CN112759651B - T cell containing chimeric antigen receptor modification and application thereof - Google Patents
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及生物医药技术领域，具体涉及一种嵌合抗原受体、表达嵌合抗原受体的T细胞以及在治疗HIV感染或AIDS中的应用。The present invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor, a T cell expressing the chimeric antigen receptor and its application in the treatment of HIV infection or AIDS.
背景技术Background technique
人类免疫缺陷病毒(Human immunodeficiency virus，HIV)，即获得性免疫缺陷综合征(艾滋病，AIDS)病毒，是一种感染人类免疫系统细胞的RNA病毒，是诱发人类产生获得性免疫缺陷综合症(AIDS)的罪魁祸首。Human immunodeficiency virus (HIV), the acquired immunodeficiency syndrome (AIDS, AIDS) virus, is an RNA virus that infects cells of the human immune system and induces acquired immunodeficiency syndrome (AIDS) in humans. ) is the culprit.
由于HIV直接侵犯人体的免疫系统，破坏人体的细胞免疫和体液免疫，HIV感染患者常常伴随着各种并发症，如脑膜炎、肺结核、癌症、肝炎等。根据联合国AIDS规划署(UNAIDS)统计显示，自艾滋病毒蔓延以来，全球已有约7610万(6520万-8800万)人感染该病毒。2016年，全球共有约3670万(3080万-4290万)名艾滋病毒感染者，其中1950万人获得抗逆转录病毒治疗。Because HIV directly invades the body's immune system and destroys the body's cellular and humoral immunity, HIV-infected patients are often accompanied by various complications, such as meningitis, tuberculosis, cancer, and hepatitis. According to the United Nations AIDS Program (UNAIDS), since the spread of HIV, about 76.1 million (65.2-88 million) people worldwide have been infected with the virus. In 2016, there were approximately 36.7 million (30.8-42.9 million) people living with HIV globally, of whom 19.5 million received antiretroviral therapy.
目前，针对HIV感染者的治疗方法主要是抗病毒药物的联合使用(combinationanti-retroviral therapy，cART)组成的高效抗逆转录病毒疗法(highly active anti-retroviral therapy，HAART)，抗病毒药物包括HIV复制抑制剂或者HIV的抗体，例如：专利CN103429595A公开了2,3,4-取代的5,6,7,8-四氢[1]苯并噻吩并[2,3-b]吡啶化合物及其药物可接受的盐，并且涉及包含这类化合物的组合物以及这类化合物作为HIV复制抑制剂的用途。专利CN103797029A公开了针对人类免疫缺陷病毒或HIV的表位的广谱中和抗体序列。专利CN106459186A公开了针对HIV-1V1V2ENV区域的广谱中和性单克隆抗体序列。专利CN107022027A、CN107033241A公开了HIV-1广谱中和抗体序列，所述抗体特异性结合HIV-gp140。At present, the treatment methods for HIV-infected patients are mainly highly active anti-retroviral therapy (HAART) composed of combination anti-retroviral therapy (cART), antiviral drugs including HIV replication Inhibitors or HIV antibodies, for example: Patent CN103429595A discloses 2,3,4-substituted 5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine compounds and their medicines acceptable salts, and relates to compositions comprising such compounds and the use of such compounds as HIV replication inhibitors. Patent CN103797029A discloses broadly neutralizing antibody sequences against epitopes of human immunodeficiency virus or HIV. Patent CN106459186A discloses the sequence of broad-spectrum neutralizing monoclonal antibody against HIV-1V1V2 ENV region. Patents CN107022027A and CN107033241A disclose HIV-1 broad-spectrum neutralizing antibody sequences that specifically bind HIV-gp140.
经过长期的实践检验，该疗法能够抑制AIDS患者体内病毒复制，从而大大减缓AIDS疾病进程。After a long-term practice test, the therapy can inhibit the virus replication in AIDS patients, thereby greatly slowing down the progress of AIDS disease.
然而，该疗法存在如下明显的缺陷：However, this therapy has the following obvious drawbacks:
(1)该疗法成本很高，且造成了更大量HIV携带者的存在，且完全依赖抗逆转录病毒药物且不得不承受其带来的严重负作用和经济负担；(1) The cost of this therapy is very high, and it has resulted in the existence of a larger number of HIV carriers, and is completely dependent on antiretroviral drugs and has to bear the serious negative effects and economic burdens brought about by them;
(2)该疗法针对部分患者无效，部分患者体内HIV仍然得不到抑制反而大量扩增；(2) The therapy is ineffective for some patients, and HIV in some patients is still not suppressed but massively expanded;
(3)该疗法不能靶向或清除患者体内静息CD4+中心记忆T细胞中存在的潜伏HIV病毒，这些被潜伏感染的细胞是非常稳定的，在体内持续增加，将导致停药之后病毒立即反弹。(3) The therapy cannot target or clear the latent HIV virus present in the resting CD4+ central memory T cells in the patient. These latently infected cells are very stable and continue to increase in the body, which will cause the virus to rebound immediately after drug withdrawal. .
由此可见，治愈AIDS病人的关键问题是如何彻底清除病人体内的HIV病毒。研究表明，CD4+中心记忆T细胞(central memory T cell，Tcm)占所有HIV细胞储存库总数的95％。找到明确的HIV细胞储存库的生物标志物(biomarker)，即可实现精确定位并最终清除HIV细胞储存库细胞。It can be seen that the key problem in curing AIDS patients is how to completely remove the HIV virus in the patient's body. Studies have shown that CD4+ central memory T cells (Tcm) account for 95% of all HIV cell reservoirs. Finding well-defined biomarkers of the HIV cellular reservoir allows precise targeting and eventual elimination of HIV cellular reservoir cells.
嵌合抗原受体(chimeric antigen receptor,CAR)修饰的T细胞免疫疗法是当前过继性细胞治疗技术中最新技术之一。嵌合抗原受体(CAR)赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相对于天然T细胞表面受体TCR能够识别更广泛的目标。近年来，CAR-T技术在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。因此，基因修饰的T细胞有可能成为清除HIV细胞储存库的重要途径。并且，对于清除HIV-1感染细胞也有一定的进展，例如：文献：Antibody 10-1074suppresses viremia in HIV-1-infected individuals，NatureMedicine，2017，通过将单克隆抗体10-1074注射给33个受试者，验证了10-1074的很好的耐受性，最高剂量可以注射30mg/kg，其中，至少有11个受试者对10-1074敏感性好并迅速降低了病毒，同时证明了单克隆抗体10-1074对人使用的安全性及活性。但是现有技术均尚未公开以10-1074的人单链抗体(scFv)作为CAR分子的胞外抗原结合结构域，并将其应用于清除HIV细胞储存库，甚至是根除AIDS。因此，研究以10-1074的人单链抗体(scFv)作为CAR分子的胞外抗原结合结构域的CAR-T细胞在清除HIV细胞储存库中的作用和阐明相关机制，具有重要的基础研究意义和临床应用价值。Chimeric antigen receptor (CAR) modified T cell immunotherapy is one of the latest technologies in the current adoptive cell therapy technology. Chimeric antigen receptors (CARs) confer T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a wider range of targets relative to the native T cell surface receptor TCR. In recent years, CAR-T technology has achieved remarkable efficacy in the treatment of acute leukemia and non-Hodgkin lymphoma, and is considered to be one of the most promising tumor treatments. Thus, genetically modified T cells have the potential to be an important pathway for clearing HIV cell reservoirs. Also, there has been some progress in clearing HIV-1-infected cells, for example: Literature: Antibody 10-1074 suppresses viremia in HIV-1-infected individuals, NatureMedicine, 2017, by injecting monoclonal antibody 10-1074 into 33 subjects , verified the good tolerance of 10-1074, the highest dose can be injected 30mg/kg, of which, at least 11 subjects had good sensitivity to 10-1074 and rapidly reduced the virus, and proved that the monoclonal antibody Safety and activity of 10-1074 in humans. However, the prior art has not yet disclosed the use of 10-1074 human single-chain antibody (scFv) as the extracellular antigen-binding domain of CAR molecules, and its application in clearing HIV cell reservoirs, or even eradicating AIDS. Therefore, it is of great basic research significance to study the role of CAR-T cells using 10-1074 human single-chain antibody (scFv) as the extracellular antigen-binding domain of CAR molecules in clearing HIV cell reservoirs and clarifying the relevant mechanisms. and clinical application value.
发明内容SUMMARY OF THE INVENTION
本发明人经过一系列创造性的研究，制备获得了以10-1074的人单链抗体(single-chain antibody fragment，scFv)为胞外抗原结合结构域的嵌合抗原受体(CAR)，该CAR可有效实现激活T细胞信号使其具有杀伤靶细胞功能，其中，10-1074在多种人来源的HIV抗体中具有较强的和HIV囊膜蛋白特异性结合的特征，将正常10-1074抗体制备成的具备VL-VH顺序的多种scFv形式的10-1074具有较强的和HIV囊膜蛋白特异性结合的特征，制备出的以10-1074的scFv为胞外抗原结合结构域的CAR可特异性识别HIV囊膜蛋白或HIV潜伏细胞。优选的，所述的以10-1074的scFv为胞外抗原结合结构域的CAR为10-1074-CD28-4-1BB-CD3ζ-CAR。进一步的，本发明制备出的10-1074-CD28-4-1BB-CD3ζ-CART细胞可有效的被HIV潜伏细胞特异性识别激活并具有杀伤能力，降低HIV病毒数量。更进一步的，本发明制备了包含10-1074-CD28-4-1BB-CD3ζ-CART的药物组合物用于清除人类免疫缺陷病毒(HIV)在HIV感染病人体内的细胞储存库，甚至达到了治疗AIDS的目的。After a series of creative researches, the inventors have prepared a chimeric antigen receptor (CAR) with a human single-chain antibody fragment (scFv) of 10-1074 as the extracellular antigen binding domain. It can effectively realize the activation of T cell signal to make it have the function of killing target cells. Among them, 10-1074 has strong specific binding characteristics with HIV envelope protein among various HIV antibodies of human origin. The normal 10-1074 antibody The prepared 10-1074 in various scFv forms with the VL-VH sequence has the characteristics of strong specific binding to HIV envelope protein, and the prepared CAR with the scFv of 10-1074 as the extracellular antigen binding domain It can specifically recognize HIV envelope protein or HIV latent cells. Preferably, the CAR using the scFv of 10-1074 as the extracellular antigen binding domain is 10-1074-CD28-4-1BB-CD3ζ-CAR. Further, the 10-1074-CD28-4-1BB-CD3ζ-CART cells prepared by the present invention can be effectively recognized and activated by HIV latent cells and have killing ability, thereby reducing the number of HIV viruses. Further, the present invention prepares a pharmaceutical composition comprising 10-1074-CD28-4-1BB-CD3ζ-CART for clearing the cell reservoir of human immunodeficiency virus (HIV) in HIV-infected patients, and even achieves treatment. Purpose of AIDS.
具体地，本发明的第一方面，提供了一种嵌合抗原受体(CAR)，所述的嵌合抗原受体包括胞外抗原结合结构域，所述的胞外抗原结合结构域为10-1074的人单链抗体。Specifically, the first aspect of the present invention provides a chimeric antigen receptor (CAR), the chimeric antigen receptor includes an extracellular antigen binding domain, and the extracellular antigen binding domain is 10 -1074 human single chain antibody.
优选的，所述的人单链抗体包括重链可变区、接头和轻链可变区。Preferably, the human single-chain antibody includes a heavy chain variable region, a linker and a light chain variable region.
更优选的，所述的重链可变区是与SEQ ID NO：3的氨基酸序列具有至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同源性的氨基酸序列。More preferably, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence of SEQ ID NO: 3 %, 97%, 98%, 99%, 99.5% homology of amino acid sequences.
在本发明的一个具体实施方式中，所述重链可变区的氨基酸序列如SEQ ID NO：3所示。In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3.
更优选的，所述的轻链可变区是与SEQ ID NO：2的氨基酸序列具有至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同源性的氨基酸序列。More preferably, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 2 %, 97%, 98%, 99%, 99.5% homology of amino acid sequences.
在本发明的一个具体实施方式中，所述轻链可变区的氨基酸序列如SEQ ID NO：2所示。In a specific embodiment of the present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.
更优选的，所述的接头为包含甘氨酸(Gly)和/或丝氨酸(Ser)的氨基酸序列，进一步优选的，所述接头的序列如SEQ ID NO：4-9任一项所示。More preferably, the linker is an amino acid sequence comprising glycine (Gly) and/or serine (Ser). Further preferably, the linker sequence is shown in any one of SEQ ID NOs: 4-9.
在本发明的一个具体实施方式中，所述接头的氨基酸序列如SEQ ID NO：4所示。In a specific embodiment of the present invention, the amino acid sequence of the linker is shown in SEQ ID NO:4.
在本发明的另一个具体实施方式中，所述的人单链抗体的氨基酸序列如SEQ IDNO：1或SEQ ID NO：10所示。In another specific embodiment of the present invention, the amino acid sequence of the human single-chain antibody is shown in SEQ ID NO: 1 or SEQ ID NO: 10.
优选的，所述的嵌合抗原受体还包括跨膜区，所述的跨膜区选自CD3ζ多肽、CD4多肽、CD8多肽、CD28多肽、CD28-4-1BB多肽、OX40多肽、ICOS多肽、CTLA-4多肽、PD-1多肽、LAG-3多肽、2B4多肽或BTLA多肽中的一种或两种以上的组合。Preferably, the chimeric antigen receptor further comprises a transmembrane region, and the transmembrane region is selected from the group consisting of CD3ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, CD28-4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, One or more combinations of CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide or BTLA polypeptide.
