【摘要】：背景脂質(zhì)運載蛋白是一種擁有30-50個成員的蛋白質(zhì)超家族，有著保守的三維結(jié)構(gòu)，但卻發(fā)揮著一系列不同的生物學(xué)功能。作為脂質(zhì)運載蛋白質(zhì)超家族的成員之一， α1-微球蛋白質(zhì)（α1-microglobulin, α1M）在進化上高度保守且分布廣泛提示該蛋白質(zhì)具有重要的生理功能，目前有研究發(fā)現(xiàn)其能結(jié)合和降解血紅素，具有還原酶活性及抗氧化性及免疫調(diào)節(jié)作用。但其功能活性位點以及具體的功能機制仍然不清楚。 目的本研究將解析人α1M蛋白質(zhì)的三維結(jié)構(gòu)并基于其結(jié)構(gòu)研究其生化和生理功能，從而了解其具體的功能機制并為基于α1M蛋白質(zhì)三維結(jié)構(gòu)的抗炎、抗氧化及免疫調(diào)節(jié)藥物的研究開發(fā)打下基礎(chǔ)。 方法通過克隆、表達純化等技術(shù)獲得足夠純度的人α1M蛋白質(zhì)，然后培養(yǎng)蛋白質(zhì)晶體，通過X衍射技術(shù)獲得晶體衍射數(shù)據(jù)隨之解析人α1M蛋白質(zhì)三維結(jié)構(gòu)；基于其三維結(jié)構(gòu)，利用定點突變，光譜分析等技術(shù)研究α1M蛋白質(zhì)的功能機制。 結(jié)果通過分子克隆、表達純化等相關(guān)技術(shù)成功獲得了純度達95%的人α1M蛋白質(zhì)；分子篩（Superdex75）純化結(jié)果顯示α1M在溶液中以單體和二聚體兩種聚集狀態(tài)存在；通過蛋白質(zhì)晶體的篩選及優(yōu)化成功獲得了適于X射線衍射的高質(zhì)量單晶；收集到了分辨率為2.0常規(guī)衍射數(shù)據(jù)及分辨率為3.0單波長反常散射衍射(single-wavelengthanomalous dispersion，，SAD)數(shù)據(jù)，成功的解析了α1M蛋白質(zhì)的三維結(jié)構(gòu)；利用ABTS法進行檢測，結(jié)果表明本研究獲得重組的α1M具有抗氧化能力，具備還原酶活性。
[Abstract]:Background Lipid transport protein is a protein superfamily with 30-50 members, which has a conserved three-dimensional structure, but plays a series of different biological functions. As one of the members of lipid transport protein superfamily, 偽 1-microglobulin (偽 1m) is highly conserved and widely distributed in evolution. It has been found that 偽 1-microglobulin (偽 1m) can bind and degrade heme. It has reductase activity, antioxidant activity and immunomodulatory effect. However, its functional active sites and specific functional mechanisms are still unclear. Objective in this study, the three-dimensional structure of human 偽 1m protein was analyzed and its biochemical and physiological functions were studied based on its structure, so as to understand its specific functional mechanism and to provide an anti-inflammatory mechanism based on the three-dimensional structure of 偽 1m protein. The research and development of antioxidation and immunomodulatory drugs lay the foundation. Methods sufficient purity of human 偽 1m protein was obtained by cloning, expression and purification techniques, and then protein crystals were cultured, and the three-dimensional structure of human 偽 1m protein was analyzed by X-ray diffraction data based on its three-dimensional structure. The functional mechanism of 偽 1m protein was studied by site-directed mutation and spectroscopic analysis. Results the purity of 95% human 偽 1m protein was obtained by molecular cloning, expression and purification, and the molecular sieve (Superdex75) purification showed that 偽 1m existed as monomer and dimer in the solution. High quality single crystals suitable for X-ray diffraction were successfully obtained by screening and optimizing protein crystals, and conventional diffraction data with a resolution of 2.0 and single-wavelengthanomalous scattering data with a resolution of 3.0 were collected. The three-dimensional structure of 偽 1m protein was successfully elucidated and detected by ABTS method. The results showed that the recombinant 偽 1m protein had antioxidant activity and reductase activity.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R341

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