-1细胞的小鼠肝脏及脾脏重量显著小于对照组(图7A)，(n＝3)，*p<0.05；且Allergin-1敲降Thp-1细胞在小鼠肝脏和脾脏细胞中的定植能力与对照组细胞相比显著降低(图7B)，(n＝3)，*p<0.05。It was further found that the liver and spleen weights of mice transplanted with Allergin-1 knockdown Thp-1 cells were significantly smaller than those of the control group (Fig. 7A), (n=3), *p<0.05; and Allergin-1 knockdown Thp-1 cells were The colonization ability in mouse liver and spleen cells was significantly reduced compared with control cells ( FIG. 7B ), (n=3), *p<0.05.

实施例6Example 6

评估Allergin-1抗体选择性清除AML细胞的可行性To evaluate the feasibility of Allergin-1 antibody to selectively clear AML cells

实验方法:experimental method:

使用外周血单核细胞(PBMC)作为效应细胞，AML细胞系Thp-1细胞(ATCC(Americantype culture collection),TIB-202)作为靶细胞来检测Allergin-1抗体的ADCC效果。使用Ficoll-Paque细胞密度梯度分离液(Pharmacia Biotech，Uppsala，Sweden)分离外周血单核细胞，在含15ng/ml重组人IL-2(货号#200-02，Peprotech)的DMEM完全培养基中将分离的外周血单核细胞培养18小时。收集Thp-1细胞作为靶细胞。将实验组的Allergin-1抗体和同型对照组的IgG1抗体分别加入到靶细胞中，抗体终浓度为2μg/ml，在37℃下孵育30分钟。然后将靶细胞悬浮液加入96孔U型板中，50μl/孔。以效/靶比为10：1加入效应细胞，50μl/孔，总体积为100μl/孔。在37℃，5％CO₂培养箱中培养5小时后，使用CellTiter

AQueous非放射性细胞增殖检测试剂盒(CytoTox/>

Non-Radioactive Cytotoxicity Assay，货号：G1780，Promega)显色读数。Peripheral blood mononuclear cells (PBMC) were used as effector cells, and AML cell line Thp-1 cells (ATCC (American type culture collection), TIB-202) were used as target cells to detect the ADCC effect of Allergin-1 antibody. Peripheral blood mononuclear cells were isolated using Ficoll-Paque cell density gradient separation medium (Pharmacia Biotech, Uppsala, Sweden), and in DMEM complete medium containing 15ng/ml recombinant human IL-2 (Catalog #200-02, Peprotech) Isolated peripheral blood mononuclear cells were cultured for 18 hours. Thp-1 cells were collected as target cells. The Allergin-1 antibody of the experimental group and the IgG1 antibody of the isotype control group were added to the target cells respectively, the final concentration of the antibody was 2 μg/ml, and incubated at 37°C for 30 minutes. Then add the target cell suspension into a 96-well U-shaped plate, 50 μl/well. Effector cells were added at an effector/target ratio of 10:1, 50 μl/well, and the total volume was 100 μl/well. After culturing for 5 hours in a 37°C, 5%_CO2 incubator, use the CellTiter

AQueous Non-radioactive Cell Proliferation Assay Kit (CytoTox/>

Non-Radioactive Cytotoxicity Assay, Cat. No.: G1780, Promega) chromogenic readout.

在NOD/SCID小鼠(北京维通利华实验动物技术有限公司，Beijing Vital RiverLaboratory Animal Technology Co.,Ltd.)体内检测Allergin-1抗体对健康脐带血CD34⁺细胞的重建造血能力或原代AML患者骨髓细胞生长的影响：健康脐带血CD34⁺细胞(1×10⁶/只)或原代AML患者骨髓细胞(1×10⁶/只)经眼窝静脉丛分别移植至NOD/SCID小鼠体内24h后，用Allergin-1或IgG抗体(静脉注射)处理小鼠，每周处理一次，每只小鼠每次用15μg抗体，持续4周。移植五周后，流式细胞仪检测小鼠骨髓细胞中hCD45⁺(Clone#J33，BeckmanCoulter)细胞所占比例，检测hCD45⁺细胞中CD34⁺CD38^-，CD34⁺，Allergin-1⁺，CD33⁺(Clone#HIM3-4，eBioscience Inc.)，CD19⁺(Clone#HIB19，eBioscience)或Allergin-1⁺CD33⁺细胞所占比例。In NOD/SCID mice (Beijing Vital RiverLaboratory Animal Technology Co., Ltd., Beijing Vital RiverLaboratory Animal Technology Co., Ltd.) to detect the ability of Allergin-1 antibody to rebuild hematopoiesis in healthy cord blood CD34⁺ cells or primary AML Effects on the growth of bone marrow cells in patients: healthy umbilical cord blood CD34⁺ cells (1×10⁶ /mouse) or primary AML patient bone marrow cells (1×10⁶ /mouse) were transplanted into NOD/SCID mice via the orbital venous plexus for 24 hours Afterwards, the mice were treated with Allergin-1 or IgG antibody (intravenous injection), once a week, and each mouse was treated with 15 μg of antibody each time for 4 weeks. Five weeks after the transplantation, the proportion of hCD45⁺ (Clone#J33, BeckmanCoulter) cells in mouse bone marrow cells was detected by flow cytometry, and the proportion of CD34⁺ CD38^- , CD34^+, Allergin-1⁺ , CD33⁺ ( Proportion of Clone#HIM3-4, eBioscience Inc.), CD19⁺ (Clone#HIB19, eBioscience) or Allergin-1⁺ CD33⁺ cells.

实验结果：Experimental results:

在体外，AML细胞系Thp-1细胞和Allergin-1抗体与外周血单核细胞混合作用之后，检测Allergin-1抗体介导的外周血单核细胞对AML细胞的细胞毒作用(ADCC)。发现Allergin-1抗体介导的外周血单核细胞对AML细胞的细胞毒作用显著高于对照组(图8)，(n＝3)，*p<0.05。In vitro, after AML cell line Thp-1 cells and Allergin-1 antibody were mixed with peripheral blood mononuclear cells, the cytotoxic effect (ADCC) of peripheral blood mononuclear cells on AML cells mediated by Allergin-1 antibody was detected. It was found that the cytotoxic effect of peripheral blood mononuclear cells on AML cells mediated by Allergin-1 antibody was significantly higher than that of the control group ( FIG. 8 ), (n=3), *p<0.05.

Allergin-1在LSCs和AML单核细胞中高表达，而在正常HSCs中不表达和正常单核细胞中低表达(图2A-2D)。因此，在体内检测Allergin-1抗体介导的免疫效应细胞对原发AML患者骨髓细胞的细胞毒作用。原发AML患者骨髓细胞经眼窝静脉丛移植至NOD/SCID小鼠体内，建立人异种移植AML小鼠模型。模型小鼠用IgG1(阴性对照)或Allergin-1抗体处理，移植5周后流式细胞仪检测不同处理组小鼠肝脏或骨髓中人AML细胞所占比例(图9A)。发现Allergin-1抗体处理组小鼠肝脏细胞中人CD45⁺细胞所占比例显著低于对照组IgG1抗体处理组(图9B)。进一步发现Allergin-1抗体处理组小鼠骨髓中人CD45⁺细胞，人CD45⁺细胞中CD34⁺AML细胞，人CD45⁺细胞中CD34⁺CD38^-和人CD45⁺细胞中Allergin-1⁺细胞所占比例均显著低于IgG1抗体处理对照组(图9C和9D)，尤其是Allergin-1抗体处理组小鼠骨髓细胞中几乎检测不到人CD34⁺CD38^-的LSCs(图9C下-9D左下)，(n＝3)，*p<0.05，提示Allergin-1抗体可以根本上消除AML的根源性LSCs。而Allergin-1抗体不影响脐带血CD34⁺细胞的体内造血重建和定植能力(图10A-10D)，(n＝3)，*p<0.05，提示Allergin-1抗体的安全性。Allergin-1 was highly expressed in LSCs and AML monocytes, but not expressed in normal HSCs and lowly expressed in normal monocytes (Fig. 2A-2D). Therefore, the cytotoxic effect of Allergin-1 antibody-mediated immune effector cells on bone marrow cells from patients with primary AML was examined in vivo. Bone marrow cells from patients with primary AML were transplanted into NOD/SCID mice through the orbital venous plexus to establish a human xenograft AML mouse model. The model mice were treated with IgG1 (negative control) or Allergin-1 antibody, and the proportion of human AML cells in the liver or bone marrow of mice in different treatment groups was detected byflow cytometry 5 weeks after transplantation ( FIG. 9A ). It was found that the proportion of human CD45⁺ cells in the mouse liver cells of the Allergin-1 antibody treatment group was significantly lower than that of the control IgG1 antibody treatment group ( FIG. 9B ). It was further found that the proportions of human CD45⁺ cells in bone marrow of mice treated with Allergin-1 antibody, CD34⁺ AML cells in human CD45⁺ cells, CD34^+CD38- in human CD45⁺ cells and Allergin-1⁺ cells in human CD45⁺ cells All were significantly lower than those in the IgG1 antibody-treated control group (Figure 9C and 9D), especially in the Allergin-1 antibody-treated mouse bone marrow cells, almost no human CD34^+CD38- LSCs could be detected (Figure 9C lower-9D lower left), ( n=3), *p<0.05, suggesting that Allergin-1 antibody can fundamentally eliminate the root-originated LSCs of AML. However, Allergin-1 antibody did not affect the in vivo hematopoietic reconstitution and colonization ability of cord blood CD34⁺ cells (Fig. 10A-10D), (n=3), *p<0.05, suggesting the safety of Allergin-1 antibody.

