人B细胞活化因子基因启动子活性研究

Analysis on the activity of promoter from human B cell activating factor gene

为探索与自身免疫病发病密切相关的B细胞活化因子(BAFF)基因高水平转录的启动序列及IFN-γ等炎性细胞因子对其活性的影响,以人BAFF基因转录起始点上游-1 349 bp至-329 bp的片段为靶序列,取长度不等的片段作为启动子与含氯霉素乙酰基转移酶(CAT)报告基因的质粒组成5个重组体,脂质体转染法转染上述重组体至人骨髓白血病细胞株:HL-60,CAT-ELISA测定细胞CAT表达水平以比较各重组体的启动子活性;同时加入细胞因子IL-10、IFN-γ、IL-4、重组人BAFF蛋白(rBAFF),以测定其对BAFF启动序列的影响。结果表明,-1 349^-329 bp、-1 349^-743 bp序列具有强启动调控活性,而-1 349^-1 099 bp片段启动活性最低。细胞因子IFN-γ、IL-10和rBAFF均能在一定程度上调人BAFF基因的启动活性,IL-4则一定程度地抑制BAFF基因的启动活性。研究提示,人BAFF基因-1349^-743 bp片段是进一步研究BAFF基因转录相关DNA结合蛋白的重要调控靶序列。细胞因子IL-10、IFN-γ、IL-4、rBAFF可以一定程度的在转录水平影响BAFF合成和分泌。

To analyze sequences that may be important for the regulation of human B cell activating factor （BAFF） gene expression, clarify the fragment of high promoter activity of BAFF gene and explore the potential effects of cytokines on promoter activity, a functional human BAFF promoter with 1 020 base pairs （bp） was cloned and investigated with serial 3＇-deletion. Five chimeric genes were constructed in which various lengths of sequences extending from -1 349 bp to -329 bp were fused to chloramphenicol acetyhransferase （CAT） reporter gene. These recombinant plasmids were transiently transfected to human HL-60 cell lines with liposomal transfection method. Cytokines including interferon-7, interleukin-4, interleukin-10 and recombinant human BAFF （rhBAFF） were added to the culture medium of transiently transfected HL-60 cells. The putative promoters activities were tested and compared by CAT measurement in those transfected cells. The results showed that sequences of -1 349 to-329 bp and -1 349 to -743 bp of human BAFF gene had high activities as promoters. IFN-γ, IL-10, rhBAFF showed increased effects while IL-4 showed inhibitory effect on sequence -1 349 to -743 bp as promoter with activities 3.5-fold, 2.3- fold, 1.4-fold and 72% of control respectively. The results indicated that the 1 020 bp fragment of the BAFF promoter would serve as a valuable mechanistic tool for better understanding of the regulation of BAFF expression, and the high potential sequence -1 349 to -743 bp is of great significance in the futher study searching for specific cis-elements and consensus DNA-binding proteins in BAFF-producing cells. The enhancement effect of IFN-γ, IL-10, rhBAFF and inhibition effect of IL-4 on BAFF synthesis are associated with their transcriptional regulation. The promoter-reporter construct may also be used as a screening tool to identify inhibitors of BAFF expression.