重型肝炎相关的人纤维介素基因启动子的克隆及功能分析

Molecular doning of hfgl2 gene promoter and function analysis of promoter sequence

目的明确在病毒蛋白HBc和HBx作用下，hfgl2基因5’端非编码区对转录激活起重要作用的转录调控序列。方法运用基因重组的方法，构建一系列5’端缺失而保留共同3’端的hfgl2基因启动子虫荧光素酶报告质粒，将其分别与病毒蛋白HBc和HBx真核表达质粒共转染CHO细胞和HepG2细胞，检测各组细胞的相对荧光素酶活性。结果酶切鉴定以及DNA测序等证实一系列hfgl2基因启动子虫荧光素酶报告基因质粒构建成功，系列启动子缺失试验证实：在hfgl2基因启动子-817位至堋位（相对于转录起始点）之间存在着激活该基因的调控序列。结论在HBV病毒蛋白HBc及HBx作用下，hfgl2基因的启动子区存在一个与其转录表达有关的调控序列，探讨了重型肝炎相关的hfgl2基因高度表达的分子机制，为下一步研究相关的顺式作用元件及反式作用因子奠定了基础。

Objective To investigate the important regulative element domain within the 5＇ flanking uncoding region of hfgl2 gene in response to HBc and HBx. Methods A series of 5＇ truncated promoter of hfgl2 gene were amplified and subcloned into the luciferase report vector to form promoter luciferase report constructs. All plasmids constructed were determined by electrophoretic analysis and DNA sequencing. A eukaryotic construct expressing HBc or HBx, a luciferase reporter construct containing hfgl2 promoter and a β-galactosidase （β-gal） plasmid were cotransfected into Chinese hamster ovary cells and hepG2 cells respectively. Results Luciferase report plasmids containing 5＇ flanking deletion of hfgl2 promoter were successfully constructed, and series deletion array of hfgl2 gene promoter showed that a strong regulatory domain from -817 to -467 （relative to the transcription start site） was responsible for transcription regulation of hfg/2 gene. Condusion The important gene regulative domain is present in the promoter region of hfgl2 gene in response to HBc and HBx. It contributes to further pursuit of cis-acting elements and transcriptional factors involved in the transcription of hfgl2 gene.