表达猪繁殖与呼吸综合征病毒(PRRSV)GP5的重组伪狂犬病毒TK^-/gG^-/GP5^+的构建及其生物学特性初步探讨

Construction of the Recombinant Pseudorabies Virus TK^-/gG^-/GP5^+ Expressing GP5 of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the Preliminary Study on Its Biological Characterization

以伪狂犬病毒基因缺失标志疫苗株TK-/gG-/LacZ+为亲本株,通过同源重组,构建了表达猪繁殖与呼吸综合征病毒(PRRSV)主要免疫原性蛋白GP5的重组伪狂犬病毒TK-/gG-/GP5+。经PCR、Southern杂交、Westernblot证实构建正确,并能表达GP5。同时,对重组病毒在PK-15细胞中的增殖特性进行了检测,结果与亲本株相比,增殖滴度无显著性差异。将重组病毒免疫BALB/c小鼠,2次免疫后4周,50%(3/6)小鼠产生了低水平的GP5特异性ELISA抗体,但中和抗体均小于1:4。

Porcine reproductive and respiratory syndrome(PRRS) is one of the most economically significant diseases of the global pork-producing community.Pseudorabies virus(PRV),a member of alphaherpesvirus,is a novel viral vector to develop bivalent or multivalent genetic engineering vaccines.In the present study,we chose ORF5 gene encoding envelope protein GP5,the major immunogenic protein of PRRSV,as the target antigen.Based on homologous recombination,a recombinant PRV TK^-/gG^-/GP5^+ expressing GP5 was constructed using PRV TK^-/gG^-/LacZ^+ as a parental strain,and the recombinant virus was confirmed by PCR,Southern blot and Western blot.In addition,the one-step growth assay showed that the recombinant virus had growth properties identical to those of the parental virus,indicating that insertion of ORF5 gene in the gG focus of PRV did not affect its amplification.We further investigated the immunogenicity of the recombinant virus in mouse model and the results showed that low level of GP5-specific ELISA antibodies were detectable at 4 weeks after a boost and no detectable neutralizing antibodies were observed throughout the whole experimental period.