牛脂联素基因的克隆及在毕赤酵母中的表达  

Cloning and expression of bovine adiponectin gene in Pichia pastoris

作　　者：黄海龙[1,2] 王哲[1] 于国健[1] 孙佳[1] 贺鹏飞[1] 吴永进 

机构地区：[1]吉林大学畜牧兽医学院 [2]吉林农业大学动物科学技术学院 [3]西藏昌都军分区77570部队

出　　处：《中国兽医学报》2009年第9期1197-1200,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571365);吉林农业大学青年启动基金

摘　　要：应用RT-PCR方法扩增牛脂联素(Bovine adiponectin,BovADPN)基因,连接到载体pMD18-T中;测序并利用BLAST工具对比证明该基因序列正确,经EcoRⅠ和NotⅠ双酶切,回收目的基因片段,并将其定向克隆到pPICZαA载体中,构建重组质粒pPICZαA-BovADPN。用SacⅠ酶切使其线性化,以化学方法(LiCl)转化入感受态毕赤酵母细胞GS115;重组子经高质量浓度Zeocin(1 000 mg/L)筛选、MDH?MMH平板筛选、PCR鉴定后,用1%甲醇诱导表达,SDS-PAGE及Western-blot分析。结果表明,所获得的酵母重组子能够分泌表达出相对分子质量为40 000的重组蛋白。The BovADPN gene was amplified by RT PCR with the specific primers, cloned into the vector pMD18-T,and confirmed by sequencing and BLAST. The BovADPN gene fragment which was recovered after the digestion with EcoR Ⅰ and Not Ⅰ was subcloned into the pPICZαA vector to construct the pPICZαA-BovADPN Pichia pastoris expression vector. Then the expression plasmid was transformed into GS115 cells using LiCI method after it was linearized with Sac Ⅰ. The multicopy transformants were selected with 0. 1 % Zeocin, MDH / MMH culture plate identified by PCR,and induced with 1 % methanol. The selected strain could specifically secret the mo lecular weight of about 40 000 BovADPN proteins which was identified by SDS-PAGE and Western-blot.

关 键 词：牛 脂联素基因 毕赤酵母 

分 类 号：Q78[生物学—分子生物学] S856.5[农业科学—临床兽医学]