共表达猪繁殖与呼吸综合征病毒修饰型GP5和M蛋白增强DNA疫苗免疫应答的研究

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。

Study on Immunological Responses to Porcine Reproductive and Respiratory Syndrome Virus DNA Vaccines Co-expressing Modified GP5 (GP5m) and M Proteins

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.