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目的:检测制备的7株抗磷脂酰肌醇蛋白聚糖3(GPC3)蛋白C端单克隆抗体是否具有辅助杀伤肝癌细胞的活性,并研究其识别的抗原表位。方法:用细胞增殖法检测制备的抗体是否具有抗体依赖细胞介导的细胞毒性(ADCC)活性;用生物信息软件分析GPC3蛋白C端(359~580残基)的结构及抗原特征,并据此将其分为4个截短片段,将克隆的各基因片段分别连接到原核表达载体pGEX-4T-1中,进行蛋白表达和纯化,用间接ELISA和Western印迹分析GPC3 C端单克隆抗体的表位识别情况。结果与结论:制备的7株单克隆抗体对肝癌细胞HepG2均具有不同程度的辅助杀伤作用,其中5号单克隆抗体的辅助杀伤效果最好;表达并纯化了GPC3 C端4个截短片段的重组蛋白;间接ELISA和Western印迹检测结果表明,7株抗体均特异性结合GPC3蛋白的473~525残基区段。
OBJECTIVE: To test whether the monoclonal antibodies against C-terminal 3 of anti-glypican 3 (GPC3) have the activity of assisting the killing of hepatoma cells and to study the epitopes recognized by them. Methods: The cell proliferation assay was used to detect the antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The bioinformatics software was used to analyze the structure and antigenic characteristics of the C-terminal of GPC3 protein (359 ~ 580 residues) The fragments were divided into four truncated fragments. The cloned gene fragments were respectively ligated into the prokaryotic expression vector pGEX-4T-1 for protein expression and purification. The expression of GPC3 C-terminal monoclonal antibodies was analyzed by indirect ELISA and Western blot Bit recognition situation. RESULTS AND CONCLUSION: Seven monoclonal antibodies against HepG2 cells had different degree of auxiliary killing effect, and the No. 5 monoclonal antibody had the best auxiliary killing effect. The expression of 4 C-terminal truncated fragments of GPC3 The results of indirect ELISA and Western blot showed that the seven antibodies all specifically bind to the 473-525 residue segment of GPC3 protein.