【作者】 张留辉；

【导师】 何庆瑜；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2010， 硕士

【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。更多还原

【Abstract】 The human pathogen Streptococcus pneumoniae is a commensal of the nasopharynx, but it can become virulent by infecting the lung, middle ear, brain, and bloodstream, causing severe diseases such as pneumonia, otitis media, meningitis, and sepsis. An important metal ion that S. pneumoniae could face is Zn2+, which is present in the human body in concentrations ranging from a fewμM to over 100μM. In the host, Zn2+is of great importance for immunity, as it is necessary for proper functioning of immune cells, and mild Zn2+ deficiency severely affects immune function. Zn2+is crucial for bacterial growth and metabolism as it is the co-factor in many key enzymes. Moreover, Zn2+is involved in bacterial adherence and virulence. This project aims to investigate the molecular basis of the bacterial growth inhibition induced by Zn2+ deficiency and to find out the mechanism of Zn2+effects on S. pnemunoniae virulence.AIM:Establish the 2DE maps of Zn2+regulatory proteins of S. pnemunoniae and investigate the molecular mechanism of Zn2+-regulated growth inhibition of S. pneumoniae.METHOD:2DE-based proteomic approach was used to compare the differences of protein expression in S. pnemunoniae with and without zinc ion.2DE-gel maps were established for the total proteins extracted from the bacteria and the sub-proteome after Zn-IMAC separation, respectively. Proteins with altered expression were identified by MALDI-TOF/TOF mass spectrometry and further characterized based on their molecular functions.RESULT:With the 2DE analysis for the total protein extracts,96 protein spots were found to have different expressions with Zn2+deficiency and 67 unique proteins including 32 down-regulated and 35 up-regulated proteins were identified. These Zn2+regulatory proteins involve in various biological processes including oxidation, protein folding and carbohydrate metabolism. By using Zn-IMAC separation, Zn2+binding proteins in S. pneumoniae were also profiled by 2DE and 10 different protein spots with 7 unique proteins were identified.CONCLUSION:Zn2+deficiency affects the growth of S. pneumoniae. Zn2+regulated proteins can be identified by 2DE-based proteomic technology. Further functional analysis revealed that these Zn2+regulated proteins involve in oxidation, proteins folding and carbohydrate metabolism. These results provide information to help us better understand the molecular basis of the growth and virulence inhibition of S. pneumoniae induced by Zn2+ deficiency.更多还原