草鱼胰岛素样生长因子-I的融合表达、纯化和抗血清制备  

Fusion expression and purification of Ctenopharyngodon idellus IGF-I and preparation of the antiserum against GST-IGF

作　　者：叶星[1] 白俊杰[1] 劳海华[1] 简清[1] 李英华[1] 罗建仁[1] 

机构地区：[1]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,广东广州510380

出　　处：《水产学报》2002年第2期122-126,共5页Journal of Fisheries of China

基　　金：农业部"九五"重点渔业科技资助项目 (渔 95 -B -96-0 2 -0 8-0 2 )

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BL2 1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glutathionesepharose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∶6 4 。Employing PCR method, the cloned grass carp IGF Ⅰgene was modified to be a mature IGF I peptide gene and then subcloned into fusion expression vector pGEX 4T 1 to construct expression vector pGEX IGF. 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. GST IGF 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.

关 键 词：草鱼 胰岛素样 生长因子-I 纯化 融合表达 抗血清 温度 凝胶双扩散试验 

分 类 号：Q959.468[生物学—动物学]