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

作　　者：陈圣毅[1,2] 刘新富[1,3] 徐永江[1,3] 刘芝亮[1,2] 柳学周[1,3] 史宝[1,3] 王妍妍[1,3] 

机构地区：[1]中国水产科学研究院黄海水产研究所农业部海洋渔业可持续发展重点实验室,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]中国水产科学研究院黄海水产研究所青岛市海水鱼类种子工程与生物技术重点实验室,山东青岛266071

出　　处：《水产学报》2013年第9期1290-1296,共7页Journal of Fisheries of China

基　　金：国家“八六三”高技术研究发展计划(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号);山东省自然科学基金项目(ZR2012CQ025);青岛市科技计划项目(11-1-1-11-hy)

摘　　要：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.

关 键 词：七带石斑鱼 类胰岛素生长因子-Ⅰ 成熟肽克隆 原核表达 生物活性分析 

分 类 号：Q785[生物学—分子生物学] S965[农业科学—水产养殖]