人干扰素调节因子3基因启动子的初步鉴定

Characterization of promoter of human interferon regulatory factor 3

目的克隆并鉴定人干扰素调节因子3（IRF3）基因启动子，为进一步研究其转录调控机制奠定基础。方法用BLAST分析软件进行序列比较和同源分析不同种系IRF3启动子序列，以人类基因组DNA为模板，通过PCR定向克隆和酶切亚克隆策略，构建了覆盖IRF3基因5’侧翼区转录起始位点上游1kb区域的一系列IRF3启动子虫荧光素酶报告基因重组体并瞬时转染宫颈癌HeLa细胞，用虫荧光素酶检测报告基因启动子的活性。结果在人、小鼠的IRF3推断的启动子区域进行同源比较发现，推断的E2F和GATA-1高度进化保守；启动子活性分析表明，IRF3启动子区域定位于主要转录起始位点区域前111～167bp的区域内。采用转录因子结合位点预测分析软件分析表明，该区域内存在E2F转录因子结合位点。结论-167～111bp区存在IRF3启动子的核心调控元件，转录因子E2F可能参与IRF3的转录调控。

Objective To clone and identify the promoter of human interferon regulatory factor 3 （ IRF3 ） and to further investigate its transcriptional regulation. Methods The promoter sequences of IRF3 from different species were analyzed by BLAST. Several overlapping genomic fragments from the 5＇-flanking region of IRF3 gene have been then cloned into pGL3-Basic vector to construct IRF3 promoter reporters. Then transfection of HeLa cells with the promoter-driven luciferase constructs was performed to induce lucif- erase gene express and calculate luciferase activity. Results Homologous. sequence comparison in IRF3 promoter area among human and mouse showed that putative E2F and GATA-I sites were highly evolutionally conserved. Luciferase reporter assay indicated that IRF3 promoter region was mainly located in 111-167 bp region before the major transcriptional start site. Transcriptional factor binding analysis indicated that IRF3 promoter included putative transcriptional factor binding sites： E2F. Conclusion The luciferase assays re- vealed that the -（ 167-111 ） bp region was the minimal promoter of the human IRF3 gene. These results sug- gested that transcriptional factors such as E2F might be involved in the transcriptional regulation of IRF3 gene.