新型抗结核化合物3277-3的作用靶标研究

发布时间：2018-04-18 02:10

本文选题：结核分枝杆菌 + 海分枝杆菌　；参考：《北京协和医学院》2014年硕士论文

【摘要】：结核病是由结核分枝杆菌引起的传染性疾病,近来由于结核分枝杆菌的耐药以及结核分枝杆菌和人免疫缺陷病毒的双重感染使得结核病的防治变得更加困难,寻找新的抗结核药物靶标,开发新型的抗结核药物变得迫在眉睫。 3277-3为本实验室筛选得到的全新结构的抗结核活性先导物,其对敏感和耐药的结核分枝杆菌均具有良好的抑制活性,但其作用靶标尚不清楚。本研究通过构建海分枝杆菌基因随机高表达文库以及3277-3耐药突变菌株基因组序列测定两种途径,寻找和确认新型抗耐药结核杆菌候选物3277-3的作用靶标。本研究分为两个部分：第一部分是构建与结核分枝杆菌同源性高、安全性好的海分枝杆菌基因随机高表达文库,为筛选3277-3的靶标奠定基础。研究利用单cos黏粒pJRD215和pJDC16构建了双cos位点黏粒；采用双cos位点黏粒和噬菌体包装的方法构建了海分枝杆菌的基因随机高表达文库,得到文库菌落数约为3000个；利用四种己知作用机制的抗结核药物对海分枝杆菌基因随机高表达文库用于药物靶标发现的可行性进行验证,筛选得到D-环丝氨酸和环丙沙星的靶标基因ddlA和gyrA,与其被证明的作用靶标相一致。初步证明海分枝杆菌基因随机高表达文库可以用于部分抗结核活性化合物的作用靶标的研究。第二部分是筛选3277-3的海分枝杆菌耐药突变菌株,通过耐药突变株全基因组序列测定,发现和确认其可能的作用靶标。研究采用紫外线诱变海分枝杆菌,筛选得到了两株耐3277-3的突变菌株Mm3277-36和Mm3277-48；对两株耐药菌株进行全基因组重测序,并与海分枝杆菌标准菌株的基因组序列相比对,发现了16个可能是作用靶标的突变基因。生物信息学分析结果表明,16个基因中有5个PE/PPE蛋白家族成员,2个PKS合成酶,3个NRPS合成酶,3个假设蛋白,1个激酶,2个膜蛋白。大部分的突变基因与细胞壁的合成相关。研究结果为进一步确认3277-3的作用靶标奠定了坚实的基础。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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