含人DNA聚合酶β基因启动子萤光素酶报告载体的构建  

Construction of a luciferase reporter vector directed by DNA polβ promoter

作　　者：李月白[1] 张贵星[1] 于雅丽[2] 赵国强[3] 王欢[1] 董子明[4] 

机构地区：[1]郑州大学基础医学院生物化学与分子生物学教研室,郑州450001 [2]郑州市骨科医院检验科,郑州450052 [3]郑州大学基础医学院微生物学与免疫学教研室,郑州450001 [4]郑州大学基础医学院病理生理学教研室,郑州450001

出　　处：《郑州大学学报（医学版）》2008年第5期869-872,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30471952

摘　　要：目的:构建含人DNA聚合酶β(DNA polymerase beta,polβ)基因启动子的萤光素酶报告基因表达载体pGL3polβ/promoter。方法:利用PCR技术扩增人类polβ基因启动子的核心片段,克隆至含新霉素抗性的萤光素酶表达载体pGL3-Neo-enhancer,构建含DNApolβ启动子的萤光素酶表达载体pGL3polβ/promoter,重组子经双酶切、PCR及测序鉴定。将构建的载体用脂质体转染体外培养的食管癌EC-1细胞株,应用萤光素酶测定系统检测萤光素酶的表达活性。结果:测序结果表明,克隆获得的polβ基因启动子序列与GenBank报道的一致,且插入方向正确。pGL3polβ/promoter组萤光素酶的表达活性(2207348.000±71626.763)与空白组(451.670±23.347)和pGL3-Neo-enhancer组(4884.330±1623.047)相比,差异有统计学意义(F=5681.596,P<0.05)。结论:成功构建了含人DNA polβ基因启动子的萤光素酶报告基因表达载体pGL3polβ/promoter。Aim： To construct the luciferase reporter gene expression vector pGL3polβ/promoter containing wild-type DNA polβ promoter. Methods：The core fragment of DNA polβgene promoter was amplified from human genomic DNA by polymerase chain reaction （PCR）. The amplified fragment was subsequently cloned into pGL3-Neo-enhancer plasmid which contain neomycin- resistant gene. Then the recombinant was confirmed by restriction enzyme digestion, PCR analysis and DNA sequencing. PIasmid pGL3polβ/promoter was transfected into EC-1 cells by lipofectamine. The active of luciferase was detected, and the effect of polβ promoter was studied. Results：The result of DNA sequencing showed that the sequence of cloned polβ promoter was identical to that GenBank had reported and the fragment was inserted in right direction. The activity of luciferase was significantly increased in the pGL3polβ/promote transfected cells over 400 times than that in the pGL3-Neo-enhancer. Conclusion：The luciferase expression vector, pGL3polβ/promoter containing polβ promoter is constructed successfully, It will be foundation for studying its tissue-specificity and target gene therapy of tumor.

关 键 词：DNA聚合酶 启动子 萤光素酶报告基因 

分 类 号：R735.1[医药卫生—肿瘤]