磷脂酰肌醇蛋白聚糖3真核表达载体的构建及在细胞中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2016.05.019

生物技术通报 ›› 2016, Vol. 32 ›› Issue (5): 146-150.doi: 10.13560/j.cnki.biotech.bull.1985.2016.05.019

磷脂酰肌醇蛋白聚糖3真核表达载体的构建及在细胞中的表达

程华¹, 方向东², 崔硕¹, 吴萌¹, 张昭军², 李泽夏²

1.河北省科学院生物研究所，石家庄 050081;
2.中国科学院北京基因组研究所，北京 100101

收稿日期:2015-07-28 出版日期:2016-05-25 发布日期:2016-05-27
作者简介:程华，男，副研究员，研究方向：肿瘤早期诊断及相关机理；E-mail：cheng_hua@sina.com
基金资助:
河北省科学院高层次人才资助项目（20150503LR62-9），河北省科学院科技计划项目（13335）

Construction of Eukaryotic Expression Vector for Glypican-3 and Its Expression in Cell

CHENG Hua¹,FANG Xiang-dong²,CUI Shuo¹,WU Meng¹,ZHANG Zhao-jun²,LI Ze-xia²

1. Institute of Biology，Hebei Academy of Sciences，Shijiazhuang 050081;
2. Beijing Institute of Genomics，Chinese Academy of Sciences，Beijing 100101

Received:2015-07-28 Published:2016-05-25 Online:2016-05-27

摘要/Abstract

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。

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

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.

Key words: glypican-3, eukaryotic expression, liposome transfection, affinity purification

程华, 方向东, 崔硕, 吴萌, 张昭军, 李泽夏. 磷脂酰肌醇蛋白聚糖3真核表达载体的构建及在细胞中的表达[J]. 生物技术通报, 2016, 32(5): 146-150.

CHENG Hua,FANG Xiang-dong,CUI Shuo,WU Meng,ZHANG Zhao-jun,LI Ze-xia. Construction of Eukaryotic Expression Vector for Glypican-3 and Its Expression in Cell[J]. Biotechnology Bulletin, 2016, 32(5): 146-150.

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磷脂酰肌醇蛋白聚糖3真核表达载体的构建及在细胞中的表达

Construction of Eukaryotic Expression Vector for Glypican-3 and Its Expression in Cell

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