PCV-2 ORF2和PRRSV GP5双基因共表达重组腺病毒的构建及小鼠免疫试验

Construction of Recombinant Adenovirus Co-expressing PCV-2 ORF2 and PRRSV GP5 Genes and Immunoassay in Mice

为构建猪圆环病毒2型(PCV-2)ORF2和猪繁殖与呼吸综合征病毒(PRRSV)GP5双基因共表达重组腺病毒,将扩增的ORF2基因和GP5基因通过IRES元件串联,构建重组腺病毒穿梭质粒。然后用PmeⅠ酶切穿梭质粒,使其线性化,将线性化质粒转化pAdeasy-1的BJ5183感受态细胞,同源重组后筛选重组腺病毒质粒,经PacⅠ酶切后转染293A细胞进行包装,获得重组腺病毒。PCR鉴定表明重组腺病毒含有ORF2和GP5基因,Western blot检测结果表明ORF2基因和GP5基因在293A细胞内获得表达。重组腺病毒经293A细胞连续传30代,TCID50稳定为10-9.0/mL。初步动物免疫试验结果表明,该重组腺病毒能够刺激机体分别产生PCV-2和PRRSV的特异性抗体免疫应答反应。该重组腺病毒可以作为PCV-2与PRRSV二联基因工程疫苗研究的候选毒株。

The recombinant shuttle plasmid was constructed successtuiiy atrer GP5 genes were linked by IRES gene. After being digested with Pine I , the linearized recombinant plasmid was transformed into Escherichia coli BJ5183 competent cells. The homologous recombination between the linearized plasmid and the supercoiled adenoviral vector pAdEasy1 occurred. The recombinant adeno virus plasmid was digested with transfected into 293A ceils to pack into viral particles. The re combinant adenovirus was acquired. The constructed recombinant adenovirus containing ORF2 and GP5 genes was identified by PCR. The Western blot results showed that ORF2 gene of PCV2 and the GP5 gene of PRRSV were expressed in 293A cells successfully. The recombinant adenovirus was passaged 30 times in 293A cells. The titer was stably 109. s/mL. Furthermore, the recombinant adenovirus could induce PCV2 and PRRSV specific humoral immunity in mice. Therefore, the recombinant adenovirus may be a candidate for PCV2 and PRRSV gene engineering vaccines.