含肝细胞生长因子(HGF)红色荧光融合蛋白基因质粒的构建

Construction of the Plasmid Encoding the Fusion Protein of Hepatocyte Growth Factor and Red Fluorescent Protein

为了更真实准确地标示真核细胞内外源肝细胞生长因子(Hepatocyte growth factor,HGF)的表达,构建含HGF红色荧光融合蛋白(Red fluorescent protein,RFP)基因的质粒。实验中以pMD19-T-HGF为模板,PCR扩增得到去除了终止密码子的HGF基因全长序列,并定向插入pDsRed-express-N1质粒。重组质粒用限制性内切酶酶切和DNA序列测定鉴定后转染293T细胞,观察细胞中HGF mRNA及其蛋白和红色荧光蛋白的表达。结果表明pDsRed-HGF质粒中所插入目的基因大小及其序列与GenBank中HGF cDNA序列完全一致,并可在293T细胞中高水平表达。本实验成功构建了在真核细胞中表达的pDsRed-HGF重组质粒。

Abstract In order to trace the expression of exogenous hepatocyte growth factor （HGF） in eukaryotie cells exactly, a recombinant plasmid that expresses the fusion protein of HGF and red fluorescent protein（RFP） was constructed. The gene encoding HGF without stop code was amplified from pMD19-T-HGF by PCR technique and then cloned in pDsRed-express-N1. The recombinant plasmid （pDsRed-HGF） was identified by restriction endonuclease enzyme analysis and DNA sequence analysis, pDsRed-HGF was transfected into 293T cells. The expression of HGF mRNA, HGF and red fluorescent protein was detected. The results showed that the target gene sequence in pDsRed- HGF was completely in conformity with HGF eDNA in GenBank, and it was expressed highly in 293T cells. In this study, pDsRed-HGF was successfully constructed and expressed in eukaryotic cells.