实施例7Example 7

检测Allergin-1对NK细胞活性及细胞毒作用的影响Detection of the effect of Allergin-1 on the activity and cytotoxicity of NK cells

实验方法:experimental method:

通过Allergin-1⁺或Allergin-1^-AML细胞与健康人外周血单核细胞来源CD3^-CD56⁺NK细胞共培养来检测Allergin-1对NK细胞活性的影响。从健康人外周血单核细胞中分离CD3^-CD56⁺NK细胞，调整细胞浓度为1×10⁶/ml。收集Allergin-1⁺或Allergin-1^-Thp-1细胞，调整细胞浓度为2×10⁵/ml。将100μl的NK细胞与100μl的Allergin-1⁺或Allergin-1^-Thp-1细胞悬浮液充分混合后加入96U孔板中，总容积为200μl/孔。含100ng/ml recombinanthuman IL-2和1ng/ml recombinant human IL-3(Catalog#200-03，Peprotech)的DMEM完全培养基中培养2天后，流式细胞仪检测活化NK细胞的标志CD25。使用的抗体分别是Anti-human CD25-PE(Clone：1D4B，BioLegend)，Anti-human CD56-PC5.5(Clone：PK136，BioLegend)和Anti-human CD3e-FITC(Clone：145-2C11，eBioscience.)。The effect of Allergin-1 on NK cell activity was detected by co-culture of Allergin-1⁺ or Allergin-1^- AML cells with CD3^- CD56⁺ NK cells derived from peripheral blood mononuclear cells of healthy people. CD3^- CD56⁺ NK cells were isolated from peripheral blood mononuclear cells of healthy people, and the cell concentration was adjusted to 1×10⁶ /ml. Collect Allergin-1⁺ or Allergin-1^- Thp-1 cells and adjust the cell concentration to 2×10⁵ /ml. Mix 100 μl of NK cells with 100 μl of Allergin-1⁺ or Allergin-1^- Thp-1 cell suspension and add to a 96U well plate with a total volume of 200 μl/well. After cultured in DMEM complete medium containing 100ng/ml recombinanthuman IL-2 and 1ng/ml recombinant human IL-3 (Catalog#200-03, Peprotech) for 2 days, CD25, a marker of activated NK cells, was detected by flow cytometry. The antibodies used were Anti-human CD25-PE (Clone: 1D4B, BioLegend), Anti-human CD56-PC5.5 (Clone: PK136, BioLegend) and Anti-human CD3e-FITC (Clone: 145-2C11, eBioscience. ).

实验结果：Experimental results:

在体外将Allergin-1⁺或Allergin-1^-Thp-1细胞与健康人外周血单核细胞来源的CD3^-CD56⁺NK细胞在体外共培养2天后，检测Allergin-1对NK细胞活性的影响。发现与Allergin-1^-Thp-1细胞共培养的NK细胞的CD25(活化NK细胞标志)表达水平显著高于与Allergin-1⁺Thp-1细胞共培养的NK细胞，且与Allergin-1^-Thp-1细胞共培养的NK细胞的细胞毒作用显著高于与Allergin-1⁺Thp-1细胞共培养的NK细胞(图11)，(n＝3)，*p<0.05。以上发现提示N K细胞的活化与共培养AML细胞的Allergin-1表达相关，因此，在AML细胞中降低Allergin-1的表达或/和Allergin-1的活性有助于更加有效的清除A M L细胞。After co-culturing Allergin-1⁺ or Allergin-1^- Thp-1 cells with CD3^- CD56⁺ NK cells derived from peripheral blood mononuclear cells of healthy people in vitro for 2 days, the effect of Allergin-1 on the activity of NK cells was detected. It was found that the expression level of CD25 (a marker of activated NK cells)ⁱⁿ NK cells co-cultured with Allergin-1^- Thp-1 cells was significantly higher than that of NK cells co-cultured with Allergin-1⁺ Thp-1 cells, and the The cytotoxic effect of NK cells co-cultured with -1 cells was significantly higher than that of NK cells co-cultured with Allergin-1⁺ Thp-1 cells ( FIG. 11 ), (n=3), *p<0.05. The above findings suggest that the activation of NK cells is related to the expression of Allergin-1 in co-cultured AML cells. Therefore, reducing the expression of Allergin-1 or/and the activity of Allergin-1 in AML cells helps to clear AML cells more effectively.

本说明书中公开的所有特征可以以任何组合进行组合。本说明书中公开的每个特征可以由具有相同，等同或相似目的的替代特征代替。因此，除非另有明确说明，否则所公开的每个特征仅是一系列等同或相似特征的示例。根据以上描述，本领域技术人员可以容易地确定本发明的基本特征，并且在不脱离本发明的精神和范围的情况下，可以对本发明进行各种改变和修改以使其适应各种用途和条件。因此，其他实施例也在所附权利要求的范围内。All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only example of a series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, they can make various changes and modifications to the present invention to adapt it to various usages and conditions . Accordingly, other implementations are within the scope of the following claims.

序列表sequence listing

<110> 大连理工大学<110> Dalian University of Technology

<120> 白血病相关标志物以及应用<120> Leukemia-related markers and their applications

<130> C21P1173<130> C21P1173

<160> 26<160> 26

<170> PatentIn version 3.5<170> PatentIn version 3.5

<210> 1<210> 1

<211> 1419<211> 1419

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 1<400> 1

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300

gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360

cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420

atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480

aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540

attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600

ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660

tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720

gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780

gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840

gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900

tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960

gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020

gtgggatcca ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta 1080gtgggatcca ggccgtgtgtttccacagcc caagatgagg ccaaacactc ccaggagcta 1080

cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140

tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200

tctcaggtga gtactaaaac ttgatcatca attacagagc cattttgact aatatggaaa 1260tctcaggtga gtactaaaac ttgatcatca attacagagc cattttgact aatatggaaa 1260

gttcatggag ggccagttgc aatccccaag atccagcaag actgcctcgc ctttccaccc 1320gttcatggag ggccagttgc aatccccaag atccagcaag actgcctcgc ctttccaccc 1320

gacacctgtt ttgaagcatg aaatcgtgaa gcataccgtg aagaaggttc tcccgtagtt 1380gacacctgtt ttgaagcatg aaatcgtgaa gcataccgtg aagaaggttc tcccgtagtt 1380

tcccagaagt ttttaatacg ttctcaagaa atgctacta 1419tcccagaagt ttttaatacg ttctcaagaa atgctacta 1419

<210> 2<210> 2

<211> 1475<211> 1475

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 2<400> 2

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300

gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360

cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420

atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480

aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540

attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600

ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660

tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720

gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780

gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840

gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900

tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960

gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020

gtgggatcca ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta 1080gtgggatcca ggccgtgtgtttccacagcc caagatgagg ccaaacactc ccaggagcta 1080

cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140

tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200

tctcaggtga gtactaaaac ttgatcatca attacagagc cattttgact aatatggaaa 1260tctcaggtga gtactaaaac ttgatcatca attacagagc cattttgact aatatggaaa 1260

gttcatggag atgtggccag tgacaaccct cctcaagcca gcatctgtga cctttttata 1320gttcatggag atgtggccag tgacaaccct cctcaagcca gcatctgtga cctttttata 1320

tgacccatta gtcttcaagc aattctttgc tttttagcac cataaagtgt ttcaaactca 1380tgacccatta gtcttcaagc aattctttgc tttttagcac cataaagtgtttcaaactca 1380

ccttgtactt tccctaaccc cagacccgga atcagccatt tctcccagta ctggttcatc 1440ccttgtactt tccctaaccc cagacccgga atcagccatt tctcccagta ctggttcatc 1440

ttagaaacca agatctggag gccgggcgca gtggc 1475ttagaaacca agatctggag gccgggcgca gtggc 1475

