【摘要】 为了增强猪繁殖与呼吸综合征病毒(PRRSV)DNA疫苗免疫效力,分别以PRRSV ORF5m(修饰型ORF5基因)及ORF6为候选基因,构建了单基因或双基因共表达的真核表达质粒pCI-ORF5m、pCI-ORF6及pCI-ORF5m/ORF6。转染BHK-21细胞,Western blot检测证实ORF5m和ORF6基因均获得正确表达,并且共表达的GP5m和M蛋白能够形成异源二聚体。将构建的表达质粒免疫小鼠,首免后6周共表达ORF5m和ORF6基因的pCI-ORF5m/ORF6免疫组小鼠血清中和抗体全部阳转,8周时最高可达到1:32,同时还可检测到较高的特异性细胞免疫应答,明显高于单独表达ORF5m基因的pCI-ORF5m免疫组。进一步将DNA疫苗免疫断奶仔猪,pCI-ORF5m/ORF6免疫组同样能诱发优于pCI-ORF5m免疫组的中和抗体和细胞免疫反应。上述研究结果表明采用ORF5m和ORF6双基因共表达策略能够显著提高PRRSV DNA疫苗效力,为PRRSV新型疫苗的研制提供了有用的数据。更多还原

【Abstract】 To enhance the immune efficiency of porcine reproductive and respiratory syndrome virus(PRRSV)DNA vaccine,the modified ORF5(ORF5m)and ORF6 genes were used as candidate genes to construct the recombinant plasmids pCI-ORF5m and pCI-ORF6 expressing GP5 and M alone,respectively,and pCI-ORF5m/ORF6 co-expressing the two proteins.Western blot analysis showed that the expressed GP5m and M proteins had good biological activities analogy to the authentic proteins and could form disulfide-linked heterodimers GP5m/M in BHK-21 cells transfected with pCI-ORF5m/ORF6.Immunological responses to the constructs were investigated in BALB/c mice and piglets.The specific detectable anti-PRRSV neutralizing antibodies were produced in the mice vaccinated with pCI-ORF5m/ORF6 at 6 weeks post primary immunization(ppi)and reached a peak titer of 1:32 at 8 weeks ppi.The specific stronger cell-mediated immune responses were also observed.In addition,the higher neutralizing antibodies and cell-mediated immune responses of the piglets vaccinated with pCI-ORF5m/ORF6 were produced when compared only with pCI-ORF5m.These data indicated an enhancement of the immunological responses of DNA vaccine co-expressing ORF5m and ORF6 and it will be a promising new vaccine for PRRSV.更多还原