猪流行性腹泻病毒Nsp7蛋白的表达与多克隆抗体制备

Prokaryotic expression of Nsp7 of porcine epidemic diarrhea virus and preparation of polyclonal antibodies against Nsp7

为了解猪流行性腹泻病毒(PEDV)非结构蛋白7(Nsp7)的结构特征和免疫原性,本研究通过生物信息学分析Nsp7蛋白序列特征及空间结构,在此基础上对Nsp7进行原核表达和纯化,并制备抗Nsp7多克隆抗体。结果显示,Nsp7基因在PEDV不同毒株间高度保守,Nsp7蛋白的三级结构主要由4个α螺旋片组成。SDS-PAGE试验表明,Nsp7在大肠杆菌中获得大量表达,重组蛋白大小为9.2 ku,经纯化后得到单一条带。采用纯化的Nsp7免疫兔制备多克隆抗体,然后经Western blot和间接免疫荧光试验鉴定,多克隆抗体能够特异性识别重组Nsp7蛋白和PEDV感染细胞的Nsp7蛋白,证明Nsp7免疫原性良好,为后续研究Nsp7的功能及研制以Nsp7为检测靶标的PEDV诊断试剂盒奠定基础。

In order to study the structure characteristics and immunogenicity of Nsp7 of porcine epidemic diarrhea virus（PEDV）,the sequence and the structure of Nsp7 were analyzed by bioinformatics.Based on this,the expression of Nsp7 was conducted in Escherichia coli,and the anti-Nsp7 polyclonal antibody was prepared.The results showed that Nsp7 gene was highly conserved in different strains of PEDV,and Nsp7 consisted of four main alpha helix.SDS-PAGE showed that Nsp7 was expressed successfully,and the relative molecular weight of the recombinant protein was about 9.2 ku.The purified Nsp7 was used for the preparation of rabbit anti-Nsp7 polyclonal antibody,and then the polyclonal antibody was identified by Western blot and indirect immunofluorescence assay.The results showed that the polyclonal antibody could specifically recognize the recombinant Nsp7 protein and Nsp7 in PEDV infected cells,indicating that the immunogenicity of Nsp7 was good.This study laid a foundation for further investigation on the function of Nsp7 and the development of early diagnosis kit of PEDV.