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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。
OBJECTIVE: To clone the promoter of rat pulmonary surfactant protein A (SPA) gene and construct a fluorescent reporter system of the promoter to investigate the activity and transcriptional targeting of the promoter. In order to further investigate the mechanism of SPA gene expression and explore the target Gene therapy to lay the foundation. Methods: ① The SPA gene sequence of rat was obtained from GenBank. The upstream genomic sequence was analyzed by computer bioinformatics. It was deduced that the region of 163bp in its upstream sequence had promoter function. (2) The upstream promoter of SPA gene was amplified by PCR and subcloned into pGL3-basic to construct pGL3-SPA plasmids which were subcloned into pGL3-control to construct pGL3-SPA-enhancer plasmids. ③ The pGL3-SPA plasmid, pGL3-SPA-enhancer plasmid, pGL3-control plasmid and pGL3-basic plasmid were co-transfected into A549 cells and H441 cells respectively with the internal control plasmid pRL-TK. The luciferase reporter system was used to detect the Plasmid expression of luciferase activity in both cells. Results: The results of digestion and sequencing confirmed that the SPA gene promoter sequence was successfully cloned and inserted into the vector of luciferase reporter gene correctly. Recombinant pGL3-SPA-enhancer plasmid, pGL3-SPA plasmid transfected H441 cells can be detected high luciferase expression. CONCLUSION: A luciferase gene reporter system containing the SPA gene promoter sequence was successfully constructed, which confirmed that it has high transcriptional activity in the cells highly expressing SPA protein. It is the next step to study the function of SPA gene and its transcriptional regulation Targeting gene therapy laid the foundation.