發(fā)布時(shí)間：2018-06-29 15:53

  本文選題：免疫識(shí)別 + CIAS1��； 參考：《暨南大學(xué)》2006年碩士論文

【摘要】：固有免疫是多細(xì)胞生物抗御微生物和抗腫瘤等的第一道防線，固有免疫中感知病原的模式識(shí)別受體能識(shí)別微生物結(jié)構(gòu)中常有的保守的病原體相關(guān)分子模式。在這些病原模式識(shí)別分子中，Toll樣受體識(shí)別細(xì)胞外的病原體信息。胞漿中新發(fā)現(xiàn)的CARTERPILLER蛋白家族可能參與了對(duì)細(xì)胞內(nèi)病原體及其產(chǎn)物的識(shí)別。由CIAS1基因編碼的蛋白cryopyrin／NALP3／CIAS1／PYPAF1為CARTERPILLER蛋白NALP亞族成員，其基因突變可引起三種自發(fā)性炎癥性疾病，分別為MWS，F(xiàn)CAS和CINCA／NOMID。由于cryopyrin同時(shí)存在PYRIN蛋白的PYD結(jié)構(gòu)域與NOD蛋白的NACHT和LRR結(jié)構(gòu)域，我們推測(cè)與自發(fā)性炎癥性疾病有關(guān)的cryopyrin是重要的細(xì)胞內(nèi)固有免疫相關(guān)的胞漿蛋白之一。但cryopyrin如何在細(xì)胞內(nèi)被激活以及它又是如何觸發(fā)免疫識(shí)別反應(yīng)目前還不清楚。本研究采用分子生物學(xué)與理論計(jì)算相結(jié)合的方式，探討cryopyrin在炎癥發(fā)病機(jī)制中的作用及其各結(jié)構(gòu)域在細(xì)胞內(nèi)病原相關(guān)分子模式識(shí)別及信號(hào)級(jí)聯(lián)反應(yīng)中的作用。本實(shí)驗(yàn)用RT-PCR擴(kuò)增正常人白細(xì)胞中CIAS1基因的NACHT區(qū)，觀察不同時(shí)間及不同濃度LPS刺激對(duì)CIAS1的mRNA表達(dá)豐度的影響。另一方面，使用包括3D-PSSM，SPDBV，，Rasmol，WHAT IF，GRAMM和ExPASy等生物數(shù)據(jù)庫(kù)資源和計(jì)算工具，對(duì)cryopyrin各結(jié)構(gòu)域的三維結(jié)構(gòu)進(jìn)行模建，尋找它們?cè)谧R(shí)別細(xì)胞內(nèi)病原相關(guān)分子模式信號(hào)傳導(dǎo)中的結(jié)構(gòu)與功能的聯(lián)系，并在結(jié)構(gòu)分析的基礎(chǔ)上初步探討cryopyrin致病性突變(多分布在NACHT結(jié)構(gòu)域)導(dǎo)致結(jié)構(gòu)與功能變化的可能分子機(jī)制。 RT-PCR結(jié)果顯示100μg／mL LPS刺激對(duì)CIAS1的mRNAL達(dá)呈現(xiàn)先降低后升高的模式。不同濃度LPS刺激2h，CIAS1 mRNA增高呈劑量依賴性(P＜0.01)。序列分析結(jié)果顯示CIAS1基因產(chǎn)物cryopyrin的NACHT結(jié)構(gòu)域存在能與ATP和Mg~(2+)結(jié)合的Walker A和Walker B基序。該基因的主要致病突變位點(diǎn)在結(jié)構(gòu)上與此基序有密切關(guān)系。在LRRs區(qū)存在與Ca~(2+)和多糖結(jié)合的位點(diǎn)。NACHT結(jié)構(gòu)域模型CASTp口袋分析表明：位于NACHT結(jié)構(gòu)域中，正好在walker A基序和walker B基序的活性中心部位有一口袋，是磷酸腺苷ATP和Mg~(2+)結(jié)合及催化的部位。利用模型對(duì)接顯示了該口袋結(jié)構(gòu)與磷酸腺苷和Mg~(2+)的結(jié)合狀態(tài)，表明存在該結(jié)構(gòu)域與腺苷結(jié)合的互補(bǔ)性，與ATP結(jié)合的狀態(tài)涉及的折疊變化目
[Abstract]:Innate immunity is the first line of defense against microorganisms and tumors in multicellular organisms. The pattern recognition receptors in innate immunity can recognize conserved pathogen-related molecular patterns in microbial structures. Toll-like receptors recognize extracellular pathogen information in these pathogenetic pattern recognition molecules. The newly discovered Cartel PILLER protein family in cytoplasm may be involved in the recognition of intracellular pathogens and their products. Cryopyrin / NALP3 / CIAS1 / PYPAF1, encoded by CIAS1 gene, is a member of CARTERPILLER protein NALP subfamily. Its gene mutation can cause three kinds of spontaneous inflammatory diseases, namely MWSFCAS and CINCAP / NOMID. Since the PYD domain of PYRIN protein and the NACHT and LRR domain of nod protein exist in cryopyrin, we speculate that cryopyrin associated with spontaneous inflammatory disease is one of the important cytoplasmic proteins associated with innate cellular immunity. But how cryopyrin is activated in cells and how it triggers immune recognition