受人E2F1启动子调控的条件复制型腺病毒载体的构建与鉴定

Construction and identification of conditionally replicative adenovirus under the control of E2F1 gene promoter

目的构建并鉴定受人E2F1基因启动子调控的条件复制型腺病毒载体。方法以人胚肾293细胞的基因组DNA为模板,PCR扩增E2F1基因启动子调控序列,构建表达质粒pE2F1 p-EGFP;PCR扩增腺病毒早期基因E1A,构建表达质粒pTERT p-E1A;将重组质粒pTERT p-E1A酶切得到的E1A基因,插入线性化的pE2F1 p-EGFP,构建pE2F1 p-E1A;酶切重组质粒pE2F1 p-E1A产生受E2F1基因启动子调控E1A的完整表达框,插入质粒pDC311中,构建腺病毒包装穿梭质粒pDC311-E2F1 p-E1A,与腺病毒骨架质粒pBHGlox△1,3Cre共转染人胚肾293细胞,得到条件复制型腺病毒Ad-E2F1 p-E1A;扩增病毒并分别感染E2F1活性增强的系列肿瘤细胞株,利用病毒空斑形成实验检测重组条件复制型病毒的条件复制能力。结果限制性内切酶鉴定显示重组质粒构建成功,受人E2F1基因启动子调控的溶瘤腺病毒载体仅在E2F1活性增强的肿瘤细胞株中实现条件性复制。结论构建的新型条件复制型腺病毒载体具有良好的选择性复制能力,在E2F1活性增强的肿瘤治疗方面有良好的应用前景。

Objective To construct and identify a conditionally replicative adenovirus（CRAd） regulated by E2F1 gene promoter.Methods Human E2F1 gene promoter and adenoviral E1A gene were amplified by PCR using the genomic DNA of human embryo kidney cells AD-293 as a template.The fragment of human E2F1 gene promoter was digested and then inserted into pEGFP-1 for cons-tructing pE2F1 p-EGFP.The open read frame sequence of E1A was digested and then cloned into pTERT promoter-EGFP to obtain pTERT p-E1A.The E1A gene was digested from recombinant pTERT p-E1A plasmid and inserted into linearized pE2F1 p-EGFP to obtain pE2F1 p-E1A.The expression cassette of E1A gene under the control of E2F1 promoter was digested from recombinant pE2F1 p-E1A plasmid and cloned into shuttle vector pDC311.The pDC311-E2F1 p-E1A plasmid was cotransfected with adenoviral genomic plasmid pBHGlox△1,3Cre into AD-293 cells to package the recombinant adenovirus Ad-E2F1 p-E1A.The Ad-E2F1 p-E1A vector was amplified and its selective replication ability was evaluated by plaque forming assay in cancer cells overexpressing E2F-1 protein. Results Positive recombinant plasmid was confirmed by restriction enzyme digestion.Ad-E2F1 p-E1A vector was successfully constructed,which could selectively replicate in E2F1 positive cancer cells. Conclusion The constructed Ad-E2F1 p-E1A in tumor cells has a good ability of selective replication,and emerge an innovative promising application in the treatment of E2F1 positive cancers.