本申请是申请号为2023102434061专利申请的分案申请(原请的申请日为2023/3/14，发明名称为一种结合Dsg3的单克隆抗体及其应用)。This application is a divisional application of patent application No. 2023102434061 (the filing date of the original application is 2023/3/14, and the title of the invention is a monoclonal antibody binding to Dsg3 and its application).
技术领域Technical field
本发明属于生物医药技术领域，具体涉及一种结合Dsg3的单克隆抗体及其应用。The invention belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody binding to Dsg3 and its application.
背景技术Background technique
天疱疮是一种自身免疫性疾病，其具有寻常型、增殖型、落叶型和红斑型这四种亚型。寻常型天疱疮(pemphigus vulgaris，PV)是一种主要累及皮肤和黏膜的大疱性皮肤病，PV在临床上较为常见。患者血清循环中存在针对角质形成细胞间蛋白结构的自身抗体，破坏细胞间黏附功能，从而出现以表皮内水疱为主的临床表现。约60％的PV患者会出现口腔黏膜的水疱、大疱，且自发破溃，糜烂面难以愈合，严重影响患者进食及日常生活，给患者身心带来极大痛苦。此外，一部分的患者仅表现为持续性的口腔黏膜糜烂而无任何皮肤损伤。因此，需要快速、准确诊断PV的有效检测手段。Pemphigus is an autoimmune disease with four subtypes: vulgaris, proliferative, deciduous, and erythematous. Pemphigus vulgaris (PV) is a bullous skin disease that mainly affects the skin and mucous membranes. PV is relatively common clinically. There are autoantibodies targeting the protein structure between keratinocytes in the patient's serum circulation, which destroys the intercellular adhesion function, resulting in clinical manifestations mainly involving intraepidermal blisters. About 60% of PV patients will develop oral mucosal blisters and bullae, which spontaneously rupture and are difficult to heal. This seriously affects the patient's eating and daily life, and causes great physical and mental pain to the patient. In addition, some patients only present with persistent oral mucosal erosion without any skin damage. Therefore, effective detection methods for rapid and accurate diagnosis of PV are needed.
PV的早期诊断对治疗和预后具有极其重要的作用，PV的诊断包括临床表现、组织病理和免疫诊断指标。临床表现：①皮肤出现松弛性水疱和大疱，易破溃；②水疱和大疱破溃后形成顽固性糜烂；③黏膜出现水疱或糜烂；④尼氏征阳性。组织病理：表皮或上皮细胞间棘层松解，形成水疱和大疱。免疫诊断指标：①皮损区域或皮损周围正常皮肤直接免疫荧光(DIF)示IgG和/或补体沉积于表皮(或上皮)细胞间；②间接免疫荧光(IIF)检测到血清中出现抗表皮细胞间抗体；③ELISA检测到血清中出现抗Dsg抗体。满足临床表现中的至少1条加上组织病理和免疫诊断指标中的至少1条即可确诊。满足临床表现中至少2条加上免疫诊断指标中2条亦可确诊。Early diagnosis of PV plays an extremely important role in treatment and prognosis. The diagnosis of PV includes clinical manifestations, histopathology and immunodiagnostic indicators. Clinical manifestations: ① Flax blisters and bullae appear on the skin, which are easy to rupture; ② Intractable erosion forms after the blister and bullae rupture; ③ Blisters or erosion appear on the mucous membrane; ④ Positive Nissl sign. Histopathology: acantholysis of epidermal or epithelial cells, forming blisters and bullae. Immunodiagnostic indicators: ① Direct immunofluorescence (DIF) in the lesion area or normal skin around the lesion shows that IgG and/or complement are deposited between epidermal (or epithelial) cells; ② Indirect immunofluorescence (IIF) detects the presence of anti-epidermal antibodies in the serum Intercellular antibodies; ③ ELISA detected anti-Dsg antibodies in the serum. The diagnosis can be confirmed by meeting at least one of the clinical manifestations and at least one of the histopathological and immunodiagnostic indicators. Diagnosis can also be confirmed if at least 2 of the clinical manifestations and 2 of the immunodiagnostic indicators are met.
在PV的诊断过程中需要高亲和力的抗Dsg3单克隆抗体，经典的杂交瘤技术需要耗费大量的时间和精力去获得高亲和力的抗体，而且需要进行后续的人源化改造，这导致难以通过杂交瘤技术获得人源单克隆抗体。High-affinity anti-Dsg3 monoclonal antibodies are required in the diagnosis process of PV. Classic hybridoma technology requires a lot of time and energy to obtain high-affinity antibodies, and requires subsequent humanization transformation, which makes it difficult to pass hybridization Tumor technology to obtain human monoclonal antibodies.
噬菌体展示技术已被广泛应用于抗原抗体库的建立、药物设计、疫苗研究、病原检测、基因治疗、抗原表位研究及细胞信号转导研究等。噬菌体抗体库技术是通过制备人源抗体文库(library)，把Fab段或单链抗体(ScFv)表达在噬菌体的表面，从而筛选并富集特异性抗体。从单pot抗体文库系统中筛选出几乎所有与抗原发生特异性反应的重组人单克隆抗体，因此，当使用噬菌体抗体技术时，能获得可应用于体内诊断或治疗的各种抗体片段(Fab或ScFv)。噬菌体抗体库技术不需要经过免疫和人源化改造步骤，依靠在完全可控的生物化学环境中进行富集筛选，实现短期内筛选到较高亲和力的全人源人类抗体序列。Phage display technology has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research. Phage antibody library technology is to screen and enrich specific antibodies by preparing a human antibody library (library) and expressing Fab segments or single-chain antibodies (ScFv) on the surface of phage. Almost all recombinant human monoclonal antibodies that specifically react with antigens are screened from a single pot antibody library system. Therefore, when using phage antibody technology, various antibody fragments (Fab or Fab) that can be used for in vivo diagnosis or treatment can be obtained. ScFv). Phage antibody library technology does not require immunization and humanization steps, and relies on enrichment screening in a completely controllable biochemical environment to achieve the short-term selection of fully human antibody sequences with higher affinity.
寻常型天疱疮的病理特点为表皮内水疱及棘层细胞松懈，导致该病理改变的直接原因是抗棘层细胞间质抗体，几乎所有的活动性天疱疮病人血清中都存在这种抗体，PV抗体作用的靶抗原位于表皮细胞间的连接结构-桥粒上，为跨膜桥粒核心蛋白(desmoglein3，Dsg3)。Dsg3分子量为64.95KD，属于钙粘素(Cadherin)的超家族成员之一，在维持表皮的完整性方面发挥重要作用，其结构的破坏可导致棘刺松懈，引起表皮内水疱。Dsg3的细胞内成分通过桥斑盘状蛋白与细胞骨架相互作用并构成连接，胞外区则介导细胞间的连接，Dsg3的胞外区提供了PV抗体识别的抗原表位。Dsg3中起主要作用的区域被称为细胞外功能区(extracellular domain，EC)，Dsg3的EC分为5个结构域(EC1-EC5)，EC-1位于氨基端，EC-5位于羧基端。Dsg3分子的天然立体构象对激发自身免疫性大疱很重要。已有的研究通过杆状病毒表达系统和哺乳动物表达系统获得人Dsg3重组蛋白，包括Dsg3的整个EC，所得人Dsg3重组蛋白可以与人PV患者血清的天然抗体特异性结合。人Dsg3重组蛋白不仅可为诊断PV提供可靠的手段，还可用于进一步研究天疱疮的发病机理。The pathological characteristics of pemphigus vulgaris are intraepidermal blisters and loose spinous cells. The direct cause of this pathological change is anti-cancer interstitial antibodies. This antibody is present in the serum of almost all patients with active pemphigus. The target antigen of PV antibodies is located on the desmosome, the connection structure between epidermal cells, and is the transmembrane desmoglein3 (Dsg3). Dsg3 has a molecular weight of 64.95KD and is a member of the Cadherin superfamily. It plays an important role in maintaining the integrity of the epidermis. The destruction of its structure can cause spines to loosen and cause intraepidermal blisters. The intracellular component of Dsg3 interacts with the cytoskeleton through plaque-disc proteins and forms a connection, while the extracellular region mediates the connection between cells. The extracellular region of Dsg3 provides the epitope recognized by PV antibodies. The region that plays a major role in Dsg3 is called the extracellular domain (EC). The EC of Dsg3 is divided into 5 domains (EC1-EC5). EC-1 is located at the amino terminus and EC-5 is located at the carboxyl terminus. The native three-dimensional conformation of the Dsg3 molecule is important for triggering autoimmune bullae. Existing studies have obtained human Dsg3 recombinant protein, including the entire EC of Dsg3, through baculovirus expression systems and mammalian expression systems. The resulting human Dsg3 recombinant protein can specifically bind to natural antibodies in the serum of human PV patients. Human Dsg3 recombinant protein not only provides a reliable means for diagnosing PV, but can also be used to further study the pathogenesis of pemphigus.
因此，利用噬菌体抗体库技术筛选一种结合寻常型天疱疮特异性抗原肽的人源抗体在天疱疮的诊断和/或治疗中具有重要的应用价值。Therefore, using phage antibody library technology to screen a human antibody that binds pemphigus vulgaris-specific antigen peptides has important application value in the diagnosis and/or treatment of pemphigus.
