表达PRRSV GP5(GP5m)、M蛋白与猪IL-18的重组PRV的构建及对小鼠免疫特性分析

Construction and immunogenicity evaluation in mice of recombinant pseudorabies viruses expressing porcine reproductive and respiratory syndrome virus GP5(GP5m),M protein and porcine IL-18

为获得有效预防猪繁殖与呼吸综合征(PRRS)和猪伪狂犬病(PR)的重组活载体疫苗,以rPRV-gE^(-)/TK^(-)/GFP^(+)为载体将猪繁殖与呼吸综合征病毒(PRRSV)感染株ATCC-VR2332的GP5、修饰型GP5(GP5m)和M蛋白基因以及猪IL-18基因以不同组合方式插入至伪狂犬病病毒(PRV)TK基因位点,构建5种重组PRV(rPRV):rPRV-gE^(-)/TK^(-)/GP5m^(+)、rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+)、rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)、rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)/IL-18^(+)和rPRV-gE^(-)/TK^(-)/IL-18^(+)。免疫印迹结果显示rPRV可表达GP5、GP5m、M和IL-18。随后将5种rPRV分别进行小鼠免疫原性试验,rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)、rPRV-gE^(-)/T K-/GP5^(+)/M^(+)/IL-18^(+)和rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+)可产生抗PRRSV和PRV的特异性中和抗体,并能刺激CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞增殖及IFN-γ、IL-4和IL-10细胞因子的表达。综上,rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)、rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)/IL-18^(+)和rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+)可有效诱导小鼠产生抗PRRSV和PRV特异性全身免疫反应,为研发有效预防PRRS和PR的重组活载体疫苗奠定了基础。

To obtain an effective recombinant live vector vaccine for the prevention of porcine reproductive and respiratory syndrome(PRRS)and porcine pseudorabies(PR),GP5,modified GP5(GP5m),and M gene of the PRRS virus(PRRSV)infetious clone ATCC-VR2332 and porcine IL-18 gene was inserted into the TK gene site of rPRV-gE^(-)/TK^(-)/GFP^(+)deletion strain by homologous recombination,five recombinant pseudorabies viruses(rPRVs)were constructed,respectively,namely rPRV-gE^(-)/TK^(-)/GP5m^(+),rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+),rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+),rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)/IL-18^(+),and rPRV-gE^(-)/TK^(-)/IL-18^(+).Immunoblotting results showed that the five rPRVs could express PRRSV GP5,GP5m,M protein or porcine IL-18.Subsequently mice were immunized with thesefiverPRVs,respectively,rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+),rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+)/IL-18^(+)and rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+)could produce anti-PRRSV and PRV specific neutralization antibodies,which could stimulate the proliferation of CD3^(+),CD4^(+),and CD8^(+) T lymphocytes and the expresstion of IFN-,IL-4,and IL-10 cytokine.In summary,rPRV-gE^(-)/TK^(-)/GP5^(+)/M^(+),rPRV-gE/TK/GP5^(+)/M^(+)/IL-18^(+),and rPRV-gE^(-)/TK^(-)/GP5m^(+)/IL-18^(+)could effectively induce specific systemic immune response against PRRSV and PRV in mice,which might lay the foundation for the development of the recombinant live vector vaccine for effectively prevention of PRRS and PR.