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目的构建含有VEGF基因的不同片段启动子的荧光素酶报告基因载体,并检测VEGF启动子不同片段的活性。方法用PCR扩增出VEGF启动子的不同片段,插入到荧光素酶报告基因载体pGL4-basic中,确定所扩增的DNA序列后,将其转染至293T细胞中,检测其不同片段的活性。结果测序结果表明,扩增的不同片段的VEGF启动子序列正确;荧光素酶活性实验表明,VEGF启动子+72～-128 bp至+72～-528 bp片段具有较高的活性。结论构建了VEGF启动子5’端缺失不同片段突变体报告基因表达载体,为VEGF转录因子的筛选及功能研究提供了重要基础。
Objective To construct a luciferase reporter vector containing different fragment promoters of VEGF gene and detect the activity of different fragments of VEGF promoter. Methods Different fragments of VEGF promoter were amplified by PCR and inserted into luciferase reporter gene vector pGL4-basic. The amplified DNA sequence was confirmed and transfected into 293T cells to detect the activity of different fragments . Results The sequencing results showed that the correct promoter sequence of VEGF in different fragments was amplified. The luciferase activity assay showed that the + 72 ~ -128 bp to + 72 ~ -528 bp fragment of VEGF promoter had high activity. Conclusion The reporter gene deletion vector containing different fragments of 5 ’end of VEGF promoter was constructed, which provided an important basis for the screening of VEGF transcription factor and its function.