发明内容Contents of the invention
针对现有技术存在的不足，本发明的目的在于提供一种结合Dsg3的单克隆抗体及其应用。本发明中以Dsg3抗体阳性患者的外周血单个核细胞(Peripheral bloodmononuclear cell，PBMC)构建噬菌体人源抗体库，通过与Dsg3蛋白特异性结合，筛选出能特异性结合Dsg3抗原的单克隆抗体。所述单克隆抗体亲和力高、特异性强，能够定量检测PV患者中抗Dsg3自身抗体水平，监测病情活动度，用于PV患者的临床诊断。本发明的研究为特异性治疗PV提供了理论与实验依据，同时也为治疗以自身抗体为主要致病机制的自身免疫病提供了一个新的研究方向。In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a monoclonal antibody that binds Dsg3 and its application. In the present invention, peripheral blood mononuclear cells (PBMC) of Dsg3 antibody-positive patients are used to construct a phage human antibody library, and by specifically binding to the Dsg3 protein, monoclonal antibodies that can specifically bind to the Dsg3 antigen are screened out. The monoclonal antibody has high affinity and strong specificity, can quantitatively detect anti-Dsg3 autoantibody levels in PV patients, monitor disease activity, and can be used for clinical diagnosis of PV patients. The research of the present invention provides theoretical and experimental basis for the specific treatment of PV, and also provides a new research direction for the treatment of autoimmune diseases in which autoantibodies are the main pathogenic mechanism.
为达到此发明目的，本发明采用以下技术方案：In order to achieve the purpose of this invention, the present invention adopts the following technical solutions:
第一方面，本发明提供一种结合Dsg3的单克隆抗体，所述单克隆抗体的重链CDR3包括SEQ ID NO:3、SEQ ID NO:13、SEQ ID NO:23、SEQ ID NO:33或SEQ ID NO:43所示的氨基酸序列；In a first aspect, the present invention provides a monoclonal antibody that binds Dsg3, and the heavy chain CDR3 of the monoclonal antibody includes SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33 or The amino acid sequence shown in SEQ ID NO:43;
所述单克隆抗体的轻链CDR3包括SEQ ID NO:6、SEQ ID NO:16、SEQ ID NO:26、SEQID NO:36或SEQ ID NO:46所示的氨基酸序列。The light chain CDR3 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36 or SEQ ID NO: 46.
本发明从噬菌体库中筛选出了特异性好、亲和力强的结合Dsg3的单克隆抗体，所述单克隆抗体为全人源抗体，所述全人源抗体具有更低的免疫原性，在抗体药物及诊断药物开发上有巨大的应用潜力。The present invention screens out monoclonal antibodies that bind Dsg3 with good specificity and strong affinity from the phage library. The monoclonal antibodies are fully human antibodies. The fully human antibodies have lower immunogenicity. It has huge application potential in the development of drugs and diagnostic drugs.
优选地，所述单克隆抗体的重链CDR1包括SEQ ID NO:1、SEQ ID NO:11、SEQ IDNO:21、SEQ ID NO:31或SEQ ID NO:41所示的氨基酸序列。Preferably, the heavy chain CDR1 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31 or SEQ ID NO: 41.
优选地，所述单克隆抗体的重链CDR2包括SEQ ID NO:2、SEQ ID NO:12、SEQ IDNO:22、SEQ ID NO:32或SEQ ID NO:42所示的氨基酸序列。Preferably, the heavy chain CDR2 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32 or SEQ ID NO: 42.
优选地，所述单克隆抗体的轻链CDR1包括SEQ ID NO:4、SEQ ID NO:14、SEQ IDNO:24、SEQ ID NO:34或SEQ ID NO:44所示的氨基酸序列。Preferably, the light chain CDR1 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 34 or SEQ ID NO: 44.
优选地，所述单克隆抗体的轻链CDR2包括SEQ ID NO:5、SEQ ID NO:15、SEQ IDNO:25、SEQ ID NO:35或SEQ ID NO:45所示的氨基酸序列。Preferably, the light chain CDR2 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 35 or SEQ ID NO: 45.
优选地，所述单克隆抗体的重链可变区包括SEQ ID NO:7、SEQ ID NO:17、SEQ IDNO:27、SEQ ID NO:37或SEQ ID NO:47所示的氨基酸序列。Preferably, the heavy chain variable region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37 or SEQ ID NO:47.
优选地，所述单克隆抗体的轻链可变区包括SEQ ID NO:8、SEQ ID NO:18、SEQ IDNO:28、SEQ ID NO:38或SEQ ID NO:48所示的氨基酸序列。Preferably, the light chain variable region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38 or SEQ ID NO: 48.
优选地，所述单克隆抗体的重链包括SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:39或SEQ ID NO:49所示的氨基酸序列。Preferably, the heavy chain of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:39 or SEQ ID NO:49.
优选地，所述单克隆抗体的轻链包括SEQ ID NO:10、SEQ ID NO:20、SEQ ID NO:30、SEQ ID NO:40或SEQ ID NO:50所示的氨基酸序列。Preferably, the light chain of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40 or SEQ ID NO: 50.
本发明分离了Dsg3抗体阳性患者的PBMC，提取RNA并对RNA进行质检。采用RT-PCR技术将质检合格的RNA反转录成cDNA，扩增其中全部的抗体VH、VL基因片段。将体外扩增的Vκ、Vλ基因片段克隆入pATA-scFv-2载体，构建成抗体组合文库。将抗体基因组合文库插入噬菌体编码的膜蛋白的基因III(g3)或基因VIII(g8)的先导系列的紧邻下游，通过辅助噬菌体的超感染，使外源抗体基因表达的多肽可以以融合蛋白的形式展示在噬菌体外壳蛋白pIII或pVIII的N端。每个噬菌体颗粒编码并呈递不同的抗体，其含有数十亿个单个克隆。在这些抗体文库中，编码可与抗原结合的那些抗体的基因通过在体外对抗原进行亲和力富集-温和洗脱-噬菌体扩增，再继续重复以上富集筛选过程，直至数个循环后获得特异性好、亲和力强的抗体噬菌体库，从抗体噬菌体库中筛选阳性克隆。通过ELISA方法对阳性克隆进行鉴定，最后从中筛选特异性好、亲和力强的全人源抗体。The present invention isolates PBMC from Dsg3 antibody-positive patients, extracts RNA, and performs quality inspection on the RNA. RT-PCR technology is used to reverse-transcribe the qualified RNA into cDNA, and amplify all antibody VH and VL gene fragments. The Vκ and Vλ gene fragments amplified in vitro were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library. The antibody gene combination library is inserted immediately downstream of the leader series of gene III (g3) or gene VIII (g8) of the membrane protein encoded by the phage. Through superinfection of the helper phage, the polypeptide expressed by the foreign antibody gene can be expressed as a fusion protein. The form is displayed at the N-terminus of the phage coat protein pill or pVIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, the genes encoding those antibodies that can bind to the antigen are affinity enriched for the antigen in vitro - gentle elution - phage amplification, and then continue to repeat the above enrichment and screening process until specificity is obtained after several cycles. Use an antibody phage library with good properties and strong affinity to screen positive clones from the antibody phage library. The positive clones were identified by ELISA method, and fully human antibodies with good specificity and strong affinity were finally screened.
经过抗体噬菌体库的筛选，最终得到了5条高亲和力的单克隆抗体，命名为76F-DSG3-IC-R2P1-G1、76F-DSG3-IC-R2P1-F2、76F-DSG3-IC-R2P1-E6、76F-DSG3-IC-R2P1-C9和76F-DSG3-IC-R2P1-H9。所述单克隆抗体活性高、稳定性好，且具有较强的特异性，能作为定性检测PV阳性的参考标准，也能定量检测PV患者中抗Dsg3自身抗体水平。After screening the antibody phage library, five high-affinity monoclonal antibodies were finally obtained, named 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, and 76F-DSG3-IC-R2P1-E6. , 76F-DSG3-IC-R2P1-C9 and 76F-DSG3-IC-R2P1-H9. The monoclonal antibody has high activity, good stability, and strong specificity. It can be used as a reference standard for qualitative detection of PV positivity and can also quantitatively detect anti-Dsg3 autoantibody levels in PV patients.
单克隆抗体76F-DSG3-IC-R2P1-G1：Monoclonal antibody 76F-DSG3-IC-R2P1-G1:
SEQ ID NO:1(重链CDR1)：GFTLANST。SEQ ID NO: 1 (heavy chain CDR1): GFTLANST.
SEQ ID NO:2(重链CDR2)：SVVGNDKT。SEQ ID NO: 2 (heavy chain CDR2): SVVGNDKT.
SEQ ID NO:3(重链CDR3)：AASSHFWSGSLDI。SEQ ID NO: 3 (heavy chain CDR3): AASSHFWSGSLDI.
SEQ ID NO:4(轻链CDR1)：QSVLYSSNNKNY。SEQ ID NO: 4 (light chain CDR1): QSVLYSSNNKNY.
SEQ ID NO:5(轻链CDR2)：WAS。SEQ ID NO: 5 (light chain CDR2): WAS.
SEQ ID NO:6(轻链CDR3)：QQYYSTPLT。SEQ ID NO: 6 (light chain CDR3): QQYYSTPLT.
SEQ ID NO:7(重链可变区)：SEQ ID NO:7 (heavy chain variable region):
EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS.
SEQ ID NO:8(轻链可变区)：SEQ ID NO:8 (light chain variable region):
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIK。DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIK.
