大肠杆菌偏嗜性人胰岛素样生长因子1基因的克隆及表达  

Cloning and Expression of E.coli Favorite Human IGF-1 Gene

作　　者：梁东春[1] 左爱军[1] 郭刚[1] 张镜宇[1] 

机构地区：[1]天津医科大学内分泌研究所,300070

出　　处：《天津医药》2006年第2期76-78,共3页Tianjin Medical Journal

基　　金：天津市自然科学基金重点项目(项目编号:033801611)

摘　　要：目的:提高以大肠杆菌为工程菌制备重组人胰岛素样生长因子1(rhIGF-1)的产率。方法:依据大肠杆菌遗传密码子使用频率表,人工合成3条DNA单链,PCR拼接扩增出适于在大肠杆菌中表达的人IGF-1基因。将此基因克隆入pGEM-T载体进行DNA序列分析后亚克隆入表达载体pBV220,诱导表达,表达产物行聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,酶联免疫分析(ELISA)检测表达产物中rhIGF-1的含量。结果:经基因改造后,rhIGF-1主要以包涵体的形式存在于碎菌后的沉淀中,其产率较改造前提高近4倍。结论:所构建的大肠杆菌偏嗜性人IGF-1基因可显著提高rhIGF-1的产率。Objective：The study is to increase the rate of production of rhlGF-1 using E.coli as engineering bacterium. Methods：Three single strand DNA fragments were synthesized according to codon-application frequency of E.coli, and a E.coli favorite human IGF-1 gene was reconstructed by linking these DNA fragments together. The reconstructed human IGF- 1 gene was cloned into plasmid pGEM-T and verified by DNA sequence analysis. Thereafter this fIGF-1 gene was subeloned into plasmid pBV220. Recombinant plasmid pBV-fIGF-1 was transformed into E.coli.DH5a and induced expression by increasing temperature to 42℃. Expression product was analysed by SDS-PAGE. The quantity of rhIGF-1 was determined by ELISA and the rate of production was calculated. Results： When using fIGF-1 gene to produce rhlGF-1 its product-efficiency was almost four times than that of control. Conclusion： It was useful to increase the yield of recombinant protein by changing the eukaryotic gene to E.coli favorite.

关 键 词：大肠杆菌 受体 IGF1型 克隆 分子 基因表达 

分 类 号：R587.101[医药卫生—内分泌] R739.41[医药卫生—内科学]