星突江鲽胰岛素样生长因子I的体外重组表达及生物活性分析

Prokaryotic expression and bioactivity analysis of growth hormone and insulin-like growth factor-I from Platichthys stellatus

为了在蛋白水平认识星突江鲽(Platichthys stellatus)胰岛素样生长因子I(IGF-I)的生长调控作用及机制,采用RT-PCR方法扩增了其成熟肽片段,利用原核表达载体p ET-28a成功构建了重组星突江鲽IGFI/p ET-28a质粒,转化至大肠杆菌BL21(DE3)后经IPTG诱导获得了N端含6个组氨酸的重组星突江鲽IGFI蛋白。获得的重组IGF-I蛋白大小为12.1 k D,37℃下用0.5 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)诱导3 h时目的蛋白表达量最高,占菌体总蛋白的39.8%,主要以包涵体形式存在。Western blotting免疫印迹表明星突江鲽IGF-I重组蛋白均可被6×His抗体特异性识别。包涵体经6 mol/L盐酸胍变性、Ni2+离子亲和柱纯化和尿素梯度复性后,可获得高纯度的IGF-I重组蛋白。细胞增殖试验结果显示0.6μg/m L的星突江鲽IGF-I重组蛋白能显著促进人胚胎肾细胞HEK293T的增殖而大于1.8μg/m L时则表现出抑制作用。本研究成功构建了星突江鲽IGF-I体外高效表达系统,并获得具有细胞水平生物活性的星突江鲽IGF-I重组蛋白,结果可为深入探究IGF-I在星突江鲽生长发育中的作用机制及研制高效绿色的促生长制剂提供基础资料。

The mature peptide domain of insulin-like growth factor-I from Platichthys stella-tus were amplified with specific primers based on its cDNA sequence. Then the matured peptide fragment was subcloned into the prokaryotic expression vector pET-28a to successfully con-struct IGF-I/pET-28a recombinant plasmid which were highly expressed in E.coli BL21（DE3） after being induced by IPTG with special fusion polypeptides containing His6 at their N-termi-nus. The obtained IGF-I polypeptide expressed in form of inclusion bodies with molecular weight of 12.1 kD and maximally accounted for 39.8%of the whole bacterial protein post 3 h induction with 0.5 mmol/L IPTG at 37℃. Western blotting analysis indicated fusion polypep-tides had the antigenicity to His6 antibody. The inclusion bodies were denaturalized using 6 mol/L guanidine HCl,purified using Ni-NTA affinity chromatography and annealed by gradient dialy-sis in urea,then purified proteins with molecular weight of 12.1 kD which was obtained from IGF-I recombinant bacterium. The proliferation experiment showed recombinant IGF-I protein could significantly promote the proliferation of human embryonic kidney cells HEK293T at 0.6 μg/mL and inhibit the proliferation with 1.8 μg/mL which verified its biological activity. Therefore,the IGF-I prokaryotic expression system was successfully constructed in the present study and biologically active IGF-I fusion protein was obtained. The present results would be helpful for better understanding the roles of IGF-I in growth regulation and development of high effective additive for growth promotion of Platichthys stellatus.