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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。
In order to recognize the physiological functions of IGF-Ⅱ at the protein level, the IGF-Ⅱ mature peptide sequence was cloned into the prokaryotic expression vector p ET-28a and the recombinant plasmid pSIFII-IGF-Ⅱ / p ET28a The recombinant IGF-Ⅱ protein of 11.4 kDa in size was induced by IPTG after it was introduced into E.coli BL21 (DE3) strain. The recombinant IGF-Ⅱ protein contained 6 histidine at its N-terminus and was specifically recognized by 6 × His antibody. Recombinant IGF-Ⅱprotein was induced at 37 ℃ for 2 h under optimal inducing conditions, and the target protein accounted for 43.7% of the total recombinant protein. The recombinant protein mainly existed as inclusion body. The obtained recombinant protein inclusion body was denatured, purified and refolded to obtain the purified IGF-Ⅱ recombinant protein, which can significantly promote the proliferation of human breast cancer MDA231 cells in vitro, indicating that the recombinant protein IGF-Ⅱ has the function of in vitro cell level Biological activity. The results of this study can provide theoretical support for understanding the physiological function of fish IGF-Ⅱ and the regulation mechanism of growth and development of the tongue sole.