人胰升血糖素样肽1突变体基因的克隆及在原核细胞的表达

Cloning of human glucagon-like peptide-1 mutant gene and its expression in E.coli cells

目的构建人胰升血糖素样肽1(hGLP-1)突变体基因的原核表达载体,并在大肠杆菌BL21中进行表达。方法用生物合成方法获得hGLP-1突变体基因8Val-hGLP-1,将其克隆于载体pGEX-4T-1中,构建pGEX-4T-1/8Val-hGLP-1融合表达载体,转化大肠杆菌BL21,酶切电泳以及测序鉴定阳性克隆,用SDS-PAGE和蛋白免疫印迹法检测其重组蛋白诱导表达情况。结果重组质粒pGEX-4T-1/8Val-hGLP-1经双酶切电泳鉴定与测序分析证实构建成功,SDS-PAGE与蛋白免疫印迹分析证实其成功诱导表达重组融合蛋白。结论成功地克隆了hGLP-1突变体基因8Val-hGLP-1,并在大肠杆菌中进行表达,为下一步获得含突变体基因的重组hGLP-1蛋白及其生物学功能研究奠定良好基础。

Objective To construct the prokaryotic expression vector of human glucagon-like peptide-1（hGLP-1） mutant gene,and express it in E.coli cells.Methods The mutant gene of hGLP-1（^8Val-hGLP-1） was obtained by biosynthesis technique.The mutant gene was cloned into the vector pGEX-4T-1 to construct the expression vector of pGEX-4T-1/^8Val-hGLP-1.The recombinant plasmid was transformed into E.coli cells（BL21）,and then the positive clone was identified by electrophoresis after enzyme digestion and DNA sequencing.The expression of fusion protein induced by IPTG was analyzed by SDS-PAGE and Western blot.Results Electrophoretic analysis and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1/^8Val-hGLP-1 had been constructed successfully,and the expression of recombinant fusion protein in E.coli cells was displayed by SDS-PAGE and testified by Western blot analysis.Conclusion The mutant gene ^8Val-hGLP-1 has been successfully cloned and expressed in E.coli cells,which may provide the foundation for further obtaining recombinant hGLP-1 mutant and experimental studies on its biological function.