SEQ ID NO:9(76F-DSG3-IC-R2P1-G1抗体的重链氨基酸序列)：SEQ ID NO:9 (Heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-G1 antibody):
MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:10(76F-DSG3-IC-R2P1-G1抗体的轻链氨基酸序列)：SEQ ID NO: 10 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-G1 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
单克隆抗体76F-DSG3-IC-R2P1-F2：Monoclonal antibody 76F-DSG3-IC-R2P1-F2:
SEQ ID NO:11(重链CDR1)：GFTLANST。SEQ ID NO: 11 (heavy chain CDR1): GFTLANST.
SEQ ID NO:12(重链CDR2)：SVVGNDKT。SEQ ID NO: 12 (heavy chain CDR2): SVVGNDKT.
SEQ ID NO:13(重链CDR3)：AASSHFWSGSLDI。SEQ ID NO: 13 (heavy chain CDR3): AASSHFWSGSLDI.
SEQ ID NO:14(轻链CDR1)：QSVLYSSNNKNY。SEQ ID NO: 14 (light chain CDR1): QSVLYSSNNKNY.
SEQ ID NO:15(轻链CDR2)：WAS。SEQ ID NO: 15 (light chain CDR2): WAS.
SEQ ID NO:16(轻链CDR3)：QQYYSTPIT。SEQ ID NO: 16 (light chain CDR3): QQYYSTPIT.
SEQ ID NO:17(重链可变区)：SEQ ID NO: 17 (heavy chain variable region):
EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS.
SEQ ID NO:18(轻链可变区)：SEQ ID NO: 18 (light chain variable region):
DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIK。DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIK.
SEQ ID NO:19(76F-DSG3-IC-R2P1-F2抗体的重链氨基酸序列)：SEQ ID NO: 19 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-F2 antibody):
MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:20(76F-DSG3-IC-R2P1-F2抗体的轻链氨基酸序列)：SEQ ID NO:20 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-F2 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
单克隆抗体76F-DSG3-IC-R2P1-E6：Monoclonal antibody 76F-DSG3-IC-R2P1-E6:
SEQ ID NO:21(重链CDR1)：GFTLANST。SEQ ID NO: 21 (heavy chain CDR1): GFTLANST.
SEQ ID NO:22(重链CDR2)：SVVGNDKT。SEQ ID NO: 22 (heavy chain CDR2): SVVGNDKT.
SEQ ID NO:23(重链CDR3)：AASSHFWSGSLDV。SEQ ID NO: 23 (heavy chain CDR3): AASSHFWSGSLDV.
SEQ ID NO:24(轻链CDR1)：QSVLYNSNNKNY。SEQ ID NO: 24 (light chain CDR1): QSVLYNSNNKNY.
SEQ ID NO:25(轻链CDR2)：WAS。SEQ ID NO: 25 (light chain CDR2): WAS.
SEQ ID NO:26(轻链CDR3)：HQYFGTPYT。SEQ ID NO: 26 (light chain CDR3): HQYFGTPYT.
SEQ ID NO:27(重链可变区)：SEQ ID NO:27 (heavy chain variable region):
QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGN DKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLV TVSS。QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGN DKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLV TVSS.
SEQ ID NO:28(轻链可变区)：SEQ ID NO:28 (light chain variable region):
EIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIK。EIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIK.
SEQ ID NO:29(76F-DSG3-IC-R2P1-E6抗体的重链氨基酸序列)：SEQ ID NO:29 (Heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-E6 antibody):
MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGNDKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGNDKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:30(76F-DSG3-IC-R2P1-E6抗体的轻链氨基酸序列)：SEQ ID NO:30 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-E6 antibody):
MVLQTQVFISLLLWISGAYGEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MVLQTQVFISLLLWISGAYGEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
单克隆抗体76F-DSG3-IC-R2P1-C9：Monoclonal antibody 76F-DSG3-IC-R2P1-C9:
SEQ ID NO:31(重链CDR1)：GGSFSGYY。SEQ ID NO: 31 (heavy chain CDR1): GGSFSGYY.
SEQ ID NO:32(重链CDR2)：INHSGST。SEQ ID NO: 32 (heavy chain CDR2): INHSGST.
SEQ ID NO:33(重链CDR3)：ARGRYLATVRHYYYYYMDV。SEQ ID NO: 33 (heavy chain CDR3): ARGRYLATVRHYYYYYMDV.
SEQ ID NO:34(轻链CDR1)：QSLENSAGNTY。SEQ ID NO: 34 (light chain CDR1): QSLENSAGNTY.
SEQ ID NO:35(轻链CDR2)：KVS。SEQ ID NO: 35 (light chain CDR2): KVS.
SEQ ID NO:36(轻链CDR3)：MQGSHWPPYT。SEQ ID NO: 36 (light chain CDR3): MQGSHWPPYT.
SEQ ID NO:37(重链可变区)：SEQ ID NO:37 (heavy chain variable region):
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKG TLVTVSS。QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTN YNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKG TLVTVSS.
SEQ ID NO:38(轻链可变区)：SEQ ID NO:38 (light chain variable region):
EIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIK。EIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIK.
SEQ ID NO:39(76F-DSG3-IC-R2P1-C9抗体的重链氨基酸序列)：SEQ ID NO:39 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-C9 antibody):
MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:40(76F-DSG3-IC-R2P1-C9抗体的轻链氨基酸序列)：SEQ ID NO:40 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-C9 antibody):
MVLQTQVFISLLLWISGAYGEIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MVLQTQVFISLLLWISGAYGEIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
单克隆抗体76F-DSG3-IC-R2P1-H9：Monoclonal antibody 76F-DSG3-IC-R2P1-H9:
SEQ ID NO:41(重链CDR1)：GFTLANST。SEQ ID NO: 41 (heavy chain CDR1): GFTLANST.
SEQ ID NO:42(重链CDR2)：SVVGNDKT。SEQ ID NO: 42 (heavy chain CDR2): SVVGNDKT.
SEQ ID NO:43(重链CDR3)：AASSHFWSGSLDI。SEQ ID NO: 43 (heavy chain CDR3): AASSHFWSGSLDI.
SEQ ID NO:44(轻链CDR1)：QSVLYSSNNKNY。SEQ ID NO: 44 (light chain CDR1): QSVLYSSNNKNY.
SEQ ID NO:45(轻链CDR2)：WAS。SEQ ID NO: 45 (light chain CDR2): WAS.
SEQ ID NO:46(轻链CDR3)：QQYYSTPFT。SEQ ID NO: 46 (light chain CDR3): QQYYSTPFT.
SEQ ID NO:47(重链可变区)：SEQ ID NO:47 (heavy chain variable region):
QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS.
SEQ ID NO:48(轻链可变区)：SEQ ID NO:48 (light chain variable region):
DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK。DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK.
SEQ ID NO:49(76F-DSG3-IC-R2P1-H9抗体的重链氨基酸序列)：SEQ ID NO:49 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-H9 antibody):
MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT Question PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:50(76F-DSG3-IC-R2P1-H9抗体的轻链氨基酸序列)：SEQ ID NO:50 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-H9 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
第二方面，本发明提供一种核酸分子，所述核酸分子编码第一方面所述的结合Dsg3的单克隆抗体。In a second aspect, the present invention provides a nucleic acid molecule encoding the Dsg3-binding monoclonal antibody described in the first aspect.
第三方面，本发明提供一种表达载体，所述表达载体含有第二方面所述的核酸分子。In a third aspect, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.
第四方面，本发明提供一种宿主细胞，所述宿主细胞中含有至少一个拷贝的第三方面所述的表达载体。In a fourth aspect, the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect.
第五方面，本发明提供一种药物组合物，所述药物组合物包括第一方面所述的结合Dsg3的单克隆抗体，以及药学上可接受的载体和/或稀释剂。In a fifth aspect, the present invention provides a pharmaceutical composition, which includes the Dsg3-binding monoclonal antibody described in the first aspect, and a pharmaceutically acceptable carrier and/or diluent.
第六方面，本发明提供一种用于检测样品中Dsg3蛋白的试剂盒，所述试剂盒包括第一方面所述的结合Dsg3的单克隆抗体。In a sixth aspect, the present invention provides a kit for detecting Dsg3 protein in a sample, which kit includes the Dsg3-binding monoclonal antibody described in the first aspect.
第七方面，本发明提供第一方面所述的结合Dsg3的单克隆抗体、第四方面所述的宿主细胞或第五方面所述的药物组合物中任意一种或至少两种的组合在制备用于诊断和/或治疗天疱疮的药物中的用途。In the seventh aspect, the present invention provides any one or a combination of at least two of the Dsg3-binding monoclonal antibodies described in the first aspect, the host cells described in the fourth aspect, or the pharmaceutical compositions described in the fifth aspect. Use in medicines for the diagnosis and/or treatment of pemphigus.
相对于现有技术，本发明具有以下有益效果：Compared with the existing technology, the present invention has the following beneficial effects:
本发明经过抗体噬菌体库的筛选，得到了5条高亲和力的结合Dsg3的单克隆抗体，所述单克隆抗体活性高、稳定性好，且具有较强的特异性，能作为定性检测PV阳性的参考标准，也能定量检测PV患者中抗Dsg3自身抗体水平。Through screening of the antibody phage library, the present invention obtains five high-affinity monoclonal antibodies that bind Dsg3. The monoclonal antibodies have high activity, good stability, and strong specificity, and can be used as a qualitative test for detecting PV positivity. A reference standard that can also quantitatively detect anti-Dsg3 autoantibody levels in PV patients.
