前凋亡因子bimS基因的重组腺病毒载体构建

Construction of Recombinant Adenovirus Vectors Carrying Proapoptotic Gene bimS

为探讨前凋亡因子bimS用于肿瘤基因治疗奠定基础，拟构建bimS的重组腺病毒载体。采用RT—PCR方法从人HL-60细胞扩增目的基因bimS；克隆至T载体测序正确。亚克隆bimS基因到腺病毒穿梭质粒pAdtrack—CMV上，形成含目的基因和绿色荧光蛋白（GFP）基因的穿梭载体。穿梭载体pAdtrack—CMV—bimS和病毒骨架载体pAdEasy-1经电穿孔法转入BJ5183大肠杆菌中行同源重组，获得重组pAdEasy—CMV—bimS，线性化后脂质体法转染人胚肾（HEK）293细胞进行病毒的包装、扩增、病毒滴度测定以及瞬时表达的观察；将病毒以MOI-100感染HEK293细胞，western blot检测bimS蛋白表达。结果显示病毒感染293细胞48～72h观察到GFP表达，7d出现典型细胞病变症状（CPE）；提取培养上清中病毒DNA，以PCR法可检测到bimS的扩增产物；western blot检测病毒感染的293细胞总蛋白，与未感染的阴性对照细胞相比，bimS蛋白表达明显高于阴性对照细胞。表明重组bimS腺病毒构建成功；为进一步进行肿瘤基因治疗的体内外试验奠定基础。

In order to further study the anti-tumor activity of bimS and the feasibility of using adenovirus vector for gene therapy, we constructed a recombinant adenovirus vector of pro-apoptotic factor bimS （pAdEasy- CMV-bimS）. Human bimS gene was amplified from HL-60 leukemic cell by RT-PCR and it was identified by sequencing. Then bimS was cloned into the shuttle vector pAdTrack-CMV that carried a green fluorescence protein （GFP） gene to generate a recombinant plasmid pAdTrack-CMV-bimS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E. coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restriction endonuclease digestion. The recombinated adenovirus pAdEasy-CMV-bimS was transferred into HEK293 cell for packaging and amplification. The virus titre was determined and the insert of bimS gene was verified by PCR method. Finally, HEK293 cell was infected by recombinated adenovirus pAdEasy-CMV-bimS （MOI =100） and bimS protein was detected by western blot. GFP expression in transfected HEK293 cells could be observed at 48-72 hours, bimS gene was detected in cultural supernatant of infected HEK293 cell by PCR and there was typical cytopathic effect（CPE） in HEK293 cell 7 days after infection. Western blot showed bimS protein expression in infected HEK293 cells was markedly higher than that in uninfected HEK293 cells. The result indicated that human bimS recombinant adenovirus was constructed successfully, which could therefore provide a sound base for anti-tumor gene therapy with bimS in vivo and in vitro.