【摘要】 目的基于腺病毒可感染禽类呼吸道和肠道上皮黏膜的特性,本文构建了包含有禽流感病毒H5N2亚型HA和NA基因的重组腺病毒,为进一步研制口服禽流感活载体疫苗奠定基础。方法通过RT-PCR方法分别扩增出AIV的HA和NA全基因片段,利用拼接接头将HA和NA定向串联并插入到腺病毒穿梭载体pAdTrack-CMV之中,然后在大肠杆菌中实现腺病毒骨架载体pAdEasy-1和重组腺病毒穿梭载体的同源重组,获得插有外源基因的重组腺病毒质粒,进而通过转染HEK293细胞,得到含有目的基因HA和NA的重组腺病毒。结果在pAdTrack-CMV载体中成功克隆了HA+NA双基因,并与腺病毒骨架载体在大肠杆菌中实现了同源重组;线性化后的重组腺病毒质粒转染HEK293细胞后可观察到典型的细胞病变和绿色荧光蛋白的表达。结论成功获得了含有HA+NA双基因重的组腺病毒,为口服活载体疫苗的研制提供了依据。更多还原

【Abstract】 To construct the recombinant adenovirus containing hemagglutinin(HA) and neuraminidase(NA) genes of avian influenza virus(AIV), the genes for HA and NA were obtained by RT-PCR. The amplification products were digested with double restriction endonuclease respectively, and orientationally subcloned into shuttle vector pAdTrack-CMV,the recombinant shuttle plasmid was named pAdTrack-CMV/HA+NA,then the recombinant adenoviral plasmid was obtained by homologous recombination of the linearized pAdTrack-CMV/HA+NA and pAdEasy-1 vector in E. coli strain BJ5183.Finally the recombinant adenoviral plasmid was cleaved with PacI and transfected into HEK293 packaging cell line. The strain of recombinant adenovirus containing HA and NA genes from H5N2 strain was successfully obtained, thus providing a basis for the study on oral AIV vaccine based on recombinant replication-defective adenovirus.更多还原