BVDV-2 E2基因的克隆及其在毕赤酵母中的表达  

Cloning of E2 Gene of Bovine Viral Diarrhoea Mucosal Disease Virus and Its Expression in Pichia pastoris

作　　者：刘昱成[1] 王国超[1] 孟庆玲[1,2] 乔军[1] 才学鹏[2] 贺志昊[1] 杨海波[1] 万鹏[1] 陈创夫[1] 

机构地区：[1]石河子大学动物遗传改良与疾病控制新疆自治区重点实验室,新疆石河子832003 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,兰州730046

出　　处：《西北农业学报》2014年第2期30-34,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：农业公益行业专项子课题(201303037-5);兵团国际科技合作计划项目(2012BC006)

摘　　要：根据GenBank登录的BVDV-2 E2基因序列,设计特异性引物,对BVDV-2新疆SW分离株E2基因进行RT-PCR扩增,克隆入T载体中测序,然后亚克隆入华赤酵母分泌型表达载体pPIC9K中,构建重组表达载体pPIC9K-E2。用SalⅠ酶切线性化重组质粒pPIC9K-E2,电转化酵母GS115,通过G418筛选重组酵母菌GS115-pPIC9K-E2,用φ=1.5%甲醇诱导,SDS-PAGE和Western blot分析表达产物。SDS-PAGE分析证实表达的重组融合蛋白分子质量为23.2ku;Western blot分析表明,该重组蛋白可与BVDV-2多克隆抗体发生特异性血清学反应,证实表达的重组E2蛋白具有良好的反应原性。According to BVDV-2 E2 gene sequences in GenBank, E2 gene of BVDV-2 SW strain am-plified using RT-PCR by specific primers. E2 gene was cloned into T vector and sequenced, and then subcloned into the yeast （P. pastoris） secretary expression vector pPIC9K to generate the recombinantexpression vector pPIC9K-E2. Recombinant plasmid pPIC9K-E2 was liberalized by Sal Ⅰ digestion and then electrically transformed into P. pastoris GSl15. GS115-pPIC9K-E2 recombinant yeast wasscreened by G418, and then induced with 1.5 % methanol. The expression product was analyzed by SDS-PAGE and western blot. SDS-PAGE analysis confirmed that the expression of recombinant fusion protein has a mass molecule of 23.2 ku; western blot showed that the recombinant protein can re- acts with BVDV-2 polyclonal antibody, confirming that the expression of recombinant E2 protein hasgood reactogenicity.

关 键 词：BVDV E2基因 载体构建 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]