【摘要】： 結核分枝桿菌(Mycobacterium tuberculosis，MTB)是影響人類健康的主要病原體之一，北京家族(Beijing family)是造成我國結核病傳播的主要基因型。多耐藥菌株CCDC5180、敏感菌株CCDC5079是從我國流行的北京家族基因型中篩選出的兩株測序株�？ń槊�(BCG)是預防和控制結核病使用最廣泛的疫苗，但對成人缺乏有效的保護力。 為從蛋白質(zhì)水平上較全面地了解結核分枝桿菌北京家族的生物學特征、耐藥機制，本研究采用十二烷基磺酸鈉變性聚丙烯酰胺凝膠電泳(Sodium dodecul sulphonate denatured Polyacrylamide gel electrophoresis，SDS-PAGE)、雙向凝膠電泳(Two-dimensional gel electrophoresis，2-DE)以及質(zhì)譜(Mass spectrometry，MS)技術對CCDC5180、CCDC5079與BCG進行了蛋白質(zhì)組學的初步研究。 SDS-PAGE結果顯示本次分析的三株菌的菌體蛋白圖譜非常相似，有14.4KD—118.1KD共40條蛋白條帶，其中14.8KD、23.6KD、66.0KD蛋白的相對表達量較大，大部分蛋白集中在250KD-84.2KD之間，用SDS-PAGE較難發(fā)現(xiàn)兩者間的明顯差異。為此又進行了2-DE圖譜比較，采用商業(yè)的24cm pH4-7(非線性)膠條進行第一向分離，第二向分離采用12.5％的聚丙烯酰胺凝膠。經(jīng)硝酸銀染色后發(fā)現(xiàn)它們的2-DE圖譜很相似，在CCDC5180、CCDC5079及BCG菌株的2-DE凝膠上分別檢測到1563、1620、1654個蛋白質(zhì)斑點，大部分蛋白質(zhì)點等電點分布在pH4.0-pH6.5范圍內(nèi)；銀染分辨率高，但質(zhì)譜鑒定率低下，目前我們只能對2-DE分離菌株的菌體蛋白進行膠體考馬斯亮藍G-250染色，然后進行MALDI-TOF-MS和MS／MS質(zhì)譜鑒定。在2-DE考染圖譜上分別檢測到718、767、1019個蛋白斑點，質(zhì)譜分析整理出了443、436、486個蛋白點信息，去冗余后最終鑒定了226、149、165種蛋白。通過生物信息學分析，整理了蛋白質(zhì)組表達庫，獲得了大部分蛋白相應的基因名、編碼框及功能分類等基本信息，并發(fā)現(xiàn)一些數(shù)據(jù)庫中未曾報道的蛋白，可能是北京家族基因型的特異蛋白，需要進一步深入研究。在CCDC5079與CCDC5180菌株的2-DE圖譜差異比較中發(fā)現(xiàn)，有7個蛋白點表達豐度增加，4個蛋白點表達豐度下降，新增加蛋白5個，缺失2個：CCDC5079與BCG相比，有5個蛋白點表達豐度增加，8個表達豐度下降，新增蛋白7個，缺失3個；CCDC5180菌株BCG相比，，4個蛋白點表達豐度增加，18個下降，新增點2個，缺失4個。通過對蛋白質(zhì)組表達庫和圖譜差異蛋白的研究，將更好地了解結核分枝桿菌的耐藥機制，為尋找新的藥物靶標蛋白、新疫苗構建及開發(fā)新的診斷試劑提供理論依據(jù)，也為結核分枝桿菌北京家族以后的蛋白質(zhì)組學研究奠定了基礎。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R378

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