NF-κB通路论文：CARD结构域对猪BCL 10表达及细胞周期的影响.doc

NF-B通路论文：CARD结构域对猪BCL 10表达及细胞周期的影响【中文摘要】在细胞的信号转导过程中,NF-B通路可以激活或者抑制核内基因的表达,从而将外界的刺激转化成细胞的信号。在免疫系统中,NF-B在控制抗原受体信号转导通路、调控诱导核转录因子中同样起关键作用。BCL 10是参与NF-B通路的一个重要功能基因。BCL 10正常的表达水平可以促进免疫因子的产生,增强机体的天然免疫和抵御感染的能力。反之,BCL 10的异常表达,比如过表达、过度磷酸化和核内表达会导致一系列细胞因子的非正常表达,从而影响正常细胞的生理功能,甚至会导致淋巴瘤等多种疾病的发生。已知BCL10的CARD结构域、磷酸化位点以及C末端序列参与几乎全部蛋白功能的发挥。参考以上的研究结果,我们开展猪BCL 10的研究工作。实验室前期工作:通过电子克隆得到猪BCL 10的全长基因,通过特异性引物在猪脾脏克隆到该基因片段,并得到该基因原核表达蛋白,掌握了其原核表达特点。同时,检测结果显示,猪BCL 10基因mRNA在脾脏中表达最高,胸腺、大脑和下颌淋巴结表达次之,肝脏只有微量表达,肾脏没有表达,初步获得该基因在猪体内的表达情况。本研究进一步探讨猪BCL 10的CARD结构域相关功能,通过该基因序列分析和真核表达观测,取得以下结果:1.根据猪BCL 10基因的特点,通过激酶的作用位点和空间结构的预测,找到了可能与其核内表达相关的位点,并成功构建出完整猪BCL 10基因的真核表达载体pEGFP-B。同时,通过大引物扩增法构建出真核表达载体pEGFP-D(缺失CARD结构域的猪BCL 10基因)和pEGFP-P(缺失Thr-Ser-Ser位点的猪BCL 10基因)。2.转染猪脐静脉血管内皮细胞系(SUVEC)后,Western Blot检测表明融合蛋白得到表达。分析发现,TSS位点(Thr-Ser-Ser)参与猪BCL 10的CARD结构域与其它蛋白的相互作用,是该基因的重要位点。同时,TSS位点的缺失会导致BCL 10出现核内表达。这些结果证实,TSS在猪BCL 10基因中具有重要作用。3.细胞周期变化测定表明,猪BCL 10的CARD结构域会引起细胞的G 1期缩短,S期和G 2/M期延长,表明该结构域在细胞生长过程中影响DNA的复制以及细胞有丝分裂,过度表达会导致细胞在有丝分裂前期停滞积累,但不会导致细胞凋亡。从而为进一步研究猪BCL 10蛋白在信号转导中的作用特点,以及淋巴瘤等疾病的诊断与治疗奠定基础。【英文摘要】NF-B pathway can activate or inhibit the expression of nucleus genes to transfer the external stimuli into the cell signal during the process of cell signal transduction. In the immune system, NF-B plays a similar role in the controlling the pathway of antigen receptor signaling transduction as well as the regulation and induction of nucleus transcription factors. BCL 10 is an important functional gene involving in NF-B pathway and its normal expression can promote the production of immune factors, enhance innate immunity and defence capacity. Conversely, the abnormal expression of BCL 10, such as over-expression, over-phosphorylation and nucleus expression will lead to abnormal expression of a series of cytokines, thereby affecting the physiological function of normal cells and even resulting in various diseases including lymphoma. It is well known that CARD domain, phosphorylation sites, and the C terminal sequence of BCL10 are involved in the fuctions of almost all of the protein. And the research of our group on swine BCL 10 was carried out based on the previous research.In a previous work of our group, full-length gene of swine BCL 10 was obtained by in silico cloning and coding sequence of this gene were cloned from swine spleen using specific primer. And its protein of prokaryotic expression was obtained and characterized. Scanning the expression profile of BCL 10 in vivo in swine indicated that the highest mRNA expression of swine BCL 10 gene was observed in spleen, followed by thymus, brain, and mandibular lymph node. Comparatively, there was only a trace of