附图说明Description of drawings
图1是VL噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图；Figure 1 is a monoclonal PCR agarose gel electrophoresis diagram of the VL phage library;
图2是κH噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图；Figure 2 is a monoclonal PCR agarose gel electrophoresis picture of the κH phage library;
图3是λH噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图；Figure 3 is a monoclonal PCR agarose gel electrophoresis diagram of the λH phage library;
图4A是轻链文库的测序质控结果；Figure 4A is the sequencing quality control result of the light chain library;
图4B是重链文库的测序质控结果；Figure 4B is the sequencing quality control result of the heavy chain library;
图5是纯化后的Dsg3 SDS-PAGE电泳图；Figure 5 is the SDS-PAGE electrophoresis pattern of purified Dsg3;
图6A是76F-DSG3-IC-R2P1-G1抗体与Dsg3的ELISA检测结果；Figure 6A is the ELISA detection result of 76F-DSG3-IC-R2P1-G1 antibody and Dsg3;
图6B是76F-DSG3-IC-R2P1-F2抗体与Dsg3的ELISA检测结果；Figure 6B is the ELISA detection result of 76F-DSG3-IC-R2P1-F2 antibody and Dsg3;
图6C是76F-DSG3-IC-R2P1-E6抗体与Dsg3的ELISA检测结果；Figure 6C is the ELISA detection result of 76F-DSG3-IC-R2P1-E6 antibody and Dsg3;
图6D是76F-DSG3-IC-R2P1-C9抗体与Dsg3的ELISA检测结果；Figure 6D is the ELISA detection result of 76F-DSG3-IC-R2P1-C9 antibody and Dsg3;
图6E是76F-DSG3-IC-R2P1-H9抗体与Dsg3的ELISA检测结果。Figure 6E is the ELISA detection result of 76F-DSG3-IC-R2P1-H9 antibody and Dsg3.
具体实施方式Detailed ways
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。The technical solution of the present invention will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.
发明详述：Detailed description of the invention:
单克隆抗体是治疗性蛋白质中数量最大且发展最快的一组，目前有500多种治疗性抗体及其衍生物正在临床试验中，重点放在癌症治疗和自身免疫性疾病上。本发明分离了Dsg3抗体阳性患者的PBMC，提取RNA并对RNA进行质检。采用RT-PCR技术将质检合格的RNA反转录成cDNA，扩增其中全部的抗体VH、VL基因片段。将体外扩增的Vκ、Vλ基因片段克隆入pATA-scFv-2载体，构建成抗体组合文库。Monoclonal antibodies are the largest and fastest growing group of therapeutic proteins, with more than 500 therapeutic antibodies and their derivatives currently in clinical trials, focusing on cancer treatment and autoimmune diseases. The present invention isolates PBMC from Dsg3 antibody-positive patients, extracts RNA, and performs quality inspection on the RNA. RT-PCR technology is used to reverse-transcribe the qualified RNA into cDNA, and amplify all antibody VH and VL gene fragments. The Vκ and Vλ gene fragments amplified in vitro were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.
将抗体基因组合文库插入噬菌体编码的膜蛋白的基因III(g3)或基因VIII(g8)的先导系列的紧邻下游，通过辅助噬菌体的超感染，使外源抗体基因表达的多肽可以以融合蛋白的形式展示在噬菌体外壳蛋白pIII或pVIII的N端。每个噬菌体颗粒编码并呈递不同的抗体，其含有数十亿个单个克隆。在这些抗体文库中，编码可与抗原结合的那些抗体的基因通过在体外对抗原进行亲和力富集-温和洗脱-噬菌体扩增，再继续重复以上富集筛选过程，直至数个循环后获得特异性好、亲和力强的抗体噬菌体库，从抗体噬菌体库中筛选阳性克隆。通过ELISA方法对阳性克隆进行鉴定，最后从中筛选特异性好、亲和力强的全人源抗体。The antibody gene combination library is inserted immediately downstream of the leader series of gene III (g3) or gene VIII (g8) of the membrane protein encoded by the phage. Through superinfection of the helper phage, the polypeptide expressed by the foreign antibody gene can be expressed as a fusion protein. The form is displayed at the N-terminus of the phage coat protein pill or pVIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, the genes encoding those antibodies that can bind to the antigen are affinity enriched for the antigen in vitro - gentle elution - phage amplification, and then continue to repeat the above enrichment and screening process until specificity is obtained after several cycles. Use an antibody phage library with good properties and strong affinity to screen positive clones from the antibody phage library. The positive clones were identified by ELISA method, and fully human antibodies with good specificity and strong affinity were finally screened.
本发明使用III 1st Strand cDNA Synthesis Kit(+gDNA wiper)反转录试剂盒将Dsg3抗体阳性患者的RNA反转录成cDNA，对DNA的VH和VL片段进行扩增。体外扩增得到的Vκ、Vλ基因片段通过克隆技术克隆入pATA-scFv-2载体，组成Vκ文库和Vλ文库。使用质粒提取试剂盒提取Vκ文库和Vλ文库的质粒载体，将体外扩增的VH基因片段插入Vκ文库和Vλ文库的质粒载体，构成κH文库和λH文库。The present invention uses III 1st Strand cDNA Synthesis Kit (+gDNA wiper) Reverse Transcription Kit reverse-transcribes RNA from Dsg3 antibody-positive patients into cDNA and amplifies the VH and VL fragments of DNA. The Vκ and Vλ gene fragments amplified in vitro were cloned into the pATA-scFv-2 vector through cloning technology to form the Vκ library and Vλ library. Use a plasmid extraction kit to extract the plasmid vectors of the Vκ library and Vλ library, and insert the in vitro amplified VH gene fragment into the plasmid vectors of the Vκ library and Vλ library to form the κH library and λH library.
将人工合成的Dsg3结构域的基因重组到表达载体质粒pFastBac1中，得到Dsg3-pFastBac1表达载体。将Dsg3-pFastBac1表达载体转染到DH10Bac感受态中培养，纯化得到Dsg3抗原蛋白。The synthetic Dsg3 domain gene was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured, and the Dsg3 antigen protein was purified.
经过三轮抗体噬菌体库的筛选，将抗原组大于3倍对照组的克隆定为阳性克隆，对这些单克隆进行测序分析。排除掉错误抗体序列和重复抗体序列，并结合ELISA实验所反映的抗原抗体特异性结合能力，最终得到了5条高亲和力的抗体，命名为76F-DSG3-IC-R2P1-G1、76F-DSG3-IC-R2P1-F2、76F-DSG3-IC-R2P1-E6、76F-DSG3-IC-R2P1-C9、76F-DSG3-IC-R2P1-H9。所述单克隆抗体活性高、稳定性好，且具有较强的特异性，能作为定性检测PV阳性的参考标准，也能定量检测PV患者中抗Dsg3自身抗体水平。After three rounds of screening of the antibody phage library, clones with an antigen group greater than 3 times the control group were determined as positive clones, and these single clones were sequenced and analyzed. After eliminating incorrect antibody sequences and repeated antibody sequences, and combining the specific binding abilities of antigens and antibodies reflected in the ELISA experiment, five high-affinity antibodies were finally obtained, named 76F-DSG3-IC-R2P1-G1, 76F-DSG3- IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, 76F-DSG3-IC-R2P1-H9. The monoclonal antibody has high activity, good stability, and strong specificity. It can be used as a reference standard for qualitative detection of PV positivity and can also quantitatively detect anti-Dsg3 autoantibody levels in PV patients.
实施例1人源ScFv噬菌体展示文库的构建方法Example 1 Construction method of human ScFv phage display library
本实施例分离了Dsg3抗体阳性患者的PBMC，提取RNA并对RNA进行质检。采用RT-PCR技术将质检合格的RNA反转录成cDNA，扩增其中全部的抗体VH、VL基因片段。将体外扩增的Vκ、Vλ基因片段克隆入pATA-scFv-2载体，构建成抗体组合文库。In this example, PBMCs from Dsg3 antibody-positive patients were isolated, RNA was extracted, and RNA quality inspection was performed. RT-PCR technology is used to reverse-transcribe the qualified RNA into cDNA, and amplify all antibody VH and VL gene fragments. The Vκ and Vλ gene fragments amplified in vitro were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.
本实施例中使用的主要试剂如表1所示：The main reagents used in this example are shown in Table 1:
表1Table 1
1、文库构建1. Library construction
1.1组装重链可变区(VH)和轻链可变区(VL)，实验步骤如下所示：PCR反应条件和步骤如表2所示。1.1 Assemble the heavy chain variable region (VH) and light chain variable region (VL). The experimental steps are as follows: PCR reaction conditions and steps are shown in Table 2.
表2Table 2
其中，变性，退火，延伸(1)这三个步骤，重复30次；引物序列如下所示：Among them, the three steps of denaturation, annealing, and extension (1) are repeated 30 times; the primer sequence is as follows:
上游引物(F)：Upstream primer (F):
5′L-VH 1(SEQ ID NO:51)：acaggtgcccactcccaggtgcag。5'L-VH 1 (SEQ ID NO:51): acaggtgcccactcccaggtgcag.
5′L-VH 3(SEQ ID NO:52)：aaggtgtccagtgtgargtgcag。5'L-VH 3 (SEQ ID NO:52): aaggtgtccagtgtgargtgcag.
5′L-VH 4/6(SEQ ID NO:53)：cccagatgggtcctgtcccaggtgcag。5'L-VH 4/6 (SEQ ID NO:53):cccagatgggtcctgtcccaggtgcag.
5′L-VH 5/7(SEQ ID NO:54)：caaggagtctgttccgaggtgcag。5'L-VH 5/7 (SEQ ID NO:54): caaggagtctgttccgaggtgcag.
