草鱼胰岛素样生长因子—I的融合表达、纯化和抗血清制备
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中国水产科学研究院珠江水产研究所农业部热带亚热带鱼类选育与养殖重点开放实验室

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Fusion expression and purification of Ctenopharyngodon idellus IGF2I and preparation of the antiserum against GST2IGF
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Pearl River Fisheries Research Institute , Chinese Academy of Fishery Science , Guangzhou 510380 , China

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    摘要:

    采用PCR方法改造已克隆到的草鱼胰岛素样生长因子-I(IGF-I)cDNA。使其成为成熟肽cDNA并亚克隆到pGEX-4T-1载体中,构建草鱼IGF-I融合蛋白表达质粒pGEX-IGF。质粒转化大肠杆菌BL21菌株,该菌株经IPTG诱导可表达分子量约34kD的特异蛋白。在不同的温度条件下分别产生以可溶性的和包涵体形式为主的特异蛋白,经30℃诱导的菌体经溶菌酶消化、超声破碎及高速离心后,裂解液上清经glutathione sepharose亲和层析纯化获得高纯度的GST-IGF。以此纯化的融合蛋白为抗原制备兔抗草鱼IGF-I的抗血清,凝胶双扩散试验显示抗血清效价为1:64,说明表达产物具免疫原性。

    Abstract:

    Employing PCR method , the cloned grass carp IGF2 Ⅰgene was modified to be a mature IGF2I peptide gene and then subcloned into fusion expression vector pGEX24T21 to construct expression vector pGEX2IGF. Restriction endonuclease analysis and sequencing confirmed the corrected construction. The vector was transformed into Escherichia coli BL21 cell which can be induced by IPTG to produce a special protein of 34kD in molecular weight. The special protein can be mainly in form of soluble protein or inclusion body when induced at 30 and 37 ℃ respectively. The cells induced at 30 ℃ were digested by lysozmye and disrupted by sonication. After being centrifuged the supernatant was purified by glutathione sepharose column. GST2IGF fusion protein could be absorbed specifically on this column. The high purified protein was used as antigen to immune rabbits directly and high quality antiserum was obtained , which showed that the fusion protein possesses good immunogenicity.

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叶星.草鱼胰岛素样生长因子—I的融合表达、纯化和抗血清制备[J].水产学报,2002,26(2):

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  • 收稿日期:2014-04-10
  • 最后修改日期:2014-04-10
  • 录用日期:2014-04-11
  • 在线发布日期: 2014-04-11
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