锌离子对肺炎链球菌影响的蛋白质组学研究

发布时间：2018-04-05 22:19

本文选题：锌离子　切入点：双向电泳　出处：《暨南大学》2010年硕士论文

【摘要】： 肺炎链球菌细菌为革兰氏阳性致病菌,能产生多种毒素,可引起急性咽炎、呼吸道感染、丹毒、脓疱病、软组织感染、心内膜炎和脑膜炎等,产毒株还可引起猩红热。由于该细菌的强大毒力性及对抗生素的抗性,这种感染的预防和治疗成为困扰国际医学界的一大难题。锌离子对于人体非常重要,人体中的锌离子浓度介于几微摩到几百摩尔之间。金属锌离子对人体的免疫系统起重要的作用,锌离子的缺失会导致机体免疫力下降。锌离子对于细菌的生长代谢是非常重要的,他们通过与相应的蛋白结合,尤其是与生物代谢催化剂-酶结合,共同在细菌体内发挥生物作用。金属锌离子对于细菌的粘附力与毒力有重要影响。因此,对锌离子在细菌的作用的研究尤为重要。目的：建立有无锌离子培养条件下的肺炎链球菌全蛋白质和锌结合蛋白质双向电泳图谱,结合质谱技术鉴定并分析差异表达蛋白,并结合生物信息手段对蛋白质相互关系进行分析,揭示锌离子对肺炎链球菌的生长代谢的调控机制,从而为预防和治疗肺炎链球菌感染提供理论基础。方法：本研究利用不同梯度的锌离子浓度处理肺炎链球菌,收集菌体并提取总蛋白。结合金属亲和层析技术,利用双向凝胶电泳技术分离蛋白质,建立锌调控蛋白差异表达双向凝胶电泳图谱,经ImageMsater图像分析软件识别分析差异表达蛋白质斑点后,切取差异蛋白质斑点胶块,胶内酶解后再用基质辅助激光解析电离飞行时间质谱仪(MALDI-TOF-MS)进行分析,数据库查询后对差异表达的蛋白质点进行鉴定,结合生物信息手段进行分析,以期找出锌离子调控的肺炎链球菌致病相关的蛋白。结果：1.建立了有无锌离子作用的锌离子调控蛋白双向凝胶电泳图谱,鉴定了96个差异表达蛋白斑点,共67个差异蛋白。其中32个表达下调,35个表达上调。锌离子调控蛋白的作用主要体现在氧化还原作用、帮助蛋白质折叠和糖代谢等方面。建立了有无锌离子作用的锌结合蛋白的双向电泳图谱,鉴定了10个差异表达蛋白斑点,共7个差异蛋白,其中在无锌离子培养条件下1个下调表达,6个上调表达。结论：本实验建立了肺炎链球菌蛋白双向电泳技术,通过分析有无锌离子处理肺炎链球菌蛋白的表达变化,对锌离子调控蛋白作用机制有了一定的了解。锌离子调控蛋白对肺炎链球菌的作用主要体现在氧化还原反应、蛋白质折叠、金属转运等方面。结合金属亲和层析技术研究锌离子结合蛋白,鉴定出在无锌离子条件下肺炎链球菌差异表达蛋白,研究结果为进一步探索肺炎链球菌的致病机制提供了理论依据,为相应的药物研发奠定了基础。
[Abstract]:Streptococcus pneumoniae bacteria are gram-positive pathogenic bacteria, can produce a variety of toxins, can cause acute pharyngitis, respiratory infection, erysipelas, pustular disease, soft tissue infection, endocarditis and meningitis, and so on, producing strains can also cause scarlet fever.Because of its strong virulence and resistance to antibiotics, the prevention and treatment of this infection has become a difficult problem in the international medical field.Zinc ion is very important to human body. The concentration of zinc ion in human body ranges from a few micromoles to a few hundred moles.Zinc ion plays an important role in the immune system of human body.Zinc ions are very important for the growth and metabolism of bacteria. They play a biological role in the bacteria by binding to the corresponding proteins, especially to the catalyst-enzyme of biological metabolism.Zinc ions play an important role in the adhesion and virulence of bacteria.Therefore, it is very important to study the action of zinc ions in bacteria.Objective: to establish a two-dimensional electrophoresis map of the whole protein and zinc binding protein of Streptococcus pneumoniae under the condition of zinc ion culture, and to identify and analyze the differentially expressed proteins by mass spectrometry.The relationship between proteins was analyzed by means of biological information to reveal the regulation mechanism of zinc ion on the growth and metabolism of Streptococcus pneumoniae so as to provide a theoretical basis for the prevention and treatment of Streptococcus pneumoniae infection.Methods: in this study, Streptococcus pneumoniae was treated with zinc ion concentration of different gradient, the bacteria were collected and the total protein was extracted.Combined with metal affinity chromatography, two-dimensional gel electrophoresis was used to isolate proteins. Two-dimensional gel electrophoresis (2-DE) map of differential expression of zinc regulatory proteins was established. The differential expression protein spots were identified by ImageMsater image analysis software.The differentially expressed protein spots were extracted, then analyzed by matrix assisted laser desorption ionization time of flight mass spectrometer (MALDI-TOF-MS). The differentially expressed protein spots were identified by database query, and analyzed with biological information.The aim of this study was to find out the proteins related to the pathogenesis of Streptococcus pneumoniae regulated by zinc ions.The result is 1: 1.Two-dimensional gel electrophoresis of zinc-regulated proteins with or without zinc ion action was established. 96 differentially expressed protein spots were identified and 67 differentially expressed proteins were identified.32 of them were down-regulated and 35 were up-regulated.The role of zinc ion regulating proteins is mainly reflected in redox, protein folding and glucose metabolism.Two-dimensional electrophoresis of zinc-binding proteins with or without zinc ion action was established. Ten differentially expressed protein spots were identified, and 7 differentially expressed proteins were identified, including one down-regulated and six up-regulated in zinc-free culture.Conclusion: Two-dimensional electrophoresis of streptococcus pneumoniae protein was established in this experiment. By analyzing the expression of streptococcus pneumoniae protein with or without zinc ion treatment, the mechanism of action of zinc ion regulating protein was understood.The effect of zinc ion regulating protein on Streptococcus pneumoniae is mainly reflected in redox reaction, protein folding, metal transport and so on.Zinc ion binding protein was studied by metal affinity chromatography, and differential expression protein of Streptococcus pneumoniae was identified in the absence of zinc ion. The results provided a theoretical basis for further exploring the pathogenic mechanism of Streptococcus pneumoniae.For the corresponding drug research and development laid the foundation.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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