鹅细小病毒主要免疫原性蛋白VP3基因遗传变异及其原核表达  

Genetic variation and prokaryotic expression of VP3 gene of goose parvovirus

作　　者：毕玉海[1] 丁壮[1] 徐明[1] 李志杰[1] 常爽[1] 黄海楠[1] 宋子运[1] 尹仁福[1] 杜眉[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2007年第2期172-175,共4页Chinese Journal of Veterinary Science

基　　金：吉林省科技厅发展计划资助项目(吉科合字第20000212-01)

摘　　要：根据鹅细小病毒(GPV)B株全基因序列,设计并合成了1对特异性引物,采用PCR技术对GPVGD-01株的VP3基因进行扩增,将目的基因克隆到pGEM○R-T后测序,分析了GPV GD-01株VP3基因的遗传变异情况,并进行原核表达。结果表明,GPV GD-01株VP3基因全长1605bp,编码534个氨基酸(DQ665790),与已发表的GPV、番鸭细小病毒(MDPV)的核苷酸、氨基酸同源性分别为96.15%、80.1%,97.78%、89.13%,说明GPV VP3基因具有较高的遗传稳定性。结合亲水性、表面极性、抗原位点分析比较GPV、MDPV VP3基因,为研究GPV和MDPV感染谱差异的分子机制提供了一定的理论基础。原核表达显示,VP3蛋白的最高表达量占整个菌体蛋白含量的29.9%,经表达条件优化可产生大量可溶性VP3表达蛋白。SDS-PAGE与Western-blot分析显示,表达的VP3蛋白的相对分子质量约60000,并能与鼠抗GPV阳性血清反应,证明具有一定的反应原性,为进一步研制基因工程疫苗及诊断制品奠定了基础。According to nucleotide sequence of GPV B strain,a pair of primers were designed and synthesized.The complete VP3 gene of a domestic isolate strain(GPV GD-01) was amplified by PCR,cloned into pGEM○R-T vector and then sequenced for investigating the genetic variation and prokaryotic expression.The result showed that the VP3 gene was 1 605 bp long and included a complete open reading frame(ORF) encoding a protein of 534 amino acids.Compared VP3 gene of strain GPV GD-01 with the published goose parvovirus(GPV) and muscovy duck parvovirus(MDPV) strains,the average homology of the nucleotide sequence is 96.15%,80.1% and the average homology of the deduced amino acids sequence is 97.78%,89.13%,these numbers showed that the VP3 gene of GPV have a high genetic stability.With the hydrophilicity,surface probability and antigenicity analysis,the results of this study have important meaning for study the infection spectrum and molecular pathogenic mechanism of GPV and MDPV.The results of prokaryotic expression analysis showed that the expressed VP3 protein is about 60 000 and amounts to 29.9% of the total protein of E.coli BL21(DE3) after induced by IPTG(1 mmol/L) at 37℃ for 5 hours and it can product a great deal of soluble proteins.Western-blot analysis showed that the expressed protein was specifically recognized by polyclonal antibody against GPV.This study laid foundation for the development of genetic engineering vaccine and the method for detection of GPV.

关 键 词：鹅细小病毒 VP3基因 遗传变异 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]