KIR3DL1基因启动子亚克隆及表达调控载体的构建

Sub-cloning of KIR3DL1 Promoter and Construction of the Reporter Plasmid

为研究KIR3DL1基因的表达调控机制,亚克隆KIR3DL1基因的核心启动子序列,并构建KIR3DL1基因启动子-荧光素酶报告载体,采用PCR法从含有KIR3DL1基因转录起始位点5′侧翼区的质粒中扩增KIR3DL1基因核心启动子序列,产物纯化回收后与pGEM-T easy载体连接.测序鉴定正确的重组子经限制性内切酶BglⅡ和NcoⅠ双酶切后.插入同样经BglⅡ和NcoⅠ双酶切的用于基因表达调控研究的pGL3-Basic报告载体,最终获得了长度为254bp的KIR3DL1基因启动子克隆,构建了调控荧光素酶报告基因的真核表达载体。通过酶切及基因测序的方法证实所构建的重组子是正确的,说明KIR3DL1基因启动子表达调控载体的构建是成功的。

To study the possible regulation mechanism of KIR3DL1 gene expression, a core promoter sequence of KIR3DL1 gene was sub-cloned and a KIR3DL1 promoter-luciferase reporter vector was constructed. A core promoter fragment of the KIR3DL1 5＂-untranslated region was amplified by PCR. PCR products were purified and connected with pGEM-T easy vector. Authenticated by DNA sequencing, the plasmid was digested with Bgl /Nco Ⅰ and cloned into Bgl Ⅱ/Nco Ⅰ-digested pGL3-basic reporter vector. A 254 bp promoter fragment of KIR3DL1 gene was cloned into the pGL3-Basic reporter vector. The reporter plasmid was authenticated by Bgl Ⅱ/Nco Ⅰ digestion and DNA sequencing. It indicated that the KIR3DL1 promoterpGL3- Basic reportor vector was successfully constructed.