犬细小病毒VP2基因的原核表达及纯化  

The Prokaryotic Expression and Purification for VP2 Gene of Canine Parvovirus

作　　者：张坤[1,2] 马丽娟[3] 李刚[1,4] 黄伟[2] 李伟[1,4] 贾凤琴[1,4] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100094 [2]西南大学动物科技学院,重庆北碚400715 [3]吉林大学畜牧兽医学院,长春130062 [4]动物营养学国家重点实验室,北京100094

出　　处：《中国动物保健》2009年第12期46-51,共6页China Animal Health

基　　金：中央级公益性科研院所基本业务费专项(0032007008);北京市科委基金项目(Z07010501780701)

摘　　要：参照GenBank中犬细小病毒VP2基因序列设计了一对添加EcoRⅠ和XhoⅠ酶切位点的引物,对CPV VP2基因进行PCR扩增,克隆至pGM-T载体,获得重组质粒pGM-T-VP2,将重组质粒亚克隆到表达载体pET-32a上,获得表达重组子pET-32a-VP2,经EcoRⅠ和XhoⅠ双酶切鉴定后,序列分析确定该重组子YM株VP2基因ORF正确,转化至E.coli Rosetta(DE3)中,获得了基因工程菌株E.coli Rosetta(CVP2),并对重组菌进行了诱导表达,鉴定及纯化。结果表明纯化样品中含有87kDa目的蛋白,Western blot检测发现该蛋白与犬细小病毒多克隆抗体发生反应,出现特异条带。E.coli Rosetta(CVP2)以包涵体形式表达出了CPV-YM株VP2,从而为进一步制备检测抗体效价的胶体金检测试剂盒的开发研究打下了良好的基础。A pair of primers added EcoR Ⅰ and Xho Ⅰ sites was designed and synthesized based on the sequence of the VP2 protein gene of Canine Parvovirus （CPV） for amplifying the full-length VP2 gene fragment. The fragment was cloned into pGM-T vector and transformed into Escherichia coli TOP10. Then the cloned fragment was sub-cloned into the expression vector pET-32a and identified by digestion of EcoR Ⅰ and Xho Ⅰ , it showed the ORF of VP2 gene and been transformed into E.coli Rosetta（DE3 ）. The recombinant bacteria E.coli Rosetta（CVP2） was selected and cultured to induce expression and purification The results showed that purified VP2 protein has 87 kDa of molecular by SDS-PAGE. The special band was found in the Western blot of the expressed VP2 protein with the polyclonal antibody of CPV. E.coli Rosetta（ CVP2 ）expressed the VP2 protein of CPV-YM and have the value in developing CPV colloidal gold kit for the detection of Ab against CPV.

关 键 词：犬细小病毒 原核表达 纯化 

分 类 号：S852.65[农业科学—基础兽医学]