猪细小病毒(PPV)SD1株NS1基因的克隆与原核表达

目的]为建立猪细小病毒的快速诊断方法提供理论依据。方法]根据GenBank上猪细小病毒基因组序列和原核表达质粒pET30a(+)多克隆位点序列设计1对引物,应用PCR技术扩增出猪细小病毒SD1株NS1基因全序列,对阳性重组质粒进行测序和同源性比较。构建原核表达重组质粒pET 30a/NS1,并在大肠杆菌中进行诱导表达。结果]通过PCR扩增获得2 208 bp目的片段。所克隆NS1基因与已报道的PPV相应基因的核苷酸同源性为97.3%~99.4%,表明NS1基因具有高度保守性,但在偏3′端连续缺失12个碱基。所克隆的PPVNS1基因在原核细胞中成功表达,其表达产物主要以包涵体形式存在。结论]SDS-PAGE检测结果表明PPVNS1蛋白的分子量为86 kD。

Cloning and Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus(PPV).Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a( ) with multiple cloning sites.The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison.The prokaryotic expression recombinant plasmid PET 30a/NS1 was constructed to make its induction expression in Escherichia coli.Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification.The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3% to 99.4%,which indicated that NS1 gene had high conservation.But it had a 12-basepair successive deletion near the hydroxyl end.The cloned PPV NS1 gene was successfully expressed in prokaryotic cell,and its expression products existed mostly in inclusion bodies.Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 kD.