猪流感病毒NP基因重组复制缺陷型腺病毒的构建与表达

Construction of a replication-defective recombinant adenovirus expressing NP gene of swine influenza virus

以含有猪流感病毒A/Swine/Guangdong/9/2005(H3N2)NP基因的重组质粒pMD18-NP为模板,利用带特定酶切位点的引物PCR扩增NP基因,将其亚克隆至质粒pIRES2-EGFP中,再次将含有NP及EGFP的基因片段克隆到腺病毒的穿梭质粒pDC315,构建重组穿梭质粒pDC315-NP-EGFP。利用脂质体转染法将穿梭质粒pDC315-NP-EGFP和腺病毒骨架质粒pBHGloxΔE1,E3Cre共转染HEK293细胞,基于腺病毒感染后形成的典型细胞病变及EGFP基因在细胞中的表达筛选到重组腺病毒rAd-NP-EGFP。经PCR和Western blotting鉴定,结果表明NP基因已被重组到腺病毒的基因组中,并能够伴随病毒的复制而表达,表达蛋白具有良好的生物学活性。重组腺病毒rAd-NP-EGFP经增殖、纯化后其TCID50可达1.26×1010/mL,为进一步研究该重组病毒的免疫原性奠定了基础。

To construct the recombinant adenovirus shuttle plasimd pDC315-NP-EGFP,the NP gene of A/Swine/Guangdong/9/2005（H3N2） amplified by PCR from the recombinant plasmid pMD18-NP,was subcloned into pIRES2-EGFP.The gene fragment containing NP and EGFP was then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing NP gene （rAd-NP-EGFP） was generated by co-transfecting the recombinant shuttle plasmid pDC315-NP-EGFP and the backbone plasmid pBHGloxE1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by the typical cytopathic effect and expression of EGFP gene in HEK293 cells.The results of identification by PCR and Western blotting indicated that the recombinant adenovirus rAd-NP-EGFP could efficiently express NP protien of SIV in vitro.The TCID50 of the rAd-NP-EGFP was evaluated as 1.26×1010/mL after propagation and purification.