大菱鲆胰岛素样生长因子-I成熟肽的克隆、重组表达及活性分析  

Cloning,recombinant expression and purification of turbot(Scophthalmus maximus) mature IGF-I

作　　者：赵晓杰[1,2] 陈松林[2,3] 王娜[2] 王贤丽[2] 邢士超[2] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]中国水产科学研究院黄海水产研究所,农业部海洋渔业资源可持续利用重点开放实验室,山东青岛266071 [3]上海海洋大学,上海高校水产养殖学E-研究院,上海201306

出　　处：《水产学报》2010年第1期1-7,共7页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2006AA10A402);山东省泰山学者工程专项经费资助;上海市教育委员会E-研究院建设项目(E03009)

摘　　要：通过RT-PCR方法从大菱鲆肝组织克隆了胰岛素样生长因子-I(IGF-I)成熟肽片段,分析表明,此成熟肽由70个氨基酸残基组成,含有3个链内二硫键。将扩增片段克隆到原核表达载体pGEX-4T-1上,实现了IGF-I成熟肽和GST蛋白在Escherichia coli BL21(DE3)plysS中的融合表达。融合蛋白分子量约为34ku,诱导4h时占菌体总蛋白的59%,主要以包涵体形式存在。Western-blotting免疫印迹表明,融合蛋白可以特异性地被anti-GST抗体识别。包涵体经6mol/L盐酸胍变性溶解及脉冲法稀释复性后,通过GSTrapFF亲和预装柱纯化,获得了电泳分析纯的融合蛋白。以细胞增殖实验检测蛋白生物活性,结果显示,纯化蛋白能促进大菱鲆肾脏细胞的增殖。Insulin-like growth factor-I （IGF-I） is a conserved peptide expressed ubiquitously, which shows highly homologous diverse effects on development, growth, and metabolism. With RT-PCR, the fragment encoding the turbot （Scophthalmus maximus） mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which may form 3 disulfide bonds. The target fragment was then successfully subcloned into the express vector pGEX-4T-1 and was highly expressed in E. coli BL21 （DE3）plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 ku and maximally amounted to 59 % of the whole protein in the E. coli cell 4 hours after being induced with IPTG. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/Lguanidine chloride followed by pulse renaturation in refolding buffer containing 0. 5 mol/L L- Arginine, 1. 0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysed, and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.

关 键 词：胰岛素样生长因子-I CDNA克隆 重组表达 包涵体 融合蛋白 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]