人结合珠蛋白cDNA克隆及其在大肠杆菌中的表达和鉴定  

Cloning, Expression and Identification of Human Hp cDNA in Escherichia Coli

作　　者：孟琴[1] 汤伟松[1] 刘亮[1] 李淑珍[1] 唐晓波[1] 

机构地区：[1]哈尔滨医科大学药学院,黑龙江哈尔滨150081

出　　处：《现代生物医学进展》2014年第24期4644-4647,共4页Progress in Modern Biomedicine

基　　金：哈尔滨市科技创新人才研究专项资金项目(2008RFXXS018)

摘　　要：目的:克隆人结合珠蛋白(haptoglobin,Hp)cDNA,并在大肠杆菌中表达和鉴定。方法:从Hela细胞中分离总RNA,采用RT-PCR方法获得人Hp cDNA,分别克隆至原核表达载体pET-32a和PGEX-4T-1,转化至大肠杆菌BL21,IPTG诱导表达,并进行SDS-PAGE及Western blot鉴定。结果:成功构建了高效原核表达质粒PET-32a-Hp和PGEX-4T1-Hp;Western印迹结果表明,经IPTG诱导,在大肠杆菌中表达了分子量约30 kD和37 kD的目的蛋白;表达产物经Ni2+-NTA离子交换树脂纯化,纯度>90%。结论:在E.coli成功表达和纯化了人Hp融合蛋白,为进一步开发人Hp诊断试剂打下基础。Objective： To clone, express and identificate human Haptoglobin cDNA in Escherichia coli（E. coli. Methods： Human Hp cDNA was obtained from total RNA isolated from Hela cells by reverse transcription PCR method, then it was inserted into prokaryotic expression vectors pET-32a and PGEX-4T-1 for the IPTG-induced expression in E. coli BL21, and it was identified by SDS-PAGE and Western blot. Results： The expression plasmid pET-32a-Hp and PGEX-4T-1-Hp were constructed successfully. Western blot analysis showed that fusion protein pET-32a-Hp with 30kD molecular weight and PGEX-4T-1-Hp with 37kD expressed in E. coll. The purification of fusion protein was more than 90% after purification using Ni2＋-NTA ion exchange resin. Conclusion： Fusion protein human Hp was successfully expressed in E.coli and purified, which settled a foundation for further development of human Hp diagnostic reagents.

关 键 词：卵巢癌 人结合珠蛋白 克隆 表达 纯化 

分 类 号：Q93-33[生物学—微生物学] Q75