猪流行性腹泻病毒Nsp5基因的原核表达及生物信息学分析

旨在对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)Nsp5基因进行原核表达,利用生物信息学软件,预测其编码蛋白的结构和功能,为了解PEDV致病机理奠定基础。本研究克隆PEDV/LY/2014/04毒株的Nsp5基因,亚克隆进pET-28a(+)原核表达载体,PCR和酶切验证重组质粒的构建,重组质粒pET-28a(+)-Nsp5转入E.coli BL21(DE3)中,SDS-PAGE检测Nsp5的表达和His band Ni+纯化情况,Vector NTI Advance等软件对Nsp5蛋白氨基酸组成、抗原表位、二级和三级结构进行预测和分析。结果表明,成功克隆并构建了重组质粒pET-28a(+)-Nsp5,表达重组蛋白大小约22 ku,且主要以包涵体形式存在,His band Ni+纯化后获得高纯度重组蛋白。Nsp5蛋白是由196个氨基酸残基组成的多肽,其分子质量的理论值为21 820.07 u,理论等电点(pI)为8.734,略偏碱;二级结构骨架中α-螺旋(h)占55.61%,β-折叠(t)占7.65%,无规则卷曲(c)占20.92%,延伸链(e)占15.82%;Nsp5蛋白质的三级结构中,中间段以α-螺旋为主作为骨架,C端多个复杂二级结构构成该蛋白的酶活性中心;B细胞抗原表位的预测,表明有15个潜在的B细胞优势表位。本研究为猪流行性腹泻病毒Nsp5蛋白质的生物学功能相关研究提供数据支持。

Prokaryotic expression and bioinformatics analysis of Nsp5 gene of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) nonstructural protein 5 (Nsp5) gene was expressed by the prokaryotic expression system and the structure of the encoded protein was predicted by bioinformatics software for understanding the pathogenecity mechanism of PEDV. In this study, the Nsp5 gene of PEDV/LY/2014/04 strain was subcloned into prokaryotic expression vector pET-28a, and the recombinant plasmid was identified by polymerase chain reaction (PCR) and restriction enzyme digestion. The recombinant plasmid pET-28a(+)-Nsp5 was transformed into competent cells of Escherichia coli BL21(DE3). The expression and purification effect of Nsp5 were detected by SDS-PAGE. The Vector NTI Advance, online software was utilized to predict and analyze the amino acid composition, epitope, secondary and tertiary structure of Nsp5 protein. The results showed that the recombinant plasmid pET-28a(+)-Nsp5 was successfully constructed, and approximately 22 ku recombinant protein was expressed and mainly existed in the form of inclusion bodies. High-purity recombinant protein was obtained after purifying by His band Ni+. The Nsp5 protein consisted of a polypeptide with 196 amino acid residues. The theoretical molecular mass of the slightly alkaline recombinant protein was 21 820.07 u, and the theoretical isoelectric point(pI) was 8.734. The secondary structure was composed of 55.61% α-helix(h), 7.65% β-fold(t), 20.92% irregular curl(c) and 15.82% extended chain(e). In the tertiary structure of the Nsp5 protein, the middle segment skeleton is mainly formed by α-helix, and a plurality of complex secondary structures at the C-end constituted the enzyme active center of the protein. The prediction of B cell epitope indicated that there were 15 potential B cell dominant epitope. This study provided data support for studying on the biological function of the porcine epidemic diarrhea virus Nsp5 protein.