人胰高血糖素样肽-1突变体基因的克隆及表达

Expression and Cloning of Human Glucagon-like Peptide-1 Mutant Gene

目的 :克隆人胰高血糖素样肽 - 1突变体 (2 Gly -hGLP - 1)基因 ,高效表达GST - 2 Gly -hGLP - 1融合蛋白。方法 :在获得重组hGLP - 1基因工程菌基础上 ,利用定点突变技术改造其第 2位丙氨酸为甘氨酸 ,经酶切克隆于pGEM - 7z(+)载体中 ,构建pGEM - 7z(+) / 2 Gly -hGLP - 1克隆载体 ,双酶切鉴定后把2 Gly-hGLP - 1基因定向插入pGEX - 4T - 3多克隆位点 ,构建pGEX- 4T - 3/ 2 Gly-hGLP - 1融合表达载体 ,转化大肠杆菌BL2 1(DE3)并进行诱导表达。结果 :经双酶切鉴定和测序分析 ,证明2 Gly-hGLP - 1基因已克隆到pGEX - 4T - 3载体中。SDS -PAGE和凝胶扫描分析 ,融合蛋白以可溶形式存在 ,其表达量占菌体总蛋白的 2 9.7%。表达产物经亲和层析纯化后纯度在 95 %以上 ,免疫印迹证实 ,该融合蛋白可被特异性hGLP - 1(7- 37)抗体所识别。结论 :为产业化规模制备hGLP -

Objective:To clone the gene of hGLP-1 mutant and express the GST-2Gly-hGLP-1 fusion protein.Methods:Using recombinant hGLP-1expression system,the 2nd alanine was changed to be glycine by means of site-directed mutagenesis.The mutant gene was cloned to the plasmid vector pGEM-7z(+) after appropriate cleavage with restrictive endonuclease and then inserted into the MCS of the fusion expression vector pGEX-4T-3.The recombinant plasmid was transformed into E.coli.BL21(DE3) and induced by IPTG.Results:The recombinant plasmid DNA was digested with BamHⅠand XhoⅠand hGLP-1 gene mutation was testified by DNA sequencing.The results demonstrated that the mutated 2Gly-hGLP-1 was successfully inserted into the pGEX-4T-3 vector.SDS-PAGE and scan analysis showed the fusion protein GST-2Gly-hGLP-1 was expressed in a soluble form and amounted to 29.7% of the total bacterial protein.The purity of GST-2Gly-hGLP-1 was more than 95% through affinity chromatography.Western blot analysis of fusion protein confirmed that it could be specially recognized by the antibody of hGLP-1(7-37).Conclusion:This study established foundation for further obtaining a larger quantity of recombinant hGLP-1 mutant for experimental and clinic studies.