文章摘要
赵晓杰,陈松林,王娜,王贤丽,邢士超.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[J].水产学报,2010,34(1):1~7
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
投稿时间:2008-09-30  修订日期:2009-01-17
DOI:10.3724/SP.J.1231.2010.06155
中文关键词: 胰岛素样生长因子-I  cDNA 克隆  重组表达  包涵体  融合蛋白
英文关键词: insulin-like growth factor I  cDNA cloning  recombinant expression  inclusion body  recombinant protein
基金项目: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)
作者单位E-mail
赵晓杰 黄海水产研究所  
陈松林 黄海水产研究所 chensl@ysfri.ac.cn 
王娜 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  
王贤丽 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  
邢士超 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  
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中文摘要:
      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34 ku,诱导4 h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6 mol/L盐酸胍变性溶解及脉冲法稀释复性后,通过GSTrap FF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。
英文摘要:
      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.
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