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.
出处
《天津医药》
CAS
北大核心
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)
关键词
大肠杆菌
受体
IGF1型
克隆
分子
基因表达
escherichia coli
receptor, IGF type 1
cloning, molecular
gene expression
