γδT细胞抗原识别机制研究—含γδTCR CDR3δ序列嵌合抗体的构建及其抗原结合特异性和SARS相关冠状病毒B细胞抗原

发布时间：2018-05-31 05:33

本文选题：细胞 + 抗原　；参考：《中国协和医科大学》2005年博士论文

【摘要】：本论文分两部分：第一部分为γδT细胞抗原识别机制的研究。γδT细胞认为是天然免疫和获得性免疫之间的桥梁，广泛地参与到抗感染免疫、肿瘤免疫和自身免疫等各个方面。但迄今为止，只有少数的γδT细胞受体(T cell receptor，TCR)所识别的配体被鉴定，γδTCR抗原识别的具体细节尚不清楚。对三种抗原受体的结构和序列多样性的研究结果提示，αβTCR、γδTCR和抗体的识别抗原特异性主要决定于其互补决定区(complementarity determining regions，，CDRs)；γδTCR在抗原识别上与αβTCR相差甚多而与抗体更类似，被认为是以“抗体类似”的方式与抗原结合。γδTCR和抗体整体结构比较以及δ链和抗体重链的CDR3长度和氨基酸多样性的比较提示，γδTCR的δ链和抗体重链在识别抗原中的作用类似；CDR3δ是γδTCR与抗原结合的重要部位。本课题组的前期工作证明，来源于卵巢癌的γδ肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes，TIL)δ链CDR3短肽OT3能特异性地结合肿瘤组织、肿瘤细胞系及其蛋白提取物和应激分子。为了进一步阐明γδTCR抗原识别的机制，本研究利用PCR搭桥技术，将抗体重链CDR3区用被OT3短肽的相应基因序列来替换，构建成嵌合抗体重链的重组基因片段，克隆到表达载体pNγ1中，转染到真核细胞中进行表达；筛选并大量培养阳性表达细胞以获得含γδTCR CDR3δ的嵌合抗体。利用流式细胞、激光共聚焦显微镜和表面等离子体共振(surface plasmon resonance，SPR)等技术对嵌合抗体的抗原结合特异性进行了研究，比较嵌合抗体与合成的OT3短肽在抗原识别特异性方面的差别，以阐明γδTCR抗原识别的机制及其结构基础。结果表明，含γδTCR CDR3δ的嵌合抗体可以与人卵巢癌细胞系SKOV3、HO8910和Hela等细胞及其蛋白提取物结合，与其他人类肿瘤细胞系及其蛋白提取物不结合或结合较弱，提示嵌合抗体的抗原结合性
[Abstract]:This thesis is divided into two parts: the first part is the study of the mechanism of 纬未 T cell antigen recognition. 纬未 T cells are considered as a bridge between innate immunity and acquired immunity, which are widely involved in anti-infective immunity, tumor immunity and autoimmunity. However, so far, only a few ligands recognized by 纬未 T cell receptor T cell receptor have been identified, and the specific details of 纬未 TCR antigen recognition are not clear. The results showed that the specificity of 伪尾 TCR, 纬未 TCR and antibodies was mainly determined by their complementary determining regions, and 纬未 TCR was more similar to 伪尾 TCR than 伪尾 TCR in antigen recognition. It is considered that it binds to antigen in an "antibody similar" manner. The comparison of the structure of 纬未 TCR and antibody, and the comparison of CDR3 length and amino acid diversity of 未 chain and antibody heavy chain suggest that the 未 chain and antibody heavy chain of 纬未 TCR are recognized in antigen. Similar to CDR3 未, CDR3 未 is an important site of binding of 纬未 TCR to antigen. Our previous work demonstrated that the 未 chain CDR3 short peptide OT3 of 纬未 tumor infiltrating lymphocytes derived from ovarian cancer can specifically bind tumor tissues, tumor cell lines and their protein extracts and stress molecules. In order to further elucidate the mechanism of 纬未 TCR antigen recognition, the heavy chain CDR3 region of antibody was replaced by the corresponding gene sequence of OT3 short peptide, and cloned into the expression vector pN 纬 1. After transfection into eukaryotic cells, the chimeric antibody containing 纬未 TCR CDR3 未 was obtained by screening and culturing the positive cells. The antigen-binding specificity of chimeric antibodies was studied by flow cytometry, laser confocal microscopy and surface plasmon resonance spectroscopy (SPR), and the differences between chimeric antibodies and synthetic OT3 short peptides in antigen recognition were compared. To elucidate the mechanism of 纬未 TCR antigen recognition and its structural basis. The results showed that the chimeric antibody containing 纬未 TCR CDR3 未 could bind to human ovarian cancer cell lines SKOV3HO8910 and Hela and its protein extracts, but not to other human tumor cell lines and their protein extracts, indicating the antigenicity of chimeric antibodies.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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