更优选的，所述的跨膜区选自如下组合中的任一组：More preferably, the transmembrane region is selected from any one of the following combinations:
(1)CD8多肽、CD28多肽和CD3ζ多肽；(1) CD8 polypeptide, CD28 polypeptide and CD3ζ polypeptide;
(2)CD4多肽、CD8多肽、CD28、4-1BB多肽和CD3ζ多肽；(2) CD4 polypeptide, CD8 polypeptide, CD28, 4-1BB polypeptide and CD3ζ polypeptide;
(3)CD4多肽、CD8多肽、CD28、4-1BB多肽和CD3ζ多肽。(3) CD4 polypeptide, CD8 polypeptide, CD28, 4-1BB polypeptide and CD3ζ polypeptide.
在本发明的一个具体实施方式中，所述的跨膜区为CD28多肽。In a specific embodiment of the present invention, the transmembrane region is a CD28 polypeptide.
在本发明的一个具体实施方式中，所述的跨膜区的氨基酸序列如SEQ ID NO：14所示。In a specific embodiment of the present invention, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 14.
优选的，所述的嵌合抗原受体还包括胞外铰链区，所述的胞外铰链区选自CD8或IgG的胞外铰链区。Preferably, the chimeric antigen receptor further comprises an extracellular hinge region, and the extracellular hinge region is selected from the extracellular hinge region of CD8 or IgG.
在本发明的一个具体实施方式中，所述的胞外铰链区为CD8。In a specific embodiment of the present invention, the extracellular hinge region is CD8.
在本发明的一个具体实施方式中，所述的胞外铰链区的氨基酸序列如SEQ ID NO：15所示。In a specific embodiment of the present invention, the amino acid sequence of the extracellular hinge region is shown in SEQ ID NO: 15.
优选的，所述的嵌合抗原受体还包括胞浆区，所述的胞浆区选自CD28-4-1BB-CD3ζ、CD28-CD3ζ或4-1BB-CD3ζ。Preferably, the chimeric antigen receptor further comprises a cytoplasmic region, and the cytoplasmic region is selected from CD28-4-1BB-CD3ζ, CD28-CD3ζ or 4-1BB-CD3ζ.
在本发明的一个具体实施方式中，所述的胞浆区的氨基酸序列如SEQ ID NO：16所示。In a specific embodiment of the present invention, the amino acid sequence of the cytoplasmic region is shown in SEQ ID NO: 16.
优选的，所述的嵌合抗原受体还包括控制细胞存活或者死亡的基因。更优选的，所述的控制细胞存活或者死亡的基因选自IL12或TK基因。当在外部加药的情况下，可以控制细胞的死亡。Preferably, the chimeric antigen receptor also includes genes that control cell survival or death. More preferably, the gene that controls cell survival or death is selected from IL12 or TK gene. When externally medicated, cell death can be controlled.
在本发明的一个具体实施方式中，所述的嵌合抗原受体包括胞外抗原结合结构域、跨膜区、胞外铰链区和胞浆区。In a specific embodiment of the present invention, the chimeric antigen receptor includes an extracellular antigen binding domain, a transmembrane region, an extracellular hinge region and a cytoplasmic region.
在本发明的一个具体实施方式中，所述的嵌合抗原受体的氨基酸序列是与SEQ IDNO：11所示序列具有至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同源性的氨基酸序列。In a specific embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is at least 80%, 85%, 90%, 91%, 92%, 93% of the sequence shown in SEQ ID NO: 11 , 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology of amino acid sequences.
在本发明的另一个具体实施方式中，所述的嵌合抗原受体的氨基酸序列如SEQ IDNO：11所示。In another specific embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 11.
本发明的第二方面，提供了一种分离的免疫应答细胞，所述的免疫应答细胞包含上述第一方面所述的嵌合抗原受体。A second aspect of the present invention provides an isolated immune response cell, the immune response cell comprising the chimeric antigen receptor described in the first aspect.
优选的，所述的免疫应答细胞表达控制细胞存活或者死亡的基因。更优选的，所述的控制细胞存活或者死亡的基因选自IL12或TK基因。当在外部加药的情况下，可以控制细胞的死亡。Preferably, the immune response cells express genes that control cell survival or death. More preferably, the gene that controls cell survival or death is selected from IL12 or TK gene. When externally medicated, cell death can be controlled.
优选的，将所述的免疫应答细胞与其他细胞混合控制该免疫应答细胞的存活或者死亡。所述的其他细胞选自表达IL12或TK基因的细胞。更优选的，所述的其他细胞为表达IL12或TK基因CAR-T细胞。Preferably, the immune response cells are mixed with other cells to control the survival or death of the immune response cells. Said other cells are selected from cells expressing IL12 or TK gene. More preferably, the other cells are CAR-T cells expressing IL12 or TK gene.
优选的，所述的免疫应答细胞选自T细胞、NK细胞、CTL、人胚胎干细胞、淋巴祖细胞和/或T细胞前体细胞。Preferably, the immune response cells are selected from T cells, NK cells, CTL, human embryonic stem cells, lymphoid progenitor cells and/or T cell precursor cells.
在本发明的一个具体实施方式中，所述的免疫应答细胞为T细胞，即CAR-T细胞。In a specific embodiment of the present invention, the immune response cells are T cells, namely CAR-T cells.
其中，所述的免疫应答细胞有效的被HIV潜伏细胞特异性识别、激活、有效降低HIV病毒以及治疗AIDS。Wherein, the immune response cells are effectively and specifically recognized by HIV latent cells, activated, and effectively reduce HIV virus and treat AIDS.
优选的，所述的免疫应答细胞为10-1074-CD28-4-1BB-CD3ζ-CART。Preferably, the immune response cells are 10-1074-CD28-4-1BB-CD3ζ-CART.
本发明的第三方面，提供了一种核酸分子，所述的核酸分子编码上述第一方面所述嵌合抗原受体。优选的，所述的核酸分子包括TK或IL12基因片段，所述的TK或IL12基因片段可以控制细胞的存活或者死亡，例如可以通过外部加药让细胞死亡。A third aspect of the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor of the first aspect above. Preferably, the nucleic acid molecule includes a TK or IL12 gene fragment, and the TK or IL12 gene fragment can control the survival or death of cells, for example, cells can be killed by external drug addition.
优选的，所述的核酸分子是与SEQ ID NO：12所示序列具有至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同源性的核苷酸序列。Preferably, the nucleic acid molecule is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the sequence shown in SEQ ID NO: 12 , 98%, 99%, 99.5% homology of nucleotide sequences.
在本发明的一个具体实施方式中，所述的核酸分子的核苷酸序列如SEQ ID NO：12所示。In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 12.
本发明的第四方面，提供了一种载体，所述的载体包含上述第三方面所述的核酸分子。A fourth aspect of the present invention provides a vector, wherein the vector comprises the nucleic acid molecule described in the third aspect.
优选的，所述的载体是与SEQ ID NO：13所示序列具有至少为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同源性的核苷酸序列。Preferably, the vector has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Nucleotide sequences with 98%, 99%, 99.5% homology.
在本发明的一个具体实施方式中，所述载体的核苷酸序列如SEQ ID NO：13所示。In a specific embodiment of the present invention, the nucleotide sequence of the vector is shown in SEQ ID NO:13.
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞包含上述第三方面所述的核酸分子或上述第四方面所述的载体。A fifth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect.
优选的，所述的宿主细胞为T细胞。Preferably, the host cells are T cells.
本发明的第六方面，提供了一种细胞群，所述的细胞群包含至少1个上述第五方面所述的宿主细胞。A sixth aspect of the present invention provides a cell population, wherein the cell population comprises at least one host cell according to the fifth aspect.
本发明的第七方面，提供了特异性结合上述第一方面所述的嵌合抗原受体的抗体或其抗原结合部分。A seventh aspect of the present invention provides an antibody or an antigen-binding portion thereof that specifically binds to the chimeric antigen receptor described in the first aspect.
本发明的第八方面，提供了一种药物组合物，所述的药物组合物包含有效量的上述第一方面所述的嵌合抗原受体、第二方面所述的免疫应答细胞、第三方面所述的核酸分子、第四方面所述的载体、第五方面所述的宿主细胞、第六方面所述的细胞群和/或第七方面所述的抗体或其抗原结合部分，以及药用载体。The eighth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the chimeric antigen receptor described in the first aspect, the immune response cell described in the second aspect, and the third The nucleic acid molecule of aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the cell population of the sixth aspect, and/or the antibody or antigen-binding portion thereof of the seventh aspect, and the drug Use a carrier.
优选的，所述的药物组合物用于治疗HIV感染或AIDS。Preferably, the pharmaceutical composition is used to treat HIV infection or AIDS.
本发明的第九方面，提供了上述第一方面所述的嵌合抗原受体、第二方面所述的免疫应答细胞、第三方面所述的核酸分子、第四方面所述的载体、第五方面所述的宿主细胞、第六方面所述的细胞群、第七方面所述的抗体或其抗原结合部分和/或第八方面所述的药物组合物在制备用于治疗和/或诊断HIV感染或AIDS的药物中的用途。The ninth aspect of the present invention provides the chimeric antigen receptor according to the first aspect, the immune response cell according to the second aspect, the nucleic acid molecule according to the third aspect, the vector according to the fourth aspect, and the third aspect. The host cell described in the fifth aspect, the cell population described in the sixth aspect, the antibody or its antigen-binding portion thereof described in the seventh aspect, and/or the pharmaceutical composition described in the eighth aspect are prepared for treatment and/or diagnosis. Use in medicines for HIV infection or AIDS.
本发明的第十方面，提供了一种用于治疗和/或诊断HIV感染或AIDS的试剂盒，所述的试剂盒包含上述第一方面所述的嵌合抗原受体、第二方面所述的免疫应答细胞、第三方面所述的核酸分子、第四方面所述的载体、第五方面所述的宿主细胞、第六方面所述的细胞群、第七方面所述的抗体或其抗原结合部分和/或第八方面所述的药物组合物。A tenth aspect of the present invention provides a kit for treating and/or diagnosing HIV infection or AIDS, the kit comprising the chimeric antigen receptor described in the first aspect, the chimeric antigen receptor described in the second aspect The immune response cell described in the third aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the cell population described in the sixth aspect, the antibody or its antigen described in the seventh aspect The combination part and/or the pharmaceutical composition of the eighth aspect.
本发明的第十一方面，提供了一种清除HIV感染细胞或治疗AIDS的方法，所述的方法包括将有效量的上述第一方面所述的嵌合抗原受体、第二方面所述的免疫应答细胞、第三方面所述的核酸分子、第四方面所述的载体、第五方面所述的宿主细胞、第七方面所述的抗体或其抗原结合部分、第六方面所述的细胞群和/或第八方面所述的药物组合物给予受试者。The eleventh aspect of the present invention provides a method for eliminating HIV-infected cells or treating AIDS, the method comprising combining an effective amount of the chimeric antigen receptor described in the first aspect, the chimeric antigen receptor described in the second aspect, Immune response cells, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody or its antigen-binding portion of the seventh aspect, the cell of the sixth aspect The population and/or the pharmaceutical composition of the eighth aspect is administered to a subject.
优选的，所述的方法可以为治疗目的，也可以为非治疗目的。其中，所述的治疗目的包括但不限于清除HIV细胞储存库或杀伤HIV潜伏细胞、增加或延长HIV感染者或AIDS患者生存期。所述的非治疗目的包括但不限于特异性识别HIV囊膜蛋白、免疫应答细胞被HIV潜伏细胞特异性识别激活。Preferably, the method may be for therapeutic purposes or non-therapeutic purposes. Wherein, the therapeutic purpose includes, but is not limited to, clearing HIV cell reservoirs or killing HIV latent cells, and increasing or prolonging the survival period of HIV-infected or AIDS patients. The non-therapeutic purposes include, but are not limited to, specific recognition of HIV envelope proteins, and specific recognition and activation of immune response cells by HIV latent cells.
本发明的第十二方面，提供了一种检测方法，所述的方法包括使包含来自所述宿主的一个或多个细胞的样品与上述第一方面所述的嵌合抗原受体、第二方面所述的免疫应答细胞、第三方面所述的核酸分子、第四方面所述的载体、第五方面所述的宿主细胞、第七方面所述的抗体或其抗原结合部分、第六方面所述的细胞群和/或第十方面所述的试剂盒相接触，形成复合物，并且，检测所述的复合物，其中检测到所述复合物指示HIV病毒在所述宿主中的存在。A twelfth aspect of the present invention provides a detection method, the method comprising combining a sample comprising one or more cells from the host with the chimeric antigen receptor, the second The immune response cell according to the aspect, the nucleic acid molecule according to the third aspect, the vector according to the fourth aspect, the host cell according to the fifth aspect, the antibody or antigen-binding portion thereof according to the seventh aspect, the sixth aspect The cell population and/or the kit of the tenth aspect are contacted to form a complex, and the complex is detected, wherein detection of the complex indicates the presence of HIV virus in the host.
