表达猪圆环病毒2型衣壳蛋白的重组猪痘病毒的构建及鉴定

摘要

为探索猪痘病毒(swinepox virus,SWPV)作为猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗载体的可行性,本试验以痘苗病毒启动子P11启动绿色荧光蛋白筛选标记,猪痘病毒TK基因为外源基因的插入位点,P28、P7.5启动子启动PCV20RF2基因,以猪痘病毒JX20G株为亲本病毒,采用同源重组技术分别构建了两株表达PCV2衣壳蛋白的重组猪痘病毒rSWPV11-28C和rSWPV11-7.5C.结果显示,重组猪痘病毒rSWPV11-28C和rSWPV11 7.5C均成功表达了PCV2衣壳蛋白,表达的蛋白能与PCV2单克隆抗体6E12发生特异性反应;痘苗病毒启动子P28的启动效果明显优于P7.5,P28适合用于启动目的基因;利用重组猪痘病毒制备的PCV2灭活疫苗免疫小鼠后,rSWPV11-28C疫苗组的PCV2抗体水平与某PCV2商品疫苗相当,该重组病毒的成功构建为PCV2相关疾病及其他疫病在猪群中的防控提供了新的方向.%In order to investigate the feasibility of swinepox virus (SWPV) as porcine circovirus type 2 (PCV2) vaccine carrier,this study utilized promoter Pll of vaccinia virus launching the green protein screening markers,and used SWPV TK gene as the insertion of exogenous gene loci.P28 and P7.5 promoters started PCV20RF2 gene to construct two recombinant plasmids of rSWPV11-28C and rSWPV11-7.5C,respectively.SWPV JX20G strain was defined as the parent viruses,homologous recombination technology was used to build two strains (rSWPV11-28C and rSWPVll-7.5C),which expressed the PCV2 recombinant swinepox virus capsid protein.The results showed that the PCV2 capsid protein,which was successfully expressed in recombinant swinepox virus rSWPV11-28C and rSWPV11-7.5C,could react specifically with PCV2 monoclonal antibody 6E12.The PCV2 capsid protein promoted by the vaccinia virus promoter P28 was significantly higher than P7.5,P28 was suitable for starting the target gene.After immunization in mice via recombinant swinepox virus of PCV2 inoculated vaccine,the PCV2 of the rSWPVll-28C vaccine group and commodity vaccine showed equivalent antibody levels.The successful construction of the recombinant virus provided a new direction for the prevention and control of PCV2 related diseases and other diseases in swine.