汉赛巴尔通体外膜蛋白质组学研究

发布时间：2018-01-05 17:31

本文关键词：汉赛巴尔通体外膜蛋白质组学研究　出处：《中国疾病预防控制中心》2009年博士论文　论文类型：学位论文

【摘要】： 猫抓病(cat-scratch disease,CSD)主要是由汉赛巴尔通体引起的人类新发人兽共患病。猫是这种巴尔通体的自然宿主,通过抓或咬伤将巴尔通体传染给人,人感染这些细菌后在伤口处会出现红斑丘疹或化脓性丘疱疹,继而发生以局部淋巴结炎为典型症状的CSD,少数患者还可并发心内膜炎、杆菌性血管瘤、骨髓炎和脑膜炎等全身性疾病。人们对汉赛巴尔通体的致病机理和宿主的免疫保护机制还不十分清楚。本研究拟应用蛋白质组学和生物信息学的研究方法对汉赛巴尔通体外膜蛋白质组进行研究,筛选可能用于诊断和疫苗研制的候选蛋白,以及发现可能与细胞粘附、侵入及毒力相关的膜蛋白成分。实验根据外膜蛋白组分不能溶解在十二烷基肌氨酸钠(简称SLS)和碳酸钠(Na_2CO_3)溶液中的特性,用2种分离方法制备汉赛巴尔通体ATCC 49882 Houston-1株外膜蛋白样品。采用双向电泳(2-DE),即进行pH 3-10非线性IPG干胶条进行第一向等电聚焦电泳、12.5%凝胶第二向SDS-PAGE电泳、考马斯亮蓝G-250染色、图像扫描及分析;比较上述2种方法制备的外膜蛋白样品2-DE图谱差异,并对不同裂解液溶解的蛋白样品结果进行比较。在获得SLS法和碳酸钠法在外膜蛋白样品制备中存在互补性的条件下,同时用两种方法制备了另外3种致病性巴尔通体(伊丽莎白巴尔通体、文森巴尔通体博格霍夫亚种和格拉汉姆巴尔通体)的外膜蛋白样品,进行2-DE实验分析。应用SLS制备的蛋白样品上样量为600μg,获得约368个蛋白质点,大量高丰度蛋白集中在pⅠ值4.3～6.5之间。等电点范围为4.0～10.0,分子量范围为15 kDa～150 kDa。其中大部分高丰度蛋白分布在pⅠ值5～6和8～9之间。应用碳酸钠的蛋白样品上样量为800μg,获得约471个蛋白质点,高丰度蛋白集中在pⅠ值5.0～6.3之间。等电点范围为4.8～10.0,分子量范围为10 kDa～180 KDa。在裂解液中增加ASB-14等两性离子去污剂使蛋白样品溶解性增强,检出蛋白点增多,碱性端检出蛋白点明显增加。重复性实验2种方法均获得稳定、一致的2-DE图谱。应用基质辅助激光解析-飞行时间质谱技术鉴定了上述2种方法检出的全部蛋白点,并对鉴定的蛋白进行信号肽、亚细胞定位和结构域搜索等生物信息学分析。SLS法制备的样品中鉴定出176点代表94种蛋白,外膜蛋白有6种;碳酸钠法制备的样品中鉴定出259点代表139种蛋白,外膜蛋白有9种;2种方法共鉴定出10种外膜蛋白,2种是本研究中首次证实存在的外膜蛋白,包括孔蛋白、血红素结合蛋白、表面抗原蛋白、药物排出相关蛋白和脂蛋白等。对汉赛巴尔通体基因组中预测出的外膜蛋白应用生物信息学方法初步预测结构和功能,结果发现43个外膜蛋白中与铁离子吸收相关毒力因子有5个、细胞粘附相关的蛋白5个、溶血素4个、血凝素6个、自转运蛋白7个、药物外排相关蛋白2个、运输相关蛋白2个、抵抗有机溶剂蛋白1个及一些与膜结构相关的蛋白;有2个细菌表面抗原蛋白,均已被分离鉴定出来,可能具有免疫保护性,需要进一步研究证实。本研究建立了巴尔通体外膜蛋白2-DE技术,并发现两种外膜蛋白样品制备方法有一定的互补性,SLS法表现出对外膜上高表达的孔蛋白的富集,Na_2CO_3法对低丰度、碱性蛋白表现出较好的分离效果,为今后巴尔通体外膜蛋白质组研究提供了参考。新发现的蛋白提示巴尔通体可能存在药物外排系统,对抗菌药物和耐药机制产生的影响有待进一步研究。外膜蛋白的质谱鉴定及生物信息学分析提示在汉赛巴尔通体外膜上存在大量与细胞粘附、侵袭等致病相关的蛋白,对汉赛巴尔通体外膜蛋白质组的全面评估不仅对今后开展诊断和疫苗技术研究有重要意义,而且为巴尔通体致病机制研究指出了新的方向。
[Abstract]:Cat scratch disease (cat-scratch disease CSD) is mainly caused by the human body Han Sabar new zoonosis. The cat is the natural host Barr was caught by Barr, or will the bite of infected people, people infected with these bacteria erythema papules or purulent papulovesicles in the wound, and then to local lymphadenitis as the typical symptoms of CSD, a few patients can be complicated by endocarditis, bacillary angiomatosis, meningitis, osteomyelitis and other systemic disease. The immune protection mechanism of pathogenic mechanism of Han Sabar quintana and host is still unclear. This study intends to research methodology and biological information application of proteomics research of Han Sabar the outer membrane proteins may be used for screening, diagnosis and development of candidate protein vaccines, and that may be related to cell adhesion, invasion and virulence related membrane protein Ingredients.
According to the experiment of outer membrane protein components can not be dissolved in twelve alkyl sodium sarcosinate (SLS) and sodium carbonate (Na_2CO_3) properties in solution, with 2 kinds of separation methods of preparation of Han Sabar was ATCC 49882 Houston-1 strain. The outer membrane protein samples by two-dimensional electrophoresis (2-DE), which is in the pH 3-10 nonlinear IPG dry strip the first to the isoelectric focusing electrophoresis, gel electrophoresis of SDS-PAGE 12.5% to second, Kaumas Coomassie brilliant blue G-250 staining, scanning and image analysis; comparison of the above 2 methods of preparation of outer membrane protein 2-DE of different samples, and the different lysates of dissolved protein samples were compared. Results in SLS method and sodium carbonate method are complementary the conditions of the membrane protein in the sample