摘要
用PCR方法扩增到抵抗素基因(RSTN)并将其亚克隆至pET-32a(+)表达载体,获得重组质粒pET-RSTN。将重组质粒转化大肠杆菌BL-21(DE3)感受态细胞,用IPTG诱导表达。SDS-PAGE检测结果表明,重组resistin蛋白分子量大小约30kDa。对表达条件如温度、IPTG浓度及诱导时间进行优化并用SDS-PAGE检测。结果表明,30℃、4h、IPTG浓度为1mmol/L时,可溶性重组resistin的含量最高。表达产物经Western blot检测证实是Resistin蛋白,并用镍离子亲和层析的方法获得纯化的Resistin蛋白。
Resistin is a newly discovered adipocyte hormone. With the method of gene engineering, resistin was got. The RSTN gene was subcloned to the expression vector pET-32a（ ＋ ） and the recombined plasmid named pET-RSTN was constructed successfully. The plasmid pET-RSTN were transformed into the E. coll. BL-21 （ DE3 ）. After inducing by IPTG the fusion protein of resistin were expressed and its molecular weight was about 30kDa. As temperature, IPTG concentration and induction time were optimized, the soluble recombinant protein was high expressed at 30℃ for 4h with 1mmol/L IPTG induction. The product was identified by SDS-PAGE and Western-blotting. The expression production purified on Ni^2＋-NTA column.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第5期16-20,共5页
China Biotechnology
关键词
抵抗素
蛋白表达
纯化
Resistin Protein expression Purification
