SPA基因启动子的克隆及转录靶向活性分析

Cloning of SPA gene promoter and analysis of its transcriptional targeting activity

目的:克隆大鼠肺表面活性蛋白A(SPA)基因启动子并构建该启动子的荧光报告系统,探讨该启动子的活性及转录靶向性,为进一步研究SPA基因的表达调控机制和探讨靶向性基因治疗奠定基础。方法:①从GenBank中获取大鼠SPA基因序列,对其上游基因组序列进行计算机生物信息学分析,推断其上游序列约163bp的区域具有启动子功能。②利用PCR技术扩增SPA基因上游启动子序列,将其亚克隆入pGL3-basic中,构建pGL3-SPA质粒,将其亚克隆于pGL3-control中,构建pGL3-SPA-enhancer质粒。③将构建pGL3-SPA质粒、pGL3-SPA-enhancer质粒、pGL3-control质粒和pGL3-basic质粒分别与内参质粒pRL-TK共转染入A549细胞和H441细胞,用双荧光素酶报告系统检测该质粒在两种细胞中的荧光素酶活性表达。结果:酶切及测序结果均证实成功克隆了SPA基因启动子序列,并已将该序列正确插入到荧光素酶报告基因系列载体中。重组的pGL3-SPA-enhancer质粒、pGL3-SPA质粒转染H441细胞后可以检测到荧光素酶的高表达。结论:成功构建了含有SPA基因启动子序列的荧光素酶基因报告系统,证实了它在高表达SPA蛋白的细胞中有较高的转录活性,为下一步研究SPA基因功能及其转录调控以及探讨基因治疗的靶向性奠定了基础。

AIM ： To clone the SPA gene promoter and construct its luciferase report vector of SPA gene and to study its transcriptional targeting activity. METHODS ： （1） The SPA gene sequence was acquired from GenBank, of which the upstream was analyzed according to bioinformatics. The results showed that the upstream region of SPA gene sequence about 163bp has the function of promoter.（2） The SPA gene promoter fragment was generated by polymerase chain reaction and then subclaned into the multiple clone site （MCS） of luciferase report gene vector pGL3 - basic to generate the recombined plasmid pGL3 - SPA. This fragment was also subcloned into pGL3 - control to generate recombined plasmid pGL3 - SPA - enhancer by replacing its primary SV40 promoter. （3） pGL3 - SPA, pGl3 - SPA - enhancer, pGL3 - control, pGL3 - basic were cotransfected with pRL - TK into A549 cells and H441 cells. The luciferase activities were measured by dual luciferase reportor （DLR） system. RESULTS： Sequencing and restricted digestive results showed that SPA gene promoter was successfully cloned and identified, and also correctly subcloned into plasmid pGL3 - basic and pGL3 - control to construct its luciferase report plasmid pGL3 -SPA and pGL3 -SPA -enhancer, respectively. The transcriptional activity was high in H441 cells. CONCLUSION： The luciferase report system of SPA gene promoter is successfully constructed with high transcriptional activity.