本文選題：類風(fēng)濕關(guān)節(jié)炎 + PTPN22　； 參考：《哈爾濱工業(yè)大學(xué)》2015年碩士論文

【摘要】：類風(fēng)濕關(guān)節(jié)炎是一種常見的、慢性的、自身免疫系統(tǒng)失調(diào)的自身性免疫疾病。臨床發(fā)現(xiàn)該病的主要特征是關(guān)節(jié)、骨骼等部位發(fā)生炎癥,發(fā)病過程中引發(fā)關(guān)節(jié)的壞死以及骨骼的侵蝕,最終將導(dǎo)致患者關(guān)節(jié)的畸形及其功能的喪失。類風(fēng)濕關(guān)節(jié)炎的病因尚不明確。PTPN22,即蛋白酪氨酸磷酸酶非受體型22,目前認(rèn)為該基因編碼的淋巴酪氨酸磷酸酶可以影響T細(xì)胞活化,從而影響免疫穩(wěn)態(tài)而導(dǎo)致自身免疫病的發(fā)生。PADI4,即肽基精氨酸脫亞胺酶4,為編碼精氨酸轉(zhuǎn)化為瓜氨酸殘基的酶家族的成員,可在粒細(xì)胞和巨噬細(xì)胞介導(dǎo)的炎癥和免疫反應(yīng)中發(fā)揮作用,因而可能與類風(fēng)濕關(guān)節(jié)炎的發(fā)病相關(guān)。PADI4是一種翻譯后修飾酶,可以在鈣離子存在的情況下把精氨酸殘基轉(zhuǎn)化成瓜氨酸殘基,此過程稱作瓜氨酸化。PADI4與類風(fēng)濕性關(guān)節(jié)炎最密切相關(guān),哺乳動物的PAD受細(xì)胞內(nèi)的鈣濃度、自動瓜氨酸化和二聚化的調(diào)控蛋白質(zhì)瓜氨酸化的改變可能可以解釋類風(fēng)濕性關(guān)節(jié)炎的風(fēng)險因素,有研究猜測,PTPN22R620可能與PADI4相互作用,從而限制了瓜氨酸化以及中性粒細(xì)胞胞外殺菌網(wǎng)絡(luò)的形成。PTPN22R620是天然的PADI4的抑制劑,但是PTPN22W620完全剝奪了PTPN22對PADI4的抑制作用導(dǎo)致高度瓜氨酸化。而目前對于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難以得到大量、均一度高的PTPN22和PADI4蛋白,從而造成其數(shù)據(jù)的缺乏。本論文針對這一難題,探索了PTPN22和PADI4在大腸桿菌這一原核表達(dá)系統(tǒng)中的表達(dá),并對二者相互作用關(guān)系進(jìn)行了研究。通過優(yōu)化大腸桿菌的培養(yǎng)溫度、培養(yǎng)時間和培養(yǎng)濃度,明顯改善了蛋白的表達(dá)量、行為及純度。通過親和層析、離子交換層析、凝膠過濾色譜層析等方法提高了蛋白純度,最終能夠在體外得到具有較高濃度、較高純度的、行為較為均一的PTPN22和PADI4蛋白。然而,個別PTPN22重組蛋白和PADI4蛋白可以實現(xiàn)共表達(dá),純化得到的PTPN22和PADI4蛋白之間并不存在相互作用。本研究加深了我們對于PTPN22和PADI4蛋白的整體認(rèn)識,為將來的結(jié)構(gòu)解析以及臨床應(yīng)用奠定了良好的基礎(chǔ)。
[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.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R593.22

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本文編號：1756869