猪繁殖与呼吸障碍综合征病毒河南分离株ORF3-7基因克隆与序列分析

Cloning and sequencing of ORF3-7 genes for porcine reproductive and respiratory syndrome virus isolated in Henan province

参照GenBank中公布的猪繁殖与呼吸综合征病毒（PRRSV）美洲株ATCC VR2332的ORF3～7基因序列,利用Pri mer软件分别设计合成了针对PRRSV ORF3、ORF4、ORF5、ORF6和ORF7基因的特异性引物,利用RT-PCR从河南分离株Hn-1/06株分别扩增得到了大小约964、675、718、566和501 bp的片段,并将扩增的片段插入PGEM-T-easy载体进行测序。利用DNAStar软件进行序列分析表明,河南分离株Hn-1/06株属于美洲型,同时将Hn-1/06株ORF3～7基因的核苷酸序列和推导氨基酸序列与ATCC VR2332、LV、Resp、CH-1a、BJ-4、S1、CC-1、HB-1、JX0612、HUB1等分离株进行了同源性分析。

Five pair of specific primers of PRRSV ORF3 to ORF7 were designed and synthesized according to the published gene sequence of PRRSV from GenBank（PRU87392）, and ORF3, ORF4, ORFS, ORF6, ORF7 genes of PRRSV Hn-1/06 strain were obtained by RT-PCR method. The RT-PCR products were directly cloned into the PGEM-T-easy vector and these recombinant plasmids were transformed into JM109 competent cell,identified by enzyme digesting and DNA sequencing. The positive clones were designated PGEM-T-ORF3, PGEM-T-ORF4 ,PGEM- T-ORFS, PGEM-T-ORF6, and PGEM-T-ORFT, respectively. After sequencing, the sequences of ORF3, ORF4, ORFS,ORF6,ORF7 genes of the PRRSV Hn-1/06 isolate were compared with sequences of other ten different PRRSV strains in GenBank by Blast and DNAStar software. The results showed that the Hn-1/06 displayed high nueleotide identities with strain VR22332, but distinct from LV, showing genetically much closer relationship to strain VR22332.