<210> 3<210> 3

<211> 1709<211> 1709

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 3<400> 3

cacagaggaa gacagattgg tcgaacaaac cagtattatg caaacctcat ccaaaccctc 60cacagaggaa gacagattgg tcgaacaaac cagtattatg caaacctcat ccaaaccctc 60

tgatttcctt aacttggcta agaaaaagag gaagttctcc gagttactca ccactgtggt 120tgatttcctt aacttggcta agaaaaagag gaagttctcc gagttactca ccactgtggt 120

tctactatgc cttctgaccc cgtcttggac ttcaactggg agaatgtgga gccatttgaa 180tctactatgc cttctgaccc cgtcttggac ttcaactggg agaatgtgga gccatttgaa 180

caggctcctc ttctggagca tattttcttc tgtcacttgt agaaaagctg tattggattg 240caggctcctc ttctggagca tattttcttc tgtcacttgt agaaaagctg tattggattg 240

tgaggcaatg aaaacaaatg aattcccttc tccatgtttg gactcaaaga ctaaggtggt 300tgaggcaatg aaaacaaatg aattcccttc tccatgtttg gactcaaaga ctaaggtggt 300

tatgaagggt caaaatgtat ctatgttttg ttcccataag aacaaatcac tgcagatcac 360tatgaagggt caaaatgtat ctatgttttg ttcccataag aacaaatcac tgcagatcac 360

ctattcattg tttcgacgta agacacacct gggaacccag gatggaaaag gtgaacctgc 420ctattcattg tttcgacgta agacacacct gggaacccag gatggaaaag gtgaacctgc 420

gatttttaac ctaagcatca cagaagccca tgaatcaggc ccctacaaat gcaaagccca 480gatttttaac ctaagcatca cagaagccca tgaatcaggc ccctacaaat gcaaagccca 480

agttaccagc tgttcaaaat acagtcgtga cttcagcttc acgattgtcg acccggtgac 540agttaccagc tgttcaaaat acagtcgtga cttcagcttc acgattgtcg acccggtgac 540

ttccccagtg ctgaacatta tggtcattca aacagaaaca gaccgacata taacattaca 600ttccccagtg ctgaacatta tggtcattca aacagaaaca gaccgacata taacattaca 600

ttgcctctca gtcaatggct cgctgcccat caattacact ttctttgaaa accatgttgc 660ttgcctctca gtcaatggct cgctgcccat caattacact ttctttgaaa accatgttgc 660

catatcacca gctatttcca agtatgacag ggagcctgct gaatttaact taaccaagaa 720catatcacca gctatttcca agtatgacag ggagcctgct gaatttaact taaccaagaa 720

gaatcctgga gaagaggaag agtataggtg tgaagctaaa aacagattgc ctaactatgc 780gaatcctgga gaagaggaag agtataggtg tgaagctaaa aacagattgc ctaactatgc 780

aacatacagt caccctgtca ccatgccctc aacaggcgga gacagctgtc ctttctgtct 840aacatacagt caccctgtca ccatgccctc aacaggcgga gacagctgtc ctttctgtct 840

gaagctacta cttccagggt tattactgtt gctggtggtg ataatcctaa ttctggcttt 900gaagctacta cttccagggt tattactgtt gctggtggtg ataatcctaa ttctggcttt 900

ttgggtactg cccaaataca aaacaagaaa agctatgaga aataatgtgc ccagggaccg 960ttgggtactg cccaaataca aaacaagaaa agctatgaga aataatgtgc ccagggaccg 960

tggagacaca gccatggaag ttggaatcta tgcaaatatc cttgaaaaac aagcaaagga 1020tggagacaca gccatggaag ttggaatcta tgcaaatatc cttgaaaaac aagcaaagga 1020

ggaatctgtg ccagaagtgg gatccaggcc gtgtgtttcc acagcccaag atgaggccaa 1080ggaatctgtg ccagaagtgg gatccaggcc gtgtgtttcc acagcccaag atgaggccaa 1080

acactcccag gagctacagt atgccacccc cgtgttccag gaggtggcac caagagagca 1140acactcccag gagctacagt atgccacccc cgtgttccag gaggtggcac caagagagca 1140

agaagcctgt gattcttata aatctggata tgtctattct gaactcaact tctgaaattt 1200agaagcctgt gattcttata aatctggata tgtctattct gaactcaact tctgaaattt 1200

acagaaacaa actacatctc aggtgagtac taaaacttga tcatcaatta cagagccatt 1260acagaaacaa actacatctc aggtgagtac taaaacttga tcatcaatta cagagccatt 1260

ttgactaata tggaaagttc atggaggtag gtagcaaaag ctgaagctgt gacaatggtc 1320ttgactaata tggaaagttc atggaggtag gtagcaaaag ctgaagctgt gacaatggtc 1320

aagggtcaaa ctctacatca gccaggaatg ctaggtgagg gctttgaatt ttaccttaca 1380aagggtcaaa ctctacatca gccaggaatg ctaggtgagg gctttgaatt ttaccttaca 1380

cagaaaggct aattaacaaa cacatctgag cccaacaaat gtttttacca aagtaacttt 1440cagaaaggct aattaacaaa cacatctgag cccaacaaat gtttttacca aagtaacttt 1440

gtaatctaga acaatgaaaa ttgttacaaa gagtttttgt atttgtataa tatattaata 1500gtaatctaga acaatgaaaa ttgttacaaa gagtttttgt atttgtataa tatattaata 1500

gctacataca tcaaatataa tttcttgtat gtcagaagaa ttaccactga cacattaaga 1560gctacataca tcaaatataa tttcttgtat gtcagaagaa ttaccactga cacattaaga 1560

caatttatgt attaactatt aaattaaagg taattattgc agtacctttc tccaccactg 1620caatttatgt attaactatt aaattaaagg taattattgc agtacctttc tccaccactg 1620

gagtcgatga cgtaaccaga aatcaagcca ctggtttgaa gttctcggag gagtaaacca 1680gagtcgatga cgtaaccaga aatcaagcca ctggtttgaa gttctcggag gagtaaacca 1680

tactaacgaa gcttcagtct tctcaccaa 1709tactaacgaa gcttcagtct tctcaccaa 1709

<210> 4<210> 4

<211> 1356<211> 1356

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 4<400> 4

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300

gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360

cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420

atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480

aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540

attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600

ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660

tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720

gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780

gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840

gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900

tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960

gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020

gtgggatcca ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta 1080gtgggatcca ggccgtgtgtttccacagcc caagatgagg ccaaacactc ccaggagcta 1080

cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140

tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200

tctcaggggc cagttgcaat ccccaagatc cagcaagact gcctcgcctt tccacccgac 1260tctcaggggc cagttgcaat ccccaagatc cagcaagact gcctcgcctt tccaccgac 1260

acctgttttg aagcatgaaa tcgtgaagca taccgtgaag aaggttctcc cgtagtttcc 1320acctgttttg aagcatgaaa tcgtgaagca taccgtgaag aaggttctcc cgtagtttcc 1320

cagaagtttt taatacgttc tcaagaaatg ctacta 1356cagaagtttt taatacgttc tcaagaaatg ctacta 1356

<210> 5<210> 5

<211> 1224<211> 1224

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 5<400> 5

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300

gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360

cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420

atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480

aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540

attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600

ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660

tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720

gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780

gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840

gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900

tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960

gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020

gtgggatcca ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta 1080gtgggatcca ggccgtgtgtttccacagcc caagatgagg ccaaacactc ccaggagcta 1080

cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140

tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200

tctcaggtag agacggggtt ttgc 1224tctcaggtag agacggggtt ttgc 1224

<210> 6<210> 6

<211> 1741<211> 1741

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 6<400> 6

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300aatgaattcc cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat 300

gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360gtatctatgt tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga 360

cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420cgtaagacac acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc 420

atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480atcacagaag cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca 480

aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540aaatacagtc gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac 540

attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600attatggtca ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat 600

ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660ggctcgctgc ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt 660

tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720tccaagtatg acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag 720

gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780gaagagtata ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct 780

gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840gtcaccatgc cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca 840

gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900gggttattac tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa 900

tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960tacaaaacaa gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg 960

gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020gaagttggaa tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa 1020

gtgggatcca ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta 1080gtgggatcca ggccgtgtgtttccacagcc caagatgagg ccaaacactc ccaggagcta 1080

cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140cagtatgcca cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct 1140

tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200tataaatctg gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca 1200

tctcaggtag agacagggtt ttgccatgtt ggccaggctg gtcttcaact cctgacctca 1260tctcaggtag agacagggtt ttgccatgtt ggccaggctg gtcttcaact cctgacctca 1260

agtgatccgc ccacctcgga ctcccaaagt gctgggatta caggcgtgag ccacctcgcc 1320agtgatccgc ccacctcgga ctcccaaagt gctgggatta caggcgtgag ccacctcgcc 1320

tggccctcca tttcctgatc tagtcttata tccacgctca ccacctcagc acgctcagac 1380tggccctcca tttcctgatc tagtcttata tccacgctca ccacctcagc acgctcagac 1380

ccacgctgct gtgggctcct ctggctcctg gaagagtgcg tccgcagatg ctgcagtctt 1440ccacgctgct gtgggctcct ctggctcctg gaagagtgcg tccgcagatg ctgcagtctt 1440

tgtgtggctc agcaattgcc actcacatca ggaactgcct ttaccctgtc aggctctact 1500tgtgtggctc agcaattgcc actcacatca ggaactgcct ttaccctgtc aggctctact 1500

gagacccgac cctggttatt aagctatagg ggagacaagg atggatctta aagaagacaa 1560gagacccgac cctggttat aagctatagg ggagacaagg atggatctta aagaagacaa 1560

gcaaaatgta gtgaagcaaa tagaatggtg gttcctgggg gatgggggca ggggacaacg 1620gcaaaatgta gtgaagcaaa tagaatggtg gttcctgggg gatgggggca ggggacaacg 1620

aggagtgact ggctaacaga tacagcgttt cagtttggaa agacaaaaaa gttctggaaa 1680aggagtgact ggctaacaga tacagcgttt cagtttggaa agacaaaaaa gttctggaaa 1680

aaagatggaa ggtggtgatg gttgcacaat aatatgagtt ttgttgttgt tgttttttga 1740aaagatggaa ggtggtgatg gttgcacaat aatatgagtt ttgttgttgt tgttttttga 1740

g 1741g 1741

<210> 7<210> 7

<211> 1247<211> 1247

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 7<400> 7

ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60ttggtcgaac aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg 60

gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120gctaagaaaa agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg 120

accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180accccgtctt ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg 180

agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240agcatatttt cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca 240

aatgaacaaa tcactgcaga tcacctattc attgtttcga cgtaagacac acctgggaac 300aatgaacaaa tcactgcaga tcacctattc attgtttcga cgtaagacac acctgggaac 300

ccaggatgga aaaggtgaac ctgcgatttt taacctaagc atcacagaag cccatgaatc 360ccaggatgga aaaggtgaac ctgcgatttt taacctaagc atcacagaag cccatgaatc 360

aggcccctac aaatgcaaag cccaagttac cagctgttca aaatacagtc gtgacttcag 420aggcccctac aaatgcaaag cccaagttac cagctgttca aaatacagtc gtgacttcag 420

cttcacgatt gtcgacccgg tgacttcccc agtgctgaac attatggtca ttcaaacaga 480cttcacgatt gtcgacccgg tgacttcccc agtgctgaac attatggtca ttcaaacaga 480

aacagaccga catataacat tacattgcct ctcagtcaat ggctcgctgc ccatcaatta 540aacagaccga catataacat tacattgcct ctcagtcaat ggctcgctgc ccatcaatta 540

cactttcttt gaaaaccatg ttgccatatc accagctatt tccaagtatg acagggagcc 600cactttcttt gaaaaccatg ttgccatatc accagctatt tccaagtatg acagggagcc 600

tgctgaattt aacttaacca agaagaatcc tggagaagag gaagagtata ggtgtgaagc 660tgctgaattt aacttaacca agaagaatcc tggagaagag gaagagtata ggtgtgaagc 660

taaaaacaga ttgcctaact atgcaacata cagtcaccct gtcaccatgc cctcaacagg 720taaaaacaga ttgcctaact atgcaacata cagtcaccct gtcaccatgc cctcaacagg 720

cggagacagc tgtcctttct gtctgaagct actacttcca gggttattac tgttgctggt 780cggagacagc tgtcctttct gtctgaagct actacttcca gggttattac tgttgctggt 780

ggtgataatc ctaattctgg ctttttgggt actgcccaaa tacaaaacaa gaaaagctat 840ggtgataatc ctaattctgg ctttttgggt actgcccaaa tacaaaacaa gaaaagctat 840

gagaaataat gtgcccaggg accgtggaga cacagccatg gaagttggaa tctatgcaaa 900gagaaataat gtgcccaggg accgtggaga cacagccatg gaagttggaa tctatgcaaa 900

tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa gtgggatcca ggccgtgtgt 960tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa gtgggatcca ggccgtgtgt960

ttccacagcc caagatgagg ccaaacactc ccaggagcta cagtatgcca cccccgtgtt 1020ttccacagcc caagatgagg ccaaacactc ccaggagcta cagtatgcca cccccgtgtt 1020

ccaggaggtg gcaccaagag agcaagaagc ctgtgattct tataaatctg gatatgtcta 1080ccaggaggtg gcaccaagag agcaagaagc ctgtgattct tataaatctg gatatgtcta 1080

ttctgaactc aacttctgaa atttacagaa acaaactaca tctcagggta agatgctttt 1140ttctgaactc aacttctgaa atttacagaa acaaactaca tctcagggta agatgctttt 1140

tatgaagctg atttccatga acaaaaagca aacttgaggc tgaggcaggt ggattacttg 1200tatgaagctg atttccatga acaaaaagca aacttgaggc tgaggcaggt ggattacttg 1200

aggtcaggag ttcgagacca gcctggccaa catggcaaaa accccat 1247aggtcaggag ttcgagacca gcctggccaa catggcaaaa accccat 1247

<210> 8<210> 8

<211> 1716<211> 1716

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 8<400> 8

accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60

agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120

gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180

actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240

ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300

ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360

tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420

gacccggtga cttccccagt gctgaacatt atggtcattc aaacagaaac agaccgacat 480gacccggtga cttccccagt gctgaacatt atggtcattc aaacagaaac agaccgacat 480

ataacattac attgcctctc agtcaatggc tcgctgccca tcaattacac tttctttgaa 540ataacattac attgcctctc agtcaatggc tcgctgccca tcaattacac tttctttgaa 540

aaccatgttg ccatatcacc agctatttcc aagtatgaca gggagcctgc tgaatttaac 600aaccatgttg ccatatcacc agctatttcc aagtatgaca gggagcctgc tgaatttaac 600

ttaaccaaga agaatcctgg agaagaggaa gagtataggt gtgaagctaa aaacagattg 660ttaaccaaga agaatcctgg agaagaggaa gagtataggt gtgaagctaa aaacagattg 660

cctaactatg caacatacag tcaccctgtc accatgccct caacaggcgg agacagctgt 720cctaactatg caacatacag tcaccctgtc accatgccct caacaggcgg aagacagctgt 720

cctttctgtc tgaagctact acttccaggg ttattactgt tgctggtggt gataatccta 780cctttctgtc tgaagctact acttccaggg ttaattactgt tgctggtggt gataatccta 780

attctggctt tttgggtact gcccaaatac aaaacaagaa aagctatgag aaataatgtg 840attctggctt tttgggtact gcccaaatac aaaacaagaa aagctatgag aaataatgtg 840

cccagggacc gtggagacac agccatggaa gttggaatct atgcaaatat ccttgaaaaa 900cccagggacc gtggagacac agccatggaa gttggaatct atgcaaatat ccttgaaaaa 900

caagcaaagg aggaatctgt gccagaagtg ggatccaggc cgtgtgtttc cacagcccaa 960caagcaaagg aggaatctgt gccagaagtg ggatccaggc cgtgtgtttc cacagcccaa 960

gatgaggcca aacactccca ggagctacag tatgccaccc ccgtgttcca ggaggtggca 1020gatgaggcca aacactccca ggagctacag tatgccaccc ccgtgttcca ggaggtggca 1020