mRNA expression and no expression in kidneys.Our present study explored CARD domain-related functions of swine BCL 10 gene through the analysis of gene sequence and eukaryotic expression. And our results obtained in the present work are as follows:1. According to the characteristics of swine BCL10 gene, the possible sites related to nuclear expression was found through the prediction of the kinase action sites and the spatial structure prediction. And the vector pEGFP-B was successfully constructed for complete swine BCL10 gene, meanwhile the vector pEGFP-D(deleting swine BCL 10 gene in CARD structure domain) and pEGFP-P(deleting swine BCL 10 gene at Thr- Ser-Ser site) were constructed through site-mutant PCR method (large primers amplification method). 2. After the above three vectors were transfected into swine umbilical vein endothelial cells (SUVEC), the analysis of Western Blot showed that the fusion protein was expressed. Our analysis showed that TSS sites (Thr-Ser-Ser) involved in CARD domain of swine BCL 10 and interacted with other proteins, which are important sites of the gene. Meanwhile, deleting TSS sites will lead to nucleus expression of BCL 10, thus proving the function of TSS in swine BCL 10 gene.3. The analysis of cell cycle showed that CARD domain of swine BCL 10 would cause shortening of G 1 phase but prolonging of S phase and G 2/M phase, which indicates that domain structure affects DNA replication and cell mitosis, and excessive expression will lead to cell arrest in early stage of cell division, but will not lead to cell apoptosis. This lays a good foundation for swine BCL 10 function in cell signal transduction as well as diagnosis and treatment of disease such as lymphoma, etc.【关键词】NF-B通路 BCL 10基因 核内表达 TSS位点 CARD结构域【英文关键词】NF-B pathway BCL 10 gene nuclear expression TSS sites CARD domain【目录】CARD结构域对猪BCL 10表达及细胞周期的影响摘要6-7ABSTRACT7-8文献综述11-20第一章 BCL 10 的研究进展11-201.1 BCL 10 蛋白结构与功能特点11-121.2 BCL 10 与 NF-B 信号通路12-161.2.1 NF-B 相关蛋白分子12-131.2.2 NF-B 信号通路13-151.2.3 BCL10 参与NF-B 通路15-161.3 BCL10 与细胞凋亡16-171.3.1 细胞凋亡161.3.2 蛋白的磷酸化16-171.3.3 BCL 10 的磷酸化参与细胞凋亡171.4 BCL10 与粘膜相关组织淋巴瘤17-20试验研究20-38第二章 CARD 结构域对猪BCL 10 表达及细胞周期的影响20-382.1 材料202.1.1 菌株、细胞和质粒202.1.2 主要试剂202.2 方法20-292.2.1 猪BCL 10 CARD 结构域相关三个真核表达载体的构建21-262.2.2 猪BCL 10 CARD 结构域相关三个真核表达载体在猪血管内皮细胞中表达26-292.3 结果与分析29-362.3.1 PCR 结果29-312.3.2 猪BCL 10 CARD 结构域相关三个克隆载体构建结果312.3.3 猪BCL 10 CARD 结构域相关三个真核表达载体构建结果312.3.4 猪BCL 10 CARD 结构域相关三个载体序列分析31-342.3.5 正常细胞与转染空载体的细胞对照342.3.6 猪BCL 10 CARD 结构域相关三个真核表达载体转染SUVEC34-352.3.7 猪BCL 10 CARD 结构域相关真核载体表达融合蛋白Western blot 鉴定352.3.8 猪BCL 10 CARD 结构域相关真核载体表达对SUVEC 细胞周期的影响35-362.4 讨论36-372.5 小结37-38结论38-39参考文献39-45缩略词45-46附录46-49致谢49-50个人简介50