天津医药

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癌胚抗原磷脂酰肌醇蛋白聚糖3单克隆抗体的制备及应用

王玉亮,刘涛   

  1. 天津市第一中心医院
  • 收稿日期:2013-09-29 修回日期:2013-12-03 出版日期:2014-03-15 发布日期:2014-03-15
  • 通讯作者: 王玉亮

Preparation of monoclonal antibody against carcinoembryonic antigen Glypican-3 and its preliminary application

yuliang wang,Tao LIU   

  • Received:2013-09-29 Revised:2013-12-03 Published:2014-03-15 Online:2014-03-15
  • Contact: yuliang wang

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摘要： 目的 制备抗癌胚抗原磷脂酰肌醇蛋白聚糖3（GPC3）单克隆抗体，并初步应用。方法 利用PCR方法，将GPC3基因重组到原核表达载体pET16b中，在大肠杆菌中表达，纯化后的GPC3融合蛋白对Balb/c小鼠脾内包埋免疫，取小鼠脾细胞与SP2/0细胞进行细胞融合，应用间接ELISA和Western blot进行筛选及鉴定分泌特异性抗GPC3单克隆抗体的杂交瘤细胞株，间接免疫荧光检测肝癌细胞中GPC3表达。结果 成功构建了GPC3原核表达载体，在大肠杆菌中的诱导表达重组GPC3蛋白。应用单克隆抗体杂交瘤技术成功制备1株稳定分泌抗GPC3单克隆抗体的杂交瘤细胞株， 通过Western blot 鉴定具有高度的特异性，间接免疫荧光实验显示单克隆抗体能与HepG2细胞内GPC3特异性结合。结论 成功制备出特异性抗人GPC3单克隆抗体。

关键词: 磷脂酰肌醇蛋白聚糖3, 包埋免疫, 单克隆抗体

Abstract: Objective To prepare monoclonal antibody specifically against carcinoembryonic antigen Glypican-3 (GPC3) and preliminary application. Methods GPC3 was cloned with PCR to pET16b vector and expressed in E. coliBL21. Spleen cells were obtained from Balb/c mice embedding immunized with purified antigen intraperitoneally, and fused with SP2/0 cells. Hybridoma cells were screened by indirect ELISA, and identified by Western blot using purified protein after the cell fusion. Indirect immunofluorescence method was used to test the GPC3 expression in HepG2 cell line. Results The prokaryotic expression vector of GPC3 was successfully constructed, and GPC3 was stablely expressed in E. coliBL21. A mice hybridoma cell line producing monoclonal antibody to GPC3 was obtained. Western blot analysis showed that monoclonal antibody specifically recognized recombinant protein. Monoclonal antibody could be used to detect GPC3 protein expression in HepG2 cell line by indirect immunofluorescence. Conclusion Monoclonal antibody against GPC3 was obtained. The research will be useful for its clinical study.

Key words: Glypican-3, Embedding immunology, Monoclonal antibody