金黄色葡萄球菌B型肠毒素的溶液构象及菌体生长环境效应
发布时间：2019-01-28 20:03
【摘要】：金黄色葡萄球菌(Saphylococcus aureus)是人类和某些动物的病原菌之一,其致病力强弱主要取决于产生的毒素和侵袭性酶的能力,其中金黄色葡萄球菌耐热性肠毒素,是引起人类食物中毒和葡萄球菌胃肠炎的主要原因,因此,这方面的研究已成为科学家们普遍关注的重要课题。本文以主要产B型肠毒素的金黄色葡萄球菌S6菌株为研究对象,采用X—射线衍射、扫描电镜、原子力显微镜、分子振动光谱(红外、激光拉曼)、圆二色谱、荧光光谱等现代分析技术表征金黄色葡萄球菌B型肠毒素(staphylococcal enterotoxin B,简称SEB)的聚集态结构、三维立体形貌、分子链构象及蛋白质二级结构组成。同时着重研究了肠毒素在稀溶液状态和外界环境改变时的构象特征及可能的去折叠过程,尝试用蛋白质构象理论揭示肠毒素耐热的根本原因,寻找其耐热的可能优势构象,建立研究蛋白质构象与生物学活性相关性的新方法。此外,还从酸奶发酵菌株和金黄色葡萄球菌的混合培养特性出发,研究了乳酸菌及代谢产物对金黄色葡萄球菌的抑制作用及其在抑制过程中热效应的微量变化,从热化学的角度探讨其作用机制。主要研究结果如下: 1.金黄色葡萄球菌B型肠毒素的分离纯化 采用玻璃纸覆盖琼脂平板法培养金黄色葡萄球菌,首先利用羧甲基纤维素阳离子CM-32柱层析,对B型肠毒素进行初步分离富集,然后依据筛分原理按分子量大小经Sephadex G-75凝胶进一步纯化,得到电泳纯B型肠毒素,得率为26.8%,紫外吸收光谱测得其特征吸收峰为276.6nm,SDS-PAGE凝胶电泳分析其相对分子质量为2.8×10~4,与文献报道值一致。 2.金黄色葡萄球菌B型肠毒素的结构表征 利用X-射线衍射、扫描电镜、原子力显微镜、傅里叶变换红外光谱、激光拉曼光谱等现代分析手段对金黄色葡萄球菌B型肠毒素的聚集态结构、分子链形态,二级结构等进行了表征。结果表明,SEB为一种单纯蛋白,其聚集态包含结晶区和无定形区,结晶度为37.8%,其分子聚集在一起,形态主要为片状和平板状,具有一定的规整性。原子力显微镜首次观察到SEB在稀溶液状态下的单分子链长度约为1500nm,分子链宽度约为20～40nm,分子链高0.6nm。SEB的傅里叶变换红外光谱和激光拉曼光谱分析表明,SEB的二级结构单元主要为β—折叠和α—螺旋,无规卷曲含量较少,侧链C—C—S—S—C—C构型为反式—扭曲—反式。 3.金黄色葡萄球菌B型肠毒素的稀溶液构象及去折叠作用 研究大分子的溶液构象和蛋白质的去折叠作用是近来揭示蛋白质生物活性及其与高级结构相关性的重要切入点。金黄色葡萄球菌B型肠毒素水溶液(浓度为
[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 伪 -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
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R378;S852.611
本文编号：2417273
[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 伪 -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
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R378;S852.611
【引证文献】
相关期刊论文 前1条
1 李荔枝;胡萍;;快速检测食品中金黄色葡萄球菌及其肠毒素型的研究进展[J];江西农业学报;2011年08期
相关博士学位论文 前1条
1 冯屹;同时检测多种环境激素污染物的液相免疫芯片技术研究[D];华中农业大学;2010年
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