IL-2Rα基因负调控元件（NRE/NIRS）相关结合蛋白的筛选及其功能的初步研究

【作者】 盛德乔；

【导师】 沈珝琲； 吴宁华； 张业；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 白细胞介素2(IL-2)与其高亲和力受体(IL-2R)的相互作用在调节T细胞免疫反应的强度和持续时间方面起着关键作用。高亲和力IL-2受体由α、β、γ三个亚基组成。其中β及γ亚基呈组成性表达，并参与IL-2的信号传递；而α亚基在激活的淋巴细胞表面呈诱导性表达，而且与IL-2呈特异结合。α链的特异性诱导表达产生高亲和力受体是淋巴细胞获得对生理浓度IL-2完全反应能力所必需的。 在体内，IL-2Rα基因的转录被严密地调控。目前已发现IL-2Rα基因5′非编码区存在四个正调控区：PRR Ⅰ，PRR Ⅱ，PRR Ⅲ、和PRR Ⅳ，每一个调控区都有相应的反式作用因子与之结合。但是，对于该基因启动子上游的负调控元件(negative regulatory element，NRE)调控机制研究的报道甚少。在IL-2Rα基因启动子上游调控区中存在一个核心序列为TTCATCCCAGG(-390／-380bp)的负调控元件。该NRE与HIV-1 LTR中的一段调控序列(TTCATCACATG)同源性达82％；而且，IL-2Rα基因NRE和HIV-1基因NILE结合同一种50kD非组织特异表达的蛋白质。我们以往研究发现IL-2Rα基因5′上游存在一段与NRE呈不完全反向重复的序列(NRE inverse repeat sequence，NIRS)CCTGGTTTGAA(-153／-143bp)，HIV基因中也有类似的NIRS序列。通过荧光素酶报告基因活性检测，发现在Jurkat细胞中只有当NRE和NIRS同时存在时才对IL-2Rα基因的转录起激活或抑制作用。通过紫外交联实验进一步发现在Jurkat细胞及HeLa细胞中均存在一种既能与NRE又能与NIRS结合的蛋白质。NRE和NIRS的共同存在不仅明显地阻遏了该基因的组成性表达，而且在PHA诱导下共同参与正调控元件使阻遏状态下该基因启动子转为激活状态。 IL-2Rα基因与HIV在调控机制上也有某些相似之处：淋巴细胞活化可以激活IL-2Rα基因的表达，HIV只能在激活的淋巴细胞中复制；HeLa细胞及淋巴细胞中持续表达的非组织特异表达蛋白SP50既可与IL-2Rα基因NRE结合，又可与HIV基因的NRE结合，并作为转录沉默子起作用。因此，研究NRE和NIRS在IL-2Rα基因调控中的作用，不但有助于阐明IL-2Rα基因的转录调控机理，还可为控制HIV病毒复制提供新的线索和手段。更多还原

【Abstract】 The magnitude and duration of T-cell immune response are critically regulated by the interaction of interleukin-2 (IL-2) and its high-affinity receptor (IL-2R) on the surface of the cell. The high-affinity IL-2R is composed of three subunits, α，β and γ . Among them, IL-2Rα is the only component that can be specifically induced in activated lymphocytes and binds to IL-2, the growth signal for T cell proliferation. While IL-2Rβ and IL-2Rγ are crucial for IL-2 signaling, induction of IL-2Rα to form high-affinity receptor is a prerequisite for the cell to be fully responsive to physiological concentrations of IL-2.The transcription of the IL-2Rα gene is tightly regulated in vivo. It was reported that human IL-2Rα gene contained at least four positive regulatory regions: PRR I, PRR II, PRR III, and PRR IV. Each regulatory region appears to play a well-defined and complementary role. Despite the negative regulatory element (NRE) within the upstream regulatory region of human IL-2 receptor α (IL-2Rα) gene has been identified for two decades, mechanisms of the NRE function on the gene is hetero unknown. The negative regulatory element (NRE) mapped at -390/-380 bp (5’ -TTCATCCCAGG-3’ ) of the IL-2Rα gene with 82% homologues to NRE sequence of HIV LTR, and the same kind of 50kD non-tissue specific expressed protein can bind to NRE either of IL-2Rα or HIV gene . In addition, an imperfect inverse repeated sequence of the NRE (NIRS) between -153 bp and -143 bp (5’ -CCTGGTTTGAA-3’) homologous to NRE has been found. It was also found that both NRE and NIRS are required simultaneously for activation or repression of IL-2R α gene transcription in Jurkat cells by enzyme activity assay of the luciferase reporter gene. In Jurkat and HeLa cells, there is one kind of protein that can bind to both NRE and NIRS sequence by using UV cross-linking experiments.They’re some similarity in regulatory mechanism between IL-2R α gene and HIV gene. The activation of lymphocytes can activate expression of IL-2R α gene, and HIV replicated only in activated lymphocytes. In HeLa cell and lymphocytes, constant更多还原