为了研制有效的PRRS疫苗,参照GenBank中公布的猪繁殖障碍与呼吸综合征病毒(PRRSV)美洲型ATCC VR-2332基因序列,用Primer.0软件分别设计合成了针对PRRSV ORF2、ORF3、ORF4、ORF、ORF6和ORF7基因的特异性引物,利用RT-PCR从HN-HW株分别扩增得到了大小约771,904,64,670,619和86 bp的片段,并将扩增的片段插入pGEM-Teasy载体,然后转化到大肠杆菌JM109感受态细胞中,挑取阳性克隆PCR和酶切鉴定后进行测序.利用DNAStar软件将HN-HW株ORF2-7基因的核苷酸序列和推导氨基酸序列与ATCC VR-2332、JX-A1、CH-1a、BJ-4、HB-1、HB-2、HEB-1、HUB-1、HUB-2、SP、P129、MN184A、RespPRRS MLV、Prime Pac、LV等毒株相应序列进行了同源性分析,并绘制系统进化树.结果显示,HN-HW株与美洲型ATCC VR-2332核苷酸同源性为88.9%～94.9%,与欧洲型LV核苷酸同源性为64.0%～69.0%;推导氨基酸序列与ATCC VR-2332株同源性为82.4%～96.0%,与LV株的同源性为34.%～78.9%.系统进化树表明,HN-HW株属于美洲型,与JXA1、HUB-1、HUB-2、HEB-1亲缘关系较近.
郝雪峰
,
关贵全
,
李有全
,
马米玲
,
刘爱红
,
马新龙
,
江丽丽
,
殷宏
,
罗建勋
. 猪繁殖障碍与呼吸综合征病毒HN-HW株ORF2-7基因的克隆与序列分析[J]. 华北农学报, 2009
, 24(4)
: 36
-40
.
DOI: 10.7668/hbnxb.2009.04.008
Specific primers of PRRSV ORF2 to ORF7 were designed and synthesized according to the published gene sequence of PRRSV from GenBank by Primer software, and ORF2, ORF3,ORF4, ORF5,ORF6, ORF7 genes of PRRSV HN-HW strain were obtained by RT-PCR method.The RT-PCR products were cloned into the pGEM-Teasy vector and these recombinant plasmid were transformed into E.coli JM109 competent cell.Identification by enzyme digesting and PCR, the positive clones were sent to sequencing.After sequencing, the sequences of ORF2,ORF3,ORF4,ORF5,ORF6, ORF7 genes of the PRRSV HN-HW isolate were compared with sequences of other different PRRSV strains (ATCC VR- 2332, JX-A1,CH-1a,BJ-4,HB-1,HB-2,HEB-1,HUB-1,HUB-2, SP, P129,MN184A, RespPRRS MLV, Prime Pac, LV) in GenBank by DNAStar software.The results showed that the ORF2-ORF7 sequences of the HN-HW strain shared 88.9 %-94.9 % of nucleotide homology and 82.4 %-96.0 % of amino acid homology with North American genotype ATCC VRR-2332 strain.And they shared only 64.0 %-69.0 % of nucleotide homology and 34.5 %-78.9 % of amino acid homology with Europe genotype LV strain.The phylogenetic trees revealed the PRRSV HN-HW strain was clustered within North American genotype and had close relationship with JXA1,HUB-1,HUB-2 and HEB-1.
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