BRCA2基因启动子的克隆和分析

Cloning and analysis of the BRCA2 promoter

目的构建BRCA2基因启动子的荧光素酶报告质粒,为BRCA2基因调控研究提供基础。方法采用高保真Taq聚合酶,从正常人外周血中扩增出BRCA2启动子并将其插入质粒中。通过基于PCR的定点突变方法在多态性位点rs3092989(-254A/G)获得含-254G等位基因的序列。亚克隆BRCA2启动子片段至pGL3-basic报告基因载体中,构建含BRCA2启动子的重组报告质粒。转染HeLa细胞,评价启动子活性以及-254A/G多态性位点对活性的影响。结果酶切和直接测序法证实所构建质粒含有pGL3-basic及BRCA2基因启动子序列,扩增的含-254A和-254G的BRCA2启动子序列正确。构建的报告基因具有启动子活性,与pGL3-basic质粒相比较,BRCA2启动子的相对荧光单位增加了413倍。rs3092989(-254A/G)多态位点未明显影响启动子活性。结论 BRCA2转录起始位点上游序列具有较强的启动子活性。成功克隆BRCA2启动子并引入相关突变,可为后续的基因转录调控研究奠定基础。

Purpose To construct the BRCA2 promoter luciferase reporter gene vector and analyze the mechanism of gene regulation. Methods By using genomic DNA from healthy people as template, the BRCA2 promoter region was amplified by high fidelity DNA polymerase. Then the fragment was cloned into a recombinant plasmid. To analyze whether polymorphism rs3092989 （-254A/G） could affect promoter activity, PCR-based mutagenesis was performed to generate haplotype containing -254G allele. After confirmed by direct sequencing, the BRCA2 promoter was subeloned into pGL3-basie vector. The constructed vector was transfected into HeLa cells to detect promoter activity. Results Direct sequencing and restriction endonuelease analysis confirmed the recombinant plasmid contained pGL3-basic vector and BRCA2 promoter sequence. The BRCA2 promoter containing -254A and -254G allele was success- fully cloned. The BRCA2 promoter exhibited strong promoter activity with an increase of 413 fold of relative luciferase activity unit （RLU） as compared with pGL3-basie vector. The -254A/G polymorphism did not affect promoter activity. Conclusion The cloned BRCA2 promoter region has strong promoter activity. The success of cloning and mutagenesis of BRCA2 promoter region could be used for further analysis of gene regulation.