PEDV Nsp10基因真核表达载体的构建及蛋白的表达

Construction of Eukaryotic Expression Vector and Protein Expression of PEDV Nsp10 Gene

猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)为猪流行性腹泻(Porcine epidemic diarrhea,PED)的病原体。Nsp10是PEDV编码的一种非结构蛋白,在病毒的复制过程中发挥着重要的作用。为研究在病毒侵染宿主细胞时Nsp10蛋白发挥的作用,通过RT-PCR技术扩增Nsp10基因片段,利用同源重组的方法将Nsp10基因连接到pCMV-Myc载体,成功构建了PEDV Nsp10基因的真核表达载体。酶切鉴定及测序结果证实,PEDV Nsp10基因成功克隆到pCMV-Myc载体中,并将重组质粒命名为pCMV-Myc-Nsp10。将重组质粒pCMV-Myc-Nsp10通过脂质体转染至IPEC-J2细胞中,利用Western Blot和间接免疫荧光技术检测pCMV-Myc-Nsp10的表达以及定位情况。结果显示,pCMV-Myc-Nsp10在IPEC-J2细胞中成功表达,并且均匀分布在细胞质和细胞核中,为后续研究PEDV Nsp10蛋白的生物学功奠定了基础。

Porcine epidemic diarrhea virus(PEDV)was the pathogen of porcine epidemic diarrhea(PED).Nsp10 was a non-structural protein encoded by PEDV,which played an essential role in the replication of the virus.To study the role of Nsp10 protein in virus infection of host cells,Nsp10 gene fragment was amplified by RT-PCR technology and connected to the pCMV-Myc vector by double enzyme digestion,the eukaryotic expression vector of PEDV Nsp10 gene was successfully constructed.Enzyme digestion identification and sequencing results confirmed that the PEDV Nsp10 gene was successfully cloned into the pCMV-Myc vector,and the recombinant plasmid was named after pCMV-Myc-Nsp10.The recombinant plasmid pCMV-Myc-Nsp10 was transfected into IPEC-J2 cells by liposome,and the expression and localization of pCMV-Myc-Nsp10 by Western Blot and indirect immunofluorescence were explored.The results showed that pCMV-Myc-Nsp10 was successfully expressed in IPEC-J2 cells and evenly distributed in cytoplasm and nucleus,which would provide a theoretical basis for subsequent studies on the biological functions of PEDV Nsp10 protein.