本文選題：結(jié)核分枝桿菌 + 海分枝桿菌　； 參考：《北京協(xié)和醫(yī)學(xué)院》2014年碩士論文

【摘要】：結(jié)核病是由結(jié)核分枝桿菌引起的傳染性疾病,近來由于結(jié)核分枝桿菌的耐藥以及結(jié)核分枝桿菌和人免疫缺陷病毒的雙重感染使得結(jié)核病的防治變得更加困難,尋找新的抗結(jié)核藥物靶標(biāo),開發(fā)新型的抗結(jié)核藥物變得迫在眉睫。 3277-3為本實(shí)驗(yàn)室篩選得到的全新結(jié)構(gòu)的抗結(jié)核活性先導(dǎo)物,其對(duì)敏感和耐藥的結(jié)核分枝桿菌均具有良好的抑制活性,但其作用靶標(biāo)尚不清楚。本研究通過構(gòu)建海分枝桿菌基因隨機(jī)高表達(dá)文庫以及3277-3耐藥突變菌株基因組序列測(cè)定兩種途徑,尋找和確認(rèn)新型抗耐藥結(jié)核桿菌候選物3277-3的作用靶標(biāo)。本研究分為兩個(gè)部分： 第一部分是構(gòu)建與結(jié)核分枝桿菌同源性高、安全性好的海分枝桿菌基因隨機(jī)高表達(dá)文庫,為篩選3277-3的靶標(biāo)奠定基礎(chǔ)。研究利用單cos黏粒pJRD215和pJDC16構(gòu)建了雙cos位點(diǎn)黏粒；采用雙cos位點(diǎn)黏粒和噬菌體包裝的方法構(gòu)建了海分枝桿菌的基因隨機(jī)高表達(dá)文庫,得到文庫菌落數(shù)約為3000個(gè)；利用四種己知作用機(jī)制的抗結(jié)核藥物對(duì)海分枝桿菌基因隨機(jī)高表達(dá)文庫用于藥物靶標(biāo)發(fā)現(xiàn)的可行性進(jìn)行驗(yàn)證,篩選得到D-環(huán)絲氨酸和環(huán)丙沙星的靶標(biāo)基因ddlA和gyrA,與其被證明的作用靶標(biāo)相一致。初步證明海分枝桿菌基因隨機(jī)高表達(dá)文庫可以用于部分抗結(jié)核活性化合物的作用靶標(biāo)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(cè)定,發(fā)現(xiàn)和確認(rèn)其可能的作用靶標(biāo)。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(duì)兩株耐藥菌株進(jìn)行全基因組重測(cè)序,并與海分枝桿菌標(biāo)準(zhǔn)菌株的基因組序列相比對(duì),發(fā)現(xiàn)了16個(gè)可能是作用靶標(biāo)的突變基因。生物信息學(xué)分析結(jié)果表明,16個(gè)基因中有5個(gè)PE/PPE蛋白家族成員,2個(gè)PKS合成酶,3個(gè)NRPS合成酶,3個(gè)假設(shè)蛋白,1個(gè)激酶,2個(gè)膜蛋白。大部分的突變基因與細(xì)胞壁的合成相關(guān)。研究結(jié)果為進(jìn)一步確認(rèn)3277-3的作用靶標(biāo)奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R52

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本文編號(hào)：1766317