FLT-1启动子荧光素酶报告基因重组体的构建和鉴定

Construction and Identification of Luciferase Reporter Recombinant Driven by FLT-1 Promoter

目的:为探索FLT-1启动子靶向调控活性分析,克隆FLT-1启动子基因序列,构建并鉴定FLT-1启动子调控的荧光素酶报告基因重组体pGL3-FLT-Basic-luc。方法:应用聚合酶链式反应(PCR)技术扩增FLT-1启动子序列,定向亚克隆至荧光素酶表达载体pGL3-Basic-luc中,构建含有正确目的基因的报告基因重组体pGL3-FLT-Basic-luc,通过限性内切酶酶切、PCR及测序进行鉴定。结果:通过酶切鉴定及基因测定证明,所克隆的基因产物与预期结果一致,序列无碱基突变。结论:成功构建了含有FLT-1启动子基因序列的荧光素酶报告基因真核表达载体,为下一步分析该启动子活性及血管疾病的基因治疗奠定基础。

Objective： To evaluate FLT- 1 gene promoter on gene transcription, we clone FLT- 1 gene promoter and construct luciferase reporter recombinant regulated by FLT- 1. Method：The FLT- 1 gene promoter was amplified by polymerase chain reaction and subdoned into pGL3 Basic vector to construct pGL3 Basic luciferase reporter expression vector containing FLT- 1 gene promoter（pGL3 - FLT- Basic - luc）, Then the expression vector was identified by restriction enzymes digestion, PCR analysis and DNA sequencing. Result： DNA sequencing and digestion confirmed that the recombinant of plamid pGL3 - Basic- luc contained FLT- 1 promoter identical to that in GeneBank. Condusion： Luciferase re- porter vectors regulated by FLT- 1 promoter were successfully constructed, which will offer experimental foundation for gene therapy study of vascular disease.