表达呼吸道合胞病毒F蛋白编码基因的重组腺病毒载体的构建  

Construction of recombinant adenovirus expressing RSV F gene

作　　者：王潇潇[1] 李秀玲[1] 赵铠[1] 

机构地区：[1]北京生物制品研究所,100024

出　　处：《中华微生物学和免疫学杂志》2009年第5期431-437,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的构建表达呼吸道合胞病毒（respiratory syncytial virus，RSV）F蛋白编码基因片段的重组腺病毒。方法以RSV RNA为模板，利用RT—PCR扩增出F基因片段，应用AdEasy腺病毒载体系统，构建含有目的基因的重组穿梭质粒pShuttle—CMV／F，再转化含有骨架质粒pAdEasy-1的大肠杆菌BJ5183-AD-1获得重组腺病毒质粒pAdEasy／F。将该质粒转染293细胞包装产生出重组腺病毒rAd／F。重组腺病毒分别经电子显微镜技术、RT—PCR、Western blot和间接免疫荧光方法进行鉴定，并对该重组腺病毒的滴度和遗传稳定性进行了初步研究。结果获得了含有RSVF基因片段的重组腺病毒rAd／F，电子显微镜下可见典型腺病毒颗粒形态，RT-PCR、Western blot和间接免疫荧光方法分析显示rAd／F中的F基因片段可以在293细胞中有效转录和表达。结论成功构建出含有RSVF基因片段的重组腺病毒rAd／F，为进一步研究重组腺病毒载体疫苗奠定了基础。Objective To construct a recombinant adenovirus contained respiratory syncytial virus F gene fragment. Methods The F gene fragment was amplified by RT-PCR. Then the recombinant shuttle plasmid pShuttle-CMV/F was constructed. The linearized pShuttle-CMV/F was transformed into BJS183- AD-1 electroporation competent cells which contained plasmid pAdEasy-1. The obtained homologous recombinant plasmid was named pAdEasy/F, and transfected into 293 cells. Then the recombinant adenovirus rAd/F was obtained, and it was tested by electron microscope, RT-PCR, Western blot and immuno-fluorescence assay（IFA）. The virus titer and genetic stability were also studied initially. Results The recombinant adenovirus rAd/F was constructed, which was visualized as typical adenovirus morphology under electron microscope. The transcription and expression of RSV F gene fragment integrated in the rAd/F were confirmed by RT-PCR, Western blot and IFA. Conclusion The recombinant adenovirus rAd/F contained RSV F gene fragment is successfully constructed and lay an important foundation for the further vaccine study.

关 键 词：呼吸道合胞病毒 融合蛋白 腺病毒载体 

分 类 号：R3[医药卫生—基础医学]