摘要
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GPC3C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GPC3C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 determine the auxiliary antitumor activity of seven anti-glypican-3 （GPC3） C-terminal monoelonal antibodies（mAb） and to identify epitopes specifically recognized by these mAb. Methods： The anti- body-dependent cell-mediated cytotoxity（ADCC） effect of the seven mAb against liver carcinoma HepG2 cell line was detected by cell proliferation method. The structure and the antigenic characteristics of 359~580 aa of GPC3 C-terminal were analyzed by biological information software Goldkey. According to the analysis, GPC3 C-terminal was divided into four fragments and their coding genes were cloned into the prokaryotic expression vector pGEX-4T-1 respectively. After these fragments were expressed and purified, they were used to detect epitope spe- cifically identified by those mAb by indirect ELISA and Western blot. Results： The results of MTT showed that all the mAb exhibited different ADCC activity against liver carcinoma HepG2 cell line and the 5tb mAb was most effective of the seven mAb. Four recombinant fragments containing possible epitopes were expressed and purified, in which the 473～525 aa fragment was recognized specifically by all the seven mAb as indicated by ELISA and Western blot. Conclusion： All the mAb have auxiliary antitumor activity and 473～525 aa region of GPC3 C-termi- nal was identified as their binding epitope.
出处
《生物技术通讯》
CAS
2013年第1期13-18,共6页
Letters in Biotechnology
基金
国家自然科学基金青年基金(30900753)
