作　　者： ; ; ; ; ; ;

机构地区： 长沙学院生物工程与环境科学系

出　　处： 《淡水渔业》 2011年第6期19-24,共6页

摘　　要： 根据GenBank数据库中登记的青鱼(Mylopharyngodon picens)生长激素(bcGH)基因序列(AF389238)设计特异性引物,采用RT-PCR技术扩增青鱼生长激素cDNA编码区,并通过定向插入含强启动子AOX I的表达载体pPIC3.5k、电击转入毕赤酵母GS115菌株、G418多克隆子筛选和测序验证等步骤进行了重组生长激素酵母菌诱导表达等相关实验,成功获得重组生长激素酵母菌(pPIC3.5k-bcGH重组酵母菌)。蛋白表达产物经SDS-PAGE和ELISA检测,在23 kD处发现一条与预期蛋白分子量大小一致的特异性条带,并且目的蛋白能与青鱼生长激素多克隆抗体特异性结合,说明bcGH在酵母胞内得到正确表达。表达条件优化过程中发现,诱导24 h已有重组蛋白表达,pH值4.5～7.0之间都能表达重组蛋白。在pH值6.5、0.5%甲醇、温度30℃和培养时间72 h的优化条件下,目的蛋白的表达量高达893 mg/L。 In order to amplify the cDNA of black carp growth hormone,the primers were designed according to the GenBank sequence of black carp growth hormone gene（AF389238）.The black carp growth hormone（bcGH） cDNA was oriently inserted into pPIC3.5k which contains AOXI promoter and Pichia wild-type gene coding for histidinol dehydrogenase.The recombinant plasmid pPIC3.5k-bcGH was linearized by restriction endonucleases Sal I,then transformed into HIS4 mutant yeast GS115 by electroporation,the recombinant growth hormone yeasts were certified by the screening of G418 and sequencing.SDS-PAGE and ELISA analysis showed that bcGH gene expression product appeared in intracellular solution.Under the optimization of fermentation conditions,the recombinant protein can be expressed in induction time 24 h and pH 4.5~7.0.Under the optimized conditions of 30 ℃,pH 6.5,fermentation time 72 hours and induced concentration 0.5%,the yield is 893 mg/L.

关 键 词： 青鱼 生长激素 毕赤酵母 表达

领　　域： [农业科学]