5′L VK 1/2(SEQ ID NO:55)：atgaggstcccygctcagctgctgg。5'L VK 1/2 (SEQ ID NO:55): atgaggstcccygctcagctgctgg.
5′L VK 3(SEQ ID NO:56)：ctcttcctcctgctactctggctcccag。5'L VK 3 (SEQ ID NO:56): ctcttcctcctgctactctggctcccag.
5′L VK 4/5(SEQ ID NO:57)：atttctctgttgctctggatctctg。5'L VK 4/5 (SEQ ID NO:57): attctctgttgctctggatctctg.
5′L Vλ1(SEQ ID NO:58)：ggtcctgggcccagtctgtgctg。5'L Vλ1 (SEQ ID NO:58): ggtcctgggcccagtctgtgctg.
5′L Vλ2(SEQ ID NO:59)：ggtcctgggcccagtctgccctg。5'L Vλ2 (SEQ ID NO:59): ggtcctgggcccagtctgccctg.
5′L Vλ3(SEQ ID NO:60)：gctctgtgacctcctatgagctg。5'L Vλ3 (SEQ ID NO:60): gctctgtgacctcctatgagctg.
5′L Vλ4/5(SEQ ID NO:61)：ggtctctctcscagcytgtgctg。5'L Vλ4/5 (SEQ ID NO:61): ggtctctctcscagcytgtgctg.
5′L Vλ6(SEQ ID NO:62)：gttcttgggccaattttatgctg。5'L Vλ6 (SEQ ID NO:62): gttcttgggccaattttatgctg.
5′L Vλ7(SEQ ID NO:63)：ggtccaattcycaggctgtggtg。5'L Vλ7 (SEQ ID NO:63): ggtccaattcycaggctgtggtg.
5′L Vλ8/9/10(SEQ ID NO:64)：gagtggattctcagactgtggtg。5'L Vλ8/9/10 (SEQ ID NO:64):gagtggattctcagactgtggtg.
下游引物(R)：Downstream primer (R):
3′CK(SEQ ID NO:65)：tgctgtccttgctgtcctgct。3'CK (SEQ ID NO:65): tgctgtccttgctgtcctgct.
3′Cλ(SEQ ID NO:66)：caccagtgtggccttgttggcttg。3'Cλ (SEQ ID NO:66): caccagtgtggccttgttggcttg.
1.2构建轻链可变区噬菌体展示文库1.2 Construction of light chain variable region phage display library
1.2.1为文库克隆准备pATA-scFv-2载体。1.2.1 Prepare pATA-scFv-2 vector for library cloning.
1.2.2消化载体和PCR产物，消化载体和PCR产物的反应体系如表3所示。1.2.2 Digest vector and PCR product. The reaction system for digesting vector and PCR product is shown in Table 3.
表3table 3
1.2.3将消化后的载体和PCR产物进行连接，连接反应体系如表4所示；将配制的连接反应体系置于16℃孵育过夜，65℃加热灭活10min终止反应。1.2.3 Ligate the digested vector and PCR product. The ligation reaction system is shown in Table 4; incubate the prepared ligation reaction system at 16°C overnight, and heat inactivate at 65°C for 10 minutes to terminate the reaction.
表4Table 4
1.2.4将连接产物电转到感受态细胞中，电转的步骤如下所示：1.2.4 Electroporate the ligation product into competent cells. The electroporation steps are as follows:
1.2.4.1TG1感受态细胞的制备。1.2.4.1 Preparation of TG1 competent cells.
1.2.4.2 37℃预温1mL SOC培养基(Sigma，S1797)；将电穿孔比色皿(0.1厘米的间隙)和微离心管放在冰上(每个转换反应一个比色皿和一个微离心管)。1.2.4.2 Pre-warm 1mL SOC medium (Sigma, S1797) at 37°C; place electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge for each conversion reaction Tube).
1.2.4.3从-80℃的冰箱中取出电转感受态细胞，放在冰上直到它们完全融化(10-15min)；细胞解冻后，轻轻混匀；将50μL细胞放入在冰上的冷冻微离心管中。1.2.4.3 Take out the electroporated competent cells from the -80°C refrigerator and place them on ice until they are completely thawed (10-15 minutes); after thawing the cells, mix gently; put 50 μL of cells into a frozen microplate on ice. in a centrifuge tube.
1.2.4.4小心地将3μL的DNA混合物加入到冷冻的电穿孔比色皿中，不要产生气泡；用手腕快速向下轻弹试管，使细胞沉积在底部。1.2.4.4 Carefully add 3 μL of DNA mixture to the frozen electroporation cuvette without creating air bubbles; flick the tube downward quickly with your wrist to allow the cells to settle at the bottom.
1.2.4.5在600Ω、10μF和1.8kV下电穿孔；在脉冲的10s内，立即在每个试管中加入1mL预热的SOC培养基；37℃，250rpm摇晃1h。1.2.4.5 Electroporate at 600Ω, 10μF and 1.8kV; within 10s of the pulse, immediately add 1mL of preheated SOC culture medium to each test tube; shake at 37°C at 250rpm for 1h.
1.2.4.6收集所有的电转化培养基；连续稀释10μL培养物到90μL SOC培养基中，涂布于LB/Amp/Glucose(LB/氨苄青霉素/葡萄糖培养基)上。37℃孵育过夜；通过计数菌落数量，乘以培养体积，除以平板接种体积，计算转化子总数。1.2.4.6 Collect all electroconversion media; serially dilute 10 μL of culture into 90 μL SOC culture medium, and spread on LB/Amp/Glucose (LB/ampicillin/glucose medium). Incubate overnight at 37°C; calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the plate inoculation volume.
1.3构建VL-VH噬菌体展示文库1.3 Construction of VL-VH phage display library
1.3.1消化载体和PCR产物，消化反应体系如表5所示。1.3.1 Digest the vector and PCR product. The digestion reaction system is shown in Table 5.
表5table 5
1.3.2连接反应体系如表6所示，将配制的连接反应体系置于16℃孵育过夜，65℃加热灭活10min终止反应。1.3.2 The ligation reaction system is shown in Table 6. The prepared ligation reaction system was incubated at 16°C overnight, and then heated to inactivate at 65°C for 10 minutes to terminate the reaction.
表6Table 6
1.3.3将连接产物电转到感受态细胞中，电转的步骤如下所示：1.3.3 Electroporate the ligation product into competent cells. The electroporation steps are as follows:
1.3.3.1TG1感受态细胞的制备。1.3.3.1 Preparation of TG1 competent cells.
1.3.3.2 37℃预温4mL SOC培养基(Sigma，S1797)。将电穿孔比色皿(0.2厘米的间隙)和微离心管放在冰上(每个转换反应一个比色皿和一个微离心管)。1.3.3.2 Pre-warm 4mL SOC culture medium (Sigma, S1797) at 37°C. Place electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).
1.3.3.3从-80℃的冰箱中取出电转感受态细胞，放在冰上直到它们完全融化(10-15min)。细胞解冻后，轻轻混匀。1.3.3.3 Take out the electroporated competent cells from the -80°C refrigerator and place them on ice until they are completely melted (10-15 minutes). After the cells are thawed, mix gently.
1.3.3.4小心地将6μL的DNA混合物加入到冷冻的电穿孔比色皿中，不要产生气泡。用你的手腕快速向下轻弹试管，使细胞沉积在底部。1.3.3.4 Carefully add 6 μL of DNA mixture into the frozen electroporation cuvette without creating air bubbles. Use your wrist to flick the tube downward quickly to settle the cells at the bottom.
1.3.3.5在600Ω，10μF和2.5kV下电穿孔。在脉冲的10s内，立即在每个试管中加入2mL预热的SOC培养基。37℃，250rpm摇晃1h。1.3.3.5 Electroporate at 600Ω, 10μF and 2.5kV. Within 10 s of the pulse, immediately add 2 mL of prewarmed SOC medium to each tube. Shake at 37℃, 250rpm for 1h.
1.3.3.6收集所有的电转化培养基。连续稀释10μL培养物到90μL SOC培养基中，涂布于LB/Amp/Glucose(LB/氨苄青霉素/葡萄糖培养基)上。37℃孵育过夜。通过计数菌落数量，乘以培养体积，除以平板接种体积，计算转化子总数。1.3.3.6 Collect all electroconversion media. Serially dilute 10 μL of culture into 90 μL SOC medium and spread on LB/Amp/Glucose (LB/ampicillin/glucose medium). Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the plating volume.
1.4文库评估1.4 Library evaluation
1.4.1菌落PCR：以构建好的文库为模板进行PCR；PCR反应条件如表7所示。1.4.1 Colony PCR: Use the constructed library as a template to perform PCR; PCR reaction conditions are shown in Table 7.
表7Table 7
其中，变性，退火，延伸(1)这三个步骤，重复30次。PCR反应的引物序列如下所示：Among them, the three steps of denaturation, annealing, and extension (1) were repeated 30 times. The primer sequence for the PCR reaction is as follows:
上游引物(F)(SEQ ID NO:67)：agcggataacaatttcacacagga。Upstream primer (F) (SEQ ID NO:67): agcggataacaatttcacacagga.
下游引物(R)(SEQ ID NO:68)：gcccccttattagcgtttgccatc。Downstream primer (R) (SEQ ID NO:68): gccccccttattagcgtttgccatc.
