鸡贫血病毒vp1和vp2基因在毕赤酵母中的表达及产物免疫学特性的初步研究

Expression of chicken anemia virus vp1 and vp2 gene in Pichia pastoris

利用特异性引物从pBluemCAV中PCR扩增得到鸡贫血病病毒(CAV)vp1、vp2基因,经EcoRⅠ和NotⅠ双酶切处理,纯化后,克隆至EcoRⅠ和NotⅠ双酶切处理的表达载体pPIC9K中,构建了真核表达质粒pPIC9K-VP1和pPIC9K-VP2。将pPIC9K-VP1和pPIC9K-VP2转化至毕赤酵母SMD1168中,在0.5%甲醇诱导下表达CAV-VP1、CAV-VP2蛋白,运用Westernblot和Dot-ELISA鉴定表达蛋白。结果表明,重组酵母菌株表达出约54ku和24ku的目的蛋白,与针对鸡贫血病毒的单克隆抗体发生特异性反应。将表达产物乳化后免疫6周龄BALB/c小鼠,用ELISA、IFA检测免疫小鼠血清,均检测到抗体。

vp1 and vp2 gene from chicken infectious anemia virus （CIAV） were amplified from pBluemCAV by PCR and cloned into pPIC9K vector, respectively. The recombinant plasmid pPIC9K-VP1 and pPIC9K-VP2 were transformed into SMD1168 cells and proteins expressions were induced with methanol. A 54 ku and 24 ku protein were successfully expressed from respective vector. The expression products were shown to react with monoclonal antibodies against CAV in DOT ELISA test. BALB/c mice were immunized with the expressed proteins and specific antibodies were produced and confirmed in ELISA and IFA assays