血管内皮细胞生长因子启动子突变体萤光素酶报告基因载体的构建

摘要

目的：构建含有不同片段血管内皮细胞生长因子(VEGF)基因的启动子的萤光素酶报告基因载体,并定位缺氧诱导因子1α(HIF-1α)结合VEGF启动子的区域。方法：以VEGF全长启动子为模板,PCR扩增VEGF启动子的不同片段,插入萤光素酶报告基因载体pGL4-basic,确定所扩增的DNA序列后,将其与HIF-1α表达载体共转染293T细胞,定位HIF-1α结合VEGF启动子的区域。结果：测序结果表明扩增的不同片段的VEGF启动子序列正确；萤光素酶活性实验表明,-1128~-728 bp片段是HIF-1α与VEGF相互作用激活VEGF启动子转录活性的区域。结论：克隆了VEGF启动子5'端缺失突变体报告基因表达载体,为调控VEGF表达的转录因子的筛选及功能研究打下了良好基础。%Objective: To clone different regions of vascular endothelial growth factor(VEGF) promoter and insert them into a luciferase reporter gene vector, and to identify the region of VEGF promoter for hypoxia inducible fac⁃tor-1α(HIF-1α) binding. Methods: Different regions of VEGF promoter were amplified from full length VEGF promoter by PCR and cloned into pGL4-basic. The resulting plasmids were examined by DNA sequencing. Then they were co-transfected with HIF-1α expression plasmid into 293T cell line to analyze promoter activity in the presence of HIF-1α. Results: DNA sequencing showed that the sequences of the promoter regions were correctly cloned. The region -1128~-728 bp of VEGF promoter played a major role in the interaction between HIF-1αand VEGF promoter. Conclusion: The VEGF promoter and 5' terminal deletion mutant reporter genes were cloned successfully, which will be contribute to screen transcription factors that regulate VEGF expression.