本文選題：結核分枝桿菌 + 海分枝桿菌��； 參考：《北京協(xié)和醫(yī)學院》2014年碩士論文

【摘要】：結核病是由結核分枝桿菌引起的傳染性疾病,近來由于結核分枝桿菌的耐藥以及結核分枝桿菌和人免疫缺陷病毒的雙重感染使得結核病的防治變得更加困難,尋找新的抗結核藥物靶標,開發(fā)新型的抗結核藥物變得迫在眉睫。 3277-3為本實驗室篩選得到的全新結構的抗結核活性先導物,其對敏感和耐藥的結核分枝桿菌均具有良好的抑制活性,但其作用靶標尚不清楚。本研究通過構建海分枝桿菌基因隨機高表達文庫以及3277-3耐藥突變菌株基因組序列測定兩種途徑,尋找和確認新型抗耐藥結核桿菌候選物3277-3的作用靶標。本研究分為兩個部分： 第一部分是構建與結核分枝桿菌同源性高、安全性好的海分枝桿菌基因隨機高表達文庫,為篩選3277-3的靶標奠定基礎。研究利用單cos黏粒pJRD215和pJDC16構建了雙cos位點黏粒；采用雙cos位點黏粒和噬菌體包裝的方法構建了海分枝桿菌的基因隨機高表達文庫,得到文庫菌落數約為3000個；利用四種己知作用機制的抗結核藥物對海分枝桿菌基因隨機高表達文庫用于藥物靶標發(fā)現(xiàn)的可行性進行驗證,篩選得到D-環(huán)絲氨酸和環(huán)丙沙星的靶標基因ddlA和gyrA,與其被證明的作用靶標相一致。初步證明海分枝桿菌基因隨機高表達文庫可以用于部分抗結核活性化合物的作用靶標的研究。 第二部分是篩選3277-3的海分枝桿菌耐藥突變菌株,通過耐藥突變株全基因組序列測定,發(fā)現(xiàn)和確認其可能的作用靶標。研究采用紫外線誘變海分枝桿菌,篩選得到了兩株耐3277-3的突變菌株Mm3277-36和Mm3277-48；對兩株耐藥菌株進行全基因組重測序,并與海分枝桿菌標準菌株的基因組序列相比對,發(fā)現(xiàn)了16個可能是作用靶標的突變基因。生物信息學分析結果表明,16個基因中有5個PE/PPE蛋白家族成員,2個PKS合成酶,3個NRPS合成酶,3個假設蛋白,1個激酶,2個膜蛋白。大部分的突變基因與細胞壁的合成相關。研究結果為進一步確認3277-3的作用靶標奠定了堅實的基礎。
[Abstract]:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which has recently been made more difficult by the drug resistance of Mycobacterium tuberculosis and the combined infection of Mycobacterium tuberculosis and human immunodeficiency virus,It is urgent to find new targets for anti-tuberculosis drugs and develop new anti-TB drugs.3277-3 is a novel antituberculous active precursor screened in our laboratory. It has good inhibitory activity against both sensitive and drug-resistant Mycobacterium tuberculosis, but its target is not clear.In this study, we constructed a random high expression library of Mycobacterium kainiensis gene and identified the target of a novel candidate for drug resistant Mycobacterium tuberculosis, 3277-3, by constructing a random high expression library and sequencing the genome of 3277-3 drug-resistant mutant.This study is divided into two parts:The first part is to construct a random high expression library with high homology and good safety with Mycobacterium tuberculosis, which lays the foundation for screening the target of 3277-3.The double cos sites were constructed by using single cos pJRD215 and pJDC16, and the random high expression library of mycobacteria gene was constructed by using double cos locus and phage packaging, and the colony number of the library was about 3 000.Four known antituberculous drugs were used to verify the feasibility of using the random overexpression library of Mycobacterium sea for drug target detection.The target genes ddlA and gyrA of D- cycloserine and ciprofloxacin were screened, which were consistent with the target proved by D- cycloserine and ciprofloxacin.It was preliminarily proved that the random high expression library of Mycobacterium seagrass gene could be used to study the target of partial antituberculous active compounds.The second part is the screening of 3277-3 strains of Mycobacterium sea resistant mutants, through the detection of the whole genome sequence of the drug resistant mutants, to find and confirm its possible target.Two mutant strains, Mm3277-36 and Mm3277-48, were screened by ultraviolet radiation mutagenesis, and the two strains were resequenced and compared with the standard strains.Sixteen mutated genes were identified as possible targets for action.Bioinformatics analysis showed that there were 5 PE/PPE family members, 2 PKS synthase, 3 NRPS synthase, 3 hypothetical proteins, 1 kinase and 2 membrane proteins in 16 genes.Most of the mutant genes are associated with cell wall synthesis.The results lay a solid foundation for further confirmation of the action target of 3277-3.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R52

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本文編號：1766317