preparation, and is prepared by two methods and 3 kinds of pathogenic Bartonella henselae (Elizabeth Barr, Vincent Hof and Graham Berg of Bartonella subspecies of Bartonella Body) of the outer membrane protein samples, analyzed by the 2-DE experiment. Protein sample preparation using SLS sample volume is 600 g, about 368 protein spots, a large number of high abundance proteins in P 1 ranged from 4.3 to 6.5. The isoelectric point ranged from 4 to 10, the range of molecular weight is 15 ~ kDa 150 kDa. most high abundance protein distribution in P I value 5 ~ 6 and 8 ~ 9. The application of sodium carbonate in protein sample was 800 g, about 471 protein spots, high abundance protein concentration in P I value 5 ~ 6.3. The isoelectric point ranged from 4.8 to 10. The molecular weight range of 10 kDa ~ 180 KDa. in lysis buffer increased ASB-14 zwitterionic detergent to protein samples enhances the solubility and protein detection point increased, alkaline end detection protein increased significantly. Repeatability of the 2 methods are stable, 2-DE of the same.
Using matrix assisted laser desorption time-of-flight mass spectrometry identification detection of the above 2 methods all proteins, and the proteins were signal peptide, subcellular localization and structure domain search and bioinformatics analysis of.SLS samples prepared by identified 176 representatives of 94 proteins, outer membrane protein of 6 samples; the preparation of sodium carbonate in the identified 259 points represent 139 proteins, outer membrane protein 9; 2 methods were identified 10 kinds of outer membrane protein, 2 is the first confirmed the presence of outer membrane proteins in this study, including hole protein, heme binding protein, protein, drug related protein and lipoprotein discharge for the application of Bioinformatics Method of outer membrane protein predicted in the genome of the Han Sabar school preliminary prediction of structure and function, results showed that the virulence factors and the iron absorption of 43 outer membrane proteins in 5, cell adhesion With related protein 5, hemolysin 4, 6 ha, 7 autotransporter proteins, drug efflux related protein 2, transport related protein 2, organic solvent resistance protein 1 protein and membrane structure and some related; there are 2 bacterial surface proteins have been isolated, identified it may have protective immunity, need further study.
This study established the Barr outer membrane protein 2-DE, and found that two kinds of membrane protein sample preparation method is complementary to each other, the SLS method showed enrichment of membrane porin foreign high expression of Na_2CO_3 on low abundance of basic protein showed a good separation effect, provide a reference for the future Barr quintana the outer membrane proteome research. The newly discovered protein that Barr the possible drug efflux system, further study on effect of antibiotics and resistance mechanism. Mass spectrometry and bioinformatics analysis of outer membrane protein in the outer membrane was that Han Sabar had a large number of cell adhesion, invasion and pathogenicity related protein, a comprehensive assessment of the Han Sabar, the outer membrane proteins not only for future development has an important significance in diagnosis and vaccine research technique, and to study the pathogenic mechanism of Barr was pointed out The new direction.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

【相似文献】