猪伪狂犬病毒gB基因在大肠杆菌中的分段表达

目的分段表达伪狂犬病毒（pseudorabies virus,PRV）gB基因,用于PRV疫苗免疫抗体评估试剂盒研究。方法分析gB抗原表位,设计4对特异性引物,从PRV新疆分离株中扩增gB基因的4个片段,分别命名为gB-1、gB-3、gB-4和gB-5基因,克隆到pGEM-T-easy载体并测序。再将4个gB基因片段融合到pGEX-6P-1原核表达载体中,表达并纯化。Western Blotting进行重组蛋白的抗原性检测。以纯化的重组蛋白为抗原建立间接ELISA方法,检测猪血清临床样品。结果构建的4种表达质粒均可在大肠杆菌中表达,经SDS-PAGE分析,表达分子量分别为75.05、53.86、49.96和49.72×10^3Da,其中gB-3、gB-4和gB-5表达量较高,表达产物主要存在于包涵体中。Western Bloting鉴定显示,4种融合蛋白均可被猪伪狂犬阳性血清特异识别。以gB-3、gB-4和gB-5建立的ELISA方法与IDEXX公司的gB-ELISA试剂盒符合率分别为40.9%、81.8%和81.8%。结论本研究成功表达了PRV的gB-1、gB-3、gB-4和gB-5重组蛋白,gB-3、gB-4和gB-5表达量较高;其中以gB-4和gB-5建立的间接ELISA方法与IDEXX公司的符合率较高。

Fractional Expression of gB Gene of Pseudorabies Virus in E.coli

Objective To express gB gene of pseudorabies virus fractionally for developing an ELISA on PRV vaccine antibodies.Method We analysed the gB epitope,and designed four pairs of specific primers,then four fragments of gB gene were amplified from PRV Xinjiang strain,which were named for gB-1、gB-3、gB-4 and gB-5 gene.The four gene fragments were cloned into PGEM-T-easy and sequenced,then the four genes were inserted into pGEX-6P-1 vector.The expressed proteins were analyzed by Western blotting.Three indirect ELISA methods were set up using purified gB-3、gB-4 and gB-5 proteins as antigen,and 22 clinical pig sera samples were detected.Results The four recombinant expression plasmids were successfully expressed in Coli,and the proteins molecular weight were 75.05、53.86、49.96 and 49.72×10^3Da,and gB-3、gB-4 and gB-5 have higher level expression,and the expression product mainly exists in inclusion body.Western blotting identification displayed that all the four fusion proteins were distinguished by pseudorabies virus positive serum.The coincidence rate between gB-3-ELISA,gB-4-ELISA,gB-5-ELISA and gB-ELISA of IDEXX company were respectively 40.9%,81.8% and 81.8%.Conclusion The study successfully expressed gB-1、gB-3、gB-4 and gB-5 of PRV gB,gB-3、gB-4 and gB-5 had higher expression.The gB-4-ELISA and gB-5-ELISA had high level coincidence with gB-ELISA of IDEXX company.