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作 者 ：程华, 方向东, 崔硕, 吴萌, 张昭军, 李泽夏

期 刊 ：生物技术通报 2016年 32卷 5期 页码：146-150

关键词：磷脂酰肌醇蛋白聚糖3；真核表达；脂质体转染；亲和纯化；

Keywords:glypican-3, eukaryotic expression, liposome transfection, affinity purification,

摘 要 ：构建载体表达人磷脂酰肌醇蛋白聚糖3（Glypican-3，GPC3）蛋白，用以获得抗GPC3单克隆抗体。用PCR技术扩增GPC3基因，利用酶切位点将该序列插入p3XFLAG-CMV-14载体，构建pCMV-gpc3表达载体。通过脂质体将该载体转染至HEK293细胞中，利用Western blot技术检测最终结果。收集稳定表达的细胞，破碎，通过亲和层析柱，获得纯度较高的GPC3蛋白。成功构建pCMV-gpc3真核表达载体，转染至HEK293细胞后，经G418筛选获得稳定表达的单克隆细胞系；Western blot分析结果表明目的蛋白高效表达。

Abstract:This work aims to construct the eukaryotic expression vector of glypican-3（GPC3）for acquiring anti-GPC3 monoclonal antibody. The GPC3 gene was amplified by PCR and was cloned into an expression vector p3XFLAG-CMV-14，and the expression vector pCMV-gpc3 was constructed. HEK293 cells were transfected with the vector by liposome，and the final results were detected by Western blot. The stably-expressed cells were collected and ground，and the high-purity GPC3 protein was obtained by affinity column. The eukaryotic expression vector of pCMV-gpc3 was constructed successfully. After the transfection to HEK293 cells，the monoclonal cell line of stably-expressed was gained by G418 screening. Western blot analysis revealed that the target protein expressed markedly. A large number of GPC3 proteins were obtained via genetic engineering and purifying technology，which laid a foundation for the biological function and application of the protein as well as the preparation of monoclonal antibody.

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