人工合成草鱼生长激素cDNA在大肠杆菌中的表达  

Prokaryotic Expression of Recombinant Grass Carp Growth Hormone

作　　者：王伟[1] 汪亚平[1] 朱作言[1] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《Acta Genetica Sinica》2001年第4期306-312,共7页

基　　金：淡水生态与生物技术国家重点实验室开放课题资助;湖北省科委重点科研项目!(991P0502)资助

摘　　要：经密码子优化的人工合成草鱼生长激素ｃＤＮＡ与表达载体ｐＥＴ－２８ａ（＋）重组，构建重组表达质粒ｐＥＴ－ＧＨ。转化大肠杆菌ＢＬ２１（ＤＥ３），筛选阳性克隆，ＩＰＴＧ诱导表达。１２．５％的ＳＤＳ－ＰＡＧＥ分析显示，大肠杆菌表达产物中含有与草鱼生长激素分子量一致的新增蛋白带，激光密度扫描，其产量约占菌体总蛋白的４０％。金属离子螯合层析柱亲和纯化，获得电泳纯的重组蛋白。Ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ和酶联免疫吸附受体法检测证实：重组蛋白与抗草鱼生长激素的多克隆抗体发生特异性结合；复性后的重组蛋白有与天然草鱼生长激素一致的生物学活性。The recombinant plasmid containing a synthetic grass carp growth hormone cDNA was transformed into Escherichia coli BL21 (DE3) and expressed efficiently, induced by 1mmol ／ L IPTG. 12.5% SDS-PAGE showed the molecular weight of new added prote in in Escherichia coli BL21 (DE3) is consistent with that of grass carp growth hormone. The recombinant protein was composed of approximately 40% of the total bacterial proteins. The target protein was purified with Ni-NTA affinity column. Western-blottin g conformed that recombinant protein could specifically react with antibody against grass carp growth hormone. ELISA-receptor assay showed the bioactivity of recombinant protein is consistent with that of nature grass carp growth hormone.

关 键 词：草鱼 生长激素 原核表达 纯化 酶联免疫吸附受体检测 表达 

分 类 号：Q57[生物学—生物化学]