本发明所述的检测方法可以为治疗目的，也可以为非治疗目的。其中，所述的治疗目的包括但不限于诊断AIDS。所述的非治疗目的包括但不限于检测HIV病毒的存在，或者用于分离HIV病毒。The detection method of the present invention may be for therapeutic purposes or non-therapeutic purposes. Wherein, the treatment purpose includes but is not limited to diagnosing AIDS. Said non-therapeutic purposes include, but are not limited to, detecting the presence of HIV virus, or for isolating HIV virus.
本发明的第十三方面，提供了一种增加或延长HIV感染或AIDS患者生存期的方法，所述的方法包括将有效量上述第二方面所述的免疫应答细胞给予受试者，从而增加或延长所述受试者的生存期。The thirteenth aspect of the present invention provides a method for increasing or prolonging the survival of HIV-infected or AIDS patients, the method comprising administering an effective amount of the immune response cells described in the second aspect to a subject, thereby increasing the or prolong the survival of the subject.
本发明的第十四方面，提供了一种清除HIV细胞储存库的方法，所述的方法包括将有效量的上述第二方面所述的免疫应答细胞给予HIV感染或AIDS的受试者，从而清除HIV细胞储存库。A fourteenth aspect of the present invention provides a method for clearing HIV cell depots, the method comprising administering an effective amount of the immune response cells described in the second aspect above to a subject infected with HIV or AIDS, thereby Clear HIV cell reservoirs.
本发明的第十五方面，提供了一种特异性识别HIV囊膜蛋白的方法，所述的方法包括将有效量的上述第二方面所述的免疫应答细胞给予受试者。A fifteenth aspect of the present invention provides a method for specifically recognizing HIV envelope protein, the method comprising administering an effective amount of the immune response cells described in the second aspect to a subject.
本发明的第十六方面，提供了一种免疫应答细胞被HIV潜伏细胞特异性识别激活的方法，所述的方法包括将有效量的上述第二方面所述的免疫应答细胞给予受试者。The sixteenth aspect of the present invention provides a method for specific recognition and activation of immune response cells by HIV latent cells, the method comprising administering an effective amount of the immune response cells described in the second aspect to a subject.
本发明的第十七方面，涉及一种杀伤HIV潜伏细胞的方法，所述的方法包括将有效量的上述第二方面所述的免疫应答细胞给予受试者，从而增加或延长所述受试者的生存期。The seventeenth aspect of the present invention relates to a method for killing HIV latent cells, the method comprising administering an effective amount of the immune response cells described in the second aspect to a subject, thereby increasing or prolonging the test lifetime of the person.
本发明所述的方法为HIV CART的细胞免疫疗法。The method of the present invention is the cellular immunotherapy of HIV CART.
本发明所述的“药用载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质。The "pharmaceutical carrier" in the present invention refers to any formulation or carrier medium capable of delivering an effective amount of the active substance of the present invention, without interfering with the biological activity of the active substance, and without toxic and side effects to the host or patient.
本发明所述的“同源性”是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的同源性。The "homology" in the present invention means that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the used sequences are compared with those obtained in the prior art. , with (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% , 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.
本发明所述的“和/或”是指包括择一列出的项目以及任何数量的项目组合。As used herein, "and/or" is meant to include alternatively listed items as well as any number of combinations of items.
本发明所述的“包括”是指开放式的描述，含有所描述的指定成分或步骤，以及不会实质上影响的其他指定成分或步骤。In the present invention, "comprising" refers to an open-ended description, containing the specified components or steps described, as well as other specified components or steps that are not substantially affected.
本发明所述的“治疗”是指在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。"Treatment" as used herein refers to slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not Must involve complete elimination of all disease-related signs, symptoms, conditions, or disorders.
本发明所述的“诊断”是指以查明患者过去、诊断时或将来是否患有疾病或病症，或者是查明疾病的进展或将来可能的进展，或者是评估患者对治疗的反应。"Diagnosing" as used herein means to ascertain whether a patient has a disease or condition in the past, at the time of diagnosis or in the future, or to ascertain the progression or possible future progression of a disease, or to assess a patient's response to treatment.
本发明所述的“有效量”是指在以单个或多个剂量给予至患者或器官之后提供所希望的治疗或预防的本发明的CAR、CAR-T、核酸分子、载体、宿主细胞、免疫应答细胞、抗体或其抗原结合部分、细胞群、药物组合物的量或剂量。本发明所述的“预防”是指通过施用本发明所述的CAR、CAR-T、核酸分子、载体、宿主细胞、免疫应答细胞、抗体或其抗原结合部分、细胞群、药物组合物来抑制症状或者延缓特定症状紧张的所有行为。The "effective amount" of the present invention refers to the CAR, CAR-T, nucleic acid molecule, vector, host cell, immune system of the present invention that provides the desired treatment or prevention after administration to a patient or organ in a single or multiple doses The amount or dose of responsive cells, antibodies or antigen-binding portions thereof, cell populations, pharmaceutical compositions. The "prevention" of the present invention refers to the inhibition of inhibition by administering the CAR, CAR-T, nucleic acid molecule, vector, host cell, immune response cell, antibody or antigen-binding portion thereof, cell population, and pharmaceutical composition of the present invention Symptoms or all behaviors that delay the tension of a particular symptom.
本发明所述的“受试者”是指哺乳动物或人，优选为灵长类动物。The "subject" in the present invention refers to a mammal or a human, preferably a primate.
附图说明Description of drawings
以下，结合附图来详细说明本发明的实施例，其中：Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:
图1：显示了实施例1构建的10-1074scFv结合HIV潜伏细胞的结果图；流式结果显示，较对照组Mers(图A)抗体，10-1074scFv(图B)可特异性识别ACH2，虽然这种识别能力较10-1074抗体(图C)略弱。Figure 1: shows the result of binding of 10-1074scFv constructed in Example 1 to HIV latent cells; flow cytometry results show that compared with the control group Mers (Figure A) antibody, 10-1074scFv (Figure B) can specifically recognize ACH2, although This recognition ability is slightly weaker than that of the 10-1074 antibody (Panel C).
图2：显示了实施例1制备10-1074scFv-CAR的流程，其中，所述的10-1074scFv-CAR具体为10-1074scFv-CD8-CD8-CD28-CD3ζ-CAR，具体的，由CD8胞外铰链区、CD28跨膜区、CD28-4-1BB胞浆区和CD3ξ胞浆区以及10-1074scFv组成了CAR。Figure 2: shows the process of preparing 10-1074scFv-CAR in Example 1, wherein, the 10-1074scFv-CAR is specifically 10-1074scFv-CD8-CD8-CD28-CD3ζ-CAR, specifically, is composed of extracellular CD8 The hinge region, CD28 transmembrane region, CD28-4-1BB cytoplasmic region and CD3ξ cytoplasmic region and 10-1074scFv constitute the CAR.
图3：显示了10-1074-CD28-4-1BB-CD3ζCAR识别结合HIV囊膜蛋白能力的结果图；结果显示，较对照组FMC63 scFv-CAR，10-1074scFv-CAR可特异性识别结合HIV囊膜蛋白gp140。Figure 3: Graph showing the ability of 10-1074-CD28-4-1BB-CD3ζCAR to recognize and bind to HIV envelope protein; the results show that compared with the control group FMC63 scFv-CAR, 10-1074scFv-CAR can specifically recognize and bind to HIV envelope Membrane protein gp140.
图4A：显示了10-1074-CD28-4-1BB-CD3ζ-CART(试验组)细胞可有效的被HIV潜伏细胞特异性识别激活的，并产生IL2细胞因子，其中，对照组为CD19scFv-CD28-4-1BB-CD3ζ-CART。Figure 4A: shows that 10-1074-CD28-4-1BB-CD3ζ-CART (test group) cells can be effectively recognized and activated by HIV latent cells and produce IL2 cytokines, wherein the control group is CD19scFv-CD28 -4-1BB-CD3ζ-CART.
图4B：显示了10-1074-CD28-4-1BB-CD3ζ-CART(试验组)细胞可有效的被HIV潜伏细胞特异性识别激活的，并产生TNFα细胞因子，其中，对照组为CD19scFv-CD28-4-1BB-CD3ζ-CART。Figure 4B: shows that 10-1074-CD28-4-1BB-CD3ζ-CART (test group) cells can be effectively recognized and activated by HIV latent cells and produce TNFα cytokines, wherein the control group is CD19scFv-CD28 -4-1BB-CD3ζ-CART.
图4C：显示了10-1074-CD28-4-1BB-CD3ζ-CART(试验组)细胞可有效的被HIV潜伏细胞特异性识别激活的，并产生INFγ细胞因子，其中，对照组为CD19scFv-CD28-4-1BB-CD3ζ-CART。Figure 4C: shows that 10-1074-CD28-4-1BB-CD3ζ-CART (test group) cells can be effectively activated by HIV latent cells and produce INFγ cytokines, wherein the control group is CD19scFv-CD28 -4-1BB-CD3ζ-CART.
图5：显示了10-1074-CD28-4-1BB-CD3ζ-CART细胞对HIV阳性细胞ACH2在整个细胞群中所占比例的影响，结果表明，10-1074-CD28-4-1BB-CD3ζ-CART细胞可特异有效杀伤HIV潜伏细胞。Figure 5: shows the effect of 10-1074-CD28-4-1BB-CD3ζ-CART cells on the proportion of HIV-positive cells ACH2 in the entire cell population, the results show that 10-1074-CD28-4-1BB-CD3ζ- CART cells can specifically and effectively kill HIV latent cells.
图6：显示了10-1074-CD28-4-1BB-CD3ζ-CART(试验组)细胞与对照组细胞对P24浓度的影响，表明10-1074-CD28-4-1BB-CD3ζ-CART细胞可特异有效降低HIV病毒水平，其中，对照组为CD19scFv-CD28-4-1BB-CD3ζ-CART。Figure 6: shows the effect of 10-1074-CD28-4-1BB-CD3ζ-CART (test group) cells and control cells on the concentration of P24, indicating that 10-1074-CD28-4-1BB-CD3ζ-CART cells can be specific Effectively reduce the level of HIV virus, among which, the control group is CD19scFv-CD28-4-1BB-CD3ζ-CART.
图7：显示了在不同效靶比情况下，来自3BNC117、N6和10-1074多种中和抗体的scFv做成的CART(试验组)与对照组细胞对P24浓度的影响，表明10-1074-CD28-4-1BB-CD3ζ-CART细胞可相对最有效的降低HIV病毒，其中，对照组为CD19scFv-CD28-4-1BB-CD3ζ-CART。Figure 7: shows the effect of CART (test group) and control cells on the concentration of P24 under different effector-target ratios from scFv of 3BNC117, N6 and 10-1074 multiple neutralizing antibodies, indicating that 10-1074 -CD28-4-1BB-CD3ζ-CART cells can reduce HIV virus relatively most effectively, and the control group is CD19scFv-CD28-4-1BB-CD3ζ-CART.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
除非另有说明，本发明的实施采用分子生物学、微生物学、细胞生物学、生物化学和免疫学的常规技术，其均在本领域技术人员知晓的范围内。Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the purview of those skilled in the art.
实施例1 10-1074-CD28-4-1BB-CD3ξ-CAR的构建Example 1 Construction of 10-1074-CD28-4-1BB-CD3ξ-CAR
制备多种10-1074scFv，并产生基于这些scFv的CAR，具体过程如下：A variety of 10-1074 scFvs were prepared and CARs based on these scFvs were generated as follows:
(一)制备多种10-1074scFv(1) Preparation of a variety of 10-1074scFv
1、基于10-1074的核苷酸序列(SEQ ID NO：17)，进行抗体重链(VH)及轻链(VL)序列合成，基于其序列设计Overlap PCR引物。1. Based on the nucleotide sequence of 10-1074 (SEQ ID NO: 17), carry out the synthesis of antibody heavy chain (VH) and light chain (VL) sequences, and design Overlap PCR primers based on their sequences.
2、利用DNA聚合酶进行PCR扩增，扩增10-1074的VL片段(其核苷酸序列为SEQ IDNO：19，氨基酸序列为SEQ ID NO：2)及VH片段(其核苷酸序列为SEQ ID NO：18，其氨基酸序列为SEQ ID NO：3)，完成后进行PCR产物回收及DNA琼脂糖凝胶电泳验证PCR条带大小是否正确。其中，扩增VH片段所用引物为，引物10-1074-VH-F序列：cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC(SEQ ID NO：20)；引物op-10-1074-VH-VL-R序列：CACTTCCGCCTCCACCAGATCCTCCACCTCCGCTAGACACTGTCACTGTTGT(SEQ ID NO：21)；扩增VL片段所用引物为，引物op-10-1074-VH-VL-F序列：TGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGG(SEQID NO：22)；引物10-1074-VL-R序列：tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA(SEQ IDNO：23)。2. Use DNA polymerase to carry out PCR amplification, amplify the VL fragment of 10-1074 (its nucleotide sequence is SEQ ID NO: 19, the amino acid sequence is SEQ ID NO: 2) and the VH fragment (its nucleotide sequence is SEQ ID NO: 18, the amino acid sequence of which is SEQ ID NO: 3), after the completion of the PCR product recovery and DNA agarose gel electrophoresis to verify whether the size of the PCR band is correct. The primers used to amplify the VH fragment are: primer 10-1074-VH-F sequence: cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC (SEQ ID NO: 20); primer op-10-1074-VH-VL-R sequence: CACTTCCGCCTCCACCAGATCCTCCACCTCCGCTAGACACTGTCACTGTTGT (SEQ ID NO: 21 ); the primers used to amplify the VL fragment are, primer op-10-1074-VH-VL-F sequence: TGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGG (SEQ ID NO: 22); primer 10-1074-VL-R sequence: tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA (SEQ ID NO: 23).
