【摘要】：金黃色葡萄球菌(Saphylococcus aureus)是人類和某些動(dòng)物的病原菌之一,其致病力強(qiáng)弱主要取決于產(chǎn)生的毒素和侵襲性酶的能力,其中金黃色葡萄球菌耐熱性腸毒素,是引起人類食物中毒和葡萄球菌胃腸炎的主要原因,因此,這方面的研究已成為科學(xué)家們普遍關(guān)注的重要課題。本文以主要產(chǎn)B型腸毒素的金黃色葡萄球菌S6菌株為研究對(duì)象,采用X—射線衍射、掃描電鏡、原子力顯微鏡、分子振動(dòng)光譜(紅外、激光拉曼)、圓二色譜、熒光光譜等現(xiàn)代分析技術(shù)表征金黃色葡萄球菌B型腸毒素(staphylococcal enterotoxin B,簡(jiǎn)稱SEB)的聚集態(tài)結(jié)構(gòu)、三維立體形貌、分子鏈構(gòu)象及蛋白質(zhì)二級(jí)結(jié)構(gòu)組成。同時(shí)著重研究了腸毒素在稀溶液狀態(tài)和外界環(huán)境改變時(shí)的構(gòu)象特征及可能的去折疊過程,嘗試用蛋白質(zhì)構(gòu)象理論揭示腸毒素耐熱的根本原因,尋找其耐熱的可能優(yōu)勢(shì)構(gòu)象,建立研究蛋白質(zhì)構(gòu)象與生物學(xué)活性相關(guān)性的新方法。此外,還從酸奶發(fā)酵菌株和金黃色葡萄球菌的混合培養(yǎng)特性出發(fā),研究了乳酸菌及代謝產(chǎn)物對(duì)金黃色葡萄球菌的抑制作用及其在抑制過程中熱效應(yīng)的微量變化,從熱化學(xué)的角度探討其作用機(jī)制。主要研究結(jié)果如下: 1.金黃色葡萄球菌B型腸毒素的分離純化 采用玻璃紙覆蓋瓊脂平板法培養(yǎng)金黃色葡萄球菌,首先利用羧甲基纖維素陽離子CM-32柱層析,對(duì)B型腸毒素進(jìn)行初步分離富集,然后依據(jù)篩分原理按分子量大小經(jīng)Sephadex G-75凝膠進(jìn)一步純化,得到電泳純B型腸毒素,得率為26.8%,紫外吸收光譜測(cè)得其特征吸收峰為276.6nm,SDS-PAGE凝膠電泳分析其相對(duì)分子質(zhì)量為2.8×10~4,與文獻(xiàn)報(bào)道值一致。 2.金黃色葡萄球菌B型腸毒素的結(jié)構(gòu)表征 利用X-射線衍射、掃描電鏡、原子力顯微鏡、傅里葉變換紅外光譜、激光拉曼光譜等現(xiàn)代分析手段對(duì)金黃色葡萄球菌B型腸毒素的聚集態(tài)結(jié)構(gòu)、分子鏈形態(tài),二級(jí)結(jié)構(gòu)等進(jìn)行了表征。結(jié)果表明,SEB為一種單純蛋白,其聚集態(tài)包含結(jié)晶區(qū)和無定形區(qū),結(jié)晶度為37.8%,其分子聚集在一起,形態(tài)主要為片狀和平板狀,具有一定的規(guī)整性。原子力顯微鏡首次觀察到SEB在稀溶液狀態(tài)下的單分子鏈長(zhǎng)度約為1500nm,分子鏈寬度約為20～40nm,分子鏈高0.6nm。SEB的傅里葉變換紅外光譜和激光拉曼光譜分析表明,SEB的二級(jí)結(jié)構(gòu)單元主要為β—折疊和α—螺旋,無規(guī)卷曲含量較少,側(cè)鏈C—C—S—S—C—C構(gòu)型為反式—扭曲—反式。 3.金黃色葡萄球菌B型腸毒素的稀溶液構(gòu)象及去折疊作用 研究大分子的溶液構(gòu)象和蛋白質(zhì)的去折疊作用是近來揭示蛋白質(zhì)生物活性及其與高級(jí)結(jié)構(gòu)相關(guān)性的重要切入點(diǎn)。金黃色葡萄球菌B型腸毒素水溶液(濃度為
[Abstract]:Staphylococcus aureus (Saphylococcus aureus) is one of the pathogenic bacteria in humans and some animals. Its pathogenicity depends mainly on the ability of producing toxins and invasive enzymes, among which Staphylococcus aureus heat-resistant enterotoxin. It is the main cause of human food poisoning and staphylococcal gastroenteritis. In this paper, Staphylococcus aureus S6 strain, which mainly produces enterotoxin B, was studied by means of X-ray diffraction, scanning electron microscope, atomic force microscope, molecular vibration spectrum (infrared, laser Raman), circular dichroism (CD). Fluorescence spectroscopy and other modern analytical techniques were used to characterize the aggregation structure, three-dimensional morphology, molecular chain conformation and protein secondary structure of staphylococcus aureus B enterotoxin (staphylococcal enterotoxin B, (abbreviated to SEB). At the same time, the conformation characteristics and possible unfolding process of enterotoxin in dilute solution and outside environment were studied. The protein conformation theory was used to reveal the fundamental cause of enterotoxin heat resistance, and the possible dominant conformation of enterotoxin heat resistance was found. A new method to study the relationship between protein conformation and biological activity was established. In addition, the inhibitory