PTPN22与PADI4相互作用的研究

发布时间：2018-04-16 02:19

本文选题：类风湿关节炎 + PTPN22　；参考：《哈尔滨工业大学》2015年硕士论文

【摘要】：类风湿关节炎是一种常见的、慢性的、自身免疫系统失调的自身性免疫疾病。临床发现该病的主要特征是关节、骨骼等部位发生炎症,发病过程中引发关节的坏死以及骨骼的侵蚀,最终将导致患者关节的畸形及其功能的丧失。类风湿关节炎的病因尚不明确。PTPN22,即蛋白酪氨酸磷酸酶非受体型22,目前认为该基因编码的淋巴酪氨酸磷酸酶可以影响T细胞活化,从而影响免疫稳态而导致自身免疫病的发生。PADI4,即肽基精氨酸脱亚胺酶4,为编码精氨酸转化为瓜氨酸残基的酶家族的成员,可在粒细胞和巨噬细胞介导的炎症和免疫反应中发挥作用,因而可能与类风湿关节炎的发病相关。PADI4是一种翻译后修饰酶,可以在钙离子存在的情况下把精氨酸残基转化成瓜氨酸残基,此过程称作瓜氨酸化。PADI4与类风湿性关节炎最密切相关,哺乳动物的PAD受细胞内的钙浓度、自动瓜氨酸化和二聚化的调控蛋白质瓜氨酸化的改变可能可以解释类风湿性关节炎的风险因素,有研究猜测,PTPN22R620可能与PADI4相互作用,从而限制了瓜氨酸化以及中性粒细胞胞外杀菌网络的形成。PTPN22R620是天然的PADI4的抑制剂,但是PTPN22W620完全剥夺了PTPN22对PADI4的抑制作用导致高度瓜氨酸化。而目前对于PTPN22和PADI4的相互作用依然不是很清晰,其中主要的限制因素是难以得到大量、均一度高的PTPN22和PADI4蛋白,从而造成其数据的缺乏。本论文针对这一难题,探索了PTPN22和PADI4在大肠杆菌这一原核表达系统中的表达,并对二者相互作用关系进行了研究。通过优化大肠杆菌的培养温度、培养时间和培养浓度,明显改善了蛋白的表达量、行为及纯度。通过亲和层析、离子交换层析、凝胶过滤色谱层析等方法提高了蛋白纯度,最终能够在体外得到具有较高浓度、较高纯度的、行为较为均一的PTPN22和PADI4蛋白。然而,个别PTPN22重组蛋白和PADI4蛋白可以实现共表达,纯化得到的PTPN22和PADI4蛋白之间并不存在相互作用。本研究加深了我们对于PTPN22和PADI4蛋白的整体认识,为将来的结构解析以及临床应用奠定了良好的基础。
[Abstract]:Rheumatoid arthritis is a common, chronic, autoimmune disorder.It is found that the main features of the disease are inflammation of joints and bones, necrosis of joints and erosion of bones, which will eventually lead to deformity and loss of function of joints.The etiology of rheumatoid arthritis is unclear. PTPN22, a protein tyrosine phosphatase non-recipient 22, is now thought to affect the activation of T cells by the lymphotyrosine phosphatase encoded by the gene.This affects the immune homeostasis and leads to the occurrence of autoimmune disease. PADI4, which is a member of the enzyme family that encodes arginine to citrullinate residue, is called peptidyl arginine deiminase 4.May play a role in inflammatory and immune responses mediated by granulocytes and macrophages, and may therefore be associated with the pathogenesis of rheumatoid arthritis. PADI4 is a post-translational modifier.Arginine residues can be converted into citrulline residues in the presence of calcium ions, a process called melamine acidification. PADI4 is most closely related to rheumatoid arthritis, and PAD in mammals receives intracellular calcium concentrations.Changes in melamine and dimeric regulation proteins may explain the risk factors of rheumatoid arthritis, and some studies suggest that PTPN 22R620 may interact with PADI4.PTPN22R620 is a natural inhibitor of PADI4, but PTPN22W620 completely deprives the inhibition of PADI4 by PTPN22.However, the interaction between PTPN22 and PADI4 is still not clear, and the main limiting factor is that it is difficult to obtain a large number of PTPN22 and PADI4 proteins, which are once high, resulting in a lack of data.In this paper, the expression of PTPN22 and PADI4 in E. coli prokaryotic expression system was investigated, and the interaction between them was studied.By optimizing the culture temperature, time and concentration of Escherichia coli, the expression, behavior and purity of protein were significantly improved.The protein purity was improved by affinity chromatography, ion exchange chromatography and gel filtration chromatography, and PTPN22 and PADI4 proteins with high concentration and high purity could be obtained in vitro.However, individual PTPN22 recombinant protein and PADI4 protein can be coexpressed, and there is no interaction between purified PTPN22 and PADI4 protein.This study has deepened our overall understanding of PTPN22 and PADI4 proteins and laid a good foundation for future structural analysis and clinical application.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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