【摘要】 对本实验室所分离的1株猪繁殖与呼吸综合征病毒(PRRSV)毒株GXYL-1403进行全基因组克隆和序列分析。参考已发表的扩增全长PRRSV序列的13对特异性引物,用RT-PCR方法对病毒基因组进行分段扩增。将扩增得到的PCR片段克隆到pMD18-T载体上,获得的重组质粒经PCR鉴定后进行序列测定。通过软件对所扩增序列进行拼接,获得GXYL-1403的全长序列,并对GXYL-1403进行比较和遗传进化分析。结果表明GXYL1403株基因序列全长为15 018bp,与国内外多株PRRSV毒株进行同源性比较发现,与VR-2332的相似性为89.1%,与TJ、JXA1、HEB1、HUB1、HUB2、TP株同源性达到了97.9%~98.1%。另外,GXYL-1403分离株nsp2蛋白含有高致病性PRRSV特异的1+29个氨基酸的缺失。特别重要的是,在缺失29个氨基酸区域的上游,含有连续20个氨基酸缺失,在下游出现了连续74个氨基酸的缺失。遗传进化分析发现美洲型PRRSV主要分为3个亚群,GXYL-1403跟高致病性PRRSV毒株属于同一分支,证实了GXYL-1403株为美洲型PRRSV毒株。更多还原

【Abstract】 To clone and analyse the full-length genome of a PRRSV strain GXYL-1403 isolated in our lab,13 pairs of primers published were used for RT-PCR to amplify the full-length genome of GXYL-1409.The PCR fragments were cloned into pMD18-T vector.The amplicons were identified by emzyme digestion and further sequenced.The full-length genome was obtained through DNA splicing by DNAstar software.The phylogenic tree of GXYL1403 and the reference strains was constructed based on the full-length genome.The results showed that the complete genome of GXYL1403 consisted of 15 018 bp.The complete genome of GXYL-1403 shared 89.1%identity with VR-2332 and shared 97.9%-98.1%identity with HPPRRSV strains TJ,JXA1,HEB1,HUB1,HUB2,TP.The phylogenic analysis based on the full-length genome revealed that PRRSV strains were classified into 3subgroups and GXYL-1403 was confirmed as HPPRRSV.It was suggested that GXYL1403 belongs to type II PRRSV.GXYL-1403 strain contained a deletion of 1+29aa in nsp2 coding region.More importantly,GXYL-1403 strain contained a deletion of 20 aa in nsp2 coding region,which was located upstream of 29 aa deletion.Beside,a continuous deletion of 74 aa emerged following the 29 aa deletion in nsp2 region.更多还原