PCR后琼脂糖凝胶电泳检测结果如图1-图3所示。VL噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图如图1所示，图1中，泳道M：DL2000；泳道1-16：21000076F pATA-Vκ，泳道17-32：21000076F pATA-Vλ。κH噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图如图2所示，图2中，泳道M：DL2000，泳道1-48：21000076F pATA-scFv-κH。λH噬菌体文库的单克隆菌PCR琼脂糖凝胶电泳图如图3所示，图3中，泳道M：DL2000，泳道1-48：21000076FpATA-scFv-λH。The results of agarose gel electrophoresis detection after PCR are shown in Figures 1-3. The monoclonal PCR agarose gel electrophoresis pattern of the VL phage library is shown in Figure 1. In Figure 1, lane M: DL2000; lanes 1-16: 21000076F pATA-Vκ, lanes 17-32: 21000076F pATA-Vλ. The monoclonal PCR agarose gel electrophoresis pattern of the κH phage library is shown in Figure 2. In Figure 2, lane M: DL2000, lanes 1-48: 21000076F pATA-scFv-κH. The single-clonal PCR agarose gel electrophoresis pattern of the λH phage library is shown in Figure 3. In Figure 3, lane M: DL2000, lanes 1-48: 21000076FpATA-scFv-λH.
1.4.2测序1.4.2 Sequencing
对噬菌体展示文库进行测序，挑选阳性克隆送到武汉擎科生物科技有限公司测序，噬菌体展示文库的测序质控结果如图4A和图4B所示。图4A为轻链文库的测序质控结果，图4B为重链文库的测序质控结果。从图4A和图4B中可知，文库序列覆盖了人源抗体大部分重链及轻链可变区的种系，文库多样性达到预计要求。The phage display library was sequenced, and positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing quality control results of the phage display library are shown in Figure 4A and Figure 4B. Figure 4A shows the sequencing quality control results of the light chain library, and Figure 4B shows the sequencing quality control results of the heavy chain library. It can be seen from Figure 4A and Figure 4B that the library sequences cover most of the heavy chain and light chain variable regions of human antibodies, and the library diversity meets the expected requirements.
1.5表达Dsg3蛋白1.5 Expression of Dsg3 protein
将人工合成的Dsg3结构域的基因重组到表达载体质粒pFastBac1中，得到Dsg3-pFastBac1表达载体。将Dsg3-pFastBac1表达载体转染到DH10Bac感受态中培养，纯化得到Dsg3抗原蛋白。人工合成Dsg3基因序列，将Dsg3基因重组到表达载体质粒pFastBac1中，得到Dsg3-pFastBac1表达载体；克隆位点BamHⅠ/XhoⅠ，Dsg3的氨基酸序列如SEQ ID NO:69所示：The synthetic Dsg3 domain gene was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured, and the Dsg3 antigen protein was purified. The Dsg3 gene sequence was artificially synthesized, and the Dsg3 gene was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector; the cloning site BamHI/XhoI, and the amino acid sequence of Dsg3 is shown in SEQ ID NO: 69:
EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEKDAVCSSSPSVVVSARTLNNRYTGPYTFALEDQPVKLPAVWSITTLNATSALLRAQEQIPPGVYHISLVLTDSQNNRCEMPRSLTLEVCQCDNRGICGTSYPTTSPGTRYGRPHSGR。EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDV NDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTG TVYVRVPDFNDNCPTAVLEKDAVCSSSPSVVVSARTLNNRYTGPYTFALEDQPVKLPAVWSITTLNATSALLRAQEQIPPGVYHISLVLTDSQNNRCEMPRSLTLEVCQCDNRGICGTSYPTTSPGTRYGRPHSGR.
将Dsg3-pFastBac1表达载体转染到DH10Bac感受态中培养，收集沉淀进行GST标签亲和层析得到Dsg3蛋白；纯化后的Dsg3还需要进行SDS-PAGE电泳(聚丙烯酰胺凝胶电泳)以验证其纯度，纯化后的Dsg3 SDS-PAGE电泳图如图5所示，蛋白大小64.95KDa，纯度大于90％。The Dsg3-pFastBac1 expression vector is transfected into DH10Bac competent cells and cultured, and the precipitate is collected and subjected to GST tag affinity chromatography to obtain the Dsg3 protein; the purified Dsg3 also needs to be subjected to SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis) to verify its Purity. The SDS-PAGE electrophoresis pattern of purified Dsg3 is shown in Figure 5. The protein size is 64.95KDa and the purity is greater than 90%.
实施例2筛选与Dsg3特异性结合的单克隆抗体Example 2 Screening of monoclonal antibodies that specifically bind to Dsg3
采用合适的淘选策略从噬菌体抗体库中富集所需的特异性抗体克隆。本实施例中使用的主要试剂如表8所示。Use an appropriate panning strategy to enrich the desired specific antibody clones from the phage antibody library. The main reagents used in this example are shown in Table 8.
表8Table 8
1、第一轮淘选1. First round of selection
1.1生物淘选1.1 Biopanning
1.1.1包被：包被离心管，4℃孵育过夜。抗原组：1mL Dsg3转染液(50μg/mL)，对照组：500μL 转染液(0μg/mL)。1.1.1 Coating: Coat the centrifuge tube and incubate at 4°C overnight. Antigen group: 1mL Dsg3 transfection solution (50μg/mL), control group: 500μL transfection solution (0μg/mL).
1.1.2洗涤：弃掉离心管中液体，用5mL 0.05％ PBST洗涤三遍。1.1.2 Washing: Discard the liquid in the centrifuge tube and wash three times with 5 mL 0.05% PBST.
1.1.3封闭：在管中加入5mL 5％脱脂牛奶或1％酪蛋白(PBST溶解)，37℃孵育2h。1.1.3 Blocking: Add 5mL of 5% skim milk or 1% casein (dissolved in PBST) to the tube and incubate at 37°C for 2 hours.
1.1.4洗涤：弃掉离心管中液体，用5mL 0.05％ PBST洗涤一遍。1.1.4 Washing: Discard the liquid in the centrifuge tube and wash once with 5 mL 0.05% PBST.
1.1.5孵育：用1％脱脂牛奶或0.2％酪蛋白(PBST溶解)稀释噬菌体文库，取1mL加入离心管中，32℃孵育2h。1.1.5 Incubation: Dilute the phage library with 1% skim milk or 0.2% casein (dissolved in PBST), add 1 mL to a centrifuge tube, and incubate at 32°C for 2 hours.
1.1.6洗涤：弃掉离心管中液体，用5mL 0.05％ PBST洗涤三遍，用PBS洗涤两遍。1.1.6 Washing: Discard the liquid in the centrifuge tube, wash three times with 5 mL 0.05% PBST, and wash twice with PBS.
1.1.7洗脱：用1mL的甘氨酸-盐酸(pH 2.2)洗脱与Dsg3结合的噬菌体，再用Tris-HCl中和至pH 7.0。1.1.7 Elution: Use 1 mL of glycine-hydrochloric acid (pH 2.2) to elute the phage bound to Dsg3, and then neutralize to pH 7.0 with Tris-HCl.
1.2测定稀释噬菌体的滴度1.2 Determine the titer of diluted phage
1.2.1用1mLDsg3转染液(50μg/mL)包被离心管，4℃孵育过夜。1.2.1 Coat the centrifuge tube with 1mL of Dsg3 transfection solution (50μg/mL) and incubate at 4°C overnight.
1.2.2弃掉离心管中液体，用5mL0.05％ PBST洗涤三遍。1.2.2 Discard the liquid in the centrifuge tube and wash three times with 5 mL 0.05% PBST.
1.2.3在管中加入5mL5％脱脂牛奶(PBST溶解)，37℃孵育2h。1.2.3 Add 5 mL of 5% skim milk (PBST dissolved) into the tube and incubate at 37°C for 2 hours.
1.2.4弃掉离心管中液体，用5mL 0.05％ PBST洗涤一遍。1.2.4 Discard the liquid in the centrifuge tube and wash once with 5 mL 0.05% PBST.
1.2.5将1.1.7的噬菌体转移至离心管中，32℃孵育2h。1.2.5 Transfer the phage from 1.1.7 to a centrifuge tube and incubate at 32°C for 2 hours.
1.2.6培养大肠杆菌TG1直到OD600＝0.4-0.6。1.2.6 Cultivate E. coli TG1 until OD600 =0.4-0.6.
1.2.7混合10μL稀释的洗脱后噬菌体和180μL大肠杆菌TG1。1.2.7 Mix 10 μL of diluted eluted phage and 180 μL of E. coli TG1.
1.2.8 37℃培养混合物30min，然后倒到2×YT-A(Amp 100μg/mL)培养基中，将培养基倒扣培养在37℃处过夜。1.2.8 Incubate the mixture at 37°C for 30 minutes, then pour it into 2×YT-A (Amp 100μg/mL) medium, and incubate the medium upside down at 37°C overnight.
1.3噬菌体文库扩增1.3 Phage library amplification
1.3.1将10μL E.coli TG1加到800μL 2YT培养液中，在37℃混合培养至OD600＝0.4-0.6。1.3.1 Add 10 μL E.coli TG1 to 800 μL 2YT culture medium, mix and culture at 37°C until OD600 = 0.4-0.6.
1.3.2将培养至对数期的TG1转入10mL 2YT-G培养液(终浓度2％葡萄糖)，在摇床上37℃培养至OD600＝0.4-0.6。1.3.2 Transfer TG1 cultured to logarithmic phase into 10 mL 2YT-G culture medium (final concentration 2% glucose), and culture on a shaking table at 37°C until OD600 = 0.4-0.6.
