猪流行性腹泻病毒E基因真核表达载体的构建与鉴定  

Construction and Identification of the E gene Eukaryotic Expression Vector for Porcine Epidemic Diarrhea Virus

作　　者：付通超[1] 万红平[1] 程渝[2] 颜其贵[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川农业大学动物生物技术中心

出　　处：《畜牧兽医杂志》2012年第4期10-13,共4页Journal of Animal Science and Veterinary Medicine

摘　　要：(目的)构建猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)E基因真核表达载体pCI-E,为探讨E基因的功能奠定基础。(方法)根据GenBank上已发表的PEDV CV777株序列,利用Primer5.0设计1对两端含限制性核酸内切酶EcoR I和Sal I酶切位点的特异引物,用反转录聚合酶链式反应(RT-PCR)扩增出完整的231bp的目的基因,将目的基因与pMD19-T载体连接转化到大肠杆菌DH5α中,用测序、PCR、双酶切鉴定阳性克隆体。然后将纯化后的E基因插入表达载体(pCI-neo)上,并经双酶切、测序鉴定。(结果)成功构建了PEDV的pCI-E重组真核表达载体。(结论)pCI-E重组真核表达载体的构建为进一步探讨PEDV E基因的功能和分子生物学特性及PED防御新途径提供依据。This study aimed to construct an eukaryotic expression vector of Porcine epidemic diarrhea virus E gene. Based on the published nucleotide sequence of PEDV in Genbank, a pair of specific primers of PEDV was designed by the Primer 5.0 software. To identify and clone the recombinant plasmid, we designed two restriction enzyme sites EcoR I and Sal I in the up- per and down primer, respectively. Following the mixture of the primers, template and the mix reaction system, the complete E gene of PEDV was amplified by RT--PCR. The fragment of the purpose gene was 231bp in length. The purpose gene was connected with pMD19-T vector, named pMD-E, transformed into E. coli DH5a. The positive colonies were confirmed by re- striction enzyme digestion, PCR and sequencing. A recombinant plasmid, named pCI-E was constructed by inserting the E gene into the expression vector pCI-neo, verified by restriction enzyme digestion and sequencing. The recombinant eukaryotic ex- pression vector of the PEDV E gene has been constructed successfully, which provided an experimental basis for the further study of the E gene functions and the PED defense.

关 键 词：猪流行性腹泻病毒 E基因 真核表达载体 构建 

分 类 号：S855.3[农业科学—临床兽医学]