共表达猪繁殖与呼吸综合征病毒GP5基因及猪γ-干扰素基因的腺病毒载体的构建与鉴定

Construction and Identification of Recombinant Adenovirus Coexpression PRRSV GP5 Gene and Porcine Interferonγ Gene

利用PCR方法分别扩增猪繁殖与呼吸综合征病毒全长GP5基因(编码E蛋白),EMCV的核糖体介入位点序列(IRES)及猪γ-干扰素基因全长序列(IFN-γ),序列测定正确后用DNA重组法将三者串联后插入pAdenoVator-CMV5-IRES-GFP穿梭质粒中,形成的穿梭质粒plRES-GP5-IFN-γ用PmeI线性化后,与腺病毒骨架载体pAdEasy-1共转化感受态大肠埃希氏菌BJ5183,经同源重组,构建成含有GP5基因和IFN-γ基因的重组腺病毒载体,pacI酶切线性化充分暴露反向末端重复序列后,脂质体转染HEK293A细胞,借助GFP的表达可以在转染后的2～3天观察到包装病毒rAdeno-GP5-IFN-γ产生,7～10天出现病毒蚀斑。经PCR法及酶切证实各中间过程载体及最终的包装病毒中携带有目的基因,Western-blot证实两基因在腺病毒中得到了表达。大肠杆菌内同源重组法能有效和较为方便地构建出含有目的基因的腺病毒载体rAdeno-GP5-IFN-γ,重组子能够在HEK293细胞中稳定扩增,病毒包装的成功为进一步研究PRRSVE蛋白的免疫效果及IFN-γ的作用奠定了基础。

The GP5 gene of PRRSV and the porcine IFN-γ were amplified by PCR, and ligated by IRES sequence, then cloned into the transfer vector pAdenoVator-CMV5-IRES-GFP. After co-transformation of Pme Ⅰ- linearized recombinant plasmid plRES-GP5-IFN-γ and the bone vector pAdEasy-1 into Escherichia coli bacteria strain BJ5183, recombinant plasmid containing GP5 gene and IFN-γ（ pad-GP5 - IFN-γ） was obtained and identified with PCR. Upon transfection of Pad-linearized plasmid pad-GP5-IFN-γ in 293 cell line, a recombinant adenovirus was obtained and named as tad- GP5- IFN -γ. The expression of the E protein and IFN in the 293 ceUs infected with rAd- GP5 - IFN-γ was confirmed with specific antibodies to E protein and IFN-γ by Western blotting. It indicated that a stable and safety replication defective recombinant adenovirus rAd-GP5- IFN-γ was produced successfully. The recombinant adenovirus might be an attractive candidate vaccine for preventing the disease of PRRS.