猪繁殖与呼吸综合征病毒ORF7基因的克隆及其在毕赤酵母中表达  

Cloning of ORF7gene of porcine reproductive and respiratory syndrome virus and its expression in Pichia pastoris

作　　者：黄小波[1] 钱洪喜[1] 曹三杰[1] 文心田[1] 马小平[1] 

机构地区：[1]四川农业大学动物医学院动物传染病与基因芯片实验室四川省动物疫病与人类健康重点实验室,四川雅安625014

出　　处：《中国兽医学报》2013年第8期1137-1141,共5页Chinese Journal of Veterinary Science

基　　金：国家公益性行业(农业)科研专项资助项目(201203056);教育部<长江学者和创新团队发展计划>创新团队资助项目(IRTO848)

摘　　要：用RT-PCR扩增猪繁殖与呼吸综合征病毒(PRRSV)重庆分离株C14-2的ORF7基因(384bp),构建克隆质粒pMD19-T-ORF7,经EcoRⅠ/NotⅠ双酶切回收ORF7基因插入酵母表达载体pPIC9K,构建了重组表达质粒pPIC9K-ORF7,进行PCR鉴定和双酶切鉴定。鉴定的pPIC9K-ORF7经SacⅠ线性化后电转化毕赤酵母宿主菌GS115,筛选获得阳性重组菌GS115(pPIC9K-ORF7),再经G-418/YPD筛选获得高拷贝重组菌,重组子经表型鉴定为Mut+。重组菌GS115(pPIC9K-ORF7)经甲醇诱导表达,在96h表达的N蛋白量最大,N蛋白经SDS-PAGE鉴定大小约为15 000;Western blot表明N蛋白能与美洲型PRRSV阳性血清发生特异性反应,具有良好的反应活性。本研究为开展PRRSV ORF7基因在毕赤酵母中表达及应用奠定基础。The 384 bp ORF7 gene of porcine reproductive and respiratory syndrome virus （PRRSV） stain C14-2 was amplified by RT-PCR,and was inserted into pMD19 T vector to construct the recombi-nant plasmid pMD19-T-ORF7. The plasmid pMD19-T-ORF7 was digested by EcoR I/Not I and the ORF7 gene was cloned into P. pastoris exrpession vector pPIC9K to construct the expression plasmid pPICgK-ORF7, the plasmid pPIC9K-ORF7 was identified by PCR and restrictive diges-tion. The expression vector pPIC9K-ORF7 was transformed into GSl15 by electroporation after linearization with Sac I. The high copy transformants was got by sequentially screening on G-418/ YPD medium plates, the transformants were identified to be Mut＋ by phenotypic identification and positive by PCR. The recombinant of P. pastoris was then induced by methanol, and the maximum expressed N protein could be got at 96 h. The expressed N protein in P. pastoris was about 15 000 by SDS-PAGE. Western blot showed the N protein could reacted to the antiserum against Ameri-can PRRSV strains, the result showed the expressed N protein in P. pastoris had good reactionoge-nicity. This study laid a foundation for expression of PRRSV ORF gene in P. pastoris.

关 键 词：猪繁殖与呼吸综合征病毒 ORF7基因 毕赤酵母 

分 类 号：S852.65[农业科学—基础兽医学]