星突江鲽胰岛素样生长因子II的原核表达与生物活性分析  

Prokaryotic expression and bioactivity analysis of insulin-like growth factor–II from Platichthys stellatus

作　　者：臧坤[1,2] 徐永江[1] 柳学周[1] 史宝[1] 王妍妍[1] 

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,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2015年第2期214-223,共10页Journal of Fishery Sciences of China

基　　金：国家863计划项目(2012AA10A413);鲆鲽类现代产业技术体系(CARS-50);中央级公益性事业单位基本科研业务费项目(20603022012022);山东省自然科学基金项目(ZR2012CQ025)

摘　　要：根据鲽形目和鲈形目鱼类的IGF-II基因保守序列设计特异性引物,利用RT-PCR技术克隆得到编码星突江鲽(Platichthys stellatus)IGF-II成熟肽的全长c DNA序列(210 bp),同源性分析显示IGF-II成熟肽在B、A和D区高度保守。利用原核表达载体p ET-28a构建了重组表达质粒(IGF-II/p ET28a),转化大肠杆菌BL21(DE3)后经IPTG诱导,获得了N端含6个组氨酸的重组蛋白。37℃条件下用1.0 mmol/L的IPTG诱导6 h,目的蛋白表达量最高,占菌体总蛋白的42.7%。重组蛋白主要以包涵体形式存在,经SDS-PAGE电泳检测,IGF-II重组蛋白大小为11.4 k D,Western-Blotting免疫印迹呈阳性。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,获得了纯化IGF-II蛋白。细胞增殖实验结果显示,重组蛋白可显著促进人胚胎肾细胞HEK293T的增殖,表明获得的IGF-II重组蛋白具有细胞水平的生物活性。研究结果为深入研究星突江鲽IGF-II的功能提供了基础资料。Platichthys stellatus is a high-valued flatfish species because of the desirable taste of its flesh and its wide tolerance to salinity and temperature. It has become an economically important farmed marine fish in China in recent years. Insulin-like growth factor（IGF）-II plays an important role in the regulation of growth and development in teleosts. Our objective was to evaluate the physiological functions of IGF-II in P. stellatus. The c DNA sequence encoding the mature peptide domain of the IGF-II gene from P. stellatus was isolated using RT-PCR amplification. A pair of primers were designed based on the conserved sequences of IGF-II from Pleuronectiformes and Perciformes species. A 210 bp fragment was obtained and verified as the mature peptide fragment. A homology comparison revealed that the B-, A-, and D-domains of the P. stellatus IGF-II mature peptide were highly conserved among vertebrates, but with changes in the C-domain. The mature peptide fragment was reconstructed by RT-PCR with a pair of primers and the endonucleases Bam H I and Hind III, then subcloned into the prokaryotic expression vector p ET-28 a with T4 DNA polymerase to successfully construct an IGF-II/p ET28 a recombinant plasmid. The recombinant IGF-II plasmid was highly expressed in E. coli BL21（DE3） after being induced by IPTG with a special fusion polypeptide containing His6 at its N-terminus. The SDS-PAGE analysis showed that the IGF-II polypeptide was expressed in the form of inclusion bodies with a molecular weight of 11.4 k D and accounted for 42.7% of the whole bacterial protein 6-h post induction with 1.0 mmol/L IPTG at 37℃. Western blotting analysis indicated the fusion polypeptide had antigenicity to His6 antibody with primary antibodies（mouse-anti-His6 monoclonal antibody） and secondary antibodies（goat-anti-mouse Ig G labeled with horseradish peroxidases）. The inclusion bodies were denaturalized using 6 mol/L guanidine HCl, filtered through a 0.45 μm filtration membrane, purified using Ni-NTA affinity chroma

关 键 词：星突江鲽 IGF-II 原核表达 生物活性 

分 类 号：S917[农业科学—水产科学]