一株特异性识别B7-H3杂交瘤单克隆抗体Y4F11的分析和鉴定  

Identification and characterization of monoclonal antibody Y4F11 against human B7-H3

作　　者：时正朋 胡玉敏[1] 陈翰卿[1] 傅丰庆[2] 张学光[2,3] SHI Zhengpeng HU Yumin CHEN Hanqing FU Fengqing ZHNAG Xueguang(College of Biology and Basic Medical Science, Soochow University Jiangsu Provincial Institute of Clinical Immunology, First Affiliated Hospital, Soochow University Jiangsu Provincial Key Laboratory of Stem Cell and Biomedical Material, Soochow University, Suzhou 215000, China)

机构地区：[1]苏州大学基础医学与生物科学学院 [2]苏州大学附属第一医院江苏省临床免疫研究所 [3]江苏省干细胞与生物医用材料重点实验室,江苏苏州215000

出　　处：《细胞与分子免疫学杂志》2016年第10期1402-1406,1411,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31170834)

摘　　要：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 generate mouse anti-human monoclonal antibodies against the molecules expressed on immune cells from malignant pleural effusions,and identify the antigen recognized by the monoclonal antibody named Y4F11.Methods The cells separated from pleural effusions of lung cancer patients were used to immunize BALB / c mice. Hybridoma technology was used for cell fusion and screening; the monoclonal antibody named Y4F11 was chosen as the object for the following experiment. Flow cytometry was performed to analyze the molecules expressed on nave and activated T,B,NK cells and monocytes; Co-immunoprecipitation was conducted to get the antigen pulled down by Y4F11. Mass spectrometry sequencing and BLASTP were used to identify the antigen molecules. Western blotting,dot-blotting and flow cytometry were utilized to identify Y4F11. Results The study obtained 78 hybridoma clones secreting antibodies stably which could recognize surface molecules on cells from pleural effusions. The hybridoma named Y4F11 was chosen as the following experiment object for further identification. The molecule which Y4F11 recognized was about 50 Kd,and further was confirmed as B7-H3 molecule. Furthermore,Y4F11 could recognize B7-H3 expressed on B7-H3 gene-transferred cells as well as B7-H3 fusion protein. Besides,the expression pattern of B7-H3 on immune cells was confirmed by Y4F11 antibody. All these results indicated that the antigen molecule recognized by Y4F11 antibody was B7-H3. Conclusion A hybridoma cel line with stable expression of monoclonal antibody B7-H3 has been successful y obtained and could be used for biological function study on B7-H3.

关 键 词：恶性胸水 B7-H3 单克隆抗体 质谱测序 

分 类 号：Q813.2[生物学—生物工程] R392.11[医药卫生—免疫学]