结核分枝杆菌北京家族多重耐药菌株CCDC5180、药物敏感菌株CCDC5079与BCG的蛋白质组学初步研究

发布时间：2018-07-24 19:03

【摘要】： 结核分枝杆菌(Mycobacterium tuberculosis，MTB)是影响人类健康的主要病原体之一，北京家族(Beijing family)是造成我国结核病传播的主要基因型。多耐药菌株CCDC5180、敏感菌株CCDC5079是从我国流行的北京家族基因型中筛选出的两株测序株。卡介苗(BCG)是预防和控制结核病使用最广泛的疫苗，但对成人缺乏有效的保护力。为从蛋白质水平上较全面地了解结核分枝杆菌北京家族的生物学特征、耐药机制，本研究采用十二烷基磺酸钠变性聚丙烯酰胺凝胶电泳(Sodium dodecul sulphonate denatured Polyacrylamide gel electrophoresis，SDS-PAGE)、双向凝胶电泳(Two-dimensional gel electrophoresis，2-DE)以及质谱(Mass spectrometry，MS)技术对CCDC5180、CCDC5079与BCG进行了蛋白质组学的初步研究。 SDS-PAGE结果显示本次分析的三株菌的菌体蛋白图谱非常相似，有14.4KD—118.1KD共40条蛋白条带，其中14.8KD、23.6KD、66.0KD蛋白的相对表达量较大，大部分蛋白集中在250KD-84.2KD之间，用SDS-PAGE较难发现两者间的明显差异。为此又进行了2-DE图谱比较，采用商业的24cm pH4-7(非线性)胶条进行第一向分离，第二向分离采用12.5％的聚丙烯酰胺凝胶。经硝酸银染色后发现它们的2-DE图谱很相似，在CCDC5180、CCDC5079及BCG菌株的2-DE凝胶上分别检测到1563、1620、1654个蛋白质斑点，大部分蛋白质点等电点分布在pH4.0-pH6.5范围内；银染分辨率高，但质谱鉴定率低下，目前我们只能对2-DE分离菌株的菌体蛋白进行胶体考马斯亮蓝G-250染色，然后进行MALDI-TOF-MS和MS／MS质谱鉴定。在2-DE考染图谱上分别检测到718、767、1019个蛋白斑点，质谱分析整理出了443、436、486个蛋白点信息，去冗余后最终鉴定了226、149、165种蛋白。通过生物信息学分析，整理了蛋白质组表达库，获得了大部分蛋白相应的基因名、编码框及功能分类等基本信息，并发现一些数据库中未曾报道的蛋白，可能是北京家族基因型的特异蛋白，需要进一步深入研究。在CCDC5079与CCDC5180菌株的2-DE图谱差异比较中发现，有7个蛋白点表达丰度增加，4个蛋白点表达丰度下降，新增加蛋白5个，缺失2个：CCDC5079与BCG相比，有5个蛋白点表达丰度增加，8个表达丰度下降，新增蛋白7个，缺失3个；CCDC5180菌株BCG相比，，4个蛋白点表达丰度增加，18个下降，新增点2个，缺失4个。通过对蛋白质组表达库和图谱差异蛋白的研究，将更好地了解结核分枝杆菌的耐药机制，为寻找新的药物靶标蛋白、新疫苗构建及开发新的诊断试剂提供理论依据，也为结核分枝杆菌北京家族以后的蛋白质组学研究奠定了基础。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium) is one of the major pathogens affecting human health. (Beijing family) in Beijing family is the main genotype causing tuberculosis transmission in China. The multidrug resistant strain CCDC5180. The sensitive strain CCDC5079 is two sequenced strains selected from the prevalent Beijing family genotypes in China. BCG (BCG) is the most widely used vaccine to prevent and control tuberculosis, but it lacks effective protection for adults. In order to fully understand the biological characteristics and drug resistance mechanism of Mycobacterium tuberculosis Beijing family from the protein level, In this study, the proteomics of CCDC5180, CCDC5079 and BCG were studied by (Sodium dodecul sulphonate denatured Polyacrylamide gel electrophoretic gel electrophoresis (SDS-PAGE), Two-dimensional gel electrophoresis2-DE and Mass spectrometric MS. SDS-PAGE results showed that the bacterial protein profiles of the three strains were very similar. There were 40 protein bands of 14.4KD-118.1KD, of which 14.8KDX 23.6KD / 66.0KD protein was relatively large, and most of the proteins were concentrated in 250KD-84.2KD. It was difficult to find the obvious difference between them by using SDS-PAGE. For this reason, the 2-DE spectra were compared. The commercial 24cm pH4-7 (nonlinear) rubber strip was used for the first separation and the second separation was made with 12.5% polyacrylamide gel. After silver nitrate staining, their 2-DE patterns were similar. 1563 protein spots were detected on the 2-DE gel of CCDC5180 and CCDC5079 and BCG strains, most of the isoelectric spots were located in the range of pH4.0-pH6.5, and the silver staining resolution was high, but the identification rate of mass spectrometry was low. At present, we can only use colloidal Coomassie brilliant blue G-250 to stain the bacterial proteins of 2-DE isolates, and then identify them by MALDI-TOF-MS and MS/MS mass spectrometry. A total of 718 767 protein spots were detected on the 2-DE chromatogram, and 443436486 protein spots were analyzed by mass spectrometry. Finally, 226149165 proteins were identified after redundancy removal. Through bioinformatics analysis, the proteome expression library was sorted out, and the basic information of gene name, coding frame and functional classification of most proteins were obtained, and some unreported proteins were found in the database. It may be a specific protein of the Beijing family genotype and needs further study. In comparison of the 2-DE profiles of CCDC5079 and CCDC5180 strains, it was found that the expression abundance of 7 protein spots increased, 4 protein spots decreased, 5 new proteins were added, and 2: CCDC5079 were missing compared with BCG. The expression abundance of 5 protein spots increased, 8 decreased, 7 new proteins were added, and 4 protein spots increased, 18 decreased, 2 new points and 4 deletions were found in BCG with 3 deletion of CCDC5180. Through the study of proteome expression library and differential protein map, the mechanism of drug resistance of Mycobacterium tuberculosis will be better understood, which will provide theoretical basis for finding new drug target protein, constructing new vaccine and developing new diagnostic reagent. It also laid a foundation for the further proteomics research of Mycobacterium tuberculosis in Beijing.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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