pcDNA-GPC3真核表达载体的构建及鉴定

Construction of recombinant eukaryotic plasmid pcDNA-GPC3 and identification

目的构建人类GPC3重组真核表达载体。方法以人类肝脏cDNA文库为模板扩增出GPC3基因序列,应用T克隆载体及pcDNA真核表达载体重构,将质粒转化进入HLE人肝癌细胞系,进行荧光免疫方法检测。结果 测序结果序列正确,荧光染色转染质粒的HLE细胞可见细胞浆和细胞膜上有GPC3的表达。结论成功构建真核表达载体pcDNA-GPC3,并在真核细胞中可以表达。

Objective To construct the eukaryotic expression vector of the human GPC3 gene. Methods To get the human GPC3 cDNA from human liver cDNA library,and then inserted the fragment of GPC3 into pMD --18 simple T vector. The amplified products were cloned into the eukaryotic expression vector peDNA 4.1. The recombinant plasmid was transfected into HLE cells,and analyzed by immunoflurescence method. Results The recombinant plasmid was identified to be right for ORF by restriction enzyme analysis and DNA sequencing. The green fluorescence could be seen in transfected HLE cell membrane and cytoplasm under fluorescence microscope. Conclusion To construct the recombinant eukaryotic expression vector and express GPC3 protein in HLE cells successfully.