犬细小病毒JS12株VP2基因的分子特征及其在大肠杆菌中的表达  

Molecular characteristics of VP2 gene of canine parvovirus isolate JS12 and its expression in Escherichia coli

作　　者：李月华[1,2] 王永山[2] 毕振威[2] 夏兴霞[2] 欧阳伟[2] 刘华洁[1,2] 贾红[3] 姚火春[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室,江苏南京210014 [3]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国兽医科学》2013年第10期991-998,共8页Chinese Veterinary Science

基　　金：江苏省农业科技自主创新资金项目[CX(12)3062];公益性行业(农业)科研专项(201303042)

摘　　要：从犬细小病毒病免疫预防失败病犬体内分离到1株犬细小病毒（CPV），命名为CPV—JSl2株。病毒可凝集猪的红细胞，对乙醚不敏感，pH3不能灭活病毒，pHl0可使病毒失去感染性，56。C作用60min病毒仍有活性，80℃作用30min可灭活病毒。序列分析时CPV—JSl2为CPV-2a亚型，VP2基因全长1755nt，编码584aa，与国外CPV毒株VP2基因的核苷酸序列和氨基酸序列的同源性分别在98．9％～99．6％和98．39／6～99．7％之间，与国内和周边地区毒株VP2基因的核苷酸序列和氨基酸序列的同源性分别在99．0％～99．7％和97．9％～99．7％之间；16个中国分离株处在不同组，CPV—JSl2株与韩国毒株DH426处在同一分支上，亲缘关系较近。对CPV—jsl2与免疫毒株VP2蛋白的立体结构分析比较，发现有9处氨基酸残基变异，其中6处位于衣壳蛋白表面，5处位于抗原表位区。将VP2基因在大肠杆菌中表达，重组VP2蛋白的分子质量为67．0ku，主要以包涵体的形式存在；Western—blot分析中重组VP2蛋白可与CPV阳性血清发生特异性反应。以纯化的重组VP2蛋白为抗原建立的CPV抗体间接ELISA检测方法具有良好的特异性。A canine parvovirus（CPV）,designated CPV-JS12, was isolated from dogs with immunoprophylaxis defeat in Jiangsu Province in 2012. The virus could agglutinate porcine erythrocytes and was stable after treatments of 56 ℃ for 1 h,pH 3.0 and lipid solvents. Sequence analysis showed that the VP2 gene of the local CPV isolate was composed of 1755 nucleotides, encoding 584 amino acids. Clustering analysis indicated that the homology of the VP2 gene were from 98.9% to 99.6% in nucleotide and from 98.3% to 99.7% in amino acid based on the sequences of CPV isolates available in GenBank. The phylogenetic tree based on the sequence of VP2 gene of CPV showed that 16 Chinese isolates were in different groups and JS12 was in the same subgroup with a Korea strain. Compared the structure of CPV-JS12 with vaccinestrain,they had nine amino acid residues variation,of which six were located on the capsid protein surface, and five located in antigenic area. The VP2 gene from JS12 was sub-cloned into pET-28a（＋） and expressed in E. coli. SDS-PAGE analysis indicated that the recombinant VP2 protein was 67.0 ku and existed as inclusion bodies in E. coli. Western-blot showed that the recombinant VP2 protein could be recognized by CPV positive serum. An indirect ELISA for the detection of antibody against CPV by using purified recombinant VP2 protein was developed and specific to CPV.

关 键 词：犬细小病毒 VP2基因 分子特征 病毒变异 原核表达 间接ELISA 

分 类 号：S852.655[农业科学—基础兽医学]