Biotechnology Bulletin ›› 2016, Vol. 32 ›› Issue (5): 146-150.doi: 10.13560/j.cnki.biotech.bull.1985.2016.05.019

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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. 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 Online:2016-05-25 Published:2016-05-27

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