3、利用鉴定正确的10-1074VL及VH片段作为模板，采用Overlap PCR引物进行overlap PCR扩增，完成后进行DNA琼脂糖凝胶电泳验证，验证正确的条带进行切胶回收得到10-1074scFv；其中扩增所用引物10-1074-VH-F序列：cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC(SEQ ID NO：24)；引物10-1074-VL-R序列：tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA(SEQ ID NO：25)。4、胶回收的10-1074scFv以及带有Fc片段的载体(实验室保存)同时进行双酶切，酶切后进行DNA琼脂糖凝胶电泳验证，验证正确的条带进行切胶回收；3. Use the correctly identified 10-1074VL and VH fragments as templates, and use Overlap PCR primers to carry out overlap PCR amplification. After completion, carry out DNA agarose gel electrophoresis verification, and verify that the correct band is cut and recovered to obtain 10-1074scFv; The sequence of primer 10-1074-VH-F used for amplification: cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC (SEQ ID NO: 24); the sequence of primer 10-1074-VL-R: tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA (SEQ ID NO: 25). 4. The 10-1074scFv recovered from the gel and the carrier with the Fc fragment (preserved in the laboratory) were subjected to double-enzyme digestion at the same time. After the enzyme digestion, DNA agarose gel electrophoresis was performed to verify that the correct band was recovered by cutting the gel;
5、将得到的酶切后10-1074scFv目的片段及载体利用T4连接酶连接，转化至DH5α菌株在氨苄平板上涂板；5. Connect the obtained 10-1074scFv target fragment and vector with T4 ligase, transform it into DH5α strain and plate it on an ampicillin plate;
6、挑选平板上长出的3个单菌落送至公司利用CMV-F进行测序，测序正确的片段即为10-1074scFv克隆(其氨基酸序列为SEQ ID NO：1或SEQ ID NO：10)；6. Select 3 single colonies grown on the plate and send them to the company for sequencing using CMV-F. The correct fragment is the 10-1074 scFv clone (its amino acid sequence is SEQ ID NO: 1 or SEQ ID NO: 10);
7、验证制备的10-1074scFv结合HIV潜伏细胞的特异性和亲合力；7. Verify the specificity and affinity of the prepared 10-1074scFv to bind HIV latent cells;
8、表达纯化带有his标签的10-1074scFv、10-1074抗体及Mers抗体，并将其分别与激活的HIV潜伏细胞ACH2孵育，再通过anti-his PE抗体检测其是否与HIV潜伏细胞ACH2特异结合。实验结果显示(见图1)，较对照Mers抗体，10-1074scFv可特异性识别ACH2，虽然这种识别能力较10-1074抗体略弱。8. Express and purify 10-1074scFv, 10-1074 antibody and Mers antibody with his tag, incubate them with activated HIV latent cell ACH2, and then detect whether it is specific to HIV latent cell ACH2 by anti-his PE antibody combine. The experimental results showed (see Figure 1) that 10-1074scFv could specifically recognize ACH2 compared with the control Mers antibody, although the recognition ability was slightly weaker than that of the 10-1074 antibody.
其中，连接VH片段及VL片段的linker如下：Among them, the linker connecting the VH fragment and the VL fragment is as follows:
Linker序列-1(SEQ ID NO：4)：GGGGSGGGGSGGGGSLinker Sequence-1 (SEQ ID NO: 4): GGGGSGGGGSGGGGS
Linker序列-2(SEQ ID NO：5)：GGGGSGGGGSLinker Sequence-2 (SEQ ID NO: 5): GGGGSGGGGS
Linker序列-3(SEQ ID NO：6)：GGGGSGGGGSGGGGSGGGGSLinker Sequence-3 (SEQ ID NO: 6): GGGGSGGGGSGGGGSGGGGS
Linker序列-4(SEQ ID NO：7)：GGGGSGGGGSHMESKYGPPCPPCPLinker Sequence-4 (SEQ ID NO: 7): GGGGSGGGGSHMESKYGPPCPPCP
Linker序列-5(SEQ ID NO：8)：GGGGSGGGGSGGGGSHMESKYGPPCPPCPLinker Sequence-5 (SEQ ID NO: 8): GGGGSGGGGSGGGGSHMESKYGPPCPPCP
Linker序列-6(SEQ ID NO：9)：GGGGSGGGGSGGGGSGGGGSHMESKYGPPCPPCPLinker Sequence-6 (SEQ ID NO: 9): GGGGSGGGGSGGGGSGGGGSHMESKYGPPCPPCP
经过实验验证，上述的SEQ ID NO：4-9的Linker序列也是有效的，但是本实验所用linker为SEQ ID NO：4。After experimental verification, the above-mentioned Linker sequences of SEQ ID NO: 4-9 are also effective, but the linker used in this experiment is SEQ ID NO: 4.
(二)制备10-1074scFv-CAR(示例性使用编码SEQ ID NO：1的10-1074scFv的核苷酸序列SEQ ID NO：17)(2) Preparation of 10-1074 scFv-CAR (exemplarily using the nucleotide sequence SEQ ID NO: 17 encoding the 10-1074 scFv of SEQ ID NO: 1)
其制备过程及质粒结构图如图2所示。The preparation process and plasmid structure are shown in Figure 2.
1、基于CD8 IgG区、CD28跨膜区(TM)、CD28-4-1BB胞浆区和CD3ξ胞浆区的核苷酸序列设计Overlap PCR引物。1. Design Overlap PCR primers based on the nucleotide sequences of CD8 IgG region, CD28 transmembrane region (TM), CD28-4-1BB cytoplasmic region and CD3ξ cytoplasmic region.
2、利用DNA聚合酶进行PCR扩增，分别扩增CD8 IgG区、CD28跨膜区、CD28-4-1BB胞浆区和CD3ξ胞浆区片段，完成后进行PCR产物回收及DNA琼脂糖凝胶电泳验证PCR条带大小是否正确。2. Use DNA polymerase to carry out PCR amplification, respectively amplify CD8 IgG region, CD28 transmembrane region, CD28-4-1BB cytoplasmic region and CD3ξ cytoplasmic region fragments. After completion, carry out PCR product recovery and DNA agarose gel. Verify the PCR band size is correct by electrophoresis.
3、利用鉴定正确的CD8 IgG区、CD28跨膜区、CD28-4-1BB胞浆区和CD3ξ胞浆区片段作为模板，采用Overlap PCR引物进行overlap PCR扩增，完成后进行DNA琼脂糖凝胶电泳验证，验证正确的条带进行切胶回收得到CD8-CD8TM-CD28-4-1BB-CD3ξ片段；3. Using the correctly identified CD8 IgG region, CD28 transmembrane region, CD28-4-1BB cytoplasmic region and CD3ξ cytoplasmic region fragments as templates, overlap PCR amplification with Overlap PCR primers, and DNA agarose gel after completion Electrophoresis verification, verifying the correct band and cutting the gel to recover the CD8-CD8TM-CD28-4-1BB-CD3ξ fragment;
4、胶回收的步骤(一)中验证过的10-1074scFv片段和这里的CD8-CD8TM-CD28-4-1BB-CD3ξ片段以及慢病毒载体phage-IRES-RFP或phage-IRES-GFP(实验室保存，本实施例使用)同时进行双酶切，酶切后进行DNA琼脂糖凝胶电泳验证，验证正确的条带进行切胶回收；4. The 10-1074scFv fragment verified in the gel recovery step (1) and the CD8-CD8TM-CD28-4-1BB-CD3ξ fragment and the lentiviral vector phage-IRES-RFP or phage-IRES-GFP (laboratory) Preservation, used in this example) simultaneously double-enzymatic digestion, DNA agarose gel electrophoresis verification was performed after the enzymatic digestion, and the correct band was verified and recovered by cutting the gel;
5、将得到的酶切10-1074scFv片段和CD8-CD8TM-CD28-4-1BB-CD3ξ片段及载体利用T4连接酶连接，转化至stable 3菌株在氨苄平板上涂板；5. The obtained 10-1074scFv fragment and CD8-CD8TM-CD28-4-1BB-CD3ξ fragment and the vector were ligated with T4 ligase, transformed into stable 3 strain and plated on ampicillin plate;
6、挑选平板上长出的3个单菌落送至公司进行测序，测序正确的片段即为10-1074-CAR，其表达质粒命名为phage-10-1074scFv-CAR；6. Select 3 single colonies grown on the plate and send them to the company for sequencing. The correctly sequenced fragment is 10-1074-CAR, and its expression plasmid is named phage-10-1074scFv-CAR;
其中，in,
(1)CD8-CD28TM-CD28-4-1BB-CD3ξ序列如下所示(SEQ ID NO：26)：(1) The sequence of CD8-CD28TM-CD28-4-1BB-CD3ξ is shown below (SEQ ID NO: 26):
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(2)CD8LS-10-1074scFv-CD28-4-1BB-CD3ξCAR的氨基酸序列如下所示(SEQ IDNO：11)；(2) The amino acid sequence of CD8LS-10-1074scFv-CD28-4-1BB-CD3ξCAR is shown below (SEQ ID NO: 11);
MGWSCIILFLVATATGVHSASQVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSSGGGGSGGGGSGGGGSSYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLHMTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMGWSCIILFLVATATGVHSASQVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSSGGGGSGGGGSGGGGSSYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLHMTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(3)CD8LS-10-1074scFv-CD28-4-1BB-CD3ξCAR的核苷酸序列如下所示(SEQ IDNO：12)；(3) The nucleotide sequence of CD8LS-10-1074scFv-CD28-4-1BB-CD3ξCAR is shown below (SEQ ID NO: 12);
atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattccgctagcCAGGTGCAGCTGCAGGAGTCAGGACCAGGACTGGTGAAGCCTAGCGAGACACTGAGCGTGACTTGCAGCGTGTCCGGCGACAGCATGAACAACTACTATTGGACTTGGATCCGGCAGAGCCCAGGCAAAGGACTCGAGTGGATCGGCTACATCAGCGACAGGGAGAGCGCCACCTACAACCCTAGCCTGAACAGCAGGGTCGTGATCAGCAGGGACACCAGCAAGAACCAGCTGAGCCTGAAGCTGAACAGCGTGACCCCAGCCGATACCGCCGTGTACTATTGCGCCACAGCCAGAAGGGGCCAGAGAATCTACGGCGTGGTGTCCTTCGGCGAGTTCTTCTACTACTACAGCATGGACGTGTGGGGCAAGGGCACAACAGTGACAGTGTCTAGCGGAGGTGGAGGATCTGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGGCTCTGGGAGAGACAGCCAGGATCTCTTGCGGCAGACAGGCTCTGGGAAGCAGAGCAGTCCAGTGGTACCAGCACAGACCAGGACAGGCCCCTATCCTGCTGATCTACAACAACCAGGACCGGCCCAGCGGAATCCCAGAGAGATTCAGCGGCACCCCAGACATCAACTTCGGCACCAGAGCCACCCTGACAATTAGCGGCGTGGAAGCCGGAGACGAGGCCGACTACTATTGCCACATGTGGGATAGCAGGAGCGGCTTCTCTTGGAGCTTCGGAGGAGCCACAAGACTGACAGTGCTGcatatgaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggggcgggcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcTAAatgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattccgctagcCAGGTGCAGCTGCAGGAGTCAGGACCAGGACTGGTGAAGCCTAGCGAGACACTGAGCGTGACTTGCAGCGTGTCCGGCGACAGCATGAACAACTACTATTGGACTTGGATCCGGCAGAGCCCAGGCAAAGGACTCGAGTGGATCGGCTACATCAGCGACAGGGAGAGCGCCACCTACAACCCTAGCCTGAACAGCAGGGTCGTGATCAGCAGGGACACCAGCAAGAACCAGCTGAGCCTGAAGCTGAACAGCGTGACCCCAGCCGATACCGCCGTGTACTATTGCGCCACAGCCAGAAGGGGCCAGAGAATCTACGGCGTGGTGTCCTTCGGCGAGTTCTTCTACTACTACAGCATGGACGTGTGGGGCAAGGGCACAACAGTGACAGTGTCTAGCGGAGGTGGAGGATCTGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGGCTCTGGGAGAGACAGCCAGGATCTCTTGCGGCAGACAGGCTCTGGGAAGCAGAGCAGTCCAGTGGTACCAGCACAGACCAGGACAGGCCCCTATCCTGCTGATCTACAACAACCAGGACCGGCCCAGCGGAATCCCAGAGAGATTCAGCGGCACCCCAGACATCAACTTCGGCACCAGAGCCACCCTGACAATTAGCGGCGTGGAAGCCGGAGACGAGGCCGACTACTATTGCCACATGTGGGATAGCAGGAGCGGCTTCTCTTGGAGCTTCGGAGGAGCCACAAGACTGACAGTGCTGcatatgaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcc tggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggggcgggcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcTAA
(4)phage-10-1074scFv-CAR的核苷酸序列如SEQ ID NO：13所示。(4) The nucleotide sequence of phage-10-1074scFv-CAR is shown in SEQ ID NO:13.
