一株特异性识别4IgB7-H3单克隆抗体的研制及其生物学特性的初步分析  

Preparation of a monoclonal antibody against human 4IgB7-H3 molecule and analysis of its biological characteristics

作　　者：何娜[1] 周迎会[2,1] 张光波[3] 陈永井[2] 王雪峰[2,1] 邱天宇[1] 顾文超[1] 崔浩杰[4] 张学光[2,3] 

机构地区：[1]苏州大学医学部生物化学与分子生物学系,苏州215123 [2]苏州大学医学生物技术研究所,苏州215007 [3]苏州大学附属第一医院临床免疫重点实验室,苏州215006 [4]苏州大学附属第一医院整形烧伤外科,苏州215006

出　　处：《现代免疫学》2009年第6期447-452,共6页Current Immunology

基　　金：国家自然科学基金资助项目(30771958)

摘　　要：研制特异性识别人4IgB7-H3的鼠单克隆抗体,并对其生物学特性和4IgB7-H3分子的表达特性进行初步分析。以高表达人4IgB7-H3的293T细胞为免疫原,常规免疫小鼠、细胞融合和筛选。L929/4IgB7-H3细胞为抗体筛选阳性细胞,L929/2IgB7-H3为抗体筛选阴性细胞,经间接免疫荧光标记和流式细胞术对杂交瘤细胞进行反复筛选和多次亚克隆化,获得l株能稳定分泌特异性识别人4IgB7-H3的鼠单克隆抗体的杂交瘤细胞株9C3。经位点竞争实验对该抗体抗原表位的识别特异性进行了鉴定。继而利用该单抗,采用流式细胞术检测4IgB7-H3在肿瘤细胞株和免疫细胞上的表达特性。结果表明,蛋白水平上4IgB7-H3非常有限地表达在一些肿瘤细胞株,人外周血单核细胞以及上调性表达在成熟的树突状细胞。而2IgB7-H3分子广泛地表达在各种肿瘤细胞株、持续性表达在单核细胞和分化不同阶段的DC。这与先前研究所揭示的4IgB7-H3在mRNA水平的表达远高于2IgB7-H3不相一致,提示2IgB7-H3分子可能是B7-H3发挥生物学功能的主要形式。鉴此,4IgB7-H3单克隆抗体9C3的成功获得为进一步分析人B7-H3两种剪切体的表达特性及生物学功能提供了新的实验手段。To prepare functional monoclonal antibodies against human 4IgB7-H3 molecule and to analyze their biological characteristics, female BALB/c mice of 6-8 weeks old were immunized with human 293T cell as immunogen. The splenic B cells of mice were fused with SP2/0 and the hybridoma cells were screened with L929/4IgB7-H3 and L929/2IgB7-H3 transfected cells by FCM. One hybridoma cell line named 9C3 was obtained, which had the property to secrete anti-human 4IgB7 H3 molecule antibody continuously and steadily. The specificity of antibodies was investigated by cell immunofluorescence and competitive inhibition test. It showed that this mAb specifically recognized the human 4IgB7-H3 transfected cells but not the 2IgB7-H3 transfected cells. Furthermore, FCM was used to analyze the expression patterns of B7-H3 on different human cell lines. The experi mental data demonstrated that the 4IgB7-H3 molecule was not the major pattern concerned and its expression was limited although the earlier studies indicated that the expression of 4IgB7-H3 was higher than that of 2IgB7-H3 on mRNA level. These results indicate that 2IgB7-H3 may be the major functional molecule. Our study gives a clue to recognize the expression patterns and biological function of B7-H3 molecule.

关 键 词：4IgB7-H3 2IgB7-H3 单克隆抗体 表达分析 

分 类 号：R392.1[医药卫生—免疫学]