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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 ,说明表达产物具免疫原性
The cloned cDNA of grass carp IGF - Ⅰ was transformed by PCR and transformed into mature peptide. The cDNA was subcloned into pGEX 4T 1 vector to construct grass carp IGF Ⅰ fusion protein expression plasmid pGEX IGF. Plasmid was transformed into E. coli BL21 strain. The strain was induced by IPTG can express specific molecular weight of about 34kD protein. Under different temperature conditions were produced in soluble and inclusion bodies in the form of specific proteins. After lysozyme digestion, sonication and high-speed centrifugation, the supernatant of lysate was purified by glutathionesepharose affinity chromatography to obtain high-purity GST IGF. The purified fusion protein was used as antigen to prepare rabbit anti-grass carp IGF I antiserum. Gel double diffusion test showed antiserum titers of 1: 64, indicating that the expression product is immunogenic