本文選題：結(jié)核分枝桿菌 + 高通量　； 參考：《吉林大學》2008年博士論文

【摘要】： 結(jié)核分枝桿菌是結(jié)核病的致病菌,據(jù)世界衛(wèi)生組織統(tǒng)計,近年來肺結(jié)核在全球各地死灰復燃,1995年全世界有300萬人死于此病,是該病死亡人數(shù)最多的一年,大大超過了肺結(jié)核流行的1900年。在2003年3月24日“世界防治結(jié)核病日”之際,“制止結(jié)核病”世界行動組織公布的數(shù)字顯示,目前全球每天仍有5000人死于結(jié)核病,而每年罹患結(jié)核病的人數(shù)超過800萬由于人口劇增、移民增加、旅游業(yè)發(fā)展、耐藥性的產(chǎn)生、人類免疫缺陷病毒感染流行、吸毒、酗酒與貧困等原因,結(jié)核病呈日益嚴重回升趨勢。因此,對結(jié)核分枝桿菌基因組學研究、蛋白質(zhì)組學研究、后基因組學研究等,表達后蛋白質(zhì)組學研究,即從一個細胞生命整體去研究,方能徹底的弄清其分子致病機制、感染的方法,弄清結(jié)核分枝桿菌胞內(nèi)生活周期,為結(jié)核分枝桿菌病早期診斷、疫苗免疫、新藥研發(fā),提供堅厚的理論基礎。 1998年英國Sanger中心和法國Pasteur研究所科學家合作完成了結(jié)核分枝桿菌H37Rv株的全基因組測序工作,并在2002年進行了重新注釋,這為結(jié)核病病原菌致病基因的研究提供了理論基礎。結(jié)核分枝桿菌全基因組序列由4.41Mb(4.411529bp)組成,包括4411個基因,具有潛在編碼能力的基因約有3977個,約占90.2%。有3924個開放閱讀框,其中約40%有功能,44%可能有功能,16%稱為孤兒序列,與其它微生物的序列無相似性�；蚪M富含GC堿基,G+C含量高達65.6%。雖然世界各地的實驗室、醫(yī)院、科研機構都在對結(jié)核分枝桿菌H37Rv做各方面的研究,但是尚無法克服結(jié)核病這一難題,越來越高的發(fā)病率和耐藥菌株及多耐藥菌株的出現(xiàn),給結(jié)核分枝桿菌病的治療增加了難度。本試驗從整個基因組出發(fā),利用Gateway技術,pDONR221為基因供體載體,pDEST17為表達載體,倆個表達菌株BL21和BL21Codonplus RP對結(jié)核分枝桿菌H37Rv進行不同誘導條件表達,丙烯酰胺凝膠電泳對表達蛋白驗證,共對3056基因進行了克隆,得到入門質(zhì)粒2903個,表達質(zhì)粒2798個,表達蛋白1660個,其中純化蛋白64個,有16個可溶性蛋白;選取重組表達,經(jīng)酶解后得到的肽片段混合物,通過兩維液相色譜分離,采用液質(zhì)連用質(zhì)譜方法對蛋白進行鑒定,確定屬于結(jié)核分枝桿菌H37Rv。
[Abstract]:Mycobacterium tuberculosis is the pathogenic bacteria of tuberculosis. According to the statistics of the World Health Organization, tuberculosis has resurfaced around the world in recent years. In 1995, 3 million people died of the disease in the world, the highest number of deaths in the year.It was much more prevalent than tuberculosis in 1900.On the occasion of World Tuberculosis Day on 24 March 2003, figures released by the World Action to stop Tuberculosis show that there are still 5000 people dying of tuberculosis every day in the world.And more than 8 million people suffer from tuberculosis every year because of the surge in population, the increase in immigration, the development of tourism, the development of drug resistance, the prevalence of HIV infection, drug abuse, alcoholism and poverty.Tuberculosis is on the rise day by day.Therefore, the study of Mycobacterium tuberculosis genomics, proteomics, post-genomics, and post-expression proteomics, that is, the study of the molecular pathogenicity of Mycobacterium tuberculosis from a whole cell life, can completely understand its molecular pathogenetic mechanism.The method of infection, to clarify the life cycle of Mycobacterium tuberculosis, to provide a solid theoretical basis for the early diagnosis of Mycobacterium tuberculosis, vaccine immunization, new drug research and development.The whole genome sequencing of Mycobacterium tuberculosis H37Rv strain was completed in 1998 by the Sanger Center of England and the scientists of the French Institute of Pasteur. It was reannotated in 2002, which provides a theoretical basis for the study of pathogenic genes of tuberculosis pathogens.The whole genome sequence of Mycobacterium tuberculosis is composed of 4.41MbGN 4.411529bp. including 4411 genes, there are about 3977 genes with potential coding ability, accounting for 90.2%.There are 3924 open reading frames, of which about 40% have function and 44% may have function. 16% of them are called orphan sequences, which are not similar to those of other microorganisms.The content of GC base G C in genome is as high as 65. 6%.Although laboratories, hospitals and scientific institutions all over the world are doing all kinds of research on Mycobacterium tuberculosis H37Rv, it is still unable to overcome the problem of tuberculosis, the increasing incidence of tuberculosis and the emergence of drug-resistant and multi-drug resistant strains.The treatment of Mycobacterium tuberculosis is more difficult.In this experiment, based on the whole genome, Gateway technique was used as gene donor vector pDEST17 as expression vector. Two expression strains BL21 and BL21Codonplus RP were used to induce the expression of Mycobacterium tuberculosis H37Rv under different conditions. Acrylamide gel electrophoresis was used to verify the expression protein.3056 gene was cloned into 2903 primer plasmids, 2798 expression plasmids and 1660 proteins, among which 64 proteins were purified and 16 soluble proteins were obtained.The protein was identified by two dimensional liquid chromatography and mass spectrometry. The protein was identified as Mycobacterium tuberculosis H37Rv.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R378

【引證文獻】

相關碩士學位論文 前1條

1 鐘女娟;基于貝葉斯網(wǎng)絡的農(nóng)村肺結(jié)核病人DOTS效果評價[D];山東大學;2013年

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