钩端螺旋体功能基因组学研究

发布时间：2018-04-19 02:32

本文选题：钩端螺旋体 + 转座　；参考：《沈阳药科大学》2005年硕士论文

【摘要】：钩端螺旋体中国有毒赖株的全基因组测序已经完成，这只是钩端螺旋体功能基因组研究的第一步。在中国有毒赖株中共有大小两个染色体，共4691184对核苷酸，预知含有4727个功能基因。大量基因的功能需要我们通过实验的方法来推知和证明。将未知功能的基因组核苷酸序列转化为有意义的基因功能信息将是一个具有挑战性的工作。本实验独立进行了钩端螺旋体无毒赖株和有毒赖株培养条件的研究。同时考察了甘氨酸，Mg~(2+)，DMSO，MTT，，温度等对钩体生长的影响，掌握了钩体各种培养条件。用钩端螺旋体无毒赖株作为研究对象摸索了钩体的电转化条件，研究了钩端螺旋体有毒赖株的抗药性，并建立了一套有效的抗性筛选方法，为钩端螺旋体有毒赖株的全基因组突变体库的建立创建了平台。应用转座子对微生物基因的插入突变是研究微生物全基因组功能的常用方法和有力手段，被广泛应用到模式生物的功能基因组学研究当中。通过单引物PCR可以将插入位点在基因组中定位，通过表型的分析可以探知突变基因在基因组中的生物学功能。本实验通过Tn5体转座系统成功摸索出了钩端螺旋体无毒赖株的突变方法，并将Tn5体转座系统加以修饰应用于钩端螺旋体有毒赖株的突变体库的建立当中去。在今后的工作中继续构建整合有带有钩体启动子的转座酶以及多转座位点的体内转座体系。本实验同时构建了大肠杆菌ET12567接合转移系统，与钩体有毒赖株共培养，以带有钩体鞭毛蛋白基因flgH和抗性筛选标记的接合载体来摸索钩体单基因的插入失活和基因敲除方法。本实验对钩体中的必须基因金属酶蛋白基因进行了研究，并以肽键去甲基化酶(PDF)蛋白为目标，应用dock分子对接软件搜索specs化合物数据库，应用体外抗菌实验的方法筛选基于PDF药物靶点的抗菌小分子有机化合物，并申请新的化合物专利十项。
[Abstract]:The complete genome sequencing of Leptospira chinensis is the first step in the study of Leptospira functional genome.A total of 4691184 pairs of nucleotides were found in two chromosomes of Chinese virulent Lai strain, and 4727 functional genes were predicted.The function of a large number of genes needs to be deduced and proved by experimental methods.It will be a challenging task to transform unknown functional nucleotide sequences into meaningful gene functional information.The culture conditions of Leptospira non-venomous and venomous Leptospira were studied in this experiment.The effects of temperature and temperature on the growth of Leptospira were investigated, and various culture conditions of Leptospira were studied.The electrotransformation conditions of leptospirosis were studied by using leptospira nontoxic Lai strain as the research object. The resistance of leptospira venomous Lai strain was studied, and a set of effective screening methods were established.A platform was established for the establishment of a whole genome mutant library of Leptospira venomous Leptospira.The insertion mutation of microbial gene by transposon has been widely used in the functional genomics of model organisms.The insertion site can be located in the genome by single primer PCR, and the biological function of the mutant gene in the genome can be found by phenotypic analysis.In this experiment, the mutation method of leptospira venomous rogue strain was successfully explored by Tn5 transposition system, and the Tn5 transposition system was modified and applied to the establishment of the mutant library of Leptospira venomous Leptospira.In the future, we will continue to construct an in vivo transposable system with Leptospira promoter and multiple transposable loci.At the same time, we constructed E. coli ET12567 conjugation transfer system, co-cultured with Leptospira venomous Leptospira strain, and explored the method of insertion inactivation and gene knockout of Leptospira single gene with Leptospira flagellin gene flgH and resistance screening marker.In this study, the essential gene metalloproteinase gene in Leptospira was studied, and the specs compound database was searched by using dock molecular docking software with peptide desmethylase (PDF) protein as the target.Antimicrobial small molecule organic compounds based on PDF drug target were screened by in vitro antibacterial experiment and ten new compounds were patented.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R377

【参考文献】