七带石斑鱼类胰岛素生长因子-Ⅰ成熟肽的克隆及原核表达与活性分析

Prokaryotic expression and activity analysis of insulin-like growth factor-Ⅰ from Epinephelus septemfasciatus

应用RT-PCR方法扩增七带石斑鱼类胰岛素生长因子-Ⅰ(esIGF-Ⅰ)成熟肽序列。该成熟肽序列由210个碱基组成,编码70个氨基酸,包括B-C-A-D 4个结构域。将此成熟肽片段导入原核表达载体pET-28a上,在IPTG诱导下成功在E.coli BL21(DE3)中融合表达。SDS-PAGE分析表明,融合蛋白大小为11 ku,在IPTG诱导后3 h表达量最高,占菌体总蛋白的51.8%,重组蛋白主要以包涵体形式存在。对重组蛋白进行变性、纯化和复性,获得了纯化的重组蛋白。Western-blotting免疫印迹分析表明,融合蛋白可特异性地被6×His抗体识别。细胞增殖实验表明,纯化的IGF-Ⅰ融合蛋白能促使人乳腺癌细胞MDA231细胞增殖,表明具有生物活性。

In order to study the underlying molecular mechanisms for growth of Epinephelus septemfasciatus,the insulin-like growth factorⅠ（IGF-Ⅰ）mature peptide sequence was amplified from E.septemfasciatus liver with RT-PCR method.It was predicted that the mature peptide was composed of 210 base pairs,which encodes 70 amino acid residues and consists of B-C-A-D four functional domains.The matured peptide fragment was subcloned into the expression vector pET-28a and was successfully expressed in E.coli BL21（DE3）cell.The result of SDS-PAGE analysis indicated that the fusion protein expressed in the form of inclusion bodies with molecular weight of 11 ku and maximally amounted to 51.8% of the whole protein in the E.coli cell 3 hours after being induced with IPTG.The purified recombinant protein was obtained through denaturation,purification and refolding.The Western-blotting indicated that the recombinant protein had specifically been recognized by 6×His antibody.The proliferation experiment showed that the purified IGF-Ⅰ fusion protein could significantly promote the proliferation of breast cancer cells MDA231,and this indicated that it has biological activity.