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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。
A murine monoclonal antibody specifically recognizing human 4IgB7-H3 was developed and its biological characteristics and the expression characteristics of 4IgB7-H3 molecules were analyzed. 293T cells highly expressing human 4IgB7-H3 were used as immunogens to routinely immunize mice, cell fusion and screening. L929 / 4IgB7-H3 cells were screened for positive cells, L929 / 2IgB7-H3 was screened for negative cells. The hybridoma cells were repeatedly screened and subcloned by indirect immunofluorescence staining and flow cytometry to obtain 1 strain Hybridoma cell line 9C3 capable of stably secreting a murine monoclonal antibody that specifically recognizes human 4IgB7-H3. The site-specific experiments were carried out to identify the specificity of the antibody epitope recognition. Then the monoclonal antibody was used to detect the expression characteristics of 4IgB7-H3 on tumor cell lines and immune cells by flow cytometry. The results show that 4IgB7-H3 is expressed at very limited levels in some tumor cell lines, human peripheral blood mononuclear cells and up-regulated expression in mature dendritic cells at the protein level. Whereas 2IgB7-H3 molecules are widely expressed in various tumor cell lines, DCs are continuously expressed in monocytes and in different stages of differentiation. This is in agreement with the previous study revealed that the expression of 4IgB7-H3 at the mRNA level is much higher than that of 2IgB7-H3, suggesting that 2IgB7-H3 may be the main form of B7-H3 playing a biological role. In view of this, the successful obtaining of 4IgB7-H3 monoclonal antibody 9C3 provided a new experimental method for further analysis of the expression characteristics and biological functions of human B7-H3.