结核分枝杆菌单链DNA结合蛋白突变文库的建立及初步应用
发布时间：2018-08-25 10:09
【摘要】：结核分枝杆菌(M. tuberculosis)是一种严重危害人类健康的病原细菌,它们在与宿主细胞相互作用过程中呈现出活跃生长-潜伏-复苏等多种生长模式,这表明,其DNA代谢可能受到严谨的调控。单链DNA结合蛋白(SSB)能够广泛参与诸如DNA复制,重组,修复等多种生化过程,但是结核分枝杆菌的SSB的功能还没有深入研究,其关键氨基酸残基还没有鉴定。 针对结核分枝杆菌的基因组GC含量高的特点,本研究采用重亚硫酸氢钠化学诱变剂随机突变其单链DNA结合蛋白(MtuSSB)基因Rv0054,成功构建了点突变文库。在随机挑选的830个突变体中,814个测序成功,共获得了单氨基酸突变体123个。另外还获得了双突变体107个,三突变体63个,四突变体44个,和C-末端缺失体13个。这些突变覆盖了SSB基因的141个氨基酸位点,覆盖率达到93.4%(141/151)。从该SSB突变文库中选择了22个进行了非变性聚丙烯酰胺凝胶电泳和细菌双杂交等分析,初步鉴定到了8个影响多聚体状态的突变位点。 这是第一次建立高覆盖率的结核分枝杆菌H37Rv单链DNA结合蛋白氨基酸位点突变文库,这对于进一步系统剖析该蛋白质的功能及结构与功能的关系创造了条件。同时,该方法的建立和成功实践也将为深入研究结核分枝杆菌的其它关键基因搭建了主要技术平台。
[Abstract]:Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that seriously endangers human health. It shows many growth patterns, such as active growth, latent resuscitation and so on, in the process of interaction with host cells. Its DNA metabolism may be strictly regulated. Single stranded DNA binding protein (SSB) can be widely involved in many biochemical processes such as DNA replication, recombination and repair. However, the function of SSB in Mycobacterium tuberculosis has not been further studied, and its key amino acid residues have not been identified. In view of the high genomic GC content of Mycobacterium tuberculosis, the point mutation library was successfully constructed by random mutation of single strand DNA binding protein (MtuSSB) gene Rv0054, with sodium bisulfite chemical mutagen. Among the 830 mutants randomly selected, 814 were sequenced and 123 single amino acid mutants were obtained. In addition, 107 double mutants, 63 triple mutants, 44 four mutants and 13 C-terminal deletions were obtained. These mutations covered 141 amino acid sites of the SSB gene, reaching 93.4% (141 / 151). Twenty-two of the SSB mutation libraries were selected for non-denaturing polyacrylamide gel electrophoresis and bacterial two-hybrid analysis, and 8 mutation sites affecting the polymer status were identified preliminarily. This is the first time to establish a H37Rv single-stranded DNA binding protein amino acid mutation library with high coverage, which creates conditions for further systematic analysis of the function of the protein and the relationship between its structure and function. At the same time, the establishment and successful practice of this method will also provide a main technical platform for further research on other key genes of Mycobacterium tuberculosis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
[Abstract]:Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that seriously endangers human health. It shows many growth patterns, such as active growth, latent resuscitation and so on, in the process of interaction with host cells. Its DNA metabolism may be strictly regulated. Single stranded DNA binding protein (SSB) can be widely involved in many biochemical processes such as DNA replication, recombination and repair. However, the function of SSB in Mycobacterium tuberculosis has not been further studied, and its key amino acid residues have not been identified. In view of the high genomic GC content of Mycobacterium tuberculosis, the point mutation library was successfully constructed by random mutation of single strand DNA binding protein (MtuSSB) gene Rv0054, with sodium bisulfite chemical mutagen. Among the 830 mutants randomly selected, 814 were sequenced and 123 single amino acid mutants were obtained. In addition, 107 double mutants, 63 triple mutants, 44 four mutants and 13 C-terminal deletions were obtained. These mutations covered 141 amino acid sites of the SSB gene, reaching 93.4% (141 / 151). Twenty-two of the SSB mutation libraries were selected for non-denaturing polyacrylamide gel electrophoresis and bacterial two-hybrid analysis, and 8 mutation sites affecting the polymer status were identified preliminarily. This is the first time to establish a H37Rv single-stranded DNA binding protein amino acid mutation library with high coverage, which creates conditions for further systematic analysis of the function of the protein and the relationship between its structure and function. At the same time, the establishment and successful practice of this method will also provide a main technical platform for further research on other key genes of Mycobacterium tuberculosis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
【共引文献】
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2 朱非,王珊,周培瑾;低温酶冷适应的分子机制及其在生物技术中的应用[J];微生物学报;2002年05期
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