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

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

中文关键词: 半滑舌鳎  IGF-Ⅱ  原核表达  生物活性

英文关键词: Cynoglossus semilaevis Günther  IGF-Ⅱ  Prokaryotic expression  Bioactivity

中文摘要:

      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pET-28a中，成功构建了重组半滑舌鳎IGF-Ⅱ/pET28a质粒，导入到E. coli BL21(DE3)菌株后经IPTG诱导，获得了大小为11.4 kDa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。

英文摘要:

      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 pET-28a (IGF-Ⅱ/pET28a). 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 kDa, 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.