半滑舌鳎类胰岛素生长因子-Ⅰ的原核表达及活性分析  

Prokaryotic expression and bioactivity analysis of insulin-like growth factor-I from Cynoglossus semilaevis Güther

作　　者：刘芝亮[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 青岛市海水鱼类种子工程与生物技术重点实验室 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》2013年第4期706-712,共7页Journal of Fishery Sciences of China

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

摘　　要：根据半滑舌鳎(Cynoglossus semilaevis Güther)类胰岛素生长因子-Ⅰ(IGF-I)基因的cDNA序列,设计引物扩增编码IGF-I成熟肽序列,此序列由210个碱基组成,包括B-C-A-D 4个功能域。利用PCR方法将扩增片段克隆到原核表达载体pET-28a上,得到重组质粒,将重组质粒导入到大肠杆菌BL21(DE3)后经IPTG诱导产生N端含6个组氨酸的融合蛋白。以SDS-PAGE电泳检测和SigmaScan软件分析获得的多肽,结果表明:IGF-I融合蛋白大小为11.4 kD,IPTG诱导3 h时目的蛋白表达量最高,占细菌总蛋白的58.5%,1 L菌液表达目的蛋白40.7 mg,主要以包涵体形式存在。利用Western-blotting方法验证融合蛋白可特异性的被6×His抗体识别。诱导表达后的菌液沉淀经6 mol/L盐酸胍变性、Ni2+离子亲和柱纯化和尿素梯度复性,获得大小为11.4 kD的纯化蛋白。细胞增殖实验表明,IGF-I融合蛋白可显著促进乳腺癌细胞MDA231细胞的增殖,具有生物学活性。本研究通过原核表达技术获得了体外重组的具有生物活性的半滑舌鳎IGF-I融合蛋白,结果可为半滑舌鳎生长调控技术研究提供基础资料。We designed a pair of primers based on the full-length cDNA sequence of IGF-Ⅰ from Cynoglossus semilaevis Guther to clone the mature peptide domain of the IGF-Ⅰ gene. The mature peptide consisted of 70 amino acids, including four functional domains B-C-A-D. The mature peptide fragment was subcloned into the prokaryotic expression vector pET-28a to construct a recombinant plasmid that was transformed into E. coli BL21 （DE3）. Induction of the plasmid by IPTG yielded a special fusion polypeptide containing His 6 at the N-terminus. SDS-PAGE analysis revealed that the IGF-Ⅰ polypeptide had a molecular weight of 11.4 kD. The recombinant IGF-Ⅰ protein proximately accounted for 58.5% of the whole bacterial protein 3 h post induction with IPTG. Thus, 1 L of broth would yield 40.7 mg of the target protein. Western-blotting analysis suggested the fusion polypeptide exhibited antigenicity to His 6 antibodies. The IPTG-induced bacterial precipitate was denaturalized using 6 mol/L guanidine HC1, purified using Ni-NTA affinity chromatography, and annealed by gradient dialysis in urea, thereby yielding the purified protein. The recombinant IGF-Ⅰ protein promoted the proliferation of breast cancer MDA231 cells, suggesting it is biologically active. Our results provide a basis and suggested methods for regulating the growth of farmed Cynoglossus semilaevis Giither.

关 键 词：半滑舌鳎 IGF-I 原核表达 生物活性 

分 类 号：S965[农业科学—水产养殖] Q959[农业科学—水产科学]