1.3.3加入洗脱后的产物，37℃孵育30min，37℃摇床培养30min。1.3.3 Add the eluted product, incubate at 37°C for 30 minutes, and incubate on a shaking table at 37°C for 30 minutes.
1.3.4加入30mL 2YT-AG培养液(终浓度0.1％ Amp，2％葡萄糖)，37℃摇床培养1h。1.3.4 Add 30 mL of 2YT-AG culture medium (final concentration 0.1% Amp, 2% glucose), and incubate on a shaking table at 37°C for 1 hour.
1.3.5加入M13KO7(M13KO7:TG1＝20:1)，37℃孵育30min，37℃摇床培养30min。1.3.5 Add M13KO7 (M13KO7:TG1=20:1), incubate at 37°C for 30 minutes, and incubate on a shaking table at 37°C for 30 minutes.
1.3.6菌液在5000rpm离心5min，用40mL 2YT-AK(终浓度Amp 100μg/mL，Kan 100μg/mL)重悬，30℃摇床孵育过夜。1.3.6 Centrifuge the bacterial solution at 5000 rpm for 5 minutes, resuspend in 40 mL of 2YT-AK (final concentration Amp 100 μg/mL, Kan 100 μg/mL), and incubate overnight on a 30°C shaker.
1.3.7 8000rpm离心10min，取出上清。上清液中加入1/5(v/v)PEG/NaCl，混合后置于冰上孵育2h。1.3.7 Centrifuge at 8000rpm for 10 minutes and remove the supernatant. Add 1/5 (v/v) PEG/NaCl to the supernatant, mix and incubate on ice for 2 hours.
1.3.8用1mL PBS重悬，12000rpm离心5min，将上清转移至新的1.5mL离心管。1.3.8 Resuspend in 1 mL PBS, centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.
1.4扩增后的噬菌体文库滴度测定，步骤同1.2。1.4 Determine the titer of the amplified phage library. The steps are the same as 1.2.
2、第二轮到第三轮淘选2. Second to third rounds of selection
2.1生物淘选2.1 Biopanning
循环重复步骤1两次，每次投入的噬菌体文库均用前一轮扩增后的洗脱噬菌体。生物淘选结果如表9所示。Repeat step 1 twice, and the phage library input each time uses the eluted phage after the previous round of amplification. The results of biopanning are shown in Table 9.
表9Table 9
从表9可知，经过三轮淘选，抗原组获得的噬菌体/投入噬菌体比例逐轮增加，说明噬菌体已有一定程度的富集。It can be seen from Table 9 that after three rounds of panning, the ratio of phages obtained from the antigen group/input phage increased each round, indicating that the phages have been enriched to a certain extent.
3、多克隆噬菌体ELISA3. Polyclonal phage ELISA
3.1包被：包被酶标板，4℃孵育过夜。抗原组：100μL/每孔Dsg3蛋白(4μg/mL)，对照组：100μL/每孔蛋白稀释液(0μg/mL)。3.1 Coating: Coat the enzyme plate and incubate at 4°C overnight. Antigen group: 100 μL/per well Dsg3 protein (4 μg/mL), control group: 100 μL/per well protein diluent (0 μg/mL).
3.2洗涤：弃掉酶标板中液体，每孔用300μL的0.05％ PBST洗涤三遍。3.2 Washing: Discard the liquid in the microplate, and wash each well three times with 300 μL of 0.05% PBST.
3.3封闭：每孔加入300μL的5％脱脂牛奶(PBS溶解)，37℃封闭2h。3.3 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.
3.4噬菌体孵育：每孔中加入100μL稀释后扩增噬菌体，如表10所示，32℃孵育2h。3.4 Phage incubation: Add 100 μL of diluted phage to each well, as shown in Table 10, and incubate at 32°C for 2 hours.
3.5洗涤：同步骤3.2。3.5 Washing: Same as step 3.2.
3.6二抗孵育：每孔加入100μL用封闭液稀释的anti-M13-HRP antibody(HRP标记的M13抗体)(1:6000)，32℃孵育1h。3.6 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution to each well, and incubate at 32°C for 1 hour.
3.7洗涤：同步骤3.2。3.7 Washing: Same as step 3.2.
3.8显色：每孔加100μL TMB，室温孵育，然后每孔加100μL 2M HCl终止反应。3.8 Color development: Add 100 μL TMB to each well, incubate at room temperature, then add 100 μL 2M HCl to each well to terminate the reaction.
3.9读板：使用酶标仪在450-630nm读取数值。多克隆噬菌体ELISA的结果如表10所示。3.9 Read the plate: Use a microplate reader to read the value at 450-630nm. The results of polyclonal phage ELISA are shown in Table 10.
表10Table 10
从表10可知，随着轮数增加，抗原组ELISA结果逐轮增大，说明阳性噬菌体逐轮富集，其中第二轮抗原组和对照组差距较大，故选用第二组的洗脱产物进行单克隆噬菌体ELISA筛选。It can be seen from Table 10 that as the number of rounds increases, the ELISA results of the antigen group increase round by round, indicating that positive phages are enriched round by round. Among them, the gap between the second round antigen group and the control group is large, so the elution product of the second group is selected. Perform monoclonal phage ELISA screening.
4、单克隆噬菌体ELISA(根据多克隆结果选用第二轮洗脱产物进行单克隆)4. Monoclonal phage ELISA (select the second round of elution products for single cloning based on the polyclonal results)
4.1从培养皿中选择96个克隆用于检测洗脱滴度；将这些克隆在37℃250rpm培养，直到OD600nm＝0.4-0.6。4.1 Select 96 clones from the culture dish for detection of elution titer; culture these clones at 37°C and 250 rpm until OD600nm = 0.4-0.6.
4.2M13KO7感染培养物(MOI＝20:1)，37℃孵育30min，37℃摇床培养30min；将菌液离心，并用等体积2×YT-AK(终浓度Amp 100μg/mL，Kan 100μg/mL)重悬沉淀，30℃培养过夜。4.2M13KO7 infection culture (MOI=20:1), incubate at 37°C for 30 minutes, and incubate on a shaking table at 37°C for 30 minutes; centrifuge the bacterial solution, and use an equal volume of 2×YT-AK (final concentration Amp 100μg/mL, Kan 100μg/mL ) Resuspend the pellet and incubate at 30°C overnight.
4.3将培养物离心，上清液可用于ELISA。4.3 Centrifuge the culture and the supernatant can be used for ELISA.
4.4包被：包被酶标板，4℃孵育过夜；抗原组：100μL/每孔Dsg3蛋白(4μg/mL)，对照组：100μL/每孔蛋白稀释液(0μg/mL)。4.4 Coating: Coat the enzyme plate and incubate at 4°C overnight; Antigen group: 100 μL/per well Dsg3 protein (4 μg/mL), control group: 100 μL/per well protein dilution (0 μg/mL).
4.5洗涤：弃掉酶标板中液体，每孔用300μL的0.05％ PBST洗涤三遍。4.5 Washing: Discard the liquid in the microplate, and wash each well three times with 300 μL of 0.05% PBST.
4.6封闭：每孔加入300μL的5％脱脂牛奶(PBS溶解)，37℃封闭2h。4.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.
4.7噬菌体孵育：每孔中加入100μL噬菌体上清，32℃孵育2h。4.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.
4.8洗涤：同步骤4.5。4.8 Washing: Same as step 4.5.
4.9二抗孵育：每孔加入100μL用封闭液稀释的anti-M13-HRP antibody(HRP标记的M13抗体)(1:6000)，32℃孵育1h。4.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution to each well, and incubate at 32°C for 1 hour.
4.10洗涤：同步骤4.5。4.10 Washing: Same as step 4.5.
4.11显色：每孔加100μL TMB，室温孵育，然后每孔加100μL 2M HCl终止反应。4.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, then add 100 μL 2M HCl to each well to terminate the reaction.
4.12读板：使用酶标仪在450-630nm读取数值，并对高特异性克隆进行测序。抗原组单克隆噬菌体ELISA的结果如表11所示。4.12 Read the plate: Use a microplate reader to read the values at 450-630nm and sequence the highly specific clones. The results of the antigen group monoclonal phage ELISA are shown in Table 11.
表11Table 11
对照组单克隆噬菌体ELISA的结果如表12所示。The results of the control monoclonal phage ELISA are shown in Table 12.
表12Table 12
从表11和表12的结果可知，G1，F2，E6，C9，F9克隆的抗原组与对照组的结果差距大于0.1，故将其5个克隆暂时认定为阳性克隆，进行二次验证。From the results in Table 11 and Table 12, it can be seen that the difference between the results of the antigen group of G1, F2, E6, C9, and F9 clones and the control group is greater than 0.1, so these 5 clones were temporarily identified as positive clones for secondary verification.
5、阳性克隆验证ELISA5. Positive clone verification ELISA
5.1将50μL阳性克隆加入2mL 2YT-AG培养基(终浓度0.1％ Amp，2％葡萄糖)中培养至OD600＝0.4-0.6。5.1 Add 50 μL of positive clones to 2 mL of 2YT-AG medium (final concentration 0.1% Amp, 2% glucose) and culture until OD600 = 0.4-0.6.
