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目的:克隆人GDDR基因的启动子;构建GDDR启动子的报告基因载体并进行活性分析。方法:设计合成GD-DR启动子引物,采用PCR技术从人基因组DNA中扩增GD-DR启动子;将扩增片段插入T载体并利用酶切与测序进行鉴定;亚克隆该基因至pGL3-Basic荧光报告基因载体;瞬时转染细胞,用双荧光素酶报告基因系统检测报告基因载体的活性。结果:PCR扩增得到人GDDR基因启动子;成功构建pGL3-GDDR-promoter报告基因载体,测序结果表明启动子序列正确;双荧光素酶报告基因检测系统证实构建的报告基因载体具有启动子活性。结论:成功地构建人GDDR基因启动子的克隆及其报告基因载体的构建,为深入研究GDDR转录表达的调控机制提供基础。
OBJECTIVE: To clone the promoter of human GDDR gene and construct the reporter gene vector of GDDR promoter for activity analysis. Methods: The GD-DR promoter was designed and synthesized. The GD-DR promoter was amplified from human genomic DNA by PCR. The amplified fragment was inserted into T vector and identified by restriction enzyme digestion and sequencing. The gene was subcloned into pGL3- Basic fluorescent reporter gene vector; cells were transiently transfected and the reporter gene vector was tested for activity using the dual luciferase reporter gene system. Results: The promoter of human GDDR gene was amplified by PCR. The pGL3-GDDR-promoter reporter gene vector was successfully constructed. The sequencing result showed that the promoter sequence was correct. The luciferase reporter gene detection system confirmed the promoter activity of the reporter gene. Conclusion: The cloning of human GDDR gene promoter and the construction of its reporter gene vector were successfully constructed, which provided a basis for further study of the regulation of GDDR transcriptional regulation.