表达H5N1亚型禽流感病毒HA基因重组腺病毒的构建及鉴定

摘要

In order to construct recombinant adenovirus expressing HA gene of clade 2.3.4 influenza virus,the HA gene of A/Chicken/Shandong/628/2011 (H5N1) was amplified by RT-PCR and inserted into adenovirus shuttle plasmid pShuttle-CMV to construct a recombinant adenovirus shuttle plasmid pShuttle-CMV-HA.A replication-defective recombinant adenovirus expressing HA gene (rAd-HA) was generated in Ad293 cells transfected with pShuttle-CMV-HA.The adenovirus particles were observed through cell ultramicroscape.Furthermore,the transcription and expression of HA gene in Ad293 cells were also verified by RT-PCR,indiect immunofluorescence assay and western blot which had a similar immunoreactivity as influenza viral protein.This study laid a foundation for the development of new H5N1 subtype avian influenza virus live vector vaccine.%为构建表达H5N1亚型clade 2.3.4禽流感病毒(AIV) HA基因的重组腺病毒,本研究通过RT-PCR扩增A/wild duck/Shandong/628/201 1(H5NI)的HA基因,克隆于腺病毒穿梭质粒pShuttle-CMV中,构建重组腺病毒穿梭质粒pShuttle-CMV-HA.采用Ad-Easy技术,制备表达AIVHA基因的复制缺陷型重组腺病毒rAd-HA.将rAd-HA感染Ad293细胞,可产生明显的细胞病变.通过细胞超薄切片观察到完整的腺病毒颗粒.经RT-PCR、间接免疫荧光及western blot证明HA基因在Ad293细胞中成功转录和表达,并且表达的蛋白具有良好的生物学活性.本研究为新型H5N1亚型AIV活载体疫苗的研发奠定了基础.