新型鸭细小病毒NS1基因的原核表达及生物信息学分析

摘要

In order to investigate the biological function of NS1 protein of Nocel duck parvovirus (NDPV), one pair of primers were designed according to the NS1 gene sequence of NDPV DS15 strain (GenBank No. KU947420). The PCR products from the NDPV NS1 gene were cloned into the prokaryotic expression vector pGEX-4T-1, resulting in the recombinant plasmid pGEX-4T-NS1. After sequencing analysis, bioinformatics software and net servers were performed to predict and analyze the structures and biochemical properties of NS1 protein. The recombinant plasmid was transformed into BL21 competent cells and then induced with IPTG. Expression of the NS1 protein was examined in SDS-PAGE and Western blot. The results showed that the recombinant protein NS1 with molecular weight of 97 kDa was expressed successfully in the prokaryotes. Bioinformatic analysis showed that the NS1 protein, a soluble and acidic protein, was mainly expressed in the cytoplasm and composed of 627 amino acids with a molecular weight of about 72 kDa. It contained one serine/threonine kinase substrate motif and one s546 potential phosphorylation site but had neither signal peptide nor transmembrane region. The main epitope mainly located at the C terminal end of NS1 polypeptide. Phylogenetic tree and homological analysis of NS1 gene showed that NDPV and goose Parvovirus (GPV) were grouped in the same evolutionary branch, which indicated that they had the closest genetic evolutionary relationship. The results of the present study have laid a foundation for revealing the role of the NS1 protein in the replication, proliferation and pathogenesis of NDPV.%为研究新型鸭细小病毒(Novel duck parvovirus,NDPV)NS1蛋白的生物学功能,根据NDPV DS15株NS1基因(GenBank登录号:KU947420)设计1对特异性引物,对NS1全基因进行PCR扩增,将PCR扩增产物克隆入pGEX-4T-1载体,构建重组质粒pGEX-4T-NS1,并进行测序鉴定.经鉴定正确后重组质粒转化到BL21感受态细胞,SDS-PAGE和Western blot进行表达鉴定.将测序获得NS1基因编码蛋白进行生物信息学分析.结果显示,本研究构建的重组质粒可在原核细胞中成功表达97 kDa的NS1融合蛋白.进一步生物信息学分析结果显示,NS1蛋白为酸性、可溶性蛋白,由627个氨基酸组成,分子质量约为72 kDa.该蛋白主要位于胞浆,不含信号肽序列,无跨膜区,含有丝/苏氨酸激酶底物模序和1个潜在磷酸化位点(s546),抗原表位主要集中在多肽的C端.NS1基因的同源性及系统发生树分析表明,NDPV与鹅细小病毒(Goose parvovirus,GPV)处于同一进化分支,遗传进化关系最近.本研究结果为揭示NDPV NS1在病毒的复制、增殖以及致病机制中的作用奠定了基础.