人胰岛素样生长因子在pET-DsbA载体中的克隆及表达  

Cloning and Expression of Recombinant Human IGF-Ⅰ Gene in pET-DsbA

作　　者：陈德珩 李体远 朱莹 刘争红 毕晓 黄瑞芳 戴勇 

机构地区：[1]暨南大学医学院第二附属医院-深圳市人民医院,广东深圳518020

出　　处：《北京生物医学工程》2006年第4期420-422,444,共4页Beijing Biomedical Engineering

基　　金：深圳市科技局课题(20034013)资助

摘　　要：目的通过重组技术,对胰岛素样生长因子IGF-Ⅰ基因进行缺失、突变以获得最有利于高水平表达的IGF-ⅠcDNA。构建原核表达载体,并进行表达,以获得能够高水平表达高活性产物的基因工程菌珠。方法提取人肝脏组织总RNA,RT-PCR后琼脂糖电泳初步鉴定产物。将cDNA回收、补平后插入克隆质粒KS,酶切鉴定后,对IGF-Ⅰ基因进行序列分析。采用PCR法从克隆质粒中扩增IGF-Ⅰ片段,亚克隆至pET-DsbA原核表达载体,酶切鉴定,并进行序列测定。转化到大肠杆菌BL21(DE3)PLysS中,IPTG诱导表达,聚丙稀酰胺凝胶电泳、WesternBlot分析目的蛋白。结果成功构建原核表达载体pET-DsbA-IGF-Ⅰ,测序结果与预期序列完全一致,并在大肠杆菌BL21(DE3)plysS表达出IGF-Ⅰ融合蛋白。结论IGF-Ⅰ融合蛋白在大肠杆菌BL21(DE3)PLysS中的表达,对进一步研究IGF-Ⅰ的功能及开发其生物制品具有重要意义。Objective To clone the human insulin like growth factor Ⅰ gene, construct its expression vector and expression it in E. coli. Method The human insulin like growth factor Ⅰ was amplified from human liver cDNA by RT-PCR and then inserted into the KS vector. IGF-Ⅰ was inserted into pET-DsbA. The recombinant plasmid was identified by enzyme digestion, sequencing and expressed in E. coli. Result IGF-Ⅰ gene in plasmid pET-DsbA-IGF-Ⅰ was successfully expressed in BL21 （DE3） plysS. Proteins were identified by SDS - PAGE and Western Blot. Conclusion The recombinant IGF-Ⅰ protein expressed with pET-DsbA-IGF-Ⅰ could be used for the development of its biological products.

关 键 词：胰岛素样生长因子 融合蛋白 表达 

分 类 号：Q78[生物学—分子生物学]