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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

DOI: 10.7668/hbnxb.2009.04.008, PP. 36-40

Keywords: 猪繁殖障碍与呼吸综合征病毒,HN-HW株,ORF2-7,克隆与序列分析

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Abstract:

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ATCCVR-2332基因序列,用Primer.0软件分别设计合成了针对PRRSVORF2、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86bp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ATCCVR-2332、JX-A1、CH-1a、BJ-4、HB-1、HB-2、HEB-1、HUB-1、HUB-2、SP、P129、MN184A、RespPRRSMLV、PrimePac、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ATCCVR-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ATCCVR-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.

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