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

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.