绵羊肺腺瘤病毒CA基因的原核表达及抗原性检测

摘要

绵羊肺腺瘤病(OPA)于1850年发现,但国内外至今尚未建立绵羊肺腺瘤病毒(JSRV)的体外培养方法及快速有效的实验室检测方法.为表达JSRV的衣壳蛋白(CA)基因,本研究根据JSRV CA基因编码序列设计特异性引物进行PCR扩增,克隆至pEASY载体中进行测序,并构建重组表达质粒pET-CA,在大肠杆菌中经IPTG诱导表达.重组蛋白经SDS-PAGE检测约为28 ku,主要以可溶形式表达.将纯化的重组蛋白免疫新西兰白兔,制备抗血清.以制备的抗血清对重组蛋白及病毒进行western blot鉴定.结果显示,该抗血清能够识别重组蛋白及天然的JSRV.本研究首次原核表达了完整的CA重组蛋白,并制备了能够与天然JSRV结合的抗CA蛋白的抗血清,为进一步建立JSRV ELISA方法奠定了基础.%To express the capsid antigen (CA) gene of Jaagsiekte sheep retrovirus (JSRV) and prepare the antiserum against the virus,the CA gene was amplified by PCR from JSRV proviral DNA with a pair of primers designed according to the CA gene of JSRV in GenBank.Subsequently,the CA gene was cloned into pET-28a(+) for expression in E.coli and the recombinant CA protein was about 28 ku,mainly in soluble form,detected by SDS-PAGE.In addition,the antiserum was prepared from rabbits immunized with the purified recombinant protein.Western blot showed that the antiserum was able to recognize both of the recombinant CA pretein and the native virus protein,which provided the basis for the establishment of the ELISA method to detect the JSRV infection in sheep.