成都地区犬细小病毒VP2基因克隆分析及基因型鉴定

Cloning and Analysis of VP2 Gene of Canine Parvovirus and Identification of Their Genotypes in Chengdu

根据GenBank中犬细小病毒(canine parvovirus,CPV)VP2基因序列设计合成两对特异性引物1F/1R和2F/2R,从疑似CPV感染病犬的粪便病料中提取DNA作为模板,分段扩增涵盖VP2基因的两个片段,大小分别为1325和758bp,并进行克隆、测序,通过拼接得到11条长度为1755bp的VP2基因完整序列。核苷酸同源性分析表明,扩增得到的11个完整VP2基因序列与GenBank中发布的21株国内外参考毒株比较,核苷酸同源性为96.2%～99.8%。采用DNAStar软件分析,预测VP2蛋白可能成为B细胞表位的区段位于Met1-Val38、Met73-Val82、Met96-Ala103、Lys151-Thr170、Gly235-Phe243、Leu294-Phe303、Ala359-Thr399、Ile469-His483、Ala503-Arg520、Lys570-Tyr584。将扩增获得的VP2基因编码的氨基酸序列与CPV参考毒株进行比较,第426位和第555位氨基酸残基分别为Asp和Val,鉴定出本试验得到的CPV基因型均为2a型,初步证实成都地区仍以CPV-2a为主要流行毒株。

According to the canine parvovirus（CPV） VP2 gene sequences in GenBank,two pairs of primers 1F/1R and 2F/2R were designed and synthesized.Taking the DNA extracted from the feces samples of dogs suspected to be infected with CPV as templates,two fragments covering VP2 gene with the length of 1325 and 758 bp were amplified respectively,after cloning and sequencing of the two fragments,eleven complete VP2 gene sequences with the length of 1755 bp were obtained through splicing.The analysis of nucleotide homology showed that compared with 21 reference strains of CPV at home and abroad in GenBank,the nucleotide homologies of these eleven VP2 gene sequences were from 96.2% to 99.8%.By using the DNAStar software,the B cell epitopes of VP2 protein were predicted to be in the sections of Met1-Val38,Met73-Val82,Met96-Ala103,Lys151-Thr170,Gly235-Phe243,Leu294-Phe303,Ala359-Thr399,IIe469-His483,Ala503-Arg520 and Lys570-Tyr584.Compared with the amino acid sequences of the reference CPV strains,the amino acid residues at the sites of 426 and 555 of the VP2 protein coding by these eleven VP2 genes were Asp and Val respectively,which indicated that the genotypes of these eleven CPV were all CPV type 2a and also primarily proved that the CPV-2a was the main prevalent strain in Chengdu area.