5.2M13KO7感染培养物(MOI＝20:1)，37℃孵育30min，37℃摇床培养30min。将菌液离心，并用等体积2×YT-AK(终浓度Amp 100μg/mL，Kan 100μg/mL)重悬沉淀，30℃培养过夜。5.2M13KO7 infected culture (MOI=20:1), incubate at 37°C for 30 minutes, and incubate on a shaking table at 37°C for 30 minutes. Centrifuge the bacterial solution, resuspend the pellet with an equal volume of 2×YT-AK (final concentration Amp 100 μg/mL, Kan 100 μg/mL), and culture at 30°C overnight.
5.3将培养物离心，上清液可用于ELISA。5.3 Centrifuge the culture and the supernatant can be used for ELISA.
5.4包被：包被酶标板，4℃孵育过夜。抗原组：100μL/每孔Dsg3蛋白(4μg/mL)，对照组：100μL/每孔蛋白稀释液(0μg/mL)。5.4 Coating: Coat the enzyme plate and incubate at 4°C overnight. Antigen group: 100 μL/per well Dsg3 protein (4 μg/mL), control group: 100 μL/per well protein diluent (0 μg/mL).
5.5洗涤：弃掉酶标板中液体，每孔用300μL的0.05％ PBST洗涤三遍。5.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.
5.6封闭：每孔加入300μL的5％脱脂牛奶(PBS溶解)，37℃封闭2h。5.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.
5.7噬菌体孵育：每孔中加入100μL噬菌体上清，32℃孵育2h。5.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.
5.8洗涤：同步骤4.5。5.8 Washing: Same as step 4.5.
5.9二抗孵育：每孔加入100μL用封闭液稀释的anti-M13-HRP antibody(HRP标记的M13抗体)(1:6000)，32℃孵育1h。5.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution to each well, and incubate at 32°C for 1 hour.
5.10洗涤：同步骤4.5。5.10 Washing: Same as step 4.5.
5.11显色：每孔加100μL TMB，室温孵育，然后每孔加100μL 2M HCl终止反应。5.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, then add 100 μL 2M HCl to each well to terminate the reaction.
5.12读板：使用酶标仪在450-630nm读取数值，并对高特异性克隆进行测序。阳性单克隆噬菌体ELISA的结果表13所示。5.12 Read the plate: Use a microplate reader to read the values at 450-630nm and sequence the highly specific clones. The results of positive monoclonal phage ELISA are shown in Table 13.
表13Table 13
从表13的结果可知，这5个阳性克隆的二次验证结果均为阳性。将阳性克隆送去测序，得到的高特异性抗体的序列。From the results in Table 13, it can be seen that the secondary verification results of these five positive clones were all positive. The positive clones were sent for sequencing to obtain the sequence of the highly specific antibody.
实施例3高特异性抗体的亲和力测定Example 3 Affinity determination of highly specific antibodies
本实施例对实施例2筛选得到的抗体进行亲和力测定，采用酶联免疫吸附反应检测不同稀释浓度条件下的OD值。酶联免疫吸附反应ELISA实验步骤如下所示：In this example, the affinity of the antibodies screened in Example 2 was measured, and enzyme-linked immunosorbent reaction was used to detect the OD values under different dilution concentrations. The experimental steps of enzyme-linked immunosorbent reaction ELISA are as follows:
1包被：100μL/每孔Dsg3蛋白(4μg/mL)包被酶标板，4℃孵育过夜。1 Coating: Coat the enzyme plate with 100 μL/well Dsg3 protein (4 μg/mL) and incubate at 4°C overnight.
2洗涤：弃掉酶标板中液体，每孔用300μL的0.05％ PBST洗涤三遍。2 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.
3封闭：每孔加入300μL的5％脱脂牛奶(PBS溶解)，37℃封闭2h。3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.
4阳性抗体孵育：将76F-DSG3-IC-R2P1-G1、76F-DSG3-IC-R2P1-F2、76F-DSG3-IC-R2P1-E6、76F-DSG3-IC-R2P1-C9、76F-DSG3-IC-R2P1-H9抗体分别进行梯度稀释，稀释梯度为(4000ng/mL、2000ng/mL、1000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.6ng/mL、7.8ng/mL、3.9ng/mL、1.95ng/mL、0.98ng/mL、0.49ng/mL和0.25ng/mL，每孔中加入100μL稀释后的抗体溶液，37℃孵育1h。4 Positive antibody incubation: 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, 76F-DSG3- The IC-R2P1-H9 antibodies were gradient diluted respectively, and the dilution gradient was (4000ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.6 ng/mL, 7.8ng/mL, 3.9ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL and 0.25ng/mL, add 100 μL of diluted antibody solution to each well, and incubate at 37°C for 1 hour.
5洗涤：同步骤4.5。5. Washing: Same as step 4.5.
6二抗孵育：用封闭液10000倍稀释Goat Anti-Human IgG(H+L)antibody(羊抗人IgG二抗)(Jackson，code：109-035-088)，每孔加入100μL稀释的二抗，37℃孵育30min。6 Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Goat anti-human IgG secondary antibody) (Jackson, code: 109-035-088) 10,000 times with blocking solution, and add 100 μL of diluted secondary antibody to each well. , incubate at 37°C for 30 minutes.
7洗涤：同步骤4.5。7 Washing: Same as step 4.5.
8显色：每孔加100μL TMB，37℃孵育10min，然后每孔加50μL 2M HCl终止反应。8 Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 min, then add 50 μL 2M HCl to each well to terminate the reaction.
9读板：使用酶标仪在450-630nm读取数值。9. Read the plate: Use a microplate reader to read the value at 450-630nm.
酶联免疫吸附反应的结果如图6A-6E所示。图6A为76F-DSG3-IC-R2P1-G1抗体与Dsg3的ELISA检测结果，图6B为76F-DSG3-IC-R2P1-F2抗体与Dsg3的ELISA检测结果，图6C为76F-DSG3-IC-R2P1-E6抗体与Dsg3的ELISA检测结果，图6D为76F-DSG3-IC-R2P1-C9抗体与Dsg3的ELISA检测结果，图6E为76F-DSG3-IC-R2P1-H9抗体与Dsg3的ELISA检测结果。从6A-6E可知，所筛选的5种人源性抗Dsg3单克隆抗体与Dsg3蛋白均具有较强的特异性结合的能力，所述抗体能作为定性检测PV阳性的参考标准，也能用于定量检测PV患者中抗Dsg3自身抗体水平。The results of enzyme-linked immunosorbent reaction are shown in Figures 6A-6E. Figure 6A shows the ELISA detection results of 76F-DSG3-IC-R2P1-G1 antibody and Dsg3. Figure 6B shows the ELISA detection results of 76F-DSG3-IC-R2P1-F2 antibody and Dsg3. Figure 6C shows the ELISA detection results of 76F-DSG3-IC-R2P1. The ELISA detection results of -E6 antibody and Dsg3, Figure 6D shows the ELISA detection results of 76F-DSG3-IC-R2P1-C9 antibody and Dsg3, and Figure 6E shows the ELISA detection results of 76F-DSG3-IC-R2P1-H9 antibody and Dsg3. It can be seen from 6A-6E that the five screened human anti-Dsg3 monoclonal antibodies have strong specific binding ability to Dsg3 protein. The antibodies can be used as reference standards for qualitative detection of PV positivity and can also be used for Quantitative detection of anti-Dsg3 autoantibody levels in PV patients.
综上，本发明以Dsg3抗体阳性患者PBMC构建噬菌体人源抗体库，通过与Dsg3特异性结合，从而筛选出特异性结合Dsg3抗原的单克隆抗体。所述单克隆抗体活性高、稳定性好，且具有较强的特异性，能作为定性检测PV阳性的参考标准，也能定量检测PV患者中抗Dsg3自身抗体水平，从而监测病情活动度，可用于PV患者的临床诊断。本发明的研究为特异性治疗PV提供了理论与实验依据，同时也为治疗以自身抗体为主要致病机制的自身免疫病提供了一个新的研究方向。In summary, the present invention uses PBMC of Dsg3 antibody-positive patients to construct a phage human antibody library, and by specifically binding to Dsg3, monoclonal antibodies that specifically bind to the Dsg3 antigen are screened. The monoclonal antibody has high activity, good stability, and strong specificity. It can be used as a reference standard for qualitative detection of PV positivity. It can also quantitatively detect anti-Dsg3 autoantibody levels in PV patients, thereby monitoring disease activity. It can be used for the clinical diagnosis of PV patients. The research of the present invention provides theoretical and experimental basis for the specific treatment of PV, and also provides a new research direction for the treatment of autoimmune diseases in which autoantibodies are the main pathogenic mechanism.
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。The applicant declares that the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the technical field should understand that any person skilled in the technical field will not use the invention disclosed in the present invention. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of the present invention.
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| US20140079691A1 (en)* | 2012-09-20 | 2014-03-20 | Anaptysbio, Inc. | Thermostable antibody framework regions |
| MA49911A (en)* | 2017-11-01 | 2020-06-24 | Juno Therapeutics Inc | ANTIBODIES AND CHEMERICAL ANTIGENIC RECEPTORS SPECIFIC TO THE B-LYMPHOCYTE MATURATION ANTIGEN |
| CN111606994A (en)* | 2020-06-08 | 2020-09-01 | 湖南远泰生物技术有限公司 | Preparation method of non-fusion type anti-desmoglein DSG3 rabbit monoclonal antibody |
| AR123997A1 (en)* | 2020-11-04 | 2023-02-01 | Univ Rockefeller | NEUTRALIZING ANTIBODIES AGAINST SARS-CoV-2 |
| CN114152753B (en)* | 2021-11-12 | 2024-03-05 | 钱华 | ELISA kit for detecting human Dsg3 IgG antibody and application thereof |
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