实施例2、CAR-T细胞的制备(示例性采用如SEQ ID NO：13所述的phage-10-1074scFv-CAR)Example 2. Preparation of CAR-T cells (exemplarily using phage-10-1074 scFv-CAR as described in SEQ ID NO: 13)
(一)慢病毒包装、浓缩及滴度检测(1) Lentivirus packaging, concentration and titer detection
1.1复苏调整293T细胞状态，转染前24小时左右重新铺板，以包装转染时细胞融合度达7成以上为宜；1.1 Resuscitate and adjust the state of 293T cells, re-plate about 24 hours before transfection, and it is advisable that the cell confluency reaches 70% or more during packaging and transfection;
1.2转染病毒包装体系所需要的质粒psPAX2、pMD2.G和表达CAR的慢病毒质粒phage-10-1074scFv-CAR，转染前将培养基换成无血清培养基，对于15cm细胞培养皿按以下体系进行操作：1.2 Plasmids psPAX2, pMD2.G and CAR-expressing lentiviral plasmid phage-10-1074scFv-CAR required for transfection of virus packaging system, change the medium to serum-free medium before transfection, for 15cm cell culture dishes as follows The system operates:
1ml DMEM中滴加混入60μl PEI，静置5分钟，Add 60μl PEI dropwise to 1ml DMEM, and let stand for 5 minutes.
另取一tube管加入Take another tube to join
phage-10-1074scFv-CAR 15μgphage-10-1074scFv-CAR 15μg
psPAX2 10μgpsPAX2 10μg
pMD2.G 5μgpMD2.G 5μg
加DMEM至1mL，Add DMEM to 1mL,
将PEI混合液滴加混入包装质粒混合液，静置20分钟；Add the PEI mixture dropwise and mix it into the packaging plasmid mixture, and let it stand for 20 minutes;
1.3将上述混合液均匀滴入细胞培养基使其覆于细胞表面，放入培养箱；1.3 Evenly drop the above mixture into the cell culture medium to cover the cell surface, and put it into the incubator;
1.4 10小时后在病毒操作台中弃去培养基，添加约20mL新鲜培养基；1.4 After 10 hours, discard the medium in the virus operation table and add about 20mL of fresh medium;
1.5 2天后收取上层培养基，另再补加20mL新鲜培养基；另1-2天后再次收取上层培养基，合并两次收取的培养基上清；1.5 Collect the upper medium after 2 days, and add 20 mL of fresh medium; collect the upper medium again after another 1-2 days, and combine the medium supernatant collected twice;
1.6离心培养基上清，3000rpm 10分钟；用0.45μm滤膜过滤，取过滤后的培养基36mL置于超速离心管中，19500rpm 2小时；1.6 Centrifuge the culture medium supernatant at 3000rpm for 10 minutes; filter with a 0.45 μm filter, take 36mL of the filtered culture medium and place it in an ultracentrifuge tube, 19500rpm for 2 hours;
1.7小心取出离心管，将上清在病毒操作台中弃除，倒扣在喷过酒精的纸上；加入360μL1640培养基，至少吹打底块含病毒结块10次；按一定体积分装病毒置于-80℃冰箱快速冷冻保存；1.7 Carefully take out the centrifuge tube, discard the supernatant on the virus workbench, and place it upside down on the alcohol-sprayed paper; add 360 μL of 1640 medium, and pipette the bottom block containing virus agglomerates at least 10 times; -80℃ refrigerator for quick freezing storage;
(二)人原代T细胞分离、激活及感染(2) Isolation, activation and infection of human primary T cells
2.1新鲜或冻存的人血液经Ficoll液分离得到人PBMC，用正常1640培养基培养1天；2.1 Human PBMCs were separated from fresh or frozen human blood by Ficoll solution and cultured in normal 1640 medium for 1 day;
2.2第2天将上述培养液中悬浮细胞按Stem cell的EasySepTMHuman T CellIsolation Kit(Catalog#17951)说明进行阴筛得到人原代T细胞；2.2 On the second day, the suspended cells in the above-mentioned culture solution were screened negatively according to the instructions of Stem cell's EasySep™ Human T CellIsolation Kit (Catalog#17951) to obtain human primary T cells;
2.3用正常1640培养基培养上述分离得到的人原代T细胞，并按说明加入Stemcell的ImmunoCultTMHuman CD3/CD28/CD2 T Cell Activator(货号#10970)及IL2(PeproTech货号96-200-02-50)，持续激活2天；2.3 Culture the human primary T cells isolated above in normal 1640 medium, and add Stemcell's ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator (Item #10970) and IL2 (PeproTech Item No. 96-200-02- 50), continue to activate for 2 days;
2.4以MOI小于20的慢病毒量感染一定数目上述激活的人原代T细胞，并加入8μg/mL polybrene(Sigma货号H9268)混匀；室温1000-1500rpm离心1-2小时；放于培养箱培养10小时后离心换液；2.4 Infect a certain number of activated human primary T cells with a lentivirus with an MOI less than 20, and add 8 μg/mL polybrene (Sigma product number H9268) to mix well; centrifuge at 1000-1500 rpm for 1-2 hours at room temperature; place in an incubator for culture After 10 hours, centrifuge to change the medium;
2.5扩增细胞，3-4天后检测感染效率，由于将10-1074-scFv CAR构建入phage-IRES-GFP载体中因此可通过载体自带的RFP标签衡量感染效率。同时检测10-1074-scFvCAR在人原代T细胞中的基因表达定位情况(见实施例3)，筛选富集CAR阳性的T细胞用于后续实验。2.5 Expand the cells, and check the infection efficiency after 3-4 days. Since the 10-1074-scFv CAR is constructed into the phage-IRES-GFP vector, the infection efficiency can be measured by the RFP tag that comes with the vector. At the same time, the gene expression and localization of 10-1074-scFvCAR in human primary T cells was detected (see Example 3), and CAR-positive T cells were screened and enriched for subsequent experiments.
实施例3、HIV囊膜蛋白与10-1074-CD28-4-1BB-CD3ζ-CAR特异性识别结合验证实验Example 3. Validation experiment of specific recognition and binding of HIV envelope protein and 10-1074-CD28-4-1BB-CD3ζ-CAR
研究HIV囊膜蛋白是否特异性识别结合10-1074-CD28-4-1BB-CD3ζ-CAR，首先将10-1074-CD28-4-1BB-CD3ζ-CAR-GFP转染到293T细胞中，24小时后，先后用带his标签的HIV囊膜蛋白gp140及抗his PE标记的抗体孵育，通过流式观测HIV囊膜蛋白被CAR-GFP阳性细胞结合的情况。To study whether HIV envelope protein specifically recognizes and binds 10-1074-CD28-4-1BB-CD3ζ-CAR, 10-1074-CD28-4-1BB-CD3ζ-CAR-GFP was first transfected into 293T cells for 24 hours After that, they were incubated with his-tagged HIV envelope protein gp140 and anti-his PE-labeled antibody successively, and the binding of HIV envelope protein by CAR-GFP positive cells was observed by flow cytometry.
(一)细胞及试剂(1) Cells and reagents
1、实验用细胞：293T细胞，购买于ATCC，并培养于清华大学细胞实验平台。1. Experimental cells: 293T cells, purchased from ATCC, and cultured on the cell experimental platform of Tsinghua University.
2、实验用HIV囊膜蛋白：将来自中国HIV感染者的HIV囊膜蛋白(编号为CNE54)基因克隆到真核载体上，转入293f细胞培养生产，经AKTA蛋白纯化系统纯化获得。2. HIV envelope protein for experiment: The HIV envelope protein (numbered as CNE54) gene from Chinese HIV-infected patients was cloned into a eukaryotic vector, transferred to 293f cells for culture and production, and purified by AKTA protein purification system.
3、实验用抗his PE标记的抗体(货号#130-092-691)购买于美天旎公司。3. The anti-his PE-labeled antibody (catalog #130-092-691) used in the experiment was purchased from Miltenyi Company.
(二)实验方法(2) Experimental method
提前一天将293T细胞铺到六孔板的两个孔中，将对照组FMC63scFv-CD28-4-1BB-CD3ζ-CAR-GFP及试验组10-1074-CD28-4-1BB-CD3ζ-CAR-GFP转染到细胞中，24小后将293T用TEN消化，每孔细胞平均分成两份，一份细胞与HIV囊膜蛋白孵育，另一份细胞与HBV囊膜蛋白孵育计半小时，离心并用PBS洗三遍，所有细胞再分别与抗his PE标记的抗体孵育计半小时，离心并用PBS洗三遍，上流式分析仪检测。One day in advance, 293T cells were plated into two wells of a six-well plate, and the control group FMC63scFv-CD28-4-1BB-CD3ζ-CAR-GFP and the experimental group 10-1074-CD28-4-1BB-CD3ζ-CAR-GFP After transfection into cells, 293T was digested with TEN after 24 hours, and the cells in each well were equally divided into two parts, one part of cells was incubated with HIV envelope protein, and the other part of cells was incubated with HBV envelope protein for half an hour, centrifuged and washed with PBS. After washing three times, all cells were incubated with anti-his PE-labeled antibody for half an hour, centrifuged, washed three times with PBS, and detected by flow analyzer.
(三)实验结果(3) Experimental results
将10-1074-scFv CAR-IRES-GFP及对照FMC63-scFv CAR-IRES-GFP通过慢病毒载体导入人原代T细胞中，并将其与带his标签的HIV囊膜蛋白BG505孵育，检测10-1074-CD28-4-1BBζCAR定位于细胞膜并特异性的结合HIV膜蛋白gp140的能力。结果显示(如图3)，较对照FMC63-scFv CAR，10-1074-scFv CAR可特异性识别结合HIV囊膜蛋白BG505，相对于对照组FMC63 scFv-CD28-4-1BB-CD3ζ-CAR-GFP，HIV囊膜蛋白近百分百可特异性结合10-1074-CD28-4-1BB-CD3ζ-CAR-GFP阳性细胞表面；而HBV囊膜蛋白不能结合10-1074-CD28-4-1BB-CD3ζCAR-GFP阳性细胞。由此，HIV囊膜蛋白特异性识别结合10-1074-CD28-4-1BB-CD3ζ-CAR，并且10-1074-CD28-4-1BB-CD3ζ-CAR不识别HBV囊膜蛋白。10-1074-scFv CAR-IRES-GFP and control FMC63-scFv CAR-IRES-GFP were introduced into human primary T cells by lentiviral vector, and incubated with his-tagged HIV envelope protein BG505 to detect 10 The ability of -1074-CD28-4-1BBζCAR to localize to the cell membrane and specifically bind HIV membrane protein gp140. The results show (as shown in Figure 3), compared with the control FMC63-scFv CAR, the 10-1074-scFv CAR can specifically recognize and bind the HIV envelope protein BG505, compared with the control group FMC63 scFv-CD28-4-1BB-CD3ζ-CAR-GFP , HIV envelope protein can specifically bind to 10-1074-CD28-4-1BB-CD3ζ-CAR-GFP positive cell surface nearly 100%; while HBV envelope protein cannot bind 10-1074-CD28-4-1BB-CD3ζCAR -GFP positive cells. Thus, HIV envelope protein specifically recognizes and binds 10-1074-CD28-4-1BB-CD3ζ-CAR, and 10-1074-CD28-4-1BB-CD3ζ-CAR does not recognize HBV envelope protein.
实施例4、HIV潜伏细胞与10-1074-CD28-4-1BB-CD3ζ-CAR特异性识别激活验证实验Example 4. Validation experiment of specific recognition and activation of HIV latent cells and 10-1074-CD28-4-1BB-CD3ζ-CAR
研究10-1074-CD28-4-1BB-CD3ζ-CART细胞是否可有效的被HIV潜伏细胞特异性识别激活，首先从人PBMC中将T细胞分离出来，激活培养后将对照组CD19scFv-CD28-4-1BB-CD3ζ-CAR-RFP及试验组10-1074-CD28-4-1BB-CD3ζ-CAR-RFP转化到T细胞中，分选获得纯化的对照组CD19scFv-CD28-4-1BB-CD3ζCART细胞及试验组10-1074CD28-4-1BB-CD3ζ-CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2分别共培养，24小时后，检测INFγ、TNFα阳性CART细胞的比率及分泌到培养基中INFγ、TNFα和IL2的水平。To study whether 10-1074-CD28-4-1BB-CD3ζ-CART cells can be effectively recognized and activated by HIV latent cells, T cells were first isolated from human PBMC, and after activation and culture, the control group CD19scFv-CD28-4 -1BB-CD3ζ-CAR-RFP and experimental group 10-1074-CD28-4-1BB-CD3ζ-CAR-RFP were transformed into T cells, and the purified control group CD19scFv-CD28-4-1BB-CD3ζCART cells and 10-1074CD28-4-1BB-CD3ζ-CART cells in the experimental group; the above CART cells were co-cultured with HIV-negative cells A3.01 or HIV-positive cells ACH2, respectively. After 24 hours, the ratio and secretion of INFγ and TNFα-positive CART cells were detected. to the levels of INFγ, TNFα and IL2 in the medium.
1、细胞及试剂1. Cells and reagents
(1)实验用细胞：A3.01和ACH2细胞，购买于NIH AIDS试剂平台，并培养于清华大学细胞实验平台；PBMC来自合作医院。(1) Experimental cells: A3.01 and ACH2 cells, purchased from the NIH AIDS reagent platform, and cultured on the Tsinghua University Cell Experiment Platform; PBMC from the cooperative hospital.
