大理地区犬细小病毒VP2基因变异研究及进化分析

Research and Evolution Analysis of Canine Parvovirus VP2 Gene Variation in Dali Area

从云南大理地区发病的松狮犬、德国牧羊犬和博美犬的粪便内和国产疫苗中分离到4株犬细小病毒，用同步法接种到F81细胞中分离、鉴定、培养。收毒后提取基因组DNA，并以此为PCR模板；根据GeneBank己发表的犬细小病毒VP2基因序列设计合成一对引物，进行PCR扩增获得VP2全序列基因1755bp片段，并进行序列测定。利用DNAStar软件对4株病毒的VP2基因序列以及氨基酸序列进行分析，与GeneBank上已发表的16株犬细小病毒比较，发现病毒的核苷酸同源性在97.9%~99.9%之间，氨基酸同源性在96.4%~99.9%之间，总体同源性在98%以上，进化树分析显示没有形成明显的CPV中国进化分枝，表明VP2基因变异相对较少。同时分离到的4株CPV病毒又遵循2a亚型的进化特征，因此确定目前云南大理地区犬细小病毒的流行情况仍以CPV-2a为主。

4 strains of CPV were isolated from faeces of dogs and home-made CPV vaccine. Feline kidney F81 cells were used to isolate,identify and cultivate CPV. A pair of primers were designed according to the sequence of VP2 gene from Genebank,the genome DNA of CPV was extracted and used as templates for polymerase chain reaction to amplify the VP2 gene. The PCR products were gathered and sequenced,the results indicated that the length of amplifyed gene fragment was 1755bp. Gene order and amino acid sequence of the 4 strains were analysed using DNAstar,the result-ing data was compared with 16 CPV strains published in GeneBank. The results showed that nucleotide homology was 97.9%~99.9%,amino acid homology was 96.4%~99.9%,and the toal homology was over 98%. Cladogram analysis demonstrated that there was no conspicuous evolution arborization of CPV in China,suggesting little gene variation of VP2 .The 4 CPV strains followed the CPV-2a evolution features,the result showed that CPV-2a was the main prevalent stain in Dali.