結(jié)核分枝桿菌單鏈DNA結(jié)合蛋白突變文庫的建立及初步應(yīng)用
發(fā)布時間：2018-08-25 10:09
【摘要】：結(jié)核分枝桿菌(M. tuberculosis)是一種嚴重危害人類健康的病原細菌,它們在與宿主細胞相互作用過程中呈現(xiàn)出活躍生長-潛伏-復(fù)蘇等多種生長模式,這表明,其DNA代謝可能受到嚴謹?shù)恼{(diào)控。單鏈DNA結(jié)合蛋白(SSB)能夠廣泛參與諸如DNA復(fù)制,重組,修復(fù)等多種生化過程,但是結(jié)核分枝桿菌的SSB的功能還沒有深入研究,其關(guān)鍵氨基酸殘基還沒有鑒定。 針對結(jié)核分枝桿菌的基因組GC含量高的特點,本研究采用重亞硫酸氫鈉化學(xué)誘變劑隨機突變其單鏈DNA結(jié)合蛋白(MtuSSB)基因Rv0054,成功構(gòu)建了點突變文庫。在隨機挑選的830個突變體中,814個測序成功,共獲得了單氨基酸突變體123個。另外還獲得了雙突變體107個,三突變體63個,四突變體44個,和C-末端缺失體13個。這些突變覆蓋了SSB基因的141個氨基酸位點,覆蓋率達到93.4%(141/151)。從該SSB突變文庫中選擇了22個進行了非變性聚丙烯酰胺凝膠電泳和細菌雙雜交等分析,初步鑒定到了8個影響多聚體狀態(tài)的突變位點。 這是第一次建立高覆蓋率的結(jié)核分枝桿菌H37Rv單鏈DNA結(jié)合蛋白氨基酸位點突變文庫,這對于進一步系統(tǒng)剖析該蛋白質(zhì)的功能及結(jié)構(gòu)與功能的關(guān)系創(chuàng)造了條件。同時,該方法的建立和成功實踐也將為深入研究結(jié)核分枝桿菌的其它關(guān)鍵基因搭建了主要技術(shù)平臺。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378
【共引文獻】
相關(guān)期刊論文 前2條
1 欒國棟;蔡真;李寅;;超突變細胞在進化工程中的開發(fā)與應(yīng)用[J];生物技術(shù)進展;2014年04期
2 朱非,王珊,周培瑾;低溫酶冷適應(yīng)的分子機制及其在生物技術(shù)中的應(yīng)用[J];微生物學(xué)報;2002年05期
相關(guān)會議論文 前1條
1 王全富;繆錦來;丁q，
本文編號：2202562
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