ccaagagagc aagaagcctg tgattcttat aaatctggat atgtctattc tgaactcaac 1080ccaagagagc aagaagcctg tgattcttat aaatctggat atgtctattc tgaactcaac 1080

ttctgaaatt tacagaaaca aactacatct cagggattta aaggggagac ctggccagag 1140ttctgaaatt tacagaaaca aactacatct cagggatta aaggggagac ctggccagag 1140

gaatcatcaa cagtggattt gtgatgactg gagttcattt ccttctttct caactgtcct 1200gaatcatcaa cagtggattt gtgatgactg gagttcattt ccttctttct caactgtcct 1200

ggaaggaaat ggactagtgc cttacctctg actcttccgc agcacacacc caccaccaga 1260ggaaggaaat ggactagtgc cttacctctg actcttccgc agcacacacc caccaccaga 1260

gacagtaact gagatctgca gtagcacaga aacttacagg ttagaaaaat tagacaccta 1320gacagtaact gagatctgca gtagcacaga aacttacagg ttagaaaaat tagacaccta 1320

aggatttcat gactccagac ctgtttcctt agcataatac cacaccagta ggagaaatac 1380aggatttcat gactccagac ctgtttcctt agcataatac cacaccagta ggagaaatac 1380

aacttacgaa atttctttgt aaataaattc tataacattt tgggtaaaaa tttatcataa 1440aacttacgaa atttctttgt aaataaattc tataacattt tgggtaaaaa tttatcataa 1440

aaaaattatt aaaagcaatt gggttggaag gaagttctgt caaaatacaa tccaggtgtg 1500aaaaattatt aaaagcaatt gggttggaag gaagttctgt caaaatacaa tccaggtgtg 1500

cagttctctg ataaatttag cagtggttac tactgaggca gtctcaaaaa aaagtgtaaa 1560cagttctctg ataaatttag cagtggttac tactgaggca gtctcaaaaa aaagtgtaaa 1560

aggactttaa ggggtgatga aaattttcta cattgccttt gtggagatgg ttatacattt 1620aggactttaa ggggtgatga aaattttcta cattgccttt gtggagatgg ttatacattt 1620

gtcaatactc atcaatttgt acatttaaaa gaggtaaatt tattgtatgt aaattgtatc 1680gtcaatactc atcaatttgt acatttaaaa gaggtaaatt tattgtatgt aaattgtatc 1680

ttaataaaac tgattaaaac agacatacaa acccca 1716ttaataaaac tgattaaaac agacatacaa acccca 1716

<210> 9<210> 9

<211> 1448<211> 1448

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 9<400> 9

accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60

agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120

gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180

actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240

ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300

ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360

tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420

gacccggtga cttccccagt gctgaacatt atggtcattc aaacagaaac agaccgacat 480gacccggtga cttccccagt gctgaacatt atggtcattc aaacagaaac agaccgacat 480

ataacattac attgcctctc agtcaatggc tcgctgccca tcaattacac tttctttgaa 540ataacattac attgcctctc agtcaatggc tcgctgccca tcaattacac tttctttgaa 540

aaccatgttg ccatatcacc agctatttcc aagtatgaca gggagcctgc tgaatttaac 600aaccatgttg ccatatcacc agctatttcc aagtatgaca gggagcctgc tgaatttaac 600

ttaaccaaga agaatcctgg agaagaggaa gagtataggt gtgaagctaa aaacagattg 660ttaaccaaga agaatcctgg agaagaggaa gagtataggt gtgaagctaa aaacagattg 660

cctaactatg caacatacag tcaccctgtc accatgccct caacaggcgg agacagctgt 720cctaactatg caacatacag tcaccctgtc accatgccct caacaggcgg aagacagctgt 720

cctttctgtc tgaagctact acttccaggg ttattactgt tgctggtggt gataatccta 780cctttctgtc tgaagctact acttccaggg ttaattactgt tgctggtggt gataatccta 780

attctggctt tttgggtact gcccaaatac aaaacaagaa aagctatgag aaataatgtg 840attctggctt tttgggtact gcccaaatac aaaacaagaa aagctatgag aaataatgtg 840

cccagggacc gtggagacac agccatggaa gttggaatct atgcaaatat ccttgaaaaa 900cccagggacc gtggagacac agccatggaa gttggaatct atgcaaatat ccttgaaaaa 900

caagcaaagg aggaatctgt gccagaagtg ggatccaggc cgtgtgtttc cacagcccaa 960caagcaaagg aggaatctgt gccagaagtg ggatccaggc cgtgtgtttc cacagcccaa 960

gatgaggcca aacactccca ggagctacag tatgccaccc ccgtgttcca ggaggtggca 1020gatgaggcca aacactccca ggagctacag tatgccaccc ccgtgttcca ggaggtggca 1020

ccaagagagc aagaagcctg tgattcttat aaatctggat atgtctattc tgaactcaac 1080ccaagagagc aagaagcctg tgattcttat aaatctggat atgtctattc tgaactcaac 1080

ttctgaaatt tacagaaaca aactacatct caggatggag tctcactctg ttgcccaggc 1140ttctgaaatt tacagaaaca aactacatct caggatggag tctcactctg ttgcccaggc 1140

tggagttcag tagcgcgatc ttggctcact tcaatctcca tcttcccagt tcaagcgatt 1200tggagttcag tagcgcgatc ttggctcact tcaatctcca tcttcccagt tcaagcgatt 1200

ctcatgcctc gacctcccga gtagctggga ttacaggtgc ccgctaccac gcccagctaa 1260ctcatgcctc gacctcccga gtagctggga ttacaggtgc ccgctaccac gcccagctaa 1260

tttttgtatt tttagtagag atggggtttc actatggtgg ccaggctggt cttgaactcc 1320tttttgtatt tttagtagag atggggtttc actatggtgg ccaggctggt cttgaactcc 1320

tgacctcaga tgatctgcct gcctcggcct cccaaagtgc tggaactaca agcctgagcc 1380tgacctcaga tgatctgcct gcctcggcct cccaaagtgc tggaactaca agcctgagcc 1380

accgtgcccg gccctgaatc gctttagtaa ataaagggtc tccaagaata aattcatccg 1440accgtgcccg gccctgaatc gctttagtaa ataaagggtc tccaagaata aattcatccg 1440

aacatgca 1448aacatgca 1448

<210> 10<210> 10

<211> 1163<211> 1163

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 10<400> 10

accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60

agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120

gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180gtattggatt gtgaggcaat gaaaacaaat gaattccctt ctccatgttt ggactcaaag 180

actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240actaaggtgg ttatgaaggg tcaaaatgta tctatgtttt gttcccataa gaacaaatca 240

ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300ctgcagatca cctattcatt gtttcgacgt aagacacacc tgggaaccca ggatggaaaa 300

ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360ggtgaacctg cgatttttaa cctaagcatc acagaagccc atgaatcagg cccctacaaa 360

tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420tgcaaagccc aagttaccag ctgttcaaaa tacagtcgtg acttcagctt cacgattgtc 420

ggcggagaca gctgtccttt ctgtctgaag ctactacttc cagggttatt actgttgctg 480ggcggagaca gctgtccttt ctgtctgaag ctactacttc cagggttatt actgttgctg 480

gtggtgataa tcctaattct ggctttttgg gtactgccca aatacaaaac aagaaaagct 540gtggtgataa tcctaattct ggctttttgg gtactgccca aatacaaaac aagaaaagct 540

atgagaaata atgtgcccag ggaccgtgga gacacagcca tggaagttgg aatctatgca 600atgagaaata atgtgcccag ggaccgtgga gacacagcca tggaagttgg aatctatgca 600

aatatccttg aaaaacaagc aaaggaggaa tctgtgccag aagtgggatc caggccgtgt 660aatatccttg aaaaacaagc aaaggaggaa tctgtgccag aagtgggatc caggccgtgt 660

gtttccacag cccaagatga ggccaaacac tcccaggagc tacagtatgc cacccccgtg 720gtttccacag cccaagatga ggccaaacac tcccaggagc tacagtatgc cacccccgtg 720

ttccaggagg tggcaccaag agagcaagaa gcctgtgatt cttataaatc tggatatgtc 780ttccaggagg tggcaccaag agagcaagaa gcctgtgatt cttataaatc tggatatgtc 780

tattctgaac tcaacttctg aaatttacag aaacaaacta catctcagga tggagtctca 840tattctgaac tcaacttctg aaatttacag aaacaaacta catctcagga tggagtctca 840

