半滑舌鳎(Cynoglossus semilaevis Günther)IGF-Ⅱ的体外重组表达  

In vitro Recombinant Expression of Insulin-Like Factor Ⅱ from Cynoglossus semilaevis Günther

作　　者：徐永江[1] 柳学周[1] 张凯 武宁宁[3] 刘芝亮[1] 李春广 

机构地区：[1]农业部海洋渔业可持续发展重点实验室中国水产科学研究院黄海水产研究所,青岛266071 [2]青岛贝宝海洋科技有限公司,青岛266400 [3]青岛市海洋与渔业局渔业技术推广站,青岛266071 [4]绍兴市鸿港农业开发有限公司,绍兴312000

出　　处：《渔业科学进展》2015年第4期51-56,共6页Progress in Fishery Sciences

基　　金：鲆鲽类现代产业技术体系(CARS-50);山东省自然科学基金项目(ZR2012CQ025);中央级公益性事业单位基本科研业务费项目(20603022012022);绍兴市院校科技合作项目(2012704)共同资助

摘　　要：为在蛋白水平认识半滑舌鳎类胰岛素生长因子Ⅱ(IGF-Ⅱ)的生理功能,将IGF-Ⅱ成熟肽序列克隆到原核表达载体p ET-28a中,成功构建了重组半滑舌鳎IGF-Ⅱ/p ET28a质粒,导入到E.coli BL21(DE3)菌株后经IPTG诱导,获得了大小为11.4 k Da的重组IGF-Ⅱ蛋白,N端含6个组氨酸,可特异性地被6×His抗体识别。重组IGF-Ⅱ蛋白在最优诱导条件37℃诱导2 h,目的蛋白表达量占重组表达菌总蛋白的43.7%,重组蛋白主要以包涵体存在。将获得的重组蛋白包涵体经变性、纯化和复性后,获得了纯化的IGF-Ⅱ重组蛋白,其可在体外显著促进人乳腺癌MDA231细胞的增殖,表明IGF-Ⅱ重组蛋白具有体外细胞水平的生物活性。本研究结果可为认识鱼类IGF-Ⅱ生理功能及半滑舌鳎生长调控机制提供理论支撑。To explore the role of insulin-like factor Ⅱ（IGF-Ⅱ） in growth regulation of Cynoglossus semilaevis Günther,the IGF-Ⅱ gene was expressed in vitro and the bioactivity was determined by methyl thiazolyl tetrazolium（MTT method）.The mature peptide domain of IGF-Ⅱ gene of C.semilaevis Günther was cloned by PCR amplification and sequenced for verification.The obtained mature peptide fragment was then subcloned into the prokaryotic expression vector p ET-28a（IGF-Ⅱ/p ET28a）.The recombinant plasmid was expressed in E.coli BL21（DE3） cells and the recombinant IGF-Ⅱ protein containing 6×His tag at N-terminus was induced by IPTG.SDS-PAGE analysis indicated that the obtained IGF-Ⅱ protein was found in the form of inclusion bodies with molecular weight of 11.4 k Da,which accounted for 43.7% of the whole bacterial protein post 2-hour induction with IPTG.Western blotting analysis indicated that the recombinant IGF-Ⅱ protein had the antigenicity to 6×His antibody.The inclusion bodies containing recombinant protein were denaturalized,purified and annealed.The recombinant IGF-Ⅱ protein significantly promoted the proliferation of human breast cancer cells MDA231.Results could provide basic information on the role of IGF-Ⅱ in fish and be helpful to better understand the endocrine mechanism of sex-based dimorphic growth performance of C.semilaevis Günther.

关 键 词：半滑舌鳎 IGF-Ⅱ 原核表达 生物活性 

分 类 号：S961[农业科学—水产养殖]