蓝舌病病毒VP5蛋白的原核表达及其抗原性分析  

Prokaryotic expression and characterization of bluetongue virus VP5 gene

作　　者：赵大维[1,2] 朱彦青[3] 陈伟业[2] 胡倩倩[1,2] 殷倩倩[2] 黄克和[1] 步志高[2] 

机构地区：[1]南京农业大学动物医学学院,江苏南京210095 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001 [3]南京农业大学公共管理学院,江苏南京210095

出　　处：《中国预防兽医学报》2012年第8期607-610,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金青年基金(30901082)

摘　　要：为表达蓝舌病病毒(BTV)VP5蛋白并检测其生物学活性,本实验采用RT-PCR方法扩增血清12型BTV的VP5基因,构建重组质粒pMAL-VP5。将重组质粒转化TB1感受态细胞,以0.4 mmol/L的IPTG进行诱导表达,SDS-PAGE电泳结果显示,融合了大肠杆菌麦芽糖结合蛋白(MBP)标签的VP5重组蛋白以可溶形式表达,分子质量约为112.2 ku。利用MBP标签对重组蛋白进行纯化,并以其作为包被抗原,间接ELISA检测山羊血清样品,结果 5份羊血清P/N值均大于1,其中有1只羊检测结果 P/N值为2.396,鉴定为阳性血清,表明重组蛋白可以与VP5抗体反应,可以作为检测BTV VP5抗体的抗原,为今后进一步开展BT诊断研究奠定了基础。Bluetongue virus （BTV） is the agent of bluetongue disease （BTD）, an emerging, arthropod-transmitted disease of ungulates. In this study, the VP5 gene of serotype 12 BTV was amplified by RT-PCR and cloned into prokaryotic expression vector pMAL-e2X. The resultant recombinant plasmid was transformed into E. coli TB1 cells and recombinant protein （MBP-VP5） was expressed by inducing with IPTG. The recombinant protein was about 112.2 ku, mainly in soluble form and purified by MBP affinity chromatography. Five serum samples prepared from goats immunized with recombinant peste des petits ruminants virus expressing BTV VP5 protein were detected by the indirect ELISA coating with the recombinant MBP-VP5 protein. The result indicated that only serum sample was positive, which demonstrated that the recombinant MBP-VP5 protein was a promising antigen for BTV detection.

关 键 词：蓝舌病病毒 VP5基因 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]