广西猪瘟病毒E0和E2基因的克隆及序列分析

通过分析目前广西猪瘟病毒(CSFV)的E0和E2基因特征,为了解广西地区CSFV的分子流行病学、遗传变异及综合防控提供科学依据。试验采用RT-PCR方法,对阳性猪瘟样品进行CSFV的E0及E2基因的扩增,经克隆、测序后,利用DNAStar软件对序列进行比对分析,同时绘制系统遗传进化树。结果表明,从阳性猪瘟样品中成功扩增CSFV的E0及E2基因。序列比对分析发现,GX2毒株与参考毒株的E0基因核苷酸同源性在83.1%~94.1%,其推导氨基酸同源性在85.9%~99.6%;与参考毒株的E2基因核苷酸同源性为81.7%~93.7%,其推导氨基酸同源性为89.0%~97.0%;E0与E2基因均属于基因Ⅱ群。氨基酸变异位点分析表明,E0蛋白的RNase活性区域氨基酸基序位点没有发生变异;E2蛋白中15个位点上的半胱氨酸均未发生变异,但单抗识别位点S734R发生变异。遗传进化分析显示测定的GX2毒株与近年来广西CSFV流行毒株的变异趋势相似,与中国传统疫苗株HCLV、经典强毒株Shimen的同源性较低,亲缘关系较远,与广西近年来的流行毒株GXWZ02株的同源性较高,亲缘关系较近。

Cloning and Sequencing of Classical Swine Fever Virus E0 and E2 Genes in Guangxi

In order to provide the scientific basis for molecular epidemiology, genetic variation, comprehensive prevention and control of classical swine fever in Guangxi, the molecular characteristic of E0 and E2 genes of classical swine fever virus (CSFV) in Guangxi was analyzed. The E0 and E2 genes of CSFV from positive samples of swine was amplified using RT-PCR, cloned into the pMD18-T vector and sequenced. The sequences were analyzed using DNAStar software and the phylogenetic trees were drawn. The result indicated that the E0 and E2 genes of CSFV were amplified from positive samples, the homologies of nucleotide sequence and amino acid sequences of the GX2 strain to E0 gene of reference strains were 83.1% to 94 1% and 85.9% to 99.6%, respectively. The homologies of nucleotide sequence and amino acid sequences of the GX2 strain to E2 gene of reference strains were 81.7% to 93.7% and 89.0% to 97.0%. The E0 and E2 genes of CSFV belonged to group Ⅱ. No variation occurred at E0 protein amino acid sequences of two domains of RNase activity. No change was observed in 15 Cys sites of E2 protein of the GX2 strain. However, the variation of amino acids at sites S734R was observed. The variation trend of GX2 strain was similar to CSFV strains which were isolated in recent years in Guangxi. GX2 strain shared low homology with HCLV and Shimen strains and was not close related to HCLV and Shimen, but shared high homology with GXWZ02.