ctctgttgcc caggctggag ttcagtagcg cgatcttggc tcacttcaat ctccatcttc 900ctctgttgcc caggctggag ttcagtagcg cgatcttggc tcacttcaat ctccatcttc 900

ccagttcaag cgattctcat gcctcgacct cccgagtagc tgggattaca ggtgcccgct 960ccagttcaag cgattctcat gcctcgacct cccgagtagc tgggattaca ggtgcccgct 960

accacgccca gctaattttt gtatttttag tagagatggg gtttcactat ggtggccagg 1020accacgccca gctaattttt gtatttttag tagagatggg gtttcactat ggtggccagg 1020

ctggtcttga actcctgacc tcagatgatc tgcctgcctc ggcctcccaa agtgctggaa 1080ctggtcttga actcctgacc tcagatgatc tgcctgcctc ggcctcccaa agtgctggaa 1080

ctacaagcct gagccaccgt gcccggccct gaatcgcttt agtaaataaa gggtctccaa 1140ctacaagcct gagccaccgt gcccggccct gaatcgcttt agtaaataaa gggtctccaa 1140

gaataaattc atccgaacat gca 1163gaataaattc atccgaacat gca 1163

<210> 11<210> 11

<211> 1178<211> 1178

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> MILR1基因的序列<223> Sequence of the MILR1 gene

<400> 11<400> 11

accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60accactgtgg ttctactatg ccttctgacc ccgtcttgga cttcaactgg gagaatgtgg 60

agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120agccatttga acaggctcct cttctggagc atattttctt ctgtcacttg tagaaaagct 120

gtattggatt gtgaggcaat gaaaacaaat gacccggtga cttccccagt gctgaacatt 180gtattggatt gtgaggcaat gaaaacaaat gacccggtga cttccccagt gctgaacatt 180

atggtcattc aaacagaaac agaccgacat ataacattac attgcctctc agtcaatggc 240atggtcattc aaacagaaac agaccgacat ataacattac attgcctctc agtcaatggc 240

tcgctgccca tcaattacac tttctttgaa aaccatgttg ccatatcacc agctatttcc 300tcgctgccca tcaattacac tttctttgaa aaccatgttg ccatatcacc agctatttcc 300

aagtatgaca gggagcctgc tgaatttaac ttaaccaaga agaatcctgg agaagaggaa 360aagtatgaca gggagcctgc tgaatttaac ttaaccaaga agaatcctgg agaagaggaa 360

gagtataggt gtgaagctaa aaacagattg cctaactatg caacatacag tcaccctgtc 420gagtataggt gtgaagctaa aaacagattg cctaactatg caacatacag tcaccctgtc 420

accatgccct caacaggcgg agacagctgt cctttctgtc tgaagctact acttccaggg 480accatgccct caacaggcgg aagacagctgt cctttctgtc tgaagctact acttccaggg 480

ttattactgt tgctggtggt gataatccta attctggctt tttgggtact gcccaaatac 540ttaattactgt tgctggtggt gataatccta attctggctt tttgggtact gcccaaatac 540

aaaacaagaa aagctatgag aaataatgtg cccagggacc gtggagacac agccatggaa 600aaaacaagaa aagctatgag aaataatgtg cccagggacc gtggagaacac agccatggaa 600

gttggaatct atgcaaatat ccttgaaaaa caagcaaagg aggaatctgt gccagaagtg 660gttggaatct atgcaaatat ccttgaaaaa caagcaaagg aggaatctgt gccagaagtg 660

ggatccaggc cgtgtgtttc cacagcccaa gatgaggcca aacactccca ggagctacag 720ggatccaggc cgtgtgtttc cacagcccaa gatgaggcca aacactccca ggagctacag 720

tatgccaccc ccgtgttcca ggaggtggca ccaagagagc aagaagcctg tgattcttat 780tatgccacccc ccgtgttcca ggaggtggca ccaagagagc aagaagcctg tgattcttat 780

aaatctggat atgtctattc tgaactcaac ttctgaaatt tacagaaaca aactacatct 840aaatctggat atgtctattc tgaactcaac ttctgaaatt tacagaaaca aactacatct 840

caggatggag tctcactctg ttgcccaggc tggagttcag tagcgcgatc ttggctcact 900caggatggag tctcactctg ttgcccaggc tggagttcag tagcgcgatc ttggctcact 900

tcaatctcca tcttcccagt tcaagcgatt ctcatgcctc gacctcccga gtagctggga 960tcaatctcca tcttcccagt tcaagcgatt ctcatgcctc gacctcccga gtagctggga 960

ttacaggtgc ccgctaccac gcccagctaa tttttgtatt tttagtagag atggggtttc 1020ttacaggtgc ccgctaccac gcccagctaa tttttgtatt tttagtagag atggggtttc 1020

actatggtgg ccaggctggt cttgaactcc tgacctcaga tgatctgcct gcctcggcct 1080actatggtgg ccaggctggt cttgaactcc tgacctcaga tgatctgcct gcctcggcct 1080

cccaaagtgc tggaactaca agcctgagcc accgtgcccg gccctgaatc gctttagtaa 1140cccaaagtgc tggaactaca agcctgagcc accgtgcccg gccctgaatc gctttagtaa 1140

ataaagggtc tccaagaata aattcatccg aacatgca 1178ataaagggtc tccaagaata aattcatccg aacatgca 1178

<210> 12<210> 12

<211> 56<211> 56

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 12<400> 12

tgcccaagtt accagctgtt ttcaagagaa acagctggta acttgggcct tttttc 56tgcccaagtt accagctgtt ttcaagagaa acagctggta acttgggcct tttttc 56

<210> 13<210> 13

<211> 60<211> 60

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 13<400> 13

tcgagaaaaa aggcccaagt taccagctgt ttctcttgaa aacagctggt aacttgggca 60tcgagaaaaa aggcccaagt taccagctgt ttctcttgaa aacagctggt aacttgggca 60

<210> 14<210> 14

<211> 55<211> 55

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 14<400> 14

tgctactact tccagggtta ttcaagagat aaccctggaa gtagtagctt ttttc 55tgctactact tccagggtta ttcaagagat aaccctggaa gtagtagctt ttttc 55

<210> 15<210> 15

<211> 59<211> 59

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 15<400> 15

tcgagaaaaa agctactact tccagggtta tctcttgaat aaccctggaa gtagtagca 59tcgagaaaaa agctactact tccagggtta tctcttgaat aaccctggaa gtagtagca 59

<210> 16<210> 16

<211> 55<211> 55

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 16<400> 16

tgctggtggt gataatccta ttcaagagat aggattatca ccaccagctt ttttc 55tgctggtggt gataatccta ttcaagagat aggattatca ccaccagctt ttttc 55

<210> 17<210> 17

<211> 59<211> 59

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> shRNA序列<223> shRNA-seq

<400> 17<400> 17

tcgagaaaaa agctggtggt gataatccta tctcttgaat aggattatca ccaccagca 59tcgagaaaaa agctggtggt gataatccta tctcttgaat aggattatca ccaccagca 59

<210> 18<210> 18

<211> 22<211> 22

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（正向）<223> Primer sequence for amplifying Allergin-1 gene (forward)

<400> 18<400> 18

cccaagttac cagctgttca aa 22cccaagttac cagctgttcaaa 22

<210> 19<210> 19

<211> 27<211> 27

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（反向）<223> Primer sequence for amplifying Allergin-1 gene (reverse)

<400> 19<400> 19

tatatgtcgg tctgtttctg tttgaat 27tatatgtcgg tctgtttctg tttgaat 27

<210> 20<210> 20

<211> 24<211> 24

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（正向）<223> Primer sequence for amplifying Allergin-1 gene (forward)

<400> 20<400> 20

taggattatc accaccagca acag 24taggattatc accaccagca acag 24

<210> 21<210> 21

<211> 22<211> 22

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（反向）<223> Primer sequence for amplifying Allergin-1 gene (reverse)

<400> 21<400> 21

agctgtattg gattgtgagg ca 22agctgtattg gattgtgaggca 22

<210> 22<210> 22

<211> 22<211> 22

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（正向）<223> Primer sequence for amplifying Allergin-1 gene (forward)

<400> 22<400> 22

aaatgcaaag cccaagttac ca 22aaatgcaaag cccaagttacca 22

<210> 23<210> 23

<211> 18<211> 18

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 扩增Allergin-1基因的引物序列（反向）<223> Primer sequence for amplifying Allergin-1 gene (reverse)

