hTERT核心启动子驱动的条件复制型腺病毒载体的构建及鉴定

Construction and verification of a novel CRAd driven by hTERT promoter

目的构建并鉴定一种新型人端粒酶逆转录酶(hTERT)启动子驱动的条件复制型腺病毒载体。方法采用PCR的方法克隆腺病毒E1区基因(E1A,E1B55K)、E1B的启动子(E1Bp)以及人端粒酶逆转录酶亚基启动子(hTERTp),以及2个目的基因:人GM-CSF和GFP(GreenFluorescenceProtein,GFP)。采用一系列分子生物学方法构建pShuttle-GFPSV55K穿梭质粒,应用此穿梭质粒与AdEasy-1腺病毒DNA在BJ5183细菌内重组得到重组腺病毒质粒,将其转染293细胞获得Ad-GFPSV55K重组腺病毒。PCR法鉴定重组腺病毒,噬斑形成实验测病毒滴度。结果经过酶切鉴定成功地构建了分别携带GM-CSF基因和GFP报告基因的腺病毒载体,病毒滴度为7×1010pfu/ml。结论成功获得由hTERT启动子驱动的条件复制型腺病毒载体,为肿瘤的基因治疗打下良好的基础。

Objective To construct a novel CRAd vectors driven by hTERT promoter and to establish the foundation for cancer gene therapy. Methods The hTERT promoter as E1A and E1Bp gene were amplified by PCR;the GFP gene and GM-CSF were also amplified by PCR, and the products were subcloned into Teasy plasmid in certain order to generate pSh-GFP-SV55K plasmid. A recombinant adenoviral genomic DNA was obtained by homologous recombinant in E. coli BJ5183 cells pretransformed with the pAdeasy-1 plasmid （BJ5183-AD-1 cells） ; the recombinant adenovirus Ad-GFP-SV55K was gained after transfecting 293 cell line with the genomic DNA using LepofectAMINE method ; PCR was used to identify the recombinant Ad, and the titer of virus was determined by plaque assay in the 293 cells. Results The structure of the adenovirus vectors carrying GM-CSF gene and GFP gene was identified by restriction enzyme analysis. The titer of the virus was 7 × 10^10 pfu/ml. Conclusion A novel recombinant adenovirus vector driven by hTERT promoter is made successfully, and the newly constructed adenovirus is useful for cancer gene therapy.