猪细小病毒YL株序列分析及其VP2基因原核表达

Sequence Analysis of Porcine Parvovirus YL Strain and Prokaryotic Expression of VP2

为对猪细小病毒(porcine parvovirus,PPV)进行分子生物学研究,将该病毒YL株基因组分为4个重叠片段进行PCR扩增,并将扩增产物克隆到pGEM-T载体中,测定病毒基因组全长序列。另外,应用其中1对引物,通过PCR方法扩增出一段包含VP2主要抗原区域的片段,构建重组质粒pET-VP2,IPTG诱导表达。序列分析结果表明,PPV YL株和国内外PPV分离毒株NS1基因核苷酸同源性为97.8%～99.4%,氨基酸同源性为96.7%～99.5%,与国内外主要毒株VP2基因核苷酸同源性为98.7%～99.7%,氨基酸同源性为97.4%～99.8%。进化树分析表明,PPV YL株与BQ株(EU790641)毒株处在同一分支上,遗传距离最近。SDS-PAGE结果显示,表达产物分子质量为56ku,经Western blotting检测具有生物学活性。

In order to study the molecular biology of PPV, the complete genome of porcine parvovirus （PPV） was amplified using polymerase chain reaction （PCR） method and cloned. The DNA fragment was sequenced and compared with reference porcine parvovirus. In addition, the major eptiope domain of porcine parvovirus structural gene VP2 was amplified by polymerase chain reaction （PCR） and in- serted into the expression plasmid, pET-32a, then the recombination was induced by IPTG in Esche- richia coli BL21. TheNS1 gene between PPV YL strain and other porcine parvovirus strains showed 97.8%-99.4% and 96.7%-99.5% identities at nucleotide and amino acid levels respectively. The VP2 genes showed 98.7%-99.7% and 97.4%-99.8% homologies at nucleotide and amino acid levels respectively. The genetic relationship of PPV YL strain with BQ strain was the closest. SDS- PAGE and Western blotting analysis revealed that the recombinant protein was 56 ku. Biological activity of the recombinant protein was detected by Western blotting. The results can be applied in the genetic variation and differential diagnosis of PPV.