人胰高血糖素样肽-1 cDNA的克隆与表达

Cloning and expression of human glucagon-like peptide-1 cDNA

目的 :克隆人胰高血糖素样肽 - 1(h GL P- 1) c DNA,构建原核表达质粒 p GEX- 4T- 3/ h GL P- 1c DNA,并诱导表达谷胱甘肽巯基转移酶 - h GL P- 1(GST- h GL P- 1)融合蛋白。方法 :采用亚磷酸二酯法合成 6个 h GL P- 1c DNA的寡核苷酸片段 ,拼接成完整的 h GL P- 1c DNA ,克隆至 p BS SK(+ / - )载体中 ,经双酶切鉴定和测序后把 h GL P- 1c DNA定向插入融合基因表达载体 p GEX- 4T- 3的多克隆位点上 ,构建重组质粒 p GEX- 4T- 3/ h GL P- 1c DNA,并转化大肠杆菌 TG1。 结果 :经限制性内切酶Bam H 、Xho 酶切和电泳 ,证明 h GL P- 1c DNA已插入到 p GEX- 4T- 3中并诱导表达出融合蛋白。结论 :成功诱导表达出融合蛋白 GST- h GL P- 1,为进一步获得大量可供实验研究和临床应用的重组 h GL P- 1创造条件。

Objective: To clone hGLP 1 cDNA in the pBS SK(+/-)vector and construct the expression vector of pGEX 4T 3/hGLP 1cDNA to express GST hGLP 1 fusion protein. Methods: The hGLP 1 cDNA was constructed by 6 synthetic oligonucleotides fragments, followed by the procedure of annealing and ligation with oligonucleotides fragments. The hGLP 1 cDNA was cloned into the pBS SK(+/-) vector, and was selected by α complementation. It was confirmed by DNA sequening, then inserted into the MCS of the fusion expression vector pGEX 4T 3. The recombinant vector was transformed into E. coli TG1. Results: The recombinant plasmid DNA was digested with restrictive endonuclease BamHⅠand XhoⅠ. The result demonstrated that the hGLP 1 cDNA was successfully inserted into the pGEX 4T 3 vector and fusion protein GST hGLP 1 had been expressed in SDS PAGE. Conclusion: Expression of GST hGLP 1 fusion protein can provide foundation for obtaining a larger quantity of recombinant hGLP 1 for experimental and clinic studies. [