人胰高血糖素样肽 期刊论文.doc

人胰高血糖素样肽-1 突变体基因原核表达质粒的构建 作者：朱清 顾云，陆伟，刘梅【摘要】 目的：对人胰高血糖素样肽-1（hGLP-1）进行基因突变，构建 GST融合蛋白原核表达质粒 pGEX-4T/（Val8）hGLP-1，并在大肠杆菌中诱导表达。方法：选择大肠杆菌偏爱密码子，将 hGLP-1（7～37）第 2 位的丙氨酸（Ala8）定点突变为缬氨酸（Val8） 直接生物合成 4 个寡聚核苷酸片段并进行基因拼接， ，形成完整的 hGLP-1 突变体基因（Val8）hGLP-1，并连接到载体 pGEX-4T-1，转化大肠杆菌 BL21，BamH I 与 Xho I 双酶切电泳鉴定与序列测定，然后诱导表达其融合蛋白。结果：经酶切电泳鉴定与测序分析，证实所合成的突变体基因（Val8）hGLP-1 已克隆到 pGEX-4T-1 中，经 SDS分析可以诱导表达出融合蛋白。结论：成功地构建了 GST 融合蛋白原核表达质粒 pGEX-4T/（Val8）hGLP-1，为进一步获得重组 hGLP-1 蛋白奠定基础，为产业化规模制备 hGLP-1突变体提供实验依据。【关键词】 人胰高血糖素样肽-1；融合蛋白；基因突变；克隆；糖尿病 Construction of expression vector of human Glucagon-likepeptide-1 mutant gene Abstract Objective: To obtain the mutant gene of humanGlucagon-like peptide-1hGLP-1construct the expression vector ofpGEX-4T/ （Val8）hGLP-1and express the GST fusion protein in E.coli cells.Methods: With the preferential codon of E.coli being selected fouroligonucleotide fragments were directly biosynthesized then werespliced to form a complete mutant gene of hGLP-1 which changed thecodon for the second amino acid of hGLP-1737-alanine Ala8 intovaline Val8 by means of site-directed mutagenesis so it named（Val8）hGLP-1. The mutant gene was inserted into the vector pGEX-4T-1 withBamH I and Xho I restriction sites.The recombinant plasmid of pGEX-4T/（Val8） hGLP-1 was transformed into E. coli cellsBL21then identified byelectrophoresis after enzyme digestion and testified by DNAsequencing further induced to express the fusion protein by IPTG.Results: The results of electrophoretic analysis and DNA sequencingconfirmed that the synthetic mutant gene Val8 hGLP-1 had beencloned into the pGEX-4T-1 successfully and induced expression offusion protein by SDSanalysis. Conclusion: The GST fusion proteinexpression plasmid of pGEX-4T / Val8 hGLP-1 was successfullyconstructed ， which laid the foundation for further obtainingrecombinant hGLP-1 mutant for experimental or clinical studies andprovided the technical routes of industrialization. Key words Human Glucagon-like peptide-1 Fusion proteinGene mutation