共表达与牛疱疹病毒1型VP22融合的猪繁殖与呼吸综合征病毒E及M蛋白的重组伪狂犬病毒的构建及其免疫原性观察

为了研制猪繁殖与呼吸综合征病毒（PRRSV）基因工程疫苗，以伪狂犬病毒（PRV）gE基因缺失标志疫苗株TK^-／gE^-／LacZ^＋为病毒载体，通过同源重组，构建了共表达与牛疱疹病毒1型VP22（BHV-1 VP22）融合的PRRSV E及M蛋白的重组伪狂犬病毒（rPRV）TK^-／gE^-／VP22E^＋／VP22M^＋。经PCR、Southern blot、Western blot证实rPRV构建正确，并能表达与BHV-1 VP22融合的PRRSV E及M蛋白。rPRV在IBRS-2、PK-15细胞中的增殖滴度与PRV亲本株相比无显著差异，表明外源基因的插入不影响rPRV增殖。用该rPRV免疫BALB／c小鼠,检测免疫小鼠抗PRRSV中和抗体及脾淋巴细胞增殖反应，并与未融合VP22的单表达PRRSV E蛋白及共表达E及M蛋白的rPRV TK^-／gE^-／E^＋与TK^-／gE^-／E^＋／M^＋进行比较。结果显示TK^-／gE^-／VP22E^＋／VP22M^＋可诱导小鼠产生更好的体液与细胞免疫反应，BHV-1 VP22发挥了佐剂效应。本研究为研制安全、有效的猪繁殖与呼吸综合征-伪狂犬病二价基因工程疫苗奠定了基础。

Construction of Recombinant Pseudorabies Virus Coexpressing E and M Protein of Porcine Reproductive and Respiratory Syndrome Virus Fused with VP22 Protein of Bovine Herpesvirus 1 and Its Immunogenicity in Mice

In the present study,the ORF5M gene(the modified ORF5 gene) and ORF6 gene,the two major immunogenicity genes of PRRSV,were used as the target genes,and bovine herpesvirus 1(BHV-1)VP22 possessing protein transduction property was chosed as the immunoadjuvant.Based on homologous recombination,a recombinant PRV(rPRV) TK~-/gE~-/VP22E~ /VP22M~ coexpressing PRRSV E and M protein fused with BHV-1 VP22 protein was constructed by using PRV TK~-/gE~-/LacZ~ as parent strain.The rPRV was confirmed by PCR,Southern blot and Western blot.The results of TCID_(50) tests showed that the insertion of the foreign genes had no influence on the propagation of rPRV in IBRS-2 or PK15 cells.BALB/c mice were immunized with rPRV TK~-/gE~-/VP22E~ /VP22M~ ,and compared with rPRV TK~-/gE~-/E~ expressing ORF5M gene and TK~-/gE~-/E~ /M~ coexpressing ORF5M and ORF6 genes respectively.Neutralizing antibodies and lymphocyte proliferative responses were evaluated at 10 weeks after primary immunization.Among three rPRVs,TK~-/gE~-/VP22E~ /VP22M~ had the best effect on enhancing PRRSV-specific humoral and cellular immune responses,and VP22 showed adjuvant efficiencies on rPRV vaccine to a certain degree.In conclusion,the study established a base for the research of bivalent genetic engineering vaccines against PRRSV and PRV.