<400> 23<400> 23

aattgatggg cagcgagc 18aattgatggg cagcgagc 18

<210> 24<210> 24

<211> 19<211> 19

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> Allergin-1基因的靶序列<223> Target sequence of Allergin-1 gene

<400> 24<400> 24

gcccaagtta ccagctgtt 19gcccaagtta ccagctgtt 19

<210> 25<210> 25

<211> 19<211> 19

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> Allergin-1基因的靶序列<223> Target sequence of Allergin-1 gene

<400> 25<400> 25

gctactactt ccagggtta 19gctactactt ccagggtta 19

<210> 26<210> 26

<211> 19<211> 19

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> Allergin-1基因的靶序列<223> Target sequence of Allergin-1 gene

<400> 26<400> 26

gctggtggtg ataatccta 19gctggtggtg ataatccta 19

Claims (23)

Translated fromChinese

1.检测Allergin-1的表达水平或活性的试剂在制备用于鉴别LSCs的产品中的应用，所述试剂用于检测受试者或来自受试者的样本中Allergin-1的表达水平或活性；1. Application of a reagent for detecting the expression level or activity of Allergin-1 in the preparation of a product for identifying LSCs, the reagent is used to detect the expression level or activity of Allergin-1 in a subject or a sample from a subject ;任选地，所述样本包含造血干细胞；Optionally, the sample comprises hematopoietic stem cells;任选地，Allergin-1在造血干细胞中的表达水平或活性显著高于正常造血干细胞中Allergin-1的表达水平或活性，是造血干细胞为LSCs的指示；Optionally, the expression level or activity of Allergin-1 in hematopoietic stem cells is significantly higher than the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the hematopoietic stem cells are LSCs;任选地，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的至少3倍以上，是造血干细胞为LSCs的指示，优选的，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的3倍-100倍，更优选地，为20-50倍。Optionally, the expression level or activity of Allergin-1 in hematopoietic stem cells is at least 3 times the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the hematopoietic stem cells are LSCs. Preferably, the The expression level or activity of Allergin-1 in hematopoietic stem cells is 3-100 times, more preferably, 20-50 times that of Allergin-1 in normal hematopoietic stem cells.2.检测Allergin-1的表达水平或活性的试剂在制备用于白血病的诊断、病程监控和预后判断或用于确定发生白血病的风险或用于确定白血病严重程度的产品中的应用，所述试剂用于检测受试者或来自受试者的样本中Allergin-1的表达水平或活性；2. The application of the reagent for detecting the expression level or activity of Allergin-1 in the preparation of products for the diagnosis, course monitoring and prognosis judgment of leukemia or for determining the risk of leukemia or for determining the severity of leukemia, said reagent For detecting the expression level or activity of Allergin-1 in a subject or a sample from a subject;任选地，所述样本包含造血干细胞或单核细胞；Optionally, the sample comprises hematopoietic stem cells or monocytes;任选地，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的至少3倍以上，是造血干细胞为LSCs的指示，优选的，所述Allergin-1在造血干细胞中的表达水平或活性是正常造血干细胞中Allergin-1的表达水平或活性的3倍-100倍，更优选地，为20-50倍；Optionally, the expression level or activity of Allergin-1 in hematopoietic stem cells is at least 3 times the expression level or activity of Allergin-1 in normal hematopoietic stem cells, which is an indication that the hematopoietic stem cells are LSCs. Preferably, the The expression level or activity of Allergin-1 in hematopoietic stem cells is 3-100 times that of Allergin-1 in normal hematopoietic stem cells, more preferably, 20-50 times;任选地，所述Allergin-1在单核细胞中的表达水平或活性是正常单核细胞中Allergin-1的表达水平或活性的至少2倍以上，是单核细胞为AML单核细胞的指示，优选的，所述Allergin-1在单核细胞中的表达水平或活性是正常单核细胞中Allergin-1的表达水平或活性的2倍-45倍；Optionally, the expression level or activity of Allergin-1 in monocytes is at least 2 times the expression level or activity of Allergin-1 in normal monocytes, which is an indication that the monocytes are AML monocytes , preferably, the expression level or activity of Allergin-1 in monocytes is 2-45 times that of Allergin-1 in normal monocytes;任选地，在M4和M5亚型AML患者中Allergin-1的表达水平与患者的生存率呈负相关，尤其在生存率极低的M5亚型中特异性异常高表达。Optionally, the expression level of Allergin-1 in M4 and M5 subtype AML patients is negatively correlated with the patient's survival rate, especially in the M5 subtype whose survival rate is very low.3.Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)在制备用于治疗白血病的受试者病症的产品中的应用。3. Use of a modulator (eg, inhibitor/antagonist) of Allergin-1 expression or activity in the manufacture of a product for treating a leukemic condition in a subject.4.Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)与一种或多种治疗剂在制备用于治疗白血病的受试者病症的产品中的应用，4. The use of a modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity and one or more therapeutic agents in the preparation of a product for the treatment of leukemia in a subject,优选的，所述一种或多种治疗剂为IFN-γ。Preferably, the one or more therapeutic agents are IFN-γ.5.Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)在制备用于治疗白血病的受试者病症的药物中的应用，其中所述调节剂(例如抑制剂/拮抗剂)与一种或多种治疗剂组合，5. Use of a modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity in the preparation of a medicament for the treatment of leukemia in a subject, wherein the modulator (such as an inhibitor/antagonist) is combined with one or more therapeutic agent combinations,优选的，所述一种或多种治疗剂为IFN-γ。Preferably, the one or more therapeutic agents are IFN-γ.6.一种组合物，药物，制剂或治疗试剂盒，其包含Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)，任选地，所述调节剂与一种或多种其他治疗剂组合使用，用于治疗白血病的受试者病症，优选的，所述一种或多种治疗剂为IFN-γ。6. A composition, medicament, formulation or therapeutic kit comprising a modulator (e.g. inhibitor/antagonist) of Allergin-1 expression or activity, optionally, said modulator in combination with one or more other Therapeutic agents are used in combination for the treatment of leukemia subjects, preferably, the one or more therapeutic agents are IFN-γ.7.一种组合物，药物，制剂或治疗试剂盒，其包含Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)和一种或多种药学上可接受的辅料、赋形剂、载体和/或溶媒。7. A composition, medicament, preparation or treatment kit comprising a modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity and one or more pharmaceutically acceptable adjuvants and excipients , carrier and/or vehicle.8.一种用于检测LSCs的检测试剂盒，其包含检测Allergin-1表达水平或活性的试剂。8. A detection kit for detecting LSCs, comprising a reagent for detecting the expression level or activity of Allergin-1.9.一种筛选治疗白血病的候选药物的方法，所述方法包括：用待筛选物质处理表达或含有Allergin-1基因的体系；和检测处理前后所述体系中Allergin-1基因和/或蛋白的表达水平或活性；9. A method for screening candidate drugs for the treatment of leukemia, said method comprising: treating a system expressing or containing Allergin-1 gene with a substance to be screened; and detecting the expression of Allergin-1 gene and/or protein in said system before and after processing expression level or activity;任选地，处理后所述体系中Allergin-1基因和/或蛋白的表达水平或活性低于处理前所述体系中Allergin-1基因/或蛋白的表达水平或活性，指示该待筛选物质是治疗白血病的受试者病症的候选药物。Optionally, the expression level or activity of Allergin-1 gene and/or protein in the system after treatment is lower than the expression level or activity of Allergin-1 gene/or protein in the system before treatment, indicating that the substance to be screened is A drug candidate for treating a condition in a subject with leukemia.10.有效量的Allergin-1表达或活性的抑制剂或拮抗剂在制备用于消除LSCs和/或AML细胞和/或提高正常NK细胞活性或免疫治疗效果的药物中的应用。10. The use of an effective dose of an inhibitor or antagonist of Allergin-1 expression or activity in the preparation of a medicament for eliminating LSCs and/or AML cells and/or improving the activity of normal NK cells or the effect of immunotherapy.11.权利要求1-10中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述白血病为急性髓性白血病AML。11. The method, use, composition, medicament, preparation, treatment kit or detection kit according to any one of claims 1-10, wherein the leukemia is acute myeloid leukemia (AML).12.