结核分枝杆菌H37Rv ORF组学研究

发布时间：2018-04-16 16:37

本文选题：结核分枝杆菌 + 高通量　；参考：《吉林大学》2008年博士论文

【摘要】： 结核分枝杆菌是结核病的致病菌,据世界卫生组织统计,近年来肺结核在全球各地死灰复燃,1995年全世界有300万人死于此病,是该病死亡人数最多的一年,大大超过了肺结核流行的1900年。在2003年3月24日“世界防治结核病日”之际,“制止结核病”世界行动组织公布的数字显示,目前全球每天仍有5000人死于结核病,而每年罹患结核病的人数超过800万由于人口剧增、移民增加、旅游业发展、耐药性的产生、人类免疫缺陷病毒感染流行、吸毒、酗酒与贫困等原因,结核病呈日益严重回升趋势。因此,对结核分枝杆菌基因组学研究、蛋白质组学研究、后基因组学研究等,表达后蛋白质组学研究,即从一个细胞生命整体去研究,方能彻底的弄清其分子致病机制、感染的方法,弄清结核分枝杆菌胞内生活周期,为结核分枝杆菌病早期诊断、疫苗免疫、新药研发,提供坚厚的理论基础。 1998年英国Sanger中心和法国Pasteur研究所科学家合作完成了结核分枝杆菌H37Rv株的全基因组测序工作,并在2002年进行了重新注释,这为结核病病原菌致病基因的研究提供了理论基础。结核分枝杆菌全基因组序列由4.41Mb(4.411529bp)组成,包括4411个基因,具有潜在编码能力的基因约有3977个,约占90.2%。有3924个开放阅读框,其中约40%有功能,44%可能有功能,16%称为孤儿序列,与其它微生物的序列无相似性。基因组富含GC碱基,G+C含量高达65.6%。虽然世界各地的实验室、医院、科研机构都在对结核分枝杆菌H37Rv做各方面的研究,但是尚无法克服结核病这一难题,越来越高的发病率和耐药菌株及多耐药菌株的出现,给结核分枝杆菌病的治疗增加了难度。本试验从整个基因组出发,利用Gateway技术,pDONR221为基因供体载体,pDEST17为表达载体,俩个表达菌株BL21和BL21Codonplus RP对结核分枝杆菌H37Rv进行不同诱导条件表达,丙烯酰胺凝胶电泳对表达蛋白验证,共对3056基因进行了克隆,得到入门质粒2903个,表达质粒2798个,表达蛋白1660个,其中纯化蛋白64个,有16个可溶性蛋白;选取重组表达,经酶解后得到的肽片段混合物,通过两维液相色谱分离,采用液质连用质谱方法对蛋白进行鉴定,确定属于结核分枝杆菌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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R378

【引证文献】