phP53-luc质粒的构建及其用于检测化学物质遗传毒性的初步分析  

Construction of phP53-luc plasmids and phP53-luc-based genotoxicity detection of chemicals

作　　者：施柯[1] 黎怀星[1] 吴丹[1] 李雅慧[1] 孙树汉[1] 

机构地区：[1]第二军医大学基础医学部医学遗传学教研室,上海200433

出　　处：《第二军医大学学报》2004年第3期277-279,共3页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 (30 370 80 2 )

摘　　要：目的 :构建 2个分别由 p5 3基因启动子和核心启动子驱动的荧光素酶报告基因表达质粒 ,并探讨其用于检测遗传毒性化学物质的可行性。方法 :通过 PCR扩增人 p5 3基因启动子片段 ,然后采用重组 DNA技术克隆入 p GL3- BASIC的荧光素酶报告基因的上游而获得 ph P5 3- luc表达质粒。用脂质体转染法将 ph P5 3- luc转染入 NIH3T3细胞并分别用 5 -氟尿嘧啶 (5 -FU)处理 ,最后裂解细胞用 Dual- L uciferase Reporter Assay System检测荧光素酶报告基因表达。 结果 :构建了 2个分别由p5 3基因启动子和核心启动子驱动的荧光素酶报告基因表达质粒 ,位于这 2个质粒上的荧光素酶报告基因在 NIH3T3细胞内的表达显著地受遗传毒性化学物质的诱导 ,其表达比对照的 p GL 3- BASIC载体上的荧光素酶报告基因高近 2 4 0倍。结论 :基于人 p5Objective:To construct human p 53 promoter driven luciferase reporter gene expression plasmids and to explore the possibility of developing a phP53 luc based reporter gene assay system for assessing genotoxicity of chemicals. Methods: phP53 luc plasmid DNAs were generated by inserting p 53 gene promoter fragments,amplified by PCR,into upstream of luciferase reporter gene of pGL3 BASIC. The resultant constructs were transfected into NIH 3T3 cells using lipofectamine and PLUS Reagent. The cells were then treated with 5 fluorouracil (5 FU) and expression of the reporter gene in the cell lysates was assayed using Dual Luciferase Reporter Assay System. Results: Two human p53 promoter driven luciferase reporter gene expression plasmids were constructed. Luciferase was greatly induced in NIH 3T3 cells by genotoxic agents, about 240 fold increase compared to negative control (pGL3 BASIC). Conclusion: The phP53 luc based reporter gene assay system we developed can be applied in the quick detection of genotoxicity of environmental toxins.

关 键 词：phP53—luc质粒 检测 化学物质遗传毒性 P53基因 5—氟尿嘧啶 

分 类 号：R446.1[医药卫生—诊断学]