犬细小病毒西宁分离株的分子鉴定及VP2基因序列分析

Molecular identification and sequence analysis of VP2 gene from Xining isolate of Canine Parvovirus

本研究首次对地处青藏高原地区的西宁的犬细小病毒分离株(命名为CPV-XN1)进行了VP2基因的克隆、测序,并与我国以及我国周边国家地区的流行株,以及参考株进行了核苷酸同源性比对和进化分析。VP2基因分子鉴定结果显示,该分离株为CPV-2b亚型株,VP2基因核苷酸序列全长1755bp,蛋白长度为584个氨基酸,分子量大小约为64.58 k Da,VP2蛋白为稳定蛋白,总体亲水性较高,可溶于水,VP2蛋白的二级结构以无规则卷曲为主;VP2基因的T-B细胞共同表位为序列的97-105位点,其序列为ALDDTHAQI,位点286-294,序列为GLPPFLNSL。西宁分离株与我国各地区流行株的核苷酸序列同源性和氨基酸同源性均在99%以上;CPV-XN1分离株VP2基因氨基酸序列中的非同义突变位点有1处,在78位氨基酸处;VP2基因进化树分析发现:CPV-XN1分离株与CPV-2b北京分离株(KT162024)和CPV-2b兰州分离株(JQ268284)组成一个微小分支,并与其它CPV-2b分离株处于同一个大支上,从进化角度上鉴定此研究分离株应属CPV-2b株。因此,本实验对青海西宁CPV-XN1分离株的VP2基因抗原表位分析和系统进化的探讨结果,有望为我国CPV分子流行病学调查提供一定的数据参考依据。

In the present study, the isolate of canine parvovirus （CPV-XN1） from Qinghai-Tibet Plateau Area was identified by molecular method, and the VP2 gene was cloned and sequenced, the nucleotide homology and evolutionary analysis was performed with epidemic and referenced strains for the first time. The CPV-VP2 gene molecular identified results showed that the CPV-XN1 isolate was belonged CPV-2b subtype. The nucleotide sequence of the VP2 gene was 1755 bp in length and 584 amino acids in length of the protein, the molecular weight was approximately 64.58 kDa, and the protein was stable, high hydrophilicity, mainly is irregular coiled-coil of the secondary structure, the T-B cell epitope was located in site 97-105 and the sequence is ALDDTHAQI, and site 286-294, sequence GLPPFLNSL. The nucleotide and amino acid sequence homology homology of the Xining isolates with other epidemic and referenced strains were all above 99 % , the non-synonymous mutation site in the amino acid sequence of the VP2 gene was only one and at 78 amino acid site. The CPV-VP2 phylogenetic tree analysis was revealed that the CPV-XN1 isolate and the CPV-2b Beijing isolate （KT162024） and the CPV-2b Lanzhou isolate （JQ268284 ） formed a tiny branch and with other CPV-2b isolates formed a big branch,and the Xining isolate was belong to the CPV-2b strain from the perspective of evolution. Therefore, the analysis results of the VP2 gene epitopes and phylogenetic evolution of the CPV-XN1 isolate from Qinghai Xining was expected to provide scientific reference data for the molecular epidemiological investigation of CPV in China.