HLA-B27启动子荧光素酶报告基因载体的构建及其在Hela细胞的转录调控研究  

Molecular cloning of HLA-B27 gene promoter and transcriptional activity detection in HeLa cells

作　　者：赵丽珂[1] 古洁若[1] 余得恩[2] 

机构地区：[1]中山大学附属第三医院风湿科,广州510630 [2]美国加州大学洛杉矶分校风湿病学中心

出　　处：《中华风湿病学杂志》2007年第8期449-452,共4页Chinese Journal of Rheumatology

基　　金：国家自然科学基金(30571735);国家教委博士点基(20060558046);国家杰出青年基金(30325019);广东省科技计划资助项目(2005A30801005)

摘　　要：目的构建荧光素酶报告基因(luc)与HLA-B27启动子融合蛋白哺乳动物细胞表达载体,观察其在Hela细胞的表达调控。方法克隆出HLA-B27启动子序列(432 bp),构建pGL4.14/ B27pro-luc融合蛋白表达载体。转染Hela细胞构建稳定转染细胞系。观察不同的细胞因子对B27转录水平的表达调控。结果首先成功构建B27启动子-luc融合蛋白表达载体:然后使用此载体转染Hela细胞构建B27启动子的稳定转染Hela细胞株。应用该细胞株,发现肿瘤坏死因子(TNF)-α、干扰素(IFN)-α、IFN-β和IFN-γ可以通过调节稳定转染的B27启动子活性显著增加B27的转录水平(P<0.05),其启动子活性比基础表达水平分别增高(1.67±0.20)倍,(1.79±0.71)倍,(2.94±0.68)倍和(1.98±0.45)倍。结论HLA-B27启动子活性在Hela细胞中只有可调节性。细胞因子TNF-α、IFN-α、IFN-β和IFN-γ可通过调控HLA-B27启动子的活性而调节HLA-B27的表达。Objective To construct pGIA.14-luc eukaryotic expression vector for HLA-B27 promoter gene and explore the activity regulation of this promoter in Hela cells. Methods The HLA-B27 gene promot- er （-419 bp-1 bp） was amplified by polymerase chain reaction and was cloned into pGL4.14-luc vector to construct eukaryotic expression vector pGL4.14/B27 pro-luc. The purified pGL4.14/B27 pro-luc was stablely transfected into HeLa cells and the activity of HLA-B27 gene promoter was detected by luminometer. Results About 432 bp gene fragment was amplified by PCR from genomic DNA and pGL4.14/B27 pro-luc vector was constructed successfully. The activity of HLA-B27 promoter was 1.67±0.20, 1.79±0.71, 2.94±0.68, 1.98±0.45 in Hela stable cells after treated with TNF-α, IFN-α, IFN-β and IFN-γ for 48 hours. Conclusion TNF-α, IFN-α, IFN-β and IFN-γ can regulate the B27 promoter activity. The high specific activity of constructed HLA-B27 promoter eukaryotic expression vector may be a good method for further research.

关 键 词：HLA-B27抗原 启动子区(遗传学) HELA细胞 表达调控 

分 类 号：R593.2[医药卫生—内科学]