感染CDV的犬病料N、H、F基因的克隆与序列分析

目的了解病料中犬瘟热病毒（CDV）的变异情况,为犬瘟热的预防与治疗提供理论依据。方法采用RT-PCR方法对犬病料中CDV的血凝素蛋白（H）、融合蛋白（F）和核衣壳蛋白（N）基因进行扩增,将扩增产物克隆到pGEM-T-easy载体中测序,并进行序列分析。结果测定一株CDV的H、F和N基因大小分别为1863 bp、1653 bp和2235 bp。同源性分析表明,未发现碱基的插入和缺失。该病毒的H、F和N基因分别与国内强毒株BJ080127株的H基因、GN株的F基因和HL株的N基因亲缘关系最近,氨基酸同源性分别为97.3%、97.7%和99.3%。与国外标准野毒株A75-17在核苷酸水平上同源性依次为94.4%、93.8%和97.2%,与疫苗株CDV3在核苷酸水平上同源性分别为88.5%、88.8%和93.9%。系统进化树表明该病毒与国内强毒株在同一谱系。糖基化分析显示,该病毒在F基因3～5位氨基酸处多出1个糖基化位点。结论本研究从犬病料中成功克隆了犬瘟热病毒血凝素蛋白、融合蛋白和核衣壳蛋白,在F基因中新增了一个糖基化位点,为犬瘟热的遗传变异和流行学研究提供了分子生物学依据。

Cloning and Sequence Analysis of Canine Distemper Virus N,F and H Genes from the Samples Infected with CDV

Objective The aim of this study was to identify the variations of canine distemper viruses（CDV） obtained from clinical samples,and provide a theoretical basis for the prevention and control of CDV infection.Methods The segments of hemagglutinin protein（H） gene,fusion protein（F） gene and nucleocapsid protein（N） gene were amplified by RT-PCR.The amplicons were cloned into pGEM-T-easy vector and the recombination clones were sequenced.Subsequently,the nucleotide and amino acid sequences were analyzed.Results The nucleotide lengths of H,F and N genes for the virus were 1863 bp,1653 bp and 2234 bp,respectively.Sequence analysis did not present any nucleotide insertion or deletion.The three sequences determined in this study showed the high amino acid identities corresponding with three Chinese highly pathogenic CDV isolates BJ080127（H gene）,GN（F gene） and HL（N gene）,which were 97.3%,97.7% and 99.3%,respectively.Meanwhile,each of them displayed the nucleotide identity of 94.4%,93.8% and 97.2%,when compared with the CDV prototypic strain A75-17.In addition,comparing with the CDV vaccine strain CDV3,they displayed the nucleotide similarities of 88.5%,88.8% and 93.9%,respectively.The glycosylation sites analyses revealed a glycosylation site aimed at the 3 ～ 5 amino acids of F gene.The phylogenetic tree displayed that the virus had the closest relationship with the highly pathogenic CDV isolates identified in China.Conclusion In this study,we cloned the H,F and N genes of a CDV determined in canine samples and identified that the virusis a highly pathogenic CDV,which contains a novel glycosylation site in F gene.This study provides evidence in studies on the molecular mutation epidemiology of CDV infection.