【摘要】： 紅色毛癬菌(Trichophyton Rubrum)感染所致的淺部真菌病在世界各地廣泛流行,紅色毛癬菌感染難以治愈,且抗真菌藥物停止治療后頻繁復(fù)發(fā)。本研究首次從整體水平對(duì)靜止期紅色毛癬菌分生孢子的蛋白質(zhì)組進(jìn)行全面的分析。 本研究首先構(gòu)建了紅色毛癬菌8個(gè)時(shí)相的cDNA文庫(kù),建立了EST編碼蛋白數(shù)據(jù)庫(kù)。然后采用液氮研磨、蝸牛酶消化后樣品預(yù)分級(jí),同時(shí)結(jié)合多種酶消化,酶切后得到的肽片段混合物通過二維液相色譜分離,采用鳥槍法對(duì)蛋白進(jìn)行鑒定。結(jié)果獲得5304個(gè)不同的肽段序列,總計(jì)1026個(gè)蛋白得到鑒定。我們建立蝸牛酶消化破壁,結(jié)合樣品預(yù)分級(jí),然后胰酶和蛋白酶K進(jìn)行酶解的蛋白提取分級(jí)分離方法,可以顯著提高鑒定的蛋白數(shù)量。 我們對(duì)所鑒定的1026個(gè)蛋白分別用NCBI non-redundant (NR)蛋白數(shù)據(jù)庫(kù), eukaryotic orthologous groups (KOG)數(shù)據(jù)庫(kù)和gene ontology (GO)數(shù)據(jù)庫(kù)進(jìn)行檢索,所鑒定的蛋白涵蓋了所有主要的細(xì)胞器,如細(xì)胞核、線粒體、細(xì)胞膜、高爾基體、內(nèi)質(zhì)網(wǎng),所鑒定的蛋白涉及到幾乎所有的生物生化過程。本研究鑒定出紅色毛癬菌分生孢子一些重要的蛋白,包括細(xì)胞壁結(jié)構(gòu)蛋白、細(xì)胞壁生物合成相關(guān)蛋白,胞內(nèi)蛋白質(zhì)合成相關(guān)蛋白,靜止期分生孢子代謝特征蛋白,信號(hào)傳導(dǎo)蛋白以及環(huán)境壓力耐受蛋白等等,并從蛋白分子水平上對(duì)其結(jié)構(gòu)功能和機(jī)制進(jìn)行了詳細(xì)的討論,紅色毛癬菌分生孢子蛋白質(zhì)組的分析結(jié)果證明了維持孢子的休眠狀態(tài)是相當(dāng)復(fù)雜和精細(xì)的過程,涉及到一系列的蛋白翻譯、信號(hào)傳導(dǎo)、環(huán)境壓力耐受機(jī)制等,對(duì)將來(lái)真菌感染的預(yù)防、診斷,治療和致病機(jī)制的研究有重要的提示作用。
[Abstract]:The superficial mycosis caused by (Trichophyton Rubrum) infection of Trichophyton rubrum is widespread all over the world. It is difficult to cure the infection of Trichophyton rubrum and recur frequently after the treatment of antifungal drugs. In this study, the proteome of conidia of Trichophyton rubrum at the whole level was analyzed for the first time. In this study, the cDNA library of 8 phases of Trichophyton rubrum was constructed, and the EST coding protein database was established. Then the samples were ground with liquid nitrogen, pre-graded by snail's enzyme digestion, and digested by many kinds of enzymes. The mixture of peptide fragments was separated by two-dimensional liquid chromatography, and the protein was identified by bird gun method. Results 5304 different peptide sequences were obtained and a total of 1026 proteins were identified. We established a method of protein extraction and fractionation of snail's enzyme digestion and broken wall, combined with sample pre-grading, and then enzymatic hydrolysis of trypsin and protease K. the number of identified proteins can be significantly increased. We searched the 1026 identified proteins using the NCBI non-redundant (NR) protein database, eukaryotic orthologous groups (KOG) database and the gene ontology (GO) database respectively. The identified proteins covered all major organelles such as nuclei and mitochondria. Membrane, Golgi body, endoplasmic reticulum, identified proteins involved in almost all biochemical processes. In this study, some important proteins in conidia of Trichophyton rubrum were identified, including cell wall structural protein, cell wall biosynthesis related protein, intracellular protein synthesis related protein, resting conidial metabolic characteristic protein. Signal transduction proteins and environmental pressure tolerance proteins are discussed in detail, and their structure, function and mechanism are discussed at the level of protein molecules. The analysis of conidial proteome of Trichophyton rubrum proved that maintaining the dormancy of spores is a very complicated and delicate process, involving a series of protein translation, signal transduction, environmental stress tolerance mechanism, etc. It has important implications for the prevention, diagnosis, treatment and pathogenesis of fungal infections in the future.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R379

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本文編號(hào)：2339162