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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 mammalian cell expression vector containing luciferase reporter gene (luc) and HLA-B27 promoter fusion protein and observe its expression in Hela cells. Methods The HLA-B27 promoter sequence (432 bp) was cloned to construct pGL4.14 / B27pro-luc fusion protein expression vector. Hela cells were transfected to construct stable transfected cell lines. To observe the different cytokines on B27 transcriptional regulation. Results The B27 promoter-luc fusion protein expression vector was successfully constructed. Hela cells were then transfected into Hela cells to construct a stable transfected Hela cell line with B27 promoter. Using this cell line, it was found that tumor necrosis factor (TNF) -α, interferon (IFN) -α, IFN-β and IFN-γ significantly increased B27 transcription by regulating the stably transfected B27 promoter activity (P < (1.67 ± 0.20) fold, (1.79 ± 0.71) fold, (2.94 ± 0.68) fold and (1.98 ± 0.45) fold respectively). Conclusion The activity of HLA-B27 promoter is only regulatable in Hela cells. The cytokines TNF-α, IFN-α, IFN-β and IFN-γ regulate the expression of HLA-B27 by regulating the activity of HLA-B27 promoter.