(2)实验用试剂：人原代T细胞分离试剂盒(货号#17951)、激活试剂盒(货号#10970)均购买于Stem cell公司，细胞因子IL2(货号96-200-02-50)购买于Pepro Tech公司；流式抗体TNFα(货号17-7349)、INFγ(货号48-7319)及ELISA试剂盒TNFα(货号88-7346-88)、INFγ(货号88-7316-88)和IL2(货号88-7025-88)均购买于eBioscience公司。(2) Experimental reagents: Human Primary T Cell Isolation Kit (Cat. No. 17951) and Activation Kit (Cat. No. 10970) were purchased from Stem Cell Company, and cytokine IL2 (Cat. No. 96-200-02-50) was purchased From Pepro Tech; Flow Antibodies TNFα (Cat. No. 17-7349), INFγ (Cat. No. 48-7319) and ELISA Kits TNFα (Cat. No. 88-7346-88), INFγ (Cat. No. 88-7316-88) and IL2 (Cat. No. 88-7316-88) 88-7025-88) were purchased from eBioscience.
2、实验方法2. Experimental method
将人PBMC细胞铺到10cm培养皿中，第2天将上述培养液中悬浮细胞按Stem cell的T细胞分离试剂盒说明进行阴筛得到人原代T细胞，并按说明加入T细胞激活剂及IL2；用一定量包装好的慢病毒感染上述激活的人原代T细胞，并经流式分选获得CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2按1:2、1:5比例分别共培养，24小时后，检测INFγ、TNFα阳性CART细胞的比率及分泌到培养基中INFγ、TNFα和IL2的水平。The human PBMC cells were plated into a 10cm culture dish, and on the second day, the suspended cells in the above-mentioned culture medium were screened negatively according to the instructions of the Stem cell T cell isolation kit to obtain human primary T cells, and T cell activator and T cell activator were added according to the instructions. IL2; infect the above activated human primary T cells with a certain amount of packaged lentivirus, and obtain CART cells by flow sorting; the above CART cells are mixed with HIV-negative cells A3.01 or HIV-positive cells ACH2 at a ratio of 1:2 The ratio of INFγ and TNFα positive CART cells and the levels of INFγ, TNFα and IL2 secreted into the medium were detected after 24 hours.
3、实验结果3. Experimental results
10-1074-CD28-4-1BB-CD3ζ-CART细胞可有效的被HIV潜伏细胞特异性识别激活如图4A、4B、4C所示，对照组CD19scFv-CD28-4-1BB-CD3ζ-CART既不能被HIV阴性细胞A3.01也不能被HIV阳性细胞ACH2激活而产生INFγ、TNFα等细胞因子；而试验验组10-1074-CD28-4-1BB-CD3ζ-CART只能被HIV阳性细胞ACH2而不能被HIV阴性细胞A3.01激活而产生INFγ、TNFα等细胞因子。10-1074-CD28-4-1BB-CD3ζ-CART cells can be effectively recognized and activated by HIV latent cells. As shown in Figure 4A, 4B, and 4C, the control group CD19scFv-CD28-4-1BB-CD3ζ-CART can neither. HIV-negative cells A3.01 cannot be activated by HIV-positive cells ACH2 to produce INFγ, TNFα and other cytokines; while the experimental group 10-1074-CD28-4-1BB-CD3ζ-CART can only be activated by HIV-positive cells ACH2 but not Activated by HIV-negative cells A3.01 to produce cytokines such as INFγ and TNFα.
实施例5、10-1074-CD28-4-1BB-CD3ζ-CART细胞有效杀伤HIV潜伏细胞验证实验Example 5. Validation experiment that 10-1074-CD28-4-1BB-CD3ζ-CART cells effectively kill HIV latent cells
研究10-1074-CD28-4-1BB-CD3ζ-CART细胞是否可有效的杀伤HIV潜伏细胞，首先从人PBMC中将T细胞分离出来，激活培养后将CD19scFv-CD28-4-1BB-CD3ζ-CAR-RFP及10-1074-CD28-4-1BB-CD3ζ-CAR-RFP转化到T细胞中，分选获得纯化的对照组CD19scFv-CD28-4-1BB-CD3ζ-CART细胞及试验组10-1074-CD28-4-1BB-CD3ζ-CART细胞；将上述CART细胞与HIV阳性细胞ACH2(GFP标记)共培养，24小时后，检测HIV阳性细胞ACH2在整个细胞群中所占比例的变化。To study whether 10-1074-CD28-4-1BB-CD3ζ-CART cells can effectively kill HIV latent cells, T cells were first isolated from human PBMCs, and then CD19scFv-CD28-4-1BB-CD3ζ-CAR was activated and cultured. -RFP and 10-1074-CD28-4-1BB-CD3ζ-CAR-RFP were transformed into T cells, and purified CD19scFv-CD28-4-1BB-CD3ζ-CART cells in the control group and 10-1074- CD28-4-1BB-CD3ζ-CART cells; the above CART cells were co-cultured with HIV-positive cells ACH2 (GFP-labeled), and after 24 hours, the changes in the proportion of HIV-positive cells ACH2 in the entire cell population were detected.
1、细胞及试剂1. Cells and reagents
(1)实验用细胞：ACH2细胞，购买于NIH AIDS试剂平台，并培养于清华大学细胞实验平台；PBMC来自合作医院。(1) Experimental cells: ACH2 cells, purchased from the NIH AIDS reagent platform, and cultured on the Tsinghua University Cell Experiment Platform; PBMC from a cooperative hospital.
(2)实验用试剂：人原代T细胞分离试剂盒(货号#17951)、激活试剂盒(货号#10970)均购买于Stem cell公司，细胞因子IL2(货号96-200-02-50)购买于Pepro Tech公司；流式抗体CD4购买于eBioscience公司。(2) Experimental reagents: Human Primary T Cell Isolation Kit (Cat. No. 17951) and Activation Kit (Cat. No. 10970) were purchased from Stem Cell Company, and cytokine IL2 (Cat. No. 96-200-02-50) was purchased from Pepro Tech Company; flow antibody CD4 was purchased from eBioscience Company.
2、实验方法2. Experimental method
将人PBMC细胞铺到10cm培养皿中，第2天将上述培养液中悬浮细胞按Stem cell的T细胞分离试剂盒说明进行阴筛得到人原代T细胞，并按说明加入T细胞激活剂及IL2；用一定量包装好的慢病毒感染上述激活的人原代T细胞，并经流式分选获得CART细胞；将上述CART细胞HIV阳性细胞ACH2按2:1比例共培养，24小时后，检测HIV阳性细胞ACH2在整个细胞群中所占比例的变化。The human PBMC cells were plated into a 10cm culture dish, and on the second day, the suspended cells in the above-mentioned culture medium were screened negatively according to the instructions of the Stem cell T cell isolation kit to obtain human primary T cells, and T cell activator and T cell activator were added according to the instructions. IL2; infect the above activated human primary T cells with a certain amount of packaged lentivirus, and obtain CART cells by flow sorting; co-culture the above CART cells HIV-positive cells ACH2 at a ratio of 2:1, 24 hours later, Changes in the proportion of HIV-positive cells ACH2 in the entire cell population were examined.
3、实验结果3. Experimental results
10-1074-CD28-4-1BB-CD3ζ-CART细胞可有效的杀伤HIV潜伏细胞如图5所示，相较对照组CD19scFv-CD28-4-1BB-CD3ζ-CART，试验组10-1074-CD28-4-1BB-CD3ζ-CART共培养的HIV阳性细胞ACH2比例明显下降，显示10-1074-CD28-4-1BB-CD3ζ-CART细胞可有效的特异地杀伤HIV潜伏细胞。10-1074-CD28-4-1BB-CD3ζ-CART cells can effectively kill HIV latent cells as shown in Figure 5. Compared with the control group CD19scFv-CD28-4-1BB-CD3ζ-CART, the experimental group 10-1074-CD28 The proportion of ACH2 in HIV-positive cells co-cultured with -4-1BB-CD3ζ-CART decreased significantly, indicating that 10-1074-CD28-4-1BB-CD3ζ-CART cells can effectively and specifically kill HIV latent cells.
实施例6、10-1074-CD28-4-1BB-CD3ζ-CART细胞有效降低HIV病毒验证实验Example 6. Validation experiment that 10-1074-CD28-4-1BB-CD3ζ-CART cells effectively reduce HIV virus
研究10-1074-CD28-4-1BB-CD3ζ-CART细胞是否可有效降低HIV病毒，首先从人PBMC中将T细胞分离出来，激活培养后将CD19scFv-CD28-4-1BB-CD3ζ-CAR-RFP及10-1074-CD28-4-1BB-CD3ζ-CAR-RFP转化到T细胞中，分选获得纯化的对照组CD19scFv-CD28-4-1BB-CD3ζ-CART细胞及试验组10-1074-CD28-4-1BB-CD3ζ-CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2(A3.01是CD4阳性，而ACH2是CD4阴性)分别共培养，24小时后，检测培养基上清中HIV病毒量(以p24为指标)的变化。To study whether 10-1074-CD28-4-1BB-CD3ζ-CART cells can effectively reduce HIV virus, T cells were first isolated from human PBMC, and after activation and culture, CD19scFv-CD28-4-1BB-CD3ζ-CAR-RFP and 10-1074-CD28-4-1BB-CD3ζ-CAR-RFP were transformed into T cells, and the purified control group CD19scFv-CD28-4-1BB-CD3ζ-CART cells and experimental group 10-1074-CD28- 4-1BB-CD3ζ-CART cells; the above CART cells were co-cultured with HIV-negative cells A3.01 or HIV-positive cells ACH2 (A3.01 was CD4 positive, while ACH2 was CD4 negative), and 24 hours later, the medium was detected Changes in HIV viral load (indicated by p24) in the supernatant.
1、细胞及试剂1. Cells and reagents
(1)实验用细胞：A3.01和ACH2细胞，购买于NIH AIDS试剂平台，并培养于清华大学细胞实验平台；PBMC来自合作医院。(1) Experimental cells: A3.01 and ACH2 cells, purchased from the NIH AIDS reagent platform, and cultured on the Tsinghua University Cell Experiment Platform; PBMC from the cooperative hospital.
(2)实验用试剂：人原代T细胞分离试剂盒(货号#17951)、激活试剂盒(货号#10970)均购买于Stem cell公司，细胞因子IL2(货号96-200-02-50)购买于Pepro Tech公司；流式抗体CD4购买于eBioscience公司。(2) Experimental reagents: Human Primary T Cell Isolation Kit (Cat. No. 17951) and Activation Kit (Cat. No. 10970) were purchased from Stem Cell Company, and cytokine IL2 (Cat. No. 96-200-02-50) was purchased from Pepro Tech Company; flow antibody CD4 was purchased from eBioscience Company.
2、实验方法2. Experimental method
将人PBMC细胞铺到10cm培养皿中，第2天将上述培养液中悬浮细胞按Stem cell的T细胞分离试剂盒说明进行阴筛得到人原代T细胞，并按说明加入T细胞激活剂及IL2；用一定量包装好的慢病毒感染上述激活的人原代T细胞，并经流式分选获得CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2按2:1比例分别共培养，24小时后，检测培养基上清中HIV病毒量(以p24为指标)的变化。The human PBMC cells were plated into a 10cm culture dish, and on the second day, the suspended cells in the above-mentioned culture medium were screened negatively according to the instructions of the Stem cell T cell isolation kit to obtain human primary T cells, and T cell activator and T cell activator were added according to the instructions. IL2; infect the above activated human primary T cells with a certain amount of packaged lentivirus, and obtain CART cells by flow sorting; the above CART cells and HIV-negative cells A3.01 or HIV-positive cells ACH2 are 2:1 The ratios were co-cultured, and after 24 hours, the changes in HIV virus content (with p24 as an indicator) in the supernatant of the medium were detected.
3、实验结果3. Experimental results
10-1074-CD28-4-1BB-CD3ζ-CART细胞可有效的有效降低HIV病毒如图6所示，存在HIV阳性细胞ACH2的情况下，相较对照组，试验组10-1074-CD28-4-1BB-CD3ζ-CART共培养能显著下调上清中的p24水平。10-1074-CD28-4-1BB-CD3ζ-CART cells can effectively reduce HIV virus. As shown in Figure 6, in the presence of HIV-positive cells ACH2, compared with the control group, the experimental group 10-1074-CD28-4 -1BB-CD3ζ-CART co-culture can significantly down-regulate the p24 level in the supernatant.
实施例7、10-1074-CD28-4-1BB-CD3ζ-CART细胞相对最有效降低HIV病毒对比实验Example 7. Comparative experiment of 10-1074-CD28-4-1BB-CD3ζ-CART cells being the most effective in reducing HIV virus
研究多种中和抗体来源的scFv做成的HIV CAR-T细胞是否可有效降低HIV病毒，首先从人PBMC中将T细胞分离出来，激活培养后将CD19scFv-CD28-4-1BB-CD3ζ-CAR-RFP及各种HIV-CD28-4-1BB-CD3ζ-CAR-RFP转化到T细胞中，分选获得纯化的对照组CD19scFv-CD28-4-1BB-CD3ζ-CART细胞及试验组HIV-CD28-4-1BB-CD3ζ-CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2(A3.01是CD4阳性，而ACH2是CD4阴性)以不同效靶比分别共培养，24小时后，检测培养基上清中HIV病毒量(以p24为指标)的变化。To study whether HIV CAR-T cells made of scFv derived from a variety of neutralizing antibodies can effectively reduce HIV virus, T cells were first isolated from human PBMC, activated and cultured, CD19scFv-CD28-4-1BB-CD3ζ-CAR -RFP and various HIV-CD28-4-1BB-CD3ζ-CAR-RFPs were transformed into T cells, and the purified control group CD19scFv-CD28-4-1BB-CD3ζ-CART cells and experimental group HIV-CD28- 4-1BB-CD3ζ-CART cells; the above CART cells were co-cultured with HIV-negative cells A3.01 or HIV-positive cells ACH2 (A3.01 is CD4 positive, while ACH2 is CD4 negative) at different effect-target ratios, respectively, for 24 hours Afterwards, the changes in HIV viral load (with p24 as an indicator) in the supernatant of the culture medium were detected.
