共表达猪细小病毒VP2蛋白和猪圆环病毒2型Cap蛋白的重组伪狂犬病毒的构建及其鉴定

Construction and characterization of a recombinant pseudorabies virus expressing VP2 of porcine parvovirus and Cap of porcine circovirus type 2

为研制猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)3联苗,本研究将PPV VP2基因和PCV2 Cap基因通过口蹄疫病毒2A基因串联得到VP2-2A-Cap(V2C)基因,将其插入pEP-CMV-in转移载体构建pEP-V2C-in重组表达质粒,再通过Red/ET两步重组法在大肠杆菌中构建了pPRV-V2C-ΔgE和pPRV-ΔgE突变体,该突变体用磷酸钙法转染BHK-21细胞获得重组病毒rPRV-V2C-ΔgE和rPRV-ΔgE。Western blot分析表明rPRV-V2C-ΔgE能够在BHK-21细胞内表达融合蛋白VP2-2A-Cap,而且目的蛋白能够被2A蛋白裂解为64 ku和28 ku两个片段。重组病毒生长曲线和噬斑大小测定结果显示rPRV-V2C-ΔgE在BHK-21细胞中的增殖滴度与亲本株rPRV无显著差异,表明外源基因的插入不影响rPRV的增殖。本研究为PRV、PPV和PCV2 3联重组活载体疫苗的研制奠定了基础。

To develop porcine pseudorabies virus（PRV）,porcine parvovirus（PPV） and porcine circovirus type 2（PCV2） trivalent vaccine,the recombinant plasmid pEP-V2C-in was constructed by inserting VP2-2A-Cap（V2C） gene,which consisted of the CDS of PPV VP2 and PCV2 Cap gene linked with 2A gene of foot and mouth disease virus（FMDV）,into expression vector pEP-CMV-in.The BAC clones of pPRV-V2C-ΔgE and pPRV-ΔgE were generated by twice Red/ET recombinations in E.coli.Then the recombinant virus of rPRV-V2C-ΔgE and rPRV-ΔgE were rescued from BHK-21 cells transfected with the pPRV-V2C-ΔgE and pPRV-ΔgE by calcium phosphate precipitation,respectively.Western blot assay showed that PPV VP2 and PCV2 Cap proteins were expressed in rPRV-V2C-ΔgE virus-infected BHK-21 cells with the molecular mass of 64 ku and 28 ku respectively,which demonstrates that the VP2-2A-Cap fused protein were cleaved by FMDA 2A.Furthermore,the results of growth kinetics and plaque formation sizes showed that insertion of the foreign genes had no influence on the propagation of rPRV-Bac-EF1 in BHK-21 cells.These studies have laid a foundation for developing PRV based vector vaccine expressing PPV VP2 and PCV2 Cap.