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目的研制小鼠抗人恶性胸水细胞表面分子的单克隆抗体(m Ab),并对其中Y4F11识别的抗原进行鉴定和验证。方法以胸水细胞为免疫原,常规免疫BALB/c小鼠、采用杂交瘤技术进行细胞融合和筛选阳性杂交瘤克隆;选取Y4F11为实验对象,采用流式细胞术分析其在初始和活化免疫细胞(T细胞、B细胞、NK细胞和单核细胞)上的结合情况;通过免疫共沉淀法获得与Y4F11特异性结合的条带、割胶、蛋白质谱测序后拼接出肽段,并进行BLAST分析比对;采用Western blot法、Dot-blot法及流式细胞术验证Y4F11与B7同源物3(B7-H3)的抗体抗原关系。结果获得78株能持续分泌识别恶性胸水细胞表面表达分子的抗体的杂交瘤细胞株,其中Y4F11与胸水细胞结合最高;Y4F11特异识别的条带相对分子质量(Mr)大小约为50 000,质谱结果经BLAST比对为B7-H3分子;继而的结合实验显示,Y4F11可以与B7-H3的转基因细胞结合,也可以与B7-H3融合蛋白有效地结合,B7-H3在免疫细胞表面表达的模式与Y4F11抗体在这些细胞上的结合一致,表明Y4F11识别的抗原分子为B7-H3。结论成功获得1株持续表达B7-H3 m Ab的杂交瘤细胞株Y4F11,为进一步研究分析B7-H3的生物学功能提供了物质基础。
Objective To develop a monoclonal antibody (m Ab) for mouse anti-human malignant pleural effusion cell surface molecules and to identify and validate the antigen recognized by Y4F11. Methods The pleural effusion cells were used as immunogen to immunize BALB / c mice routinely. The hybridoma technique was used to fuse the cells and screen the positive hybridoma clones. Y4F11 was selected as the experimental subject and analyzed by flow cytometry in primary and activated immune cells T cells, B cells, NK cells and monocytes); Y4F11-specific bands were obtained by co-immunoprecipitation; the peptides were excised and sequenced; the protein fragments were sequenced and compared by BLAST analysis Western blot, Dot-blot and flow cytometry were used to verify the antigen-antibody relationship of Y4F11 and B7-H3. Results 78 strains of hybridoma cell lines that could consistently secrete antibodies that recognize the surface of malignant pleural effusions were obtained. Y4F11 had the highest binding to pleural effusion cells. The relative molecular mass of Y4F11 was about 50,000, The B7-H3 molecules were BLAST-aligned. The binding experiments showed that Y4F11 could bind to B7-H3 transgenic cells and effectively bind to B7-H3 fusion protein. The pattern of B7-H3 expression on immune cells was similar to that of B7- The binding of the Y4F11 antibody on these cells was consistent, indicating that the antigen molecule recognized by Y4F11 is B7-H3. Conclusion A hybridoma cell line Y4F11 expressing B7-H3 m Ab was successfully obtained, which provided the material basis for further study on the biological function of B7-H3.