猪繁殖与呼吸综合征病毒贵州株GZ-RNSP2基因的克隆与遗传变异分析

Cloning and Genetic Variation Analysis of NSP2 Gene of Porcine Reproductive and Respiratory Syndrome Virus Guizhou Strain GZ-R

为了解猪繁殖与呼吸综合征病毒(PRRSV)贵州株GZ-R的遗传变异情况及其NSP2基因的特征,本试验基于NSP2基因设计了2对特异性引物对目的基因进行巢式RT-PCR、克隆和序列分析。结果显示,第二轮巢式PCR除扩增出约3000 bp的预期条带外,还扩增出另一条约1200 bp的条带;将两DNA片段克隆至pMD19-T载体后经测序得到两条片段,大片段长2980 bp,命名为GZR-NSP2-L;小片段长1131 bp,命名为,GZR-NSP2-S;两片段同源性比对发现,GZR-NSP2-S具有GZR-NSP2-L N端第1-945位核苷酸与C端第2795-2980位核苷酸,缺失GZR-NSP2-L第946-2794位之间的核苷酸;GZR-NSP2-L与VR-2332株、Lelystad virus株的核苷酸同源性分别为81.2%、48.6%;编码氨基酸同源性比对结果与核苷酸同源性结果一致,揭示PRRSV GZ-R株属于美洲型毒株;氨基酸缺失位点比对发现,GZ-R NSP2在其第481位、第533-561位存在30个不连续氨基酸的缺失,缺失位点与PRRSV高致病性毒株一致。综上所述,可鉴定PRRSV GZ-R株为美洲型高致病性毒株。试验结果可为进一步探索PRRSV在贵州省的流行趋势以及NSP2在PRRSV复制过程中的作用机制提供一定的参考。

In order to investigate the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV)strain GZ-R and the characteristics of NSP2 gene,in this study,two specific primers were designed based on NSP2 gene for nested RT-PCR,cloning and sequence analysis of the target gene.The results showed that the second round of nested PCR not only amplified the expected band of about 3000 bp,but also amplified another band of 1200 bp.The two DNA fragments were cloned into the pMD19-T vector,and two fragments were obtained by sequencing.The large fragment was 2980 bp long and named as GZR-NSP2-L.The small fragment was 1131 bp long and named as GZR-NSP2-S.The homology comparison results of the two fragments showed that GZR-NSP2-S had GZR-NSP2-L N-terminal nucleotide 1-945 and C-terminal 2795-2980 nucleotide,and lack of nucleotide between bits 946-2794 in GZR-NSP2-L.The nucleotide homology of GZR-NSP2-L with VR-2332 strain and Lelystad virus strain was 81.2%and 48.6%.The results of homology comparison of encoded amino acids were consistent with the results of nucleotide homology,revealing that the PRRSV GZ-R strain belonged to the American-type strain.Comparison of amino acid deletion sites revealed that GZ-R NSP2 had 30 discontinuous amino acid deletions at positions 481 and 533-561.The deletion sites were consistent with PRRSV highly pathogenic strains.In summary,the PRRSV GZ-R strain could be identified as American-type highly pathogenic strain.The experimental results could provide some references for further exploring the epidemic trend of PRRSV in Guizhou province and the mechanism of NSP2 in PRRSV replication.