1、细胞及试剂1. Cells and reagents
(1)实验用细胞：A3.01和ACH2细胞，购买于NIH AIDS试剂平台，并培养于清华大学细胞实验平台；PBMC来自合作医院。(1) Experimental cells: A3.01 and ACH2 cells, purchased from the NIH AIDS reagent platform, and cultured on the Tsinghua University Cell Experiment Platform; PBMC from the cooperative hospital.
(2)实验用试剂：人原代T细胞分离试剂盒(货号#17951)、激活试剂盒(货号#10970)均购买于Stem cell公司，细胞因子IL2(货号96-200-02-50)购买于Pepro Tech公司；流式抗体CD4购买于eBioscience公司。(2) Experimental reagents: Human Primary T Cell Isolation Kit (Cat. No. 17951) and Activation Kit (Cat. No. 10970) were purchased from Stem Cell Company, and cytokine IL2 (Cat. No. 96-200-02-50) was purchased from Pepro Tech Company; flow antibody CD4 was purchased from eBioscience Company.
2、实验方法2. Experimental method
将人PBMC细胞铺到10cm培养皿中，第2天将上述培养液中悬浮细胞按Stem cell的T细胞分离试剂盒说明进行阴筛得到人原代T细胞，并按说明加入T细胞激活剂及IL2；用一定量包装好的慢病毒感染上述激活的人原代T细胞，并经流式分选获得CART细胞；将上述CART细胞与HIV阴性细胞A3.01或HIV阳性细胞ACH2按不同比例分别共培养，24小时后，检测培养基上清中HIV病毒量(以p24为指标)的变化。The human PBMC cells were plated into a 10cm culture dish, and on the second day, the suspended cells in the above-mentioned culture medium were screened negatively according to the instructions of the Stem cell T cell isolation kit to obtain human primary T cells, and T cell activator and T cell activator were added according to the instructions. IL2; infect the above activated human primary T cells with a certain amount of packaged lentivirus, and obtain CART cells by flow sorting; separate the above CART cells with HIV-negative cells A3.01 or HIV-positive cells ACH2 in different proportions After co-cultivation, 24 hours later, the change of HIV virus amount (with p24 as an indicator) in the supernatant of the medium was detected.
3、实验结果3. Experimental results
如图7所示，在不同效靶比情况下，来自3BNC117、N6和10-1074多种中和抗体的scFv做成的HIV CAR-T细胞较对照组，均可有效地下调p24水平，并且这一下调趋随着效靶比的增加而增加。其中，在所有效靶比条件下，10-1074-CD28-4-1BB-CD3ζ-CART细胞显现出比其它中和抗体的scFv做成的HIV CAR-T细胞更强的下调p24水平的能力。As shown in Figure 7, HIV CAR-T cells made of scFv from 3BNC117, N6 and 10-1074 neutralizing antibodies can effectively down-regulate p24 levels compared with the control group under different effector-target ratios, and This downregulation tends to increase with increasing effector-target ratio. Among them, under all effective target ratio conditions, 10-1074-CD28-4-1BB-CD3ζ-CART cells showed stronger ability to down-regulate p24 levels than HIV CAR-T cells made of other neutralizing antibody scFvs.
综上所述，10-1074-CD28-4-1BB-CD3ζ-CAR可特异性识别HIV囊膜蛋白，10-1074-CD28-4-1BB-CD3ζ-CART细胞可相对最有效的被HIV潜伏细胞特异性识别激活并杀伤HIV潜伏细胞且有效降低病毒水平。In summary, 10-1074-CD28-4-1BB-CD3ζ-CAR can specifically recognize HIV envelope protein, and 10-1074-CD28-4-1BB-CD3ζ-CART cells can be relatively most effective by HIV latent cells. Specific recognition activates and kills HIV latent cells and effectively reduces viral levels.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present invention provides The combination method will not be specified otherwise.
序列表sequence listing
<110> 清华大学<110> Tsinghua University
北京华夏清医治疗科技有限公司Beijing Huaxia Qingyi Therapeutic Technology Co., Ltd.
<120> 一种包含嵌合抗原受体修饰的T细胞及其应用<120> A T cell containing chimeric antigen receptor modification and application thereof
<130> 1<130> 1
<160> 26<160> 26
<170> PatentIn version 3.5<170> PatentIn version 3.5
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Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp IleTyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
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Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu AsnGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
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Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser LeuSer Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
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Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly GluThr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
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Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr ValPhe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
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Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyThr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140 130 135 140
Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu ThrGly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr
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Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val GlnAla Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln
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Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr AsnTrp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn
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Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr ProAsn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro
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Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val GluAsp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu
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Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr GlnSer Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln
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Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp Ile AsnArg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp Ile Asn
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Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly AspPhe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly Asp
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Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp IleTyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
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Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu AsnGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
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Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser LeuSer Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
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Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys AlaLys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala
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Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly GluThr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
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Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr ValPhe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
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Thr Val Ser SerThr Val Ser Ser
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly SerGly Gly Gly Ser
20 20
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Met Glu Ser Lys TyrGly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Met Glu Ser Lys Tyr
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Gly Pro Pro Cys Pro Pro Cys ProGly Pro Pro Cys Pro Pro Cys Pro
20 20
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser HisGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser His Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro ProGly Gly Gly Ser His Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GluGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 151 5 10 15
Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn TyrThr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn Tyr
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Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp IleTyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
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Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu AsnGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
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Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser LeuSer Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
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Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys AlaLys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala
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Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly GluThr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
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Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr ValPhe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
115 120 125 115 120 125
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyThr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
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Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu ThrGly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr
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Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val GlnAla Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln
165 170 175 165 170 175
Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr AsnTrp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn
180 185 190 180 185 190
Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr ProAsn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro
195 200 205 195 200 205
Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val GluAsp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu
210 215 220 210 215 220
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225 230 235 240225 230 235 240
Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val LeuGly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu
245 250 255 245 250 255
<210> 11<210> 11
<211> 545<211> 545
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr GlyMet Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 151 5 10 15
Val His Ser Ala Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly LeuVal His Ser Ala Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30 20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly AspVal Lys Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp
35 40 45 35 40 45
Ser Met Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly LysSer Met Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys
50 55 60 50 55 60
Gly Leu Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr TyrGly Leu Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr
65 70 75 8065 70 75 80
Asn Pro Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser LysAsn Pro Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys
85 90 95 85 90 95
Asn Gln Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr AlaAsn Gln Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala
100 105 110 100 105 110
Val Tyr Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly ValVal Tyr Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val
115 120 125 115 120 125
Val Ser Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp GlyVal Ser Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly
130 135 140 130 135 140
Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly GlyLys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
145 150 155 160145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser ValGly Gly Ser Gly Gly Gly Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val
165 170 175 165 170 175
Ala Leu Gly Glu Thr Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu GlyAla Leu Gly Glu Thr Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly
180 185 190 180 185 190
Ser Arg Ala Val Gln Trp Tyr Gln His Arg Pro Gly Gln Ala Pro IleSer Arg Ala Val Gln Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile
195 200 205 195 200 205
Leu Leu Ile Tyr Asn Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu ArgLeu Leu Ile Tyr Asn Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg
210 215 220 210 215 220
Phe Ser Gly Thr Pro Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu ThrPhe Ser Gly Thr Pro Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr
225 230 235 240225 230 235 240
Ile Ser Gly Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His MetIle Ser Gly Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met
245 250 255 245 250 255
Trp Asp Ser Arg Ser Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr ArgTrp Asp Ser Arg Ser Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg
260 265 270 260 265 270
Leu Thr Val Leu His Met Thr Thr Thr Pro Ala Pro Arg Pro Pro ThrLeu Thr Val Leu His Met Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
275 280 285 275 280 285
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu AlaPro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
290 295 300 290 295 300
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp PheCys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
305 310 315 320305 310 315 320
Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala CysAla Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
325 330 335 325 330 335
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg SerTyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
340 345 350 340 345 350
Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro ArgLys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg
355 360 365 355 360 365
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro ArgArg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
370 375 380 370 375 380
Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu TyrAsp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr
385 390 395 400385 390 395 400
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu GluIle Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
405 410 415 405 410 415
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys GluAsp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Glu Gly Gly Cys Glu
420 425 430 420 425 430
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln GlnLeu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
435 440 445 435 440 445
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu GluGly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
450 455 460 450 455 460
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly GlyTyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
465 470 475 480465 470 475 480
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu GlnLys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
485 490 495 485 490 495
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly GluLys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
500 505 510 500 505 510
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrArg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
515 520 525 515 520 525
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro ProAla Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
530 535 540 530 535 540
ArgArg
545545
<210> 12<210> 12
<211> 1638<211> 1638
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgct 60atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgct 60
agccaggtgc agctgcagga gtcaggacca ggactggtga agcctagcga gacactgagc 120agccaggtgc agctgcagga gtcaggacca ggactggtga agcctagcga gacactgagc 120
gtgacttgca gcgtgtccgg cgacagcatg aacaactact attggacttg gatccggcag 180gtgacttgca gcgtgtccgg cgacagcatg aacaactact attggacttg gatccggcag 180
agcccaggca aaggactcga gtggatcggc tacatcagcg acagggagag cgccacctac 240agcccaggca aaggactcga gtggatcggc tacatcagcg acagggagag cgccacctac 240
aaccctagcc tgaacagcag ggtcgtgatc agcagggaca ccagcaagaa ccagctgagc 300aaccctagcc tgaacagcag ggtcgtgatc agcagggaca ccagcaagaa ccagctgagc 300
ctgaagctga acagcgtgac cccagccgat accgccgtgt actattgcgc cacagccaga 360ctgaagctga acagcgtgac cccagccgat accgccgtgt actattgcgc cacagccaga 360
aggggccaga gaatctacgg cgtggtgtcc ttcggcgagt tcttctacta ctacagcatg 420aggggccaga gaatctacgg cgtggtgtcc ttcggcgagt tcttctacta ctacagcatg 420
gacgtgtggg gcaagggcac aacagtgaca gtgtctagcg gaggtggagg atctggtgga 480gacgtgtggg gcaagggcac aacagtgaca gtgtctagcg gaggtggagg atctggtgga 480
ggcggaagtg gcggaggggg atctagctat gtgcgacctc tgagcgtggc tctgggagag 540ggcggaagtg gcggaggggg atctagctat gtgcgacctc tgagcgtggc tctgggagag 540
acagccagga tctcttgcgg cagacaggct ctgggaagca gagcagtcca gtggtaccag 600acagccagga tctcttgcgg cagacaggct ctgggaagca gagcagtcca gtggtaccag 600
cacagaccag gacaggcccc tatcctgctg atctacaaca accaggaccg gcccagcgga 660cacagaccag gacaggcccc tatcctgctg atctacaaca accaggaccg gcccagcgga 660
atcccagaga gattcagcgg caccccagac atcaacttcg gcaccagagc caccctgaca 720atcccagaga gattcagcgg caccccagac atcaacttcg gcaccagagc caccctgaca 720
attagcggcg tggaagccgg agacgaggcc gactactatt gccacatgtg ggatagcagg 780attagcggcg tggaagccgg agacgaggcc gactactatt gccacatgtg ggatagcagg 780
agcggcttct cttggagctt cggaggagcc acaagactga cagtgctgca tatgaccacg 840agcggcttct cttggagctt cggaggagcc acaagactga cagtgctgca tatgaccacg 840
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 900acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 900
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 960cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 960
gcctgtgatt tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 1020gcctgtgatt tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 1020
gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcagggg cgggcacagt 1080gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcagggg cgggcacagt 1080
gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1140gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1140
gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa actcctgtat 1200gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa actcctgtat 1200
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1260atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1260
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1320tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1320
gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1380gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1380
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1440cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1440
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1500aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1500
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1560gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1560
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1620ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1620
gccctgcccc ctcgctaa 1638gccctgcccc ctcgctaa 1638
<210> 13<210> 13
<211> 8687<211> 8687
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 13<400> 13
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 2040cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 2040
gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2100gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2100
agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2160agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2160
aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt 2220aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt 2220
gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 2280gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 2280
ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 2340ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 2340
gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2400gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2400
cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 2460cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 2460
ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 2520ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 2520
ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 2580ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 2580
gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 2640gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 2640
gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 2700gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 2700
gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 2760gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 2760
ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 2820ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 2820
ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 2880ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 2880
ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 2940ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 2940
gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 3000gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 3000
gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 3060gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 3060
tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 3120tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 3120
tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 3180tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 3180
ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 3240ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 3240
gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg 3300gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagt ttggcacttg 3300
atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 3360atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 3360
cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagcggc cgcggatccg 3420cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagcggc cgcggatccg 3420
ccaccatggg atggtcatgt atcatccttt ttctagtagc aactgcaacc ggtgtacatt 3480ccaccatggg atggtcatgt atcatccttt ttctagtagc aactgcaacc ggtgtacatt 3480
