人B7H4启动子荧光素酶报告基因载体的构建及活性分析

Construction and functional analysis of human B7H4 promoter luciferase reporter gene vectors

为构建人B7H4基因启动子荧光素酶报告基因载体,以人外周血单个核细胞基因组DNA为模板,PCR扩增3条不同长度人B7H4启动子序列,并插入PGL3-Basic荧光素酶报告基因载体中;待测序验证后,将3个重组质粒及pRL-TK内参质粒分别共转染HEK-293T细胞,采用双荧光素酶报告基因系统检测其启动子活性。测序结果显示构建的3个人B7H4基因启动子重组载体序列正确;重组载体转染HEK-293T细胞,经双荧光素酶报告基因检测确定重组载体有启动子活性,其中PGL3-hB7H4-0.5kb重组载体的转录活性较高。本研究成功构建了3条含不同长度的B7H4启动子序列的荧光素酶报告基因系统,为后续分析人B7H4启动子的转录调控元件及肿瘤微环境中调控B7H4表达的作用因素等研究奠定了实验基础。

To construct the luciferase reporter gene vectors containing human B7 homology 4 gene promoter sequence, three fragments of B7H4 gene promoter were amplified from genomic DNA of human peripheral blood mononuclear cells by PCR, then the fragments were cloned into pGL3-Basic luciferase reporter vector respectively. After sequencing analysis, the recombi- nant vectors and reference vector pRL-TK were transiently cotransfected into HEK-293T cells and dual-luciferase reporter gene system was used to test the activity of the promotors. The sequencing results showed the recombined plasmids containing different length B7H4 gene promoters（pGL3-hBTH4-2 kb, pGL3-hBTH4-1 kb and pGL3-hBTH4-0. 5 kb） were correctly constructed. The dual-luciferase reporter assay demonstrated that the recombinant vectors had the promoter activities in HEK- 293T cells, and pGL3-hBTH4-0. 5 kb showed the highest transcription activity. In conclusion, the recombinant luciferase reporter vectors containing different length human B7H4 promoters were successfully constructed, providing a tool to investigate the transcriptional elements of human B7H4 promoter and identify the regulatory factors of B7H4 expression in tumor microen- vironment.