细菌内同源重组法高效制备含CD、TK融合自杀基因重组体腺病毒  

High efficient generation of recombinant adenovirus containing CD、TK fusion gene by using a method of homologous recombination in bacteria

作　　者：魏道严[1] 糜军 陈诗书[1] 

机构地区：[1]上海第二医科大学人类基因治疗研究中心,上海200025

出　　处：《癌症》2000年第5期399-403,共5页Chinese Journal of Cancer

基　　金：国家"863"高技术计划基金!(86 3- 10 2 - 45 2 );国家自然科学基金!(3970 44 0 )资助

摘　　要：目的 :采用细菌内同源重组法高效制备含CD、TK融合自杀基因重组体腺病毒。方法 :CD、TK融合基因CDglyTK自载体 pWZLneoCDglyTK中切出 ,亚克隆至腺病毒穿梭质粒中 ,形成转移质粒 pAdtrackCMV CDglyTK ,将之PmeI酶切线性化后与腺病毒基因组质粒 pAdeasy 1共转化BJ5 183菌 ,抽提重组体腺病毒基因组质粒DNA ,PacI酶切后转染 2 93细胞包装成腺病毒颗粒 ,采用PCR方法对重组体腺病毒进行鉴定 ,利用报告GFP基因对病毒滴度和感染效率进行测定 ,WesternBlot检测感染腺病毒的肿瘤细胞中有无目的基因的表达。结果 :由pAdTrack CMV CDglyTK和 pAdeasy 1共转化BJ5 183菌 16～ 2 0h后 ,可获得 30 %左右的阳性重组体细菌克隆 ,由重组体克隆质粒DNA转染 2 93细胞包装产生的重组体腺病毒 ,经PCR检测表明已含有目的基因 ,且无复制能力腺病毒的存在。纯化所得腺病毒滴度约为 8× 10 12 pfu/L ,当MOI =10 0时 ,腺病毒感染HepG1细胞的效率 >75 % ,WesternBlot证实在感染重组体腺病毒的细胞中有相应基因产物的表达。结论 :细菌内同源重组法是一种高效、简便、快捷的重组体腺病毒载体制备方法。所制备的Ad CDglyTK在体外能有效表达相应的基因产物 ,为今后的深入研究奠定了基础。Objective:To generate recombinant adenovirus (Ad) by using a novel and high efficient method of homologous recombination in bacteria with CDglyTK as target gene Methods:CDglyTK gene was liberated from plasmid of pWZLneoCDglyTK and subcloned into shuttle plasmid and formed transfer plasmid of pAdTrackCMV CDglyTK Then it was linealized with PmeI and cotransformed into BJ5183 bacterial cells with adenovirus genomic DNA plasmid of pAdeasy 1 The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected to 293 cells to package recombinant adenovirus particles The PCR technique was used to detect target gene and replication competent Ad (RCA) contamination The titre and its infection rate of the recombinant Ad were measured with the aid of GFP expression Western Blot technique was used to analyze the target gene expression in infected tumor cells Results:There were over 30%positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack CMV CDglyTK and pAdeasy 1 Recombinant adenovirus particles were produced after 293 cells transfected with recombinant Ad genomic plasmid DNA digested with PacI, then they were further amplified in 293 cells PCR test indicated that the recombinant Ad contained the insert of CDglyTK gene and there was no contamination of RCA in high titer viral stocks The titre of purified Ad was 8×10 12 pfu/L, and more than 75%HepG1 cells were infected with Ad CDglyTK adenoviruses when MOI reached 100 Meanwhile, protein product of CDglyTK gene was detected in the infected tumor cells Conclusion:The method of homologous recombination in bacteria is a conve nient and high efficient method to prepare recombinant Adenovirus, and the prepared adenoviruses can effectively madiate target gene expression in infected tumor cells, thus paves a sound foundation for further study

关 键 词：肿瘤 基因治疗 腺病毒载体 CD TK 融合自杀基因 

分 类 号：R730.2[医药卫生—肿瘤]