权利要求1-11中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，Allergin-1在AML患者中高表达，优选的，在M5亚型中异常高表达。12. The method, application, composition, medicament, preparation, treatment kit or detection kit according to any one of claims 1-11, wherein Allergin-1 is highly expressed in AML patients, preferably, in the M5 subtype abnormally high expression.13.权利要求1-12中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1和至少一种生物标志物组合，优选的，所述组合为Allergin-1⁺CD34⁺CD38^-。13. The method, use, composition, medicament, preparation, treatment kit or detection kit of any one of claims 1-12, wherein said Allergin-1 is combined with at least one biomarker, preferably, The combination is Allergin-1⁺ CD34⁺ CD38^- .14.权利要求1-13中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述产品包括：芯片、试剂、试纸、药物、制剂、试剂盒或高通量筛选平台。14. The method, application, composition, medicament, preparation, treatment kit or detection kit according to any one of claims 1-13, wherein the product comprises: chip, reagent, test strip, medicament, preparation, test kit or high-throughput screening platforms.15.权利要求1或2所述的应用或权利要求8所述的试剂盒，其中，所述试剂是适于通过RT-PCR、实时定量PCR、免疫检测(例如ELISA、RIA、多重免疫实验、免疫荧光实验、蛋白印记、或斑点印记实验)、原位杂交或芯片技术检测Allergin-1表达水平或活性的试剂，优选的，所述试剂包括针对Allergin-1基因的引物/探针、分子信标、或针对Allergin-1蛋白的抗体和/或配体以及小分子化合物，尤其优选的，所述针对Allergin-1基因的引物具有SEQ IDNO：18、19、20、21、22或23所示的序列。15. The application of claim 1 or 2 or the kit of claim 8, wherein the reagent is suitable for RT-PCR, real-time quantitative PCR, immunoassay (such as ELISA, RIA, multiplex immunoassay, Immunofluorescence experiment, western blotting, or dot blot experiment), in situ hybridization or a reagent for detecting the expression level or activity of Allergin-1 by chip technology, preferably, the reagent includes primers/probes for the Allergin-1 gene, molecular signal Target, or antibodies and/or ligands and small molecule compounds against Allergin-1 protein, especially preferably, the primers against Allergin-1 gene have SEQ ID NO: 18, 19, 20, 21, 22 or 23 the sequence of.16.权利要求1-15中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述抑制剂或拮抗剂与IFN-γ组合使用；或者所述组合物、药物、制剂、治疗试剂盒、检测试剂盒包含IFN-γ。16. The method, use, composition, medicament, preparation, treatment kit or detection kit of any one of claims 1-15, wherein said inhibitor or antagonist is used in combination with IFN-γ; or said Compositions, medicaments, preparations, treatment kits, detection kits comprise IFN-γ.17.权利要求1-16中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述抑制剂和/或拮抗剂是指任何可降低编码Allergin-1的核酸的表达、降低Allergin-1蛋白质水平，或抑制Allergin-1的活性的物质，优选为降低Allergin-1蛋白的活性、降低Allergin-1基因或蛋白的稳定性、下调Allergin-1的表达、减少Allergin-1蛋白有效作用时间、或抑制Allergin-1的转录和翻译的物质。17. The method, application, composition, medicament, preparation, treatment kit or detection kit of any one of claims 1-16, wherein, said inhibitor and/or antagonist refers to any that can reduce the encoding Allergin- The expression of the nucleic acid of 1, the reduction of the Allergin-1 protein level, or the substance that inhibits the activity of Allergin-1, preferably reducing the activity of the Allergin-1 protein, reducing the stability of the Allergin-1 gene or protein, and down-regulating the expression of Allergin-1 , Substances that reduce the effective action time of Allergin-1 protein, or inhibit the transcription and translation of Allergin-1.18.权利要求1-17中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)包括：核酸抑制物、蛋白抑制剂、蛋白水解酶、蛋白结合分子；及其组合。18. The method, use, composition, medicament, preparation, therapeutic kit or detection kit of any one of claims 1-17, wherein the modulator (eg inhibitor/antagonist) of Allergin-1 expression or activity Agents) include: nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules; and combinations thereof.19.权利要求1-18中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)选自：以Allergin-1或其转录本为靶序列、且能够抑制Allergin-1基因表达或基因转录的干扰分子，优选的，包括：shRNA(小发夹RNA)、小干扰RNA(siRNA)、dsRNA、微小RNA，锌指，和CRISPR/Cas9的至少之一。19. The method, use, composition, medicament, formulation, therapeutic kit or detection kit of any one of claims 1-18, wherein the modulator (eg inhibitor/antagonist) of Allergin-1 expression or activity agent) is selected from: interfering molecules that take Allergin-1 or its transcripts as target sequences and can inhibit Allergin-1 gene expression or gene transcription, preferably, include: shRNA (small hairpin RNA), small interfering RNA (siRNA ), dsRNA, microRNA, zinc finger, and at least one of CRISPR/Cas9.20.权利要求18所述的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述蛋白结合分子选自：与Allergin-1蛋白特异性结合的物质，如能够抑制Allergin-1蛋白活性的抗体或配体；Allergin-1配体结合位点的竞争剂，包括Allergin-1受体与其配体结合片段、可溶性截短的Allergin-1受体、可溶性Allergin-1受体融合蛋白，例如包含IgG免疫球蛋白的Fc部分的Allergin-1融合蛋白、配体融合蛋白；拟肽；肽抑制剂；小分子化合物；及其组合。20. The method, application, composition, medicament, preparation, treatment kit or detection kit of claim 18, wherein the protein-binding molecule is selected from the group consisting of substances that specifically bind to the Allergin-1 protein, such as substances capable of Antibodies or ligands that inhibit the activity of Allergin-1 protein; competitors for the ligand-binding site of Allergin-1, including Allergin-1 receptor and its ligand-binding fragment, soluble truncated Allergin-1 receptor, soluble Allergin-1 Receptor fusion proteins, such as Allergin-1 fusion proteins comprising the Fc portion of an IgG immunoglobulin, ligand fusion proteins; peptidomimetics; peptide inhibitors; small molecule compounds; and combinations thereof.21.权利要求20所述的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)为shRNA；尤其优选的，所述shRNA序列具有SEQ ID NO：12、13、14、15、16或17所示的序列。21. The method, application, composition, medicament, preparation, treatment kit or detection kit of claim 20, wherein the regulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity is shRNA particularly preferably, the shRNA sequence has the sequence shown in SEQ ID NO: 12, 13, 14, 15, 16 or 17.22.权利要求21所述的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)为特异性与Allergin-1结合的抗体；优选的，例如，所述特异性抗体包括单克隆抗体、多克隆抗体、中和抗体、抗原结合片段、偶联Allergin-1抗体，22. The method, application, composition, medicament, preparation, treatment kit or detection kit of claim 21, wherein the modulator (such as an inhibitor/antagonist) of Allergin-1 expression or activity is a specific Antibodies that specifically bind to Allergin-1; preferably, for example, the specific antibodies include monoclonal antibodies, polyclonal antibodies, neutralizing antibodies, antigen-binding fragments, and conjugated Allergin-1 antibodies,更优选的，所述抗体为单克隆抗体和/或中和抗体。More preferably, the antibody is a monoclonal antibody and/or a neutralizing antibody.23.权利要求1-22中任一项的方法、应用、组合物、药物、制剂、治疗试剂盒或检测试剂盒，其中，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)以约0.01mg/kg至约100mg/kg，例如，约0.01mg/kg-0.2mg/kg、约0.1mg/kg-10mg/kg、约0.5mg/kg-5mg/kg、或约1mg/kg-约2mg/kg给药；23. The method, use, composition, medicament, formulation, therapeutic kit or detection kit of any one of claims 1-22, wherein the modulator (eg inhibitor/antagonist) of Allergin-1 expression or activity agent) at about 0.01 mg/kg to about 100 mg/kg, for example, about 0.01 mg/kg-0.2 mg/kg, about 0.1 mg/kg-10 mg/kg, about 0.5 mg/kg-5 mg/kg, or about 1 mg /kg-about 2mg/kg administration;优选的，所述Allergin-1表达或活性的调节剂(例如抑制剂/拮抗剂)的单位剂量为约1mg至约100mg的单位剂量，优选为约5mg-50mg、约10mg-40mg、15mg-30mg、或20mg-25mg。Preferably, the unit dose of the regulator (such as inhibitor/antagonist) of the expression or activity of Allergin-1 is about 1 mg to about 100 mg, preferably about 5 mg-50 mg, about 10 mg-40 mg, 15 mg-30 mg , or 20mg-25mg.

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