ccgctagcca ggtgcagctg caggagtcag gaccaggact ggtgaagcct agcgagacac 3540ccgctagcca ggtgcagctg caggagtcag gaccaggact ggtgaagcct agcgagacac 3540
tgagcgtgac ttgcagcgtg tccggcgaca gcatgaacaa ctactattgg acttggatcc 3600tgagcgtgac ttgcagcgtg tccggcgaca gcatgaacaa ctactattgg acttggatcc 3600
ggcagagccc aggcaaagga ctcgagtgga tcggctacat cagcgacagg gagagcgcca 3660ggcagagccc aggcaaagga ctcgagtgga tcggctacat cagcgacagg gagagcgcca 3660
cctacaaccc tagcctgaac agcagggtcg tgatcagcag ggacaccagc aagaaccagc 3720cctacaaccc tagcctgaac agcagggtcg tgatcagcag ggacaccagc aagaaccagc 3720
tgagcctgaa gctgaacagc gtgaccccag ccgataccgc cgtgtactat tgcgccacag 3780tgagcctgaa gctgaacagc gtgaccccag ccgataccgc cgtgtactat tgcgccacag 3780
ccagaagggg ccagagaatc tacggcgtgg tgtccttcgg cgagttcttc tactactaca 3840ccagaagggg ccagagaatc tacggcgtgg tgtccttcgg cgagttcttc tactactaca 3840
gcatggacgt gtggggcaag ggcacaacag tgacagtgtc tagcggaggt ggaggatctg 3900gcatggacgt gtggggcaag ggcacaacag tgacagtgtc tagcggaggt ggaggatctg 3900
gtggaggcgg aagtggcgga gggggatcta gctatgtgcg acctctgagc gtggctctgg 3960gtggaggcgg aagtggcgga gggggatcta gctatgtgcg acctctgagc gtggctctgg 3960
gagagacagc caggatctct tgcggcagac aggctctggg aagcagagca gtccagtggt 4020gagagacagc caggatctct tgcggcagac aggctctggg aagcagagca gtccagtggt 4020
accagcacag accaggacag gcccctatcc tgctgatcta caacaaccag gaccggccca 4080accagcacag accaggacag gcccctatcc tgctgatcta caacaaccag gaccggccca 4080
gcggaatccc agagagattc agcggcaccc cagacatcaa cttcggcacc agagccaccc 4140gcggaatccc agagagattc agcggcaccc cagacatcaa cttcggcacc agagccaccc 4140
tgacaattag cggcgtggaa gccggagacg aggccgacta ctattgccac atgtgggata 4200tgacaattag cggcgtggaa gccggagacg aggccgacta ctattgccac atgtgggata 4200
gcaggagcgg cttctcttgg agcttcggag gagccacaag actgacagtg ctgcatatga 4260gcaggagcgg cttctcttgg agcttcggag gagccacaag actgacagtg ctgcatatga 4260
ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt 4320ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt 4320
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg 4380ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg 4380
acttcgcctg tgatttttgg gtgctggtgg tggttggtgg agtcctggct tgctatagct 4440acttcgcctg tgatttttgg gtgctggtgg tggttggtgg agtcctggct tgctatagct 4440
tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc aggggcgggc 4500tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc aggggcgggc 4500
acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag cattaccagc 4560acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag cattaccagc 4560
cctatgcccc accacgcgac ttcgcagcct atcgctccaa acggggcaga aagaaactcc 4620cctatgcccc accacgcgac ttcgcagcct atcgctccaa acggggcaga aagaaactcc 4620
tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag gaagatggct 4680tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag gaagatggct 4680
gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg aagttcagca 4740gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg aagttcagca 4740
ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 4800ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 4800
taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 4860taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 4860
ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 4920ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 4920
agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 4980agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 4980
acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 5040acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 5040
tgcaggccct gccccctcgc taaatcgata gatcctaatc aacctctgga ttacaaaatt 5100tgcaggccct gccccctcgc taaatcgata gatcctaatc aacctctgga ttacaaaatt 5100
tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 5160tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 5160
gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 5220gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 5220
tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 5280tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 5280
gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 5340gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 5340
cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 5400cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 5400
gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5460gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5460
ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 5520ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 5520
cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 5580cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 5580
ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 5640ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 5640
atctcccttt gggccgcctc cccgcctgag atcctttaag accaatgact tacaaggcag 5700atctcccttt gggccgcctc cccgcctgag atcctttaag accaatgact tacaaggcag 5700
ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 5760ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 5760
aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 5820aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 5820
cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 5880cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 5880
gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 5940gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 5940
gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 6000gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 6000
agtatttata acttgcaaag aaatgaatat cagagagtga gaggcccggg ttaattaagg 6060agtatttata acttgcaaag aaatgaatat cagagagtga gaggccccggg ttaattaagg 6060
aaagggctag atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt 6120aaagggctag atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt 6120
aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 6180aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 6180
ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 6240ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 6240
taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 6300taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 6300
cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 6360cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 6360
acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 6420acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 6420
ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 6480ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 6480
atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa 6540atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa 6540
gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 6600gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 6600
acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 6660acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 6660
atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 6720atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 6720
accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 6780accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 6780
ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 6840ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 6840
acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 6900acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 6900
gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 6960gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 6960
tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 7020tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 7020
ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 7080ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 7080
actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 7140actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 7140
taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 7200taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 7200
tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 7260tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 7260
gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 7320gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 7320
cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 7380cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 7380
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 7440gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 7440
gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 7500gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 7500
tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 7560tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 7560
ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 7620ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 7620
cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 7680cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 7680
gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 7740gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 7740
gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 7800gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 7800
gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 7860gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 7860
cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 7920cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 7920
tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 7980tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 7980
cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 8040cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 8040
cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 8100cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 8100
ccgcctctcc ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt 8160ccgcctctcc ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt 8160
tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 8220ttttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 8220
gaggcttttt tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc 8280gaggcttttt tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc 8280
gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 8340gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 8340
ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 8400ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 8400
caatttcaca caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt 8460caatttcaca caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt 8460
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 8520tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 8520
aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc 8580aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc 8580
actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca 8640actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca 8640
ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagt 8687ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagt 8687
<210> 14<210> 14
<211> 27<211> 27
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 14<400> 14
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser LeuPhe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 151 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp ValLeu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25 20 25
<210> 15<210> 15
<211> 45<211> 45
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 15<400> 15
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile AlaThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 151 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala GlySer Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30 20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys AspGly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45 35 40 45
<210> 16<210> 16
<211> 195<211> 195
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 16<400> 16
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met ThrArg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 151 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala ProPro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30 20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys LeuPro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu
35 40 45 35 40 45
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr GlnLeu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
50 55 60 50 55 60
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly GlyGlu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
65 70 75 8065 70 75 80
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala TyrCys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
85 90 95 85 90 95
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg ArgGln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
100 105 110 100 105 110
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu MetGlu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
115 120 125 115 120 125
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn GluGly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
130 135 140 130 135 140
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met LysLeu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
145 150 155 160145 150 155 160
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly LeuGly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
165 170 175 165 170 175
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala LeuSer Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
180 185 190 180 185 190
Pro Pro ArgPro Pro Arg
195 195
<210> 17<210> 17
<211> 765<211> 765
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 17<400> 17
caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60
acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120
ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180
cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240
aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300
ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360
gtgtggggca agggcacaac agtgacagtg tctagcggag gtggaggatc tggtggaggc 420gtgtggggca agggcacaac agtgacagtg tctagcggag gtggaggatc tggtggaggc 420
ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtggctct gggagagaca 480ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtggctct gggagagaca 480
gccaggatct cttgcggcag acaggctctg ggaagcagag cagtccagtg gtaccagcac 540gccaggatct cttgcggcag acaggctctg ggaagcagag cagtccagtg gtaccagcac 540
agaccaggac aggcccctat cctgctgatc tacaacaacc aggaccggcc cagcggaatc 600agaccaggac aggcccctat cctgctgatc tacaacaacc aggaccggcc cagcggaatc 600
ccagagagat tcagcggcac cccagacatc aacttcggca ccagagccac cctgacaatt 660ccagagagat tcagcggcac cccagacatc aacttcggca ccagagccac cctgacaatt 660
agcggcgtgg aagccggaga cgaggccgac tactattgcc acatgtggga tagcaggagc 720agcggcgtgg aagccggaga cgaggccgac tactattgcc acatgtggga tagcaggagc 720
ggcttctctt ggagcttcgg aggagccaca agactgacag tgctg 765ggcttctctt ggagcttcgg aggagccaca agactgacag tgctg 765
<210> 18<210> 18
<211> 396<211> 396
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 18<400> 18
caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60
acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120
ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180
cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240
aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300
ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360
gtgtggggca agggcacaac agtgacagtg tctagc 396gtgtggggca agggcacaac agtgacagtg tctagc 396
<210> 19<210> 19
<211> 324<211> 324
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 19<400> 19
agctatgtgc gacctctgag cgtggctctg ggagagacag ccaggatctc ttgcggcaga 60agctatgtgc gacctctgag cgtggctctg ggagagacag ccaggatctc ttgcggcaga 60
caggctctgg gaagcagagc agtccagtgg taccagcaca gaccaggaca ggcccctatc 120caggctctgg gaagcagagc agtccagtgg taccagcaca gaccaggaca ggcccctatc 120
ctgctgatct acaacaacca ggaccggccc agcggaatcc cagagagatt cagcggcacc 180ctgctgatct acaacaacca ggaccggccc agcggaatcc cagagagatt cagcggcacc 180
ccagacatca acttcggcac cagagccacc ctgacaatta gcggcgtgga agccggagac 240ccagacatca acttcggcac cagagccacc ctgacaatta gcggcgtgga agccggagac 240
gaggccgact actattgcca catgtgggat agcaggagcg gcttctcttg gagcttcgga 300gaggccgact actattgcca catgtgggat agcaggagcg gcttctcttg gagcttcgga 300
ggagccacaa gactgacagt gctg 324ggagccacaa gactgacagt gctg 324
<210> 20<210> 20
<211> 37<211> 37
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 20<400> 20
cattccgcta gccaggtgca gctgcaggag tcaggac 37cattccgcta gccaggtgca gctgcaggag tcaggac 37
<210> 21<210> 21
<211> 52<211> 52
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 21<400> 21
cacttccgcc tccaccagat cctccacctc cgctagacac tgtcactgtt gt 52cacttccgcc tccaccagat cctccacctc cgctagacac tgtcactgtt gt 52
<210> 22<210> 22
<211> 56<211> 56
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 22<400> 22
tggtggaggc ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtgg 56tggtggaggc ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtgg 56
<210> 23<210> 23
<211> 39<211> 39
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 23<400> 23
tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39
<210> 24<210> 24
<211> 37<211> 37
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 24<400> 24
cattccgcta gccaggtgca gctgcaggag tcaggac 37cattccgcta gccaggtgca gctgcaggag tcaggac 37
<210> 25<210> 25
<211> 39<211> 39
<212> DNA/RNA<212> DNA/RNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 25<400> 25
tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39
<210> 26<210> 26
<211> 267<211> 267
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 26<400> 26
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile AlaThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 151 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala GlySer Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30 20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp ValGly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val
35 40 45 35 40 45
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val ThrLeu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
50 55 60 50 55 60
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly GlyVal Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly
65 70 75 8065 70 75 80
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr ArgHis Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
85 90 95 85 90 95
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr ArgLys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
100 105 110 100 105 110
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro PheSer Lys Arg Gly Arg Lys Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
115 120 125 115 120 125
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys ArgMet Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
130 135 140 130 135 140
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe SerPhe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
145 150 155 160145 150 155 160
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu TyrArg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
165 170 175 165 170 175
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp LysAsn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
180 185 190 180 185 190
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys AsnArg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
195 200 205 195 200 205
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala GluPro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
210 215 220 210 215 220
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys GlyAla Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
225 230 235 240225 230 235 240
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr TyrHis Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
245 250 255 245 250 255
Asp Ala Leu His Met Gln Ala Leu Pro Pro ArgAsp Ala Leu His Met Gln Ala Leu Pro Pro Arg
260 265 260 265
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Citations (3)
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| CN104918658A (en)* | 2012-10-18 | 2015-09-16 | 洛克菲勒大学 | Broadly neutralizing anti-HIV antibodies |
| WO2019112347A2 (en)* | 2017-12-06 | 2019-06-13 | 앱클론(주) | Antibody or antigen binding fragment thereof for specifically recognizing b cell malignancy, chimeric antigen receptor comprising same, and use thereof |
| CN110305219A (en)* | 2018-03-27 | 2019-10-08 | 清华大学 | Use of a T cell containing chimeric antigen receptor (CAR) modification in the preparation of cellular medicine |
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Patent Citations (3)
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|---|---|---|---|---|
| CN104918658A (en)* | 2012-10-18 | 2015-09-16 | 洛克菲勒大学 | Broadly neutralizing anti-HIV antibodies |
| WO2019112347A2 (en)* | 2017-12-06 | 2019-06-13 | 앱클론(주) | Antibody or antigen binding fragment thereof for specifically recognizing b cell malignancy, chimeric antigen receptor comprising same, and use thereof |
| CN110305219A (en)* | 2018-03-27 | 2019-10-08 | 清华大学 | Use of a T cell containing chimeric antigen receptor (CAR) modification in the preparation of cellular medicine |
Non-Patent Citations (1)
| Title |
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| HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies;Ayub Ali等;《Journal of Virology》;20160525;第90卷(第15期);摘要，Fig. 1，Table 1，第7000页左栏第4段至右栏第2段* |
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