LR重组法构建重组腺病毒rAd5-Vpr

Construction of Recombinant Adenovirus Carrying HIV-1 Vpr Gene via LR Recombination

目的:构建携带HIV-1Vpr基因的重组腺病毒。方法:采用限制性内切酶消化和T4DNA连接酶连接的方法,将Vpr基因克隆至腺病毒穿梭质粒pTrack-C上,然后通过重组穿梭质粒pTrack-C-Vpr和腺病毒骨架质粒pAdeno体外LR位点特异性重组的方法,将Vpr基因转移至腺病毒骨架质粒pAdeno上,最后重组骨架质粒pAdeno-Vpr鉴定正确后经PacI酶切,转染HEK293细胞,包装成重组腺病毒rAd5-Vpr。同时在HEK293细胞中进行病毒扩增,利用PCR方法对重组腺病毒进行鉴定,用微量全细胞病变法检测病毒滴度。结果:PCR鉴定重组腺病毒rAd5-Vpr构建成功;扩增后检测病毒滴度约为5×108PFU/mL。结论:应用LR重组法能成功构建携带HIV-1 Vpr基因的重组腺病毒,扩增后滴度能满足实验需要,为进一步相关研究的开展奠定了基础。

Objective：To construct a recombinant adenovirus carrying HIV-1 viral protein R（Vpr）gene.Methods：First,by the method of restriction endonuclease digestion and by using T4 DNA ligase,the HIV-1 Vpr gene was cloned to the shuttle plasmid pTrack-C,then LR site-specific recombination was performed in vitro between recombinant shuttle plasmid pTrack-C-Vpr and adenovirus backbone plasmid pAdeno.Finally,after correct identification of the recombinant backbone plasmid pAdeno-Vpr,it was digested by PacI and transfected into HEK 293 cells and packaged out recombinant adenovirus rAd5-Vpr.Meanwhile,it was amplified in HEK 293 cells and identified by PCR analysis.The titer was detected by the method of trace total cytopathic effect.Results：PCR identification showed the construction of recombinant adenovirus rAd5-Vpr was successful.The titer was about 5×108 PFU/mL after amplification.Conclusion：The recombinant adenovirus carrying Vpr gene was constructed successfully via LR recombination,the titer after amplification meets the need of experiment,so it laid a foundation for further relevant study.