is unclear. In this study, molecular biology and theoretical calculations were used to investigate the role of cryopyrin in the pathogenesis of inflammation and the role of various domains in intracellular pathogen-related molecular pattern recognition and signal cascade reaction. The NACHT region of CIAS1 gene in normal human leukocytes was amplified by RT-PCR and the effects of LPS stimulation at different time and concentration on the mRNA expression abundance of CIAS1 were observed. On the other hand, using biological database resources and computing tools, such as 3D-PSSMN SPDBVN Rasmol-WHAT Ram and ExPASy, the three-dimensional structures of each domain of cryopyrin were modeled to find the relationship between their structures and functions in identifying the signal transduction of pathogen-associated molecular patterns in cells. On the basis of structural analysis, the possible molecular mechanism of structural and functional changes caused by cryopyrin pathogenicity mutations (mostly distributed in the NACHT domain) was preliminarily discussed. RT-PCR results showed that 100 渭 g / mL LPS stimulated CIAS1 mRNAL reached the first decreased and then increased. The increase of CIAS1 mRNA at different concentrations of LPS was dose-dependent (P < 0.01). Sequence analysis showed that the NACHT domain of CIAS1 gene product cryopyrin had Walker A and Walker B motifs that could bind to ATP and Mg2. The main pathogenicity mutation site of this gene is closely related to this motif in structure. In the LRRs region, there is a site binding to Ca ~ (2) and polysaccharides. The CASTP pocket analysis of NACHT domain model shows that there is a pocket in the active center of walker A motif and walker B motif, which is located in the NACHT domain. Adenosine phosphate ATP and mg ~ (2) binding and catalytic site. The binding states of the pocket structure to adenosine phosphate and mg ~ (2) were demonstrated by using the model docking, indicating the complementarity between the domain and adenosine, and the folding change of ATP binding state.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【共引文獻(xiàn)】

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1 軒東英;金巖;金明;軒昆;邢向輝;鄭梁;趙征;;新基因mcpr1在小鼠牙胚發(fā)育中的時(shí)空表達(dá)和意義[J];實(shí)用口腔醫(yī)學(xué)雜志;2005年06期

2 李鑫;金巖;劉源;王新文;;MCPR-1基因在小鼠腭突組織及各臟器中的表達(dá)及意義[J];現(xiàn)代口腔醫(yī)學(xué)雜志;2006年01期

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1 李鑫;小鼠腭裂相關(guān)基因的克隆、篩選及功能的研究[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

2 軒昆;先天性牙根發(fā)育不良致病相關(guān)基因的克隆研究[D];第四軍醫(yī)大學(xué);2004年

3 趙遠(yuǎn);家蠶抗核型多角體病毒病的微衛(wèi)星分子標(biāo)記篩選、定位及其病毒侵染家蠶中腸組織的差異蛋白質(zhì)表達(dá)圖譜研究[D];江蘇大學(xué);2007年

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1 熊興東;食管癌細(xì)胞差異表達(dá)核基質(zhì)蛋白的分離與鑒定[D];汕頭大學(xué);2003年

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