effect of lactic acid bacteria and its metabolites on Staphylococcus aureus and its thermal effect in the process of inhibition were studied based on the mixed culture characteristics of yoghurt fermentation strain and Staphylococcus aureus. The mechanism of its action is discussed from the point of view of thermochemistry. The main results are as follows: 1. Staphylococcus aureus B enterotoxin was isolated and purified by cellophane coated Agar plate method. Firstly, carboxymethyl cellulose cationic CM-32 column chromatography was used to culture Staphylococcus aureus. The enterotoxin B was isolated and enriched preliminarily, then purified by Sephadex G-75 gel according to the molecular weight according to the screening principle, and the electrophoretic pure enterotoxin B was obtained in the yield of 26.8%. The characteristic absorption peak measured by UV absorption spectrum was 276.6nmGE-PAGE, and the relative molecular weight was 2.8 脳 10 ~ (4), which was in agreement with the reported value in literature. 2. The structure of staphylococcus aureus enterotoxin B was characterized by X-ray diffraction, scanning electron microscope, atomic force microscope and Fourier transform infrared spectroscopy. The aggregation structure, molecular chain morphology and secondary structure of staphylococcus aureus enterotoxin B were characterized by laser Raman spectroscopy. The results show that SEB is a simple protein, its aggregation state includes crystalline region and amorphous region, the crystallinity is 37.8%, and its molecule aggregates together, its morphology is mainly flaky and plate-like, and it has certain regularity. Atomic force microscope (AFM) for the first time observed that the length of single molecular chain and the width of molecular chain of SEB in dilute solution were about 1500 nm and 20 ~ 40 nm respectively. The Fourier transform infrared spectroscopy and laser Raman spectroscopy of 0.6nm.SEB with high molecular chain height showed that the molecular chain height of 0.6nm.SEB was analyzed by Fourier transform infrared spectroscopy and laser Raman spectroscopy. The secondary structure units of SEB are mainly 尾 -folding and 偽 -helix, and the content of random coils is less. The configuration of side-chain C-S-C-C is trans-twisting and trans-type. 3. Dilute solution conformation and Defolding of Staphylococcus aureus B Enterotoxin; solution conformation of macromolecules and unfolding of proteins An important entry point for the relevance of high-level structures. Staphylococcus aureus type B enterotoxin aqueous solution
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R378;S852.611

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李荔枝;胡萍;;快速檢測(cè)食品中金黃色葡萄球菌及其腸毒素型的研究進(jìn)展[J];江西農(nóng)業(yè)學(xué)報(bào);2011年08期

相關(guān)博士學(xué)位論文 前1條

1 馮屹;同時(shí)檢測(cè)多種環(huán)境激素污染物的液相免疫芯片技術(shù)研究[D];華中農(nóng)業(yè)大學(xué);2010年

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本文編號(hào)：2417273