丙型肝炎病毒重要编码蛋白重组腺病毒载体的构建

Construction of Recombinant Adenoviral Vectors Expressing Important Hepatitis C Virus-encoded Proteins

旨在构建含HCV core、E1、E2、F、NS3、NS5a和NS5b基因的重组腺病毒载体。运用PCR技术扩增目的基因片段,通过酶切连接方法将上述7种基因片段克隆至穿梭质粒pAdTrack-TO4中,然后将重组质粒用PmeⅠ线性化后与骨架质粒pAdEasy-1在BJ5183细菌中同源重组。用PacⅠ酶把重组腺病毒载体线性化后转染HEK293细胞后产生重组腺病毒颗粒。利用穿梭质粒中带有绿色荧光蛋白GFP对其感染效率进行监测。结果显示,酶切鉴定和测序结果均证实含core、E1、E2、F、NS3、NS5a和NS5b基因的重组腺病毒构建成功,在感染的Huh7细胞中观察到绿色荧光蛋白GFP。成功制备了高滴度的腺病毒Ad-core、Ad-E1、Ad-E2、Ad-F、Ad-NS3、Ad-NS5a和Ad-NS5b,为后续研究奠定了基础。

The study aimed to construct recombinant adenoviral vectors respectively expressing HCV core,E1,E2,F,NS3,NS5a and NS5b protein for further biofunctional analysis.The genes of core,E1,E2,F,NS3,NS5a,and NS5b were amplified by PCR,and then cloned into shuttle plasmid pAdTrack-TO4 by enzyme and ligation.The recombinant plasmids were linearized using PmeⅠ and then homogeneously recombinized with backbone plasmid pAdEasy-1 in BJ5183 bacteria.Subsequently,the plasmids were linearized with PacⅠ and transfected into HEK293 cells to be packed into recombined adenoviral particles,and their infectious efficiency was examined using green fluorescent protein（GFP）expression in the shuttle plasmid.Results showed that through restriction endonuclease digestion analysis and DNA sequencing,the recombined adenoviral vectors Ad-core,Ad-E1,Ad-E2,Ad-F,Ad-NS3,Ad-NS5a and Ad-NS5b were successfully constructed.All the adenovirus carrying virus-encoded proteins can effectively infect hepatoma cells.We successfully prepare high-titer adenoviruses Ad-core,Ad-E1,Ad-E2,Ad-F,Ad-NS3,Ad-NS5a,and Ad-NS5b,which could lay down the footstone for the following biofunctional study.