本文選題：肺炎鏈球菌 + 毒力因子�。� 參考：《重慶醫(yī)科大學(xué)》2011年博士論文

【摘要】：由于抗生素耐藥的增加和疫苗本身存在的缺陷,肺炎鏈球菌的預(yù)防和治療面臨著很大的挑戰(zhàn)。這就需要我們進(jìn)一步深入研究肺炎鏈球菌的致病機(jī)理,為抗菌藥物的開(kāi)發(fā)和疫苗的研制提供理論基礎(chǔ)。前期工作中課題組研究發(fā)現(xiàn),肺炎鏈球菌的一個(gè)體內(nèi)誘導(dǎo)基因sp0987編碼的假想蛋白質(zhì)SP0987是一個(gè)潛在的毒力因子。到目前為止,SP0987的結(jié)構(gòu)和功能都不清楚,需要我們做進(jìn)一步的研究。 目的本研究將用X-射線(xiàn)晶體學(xué)方法首次解析假想蛋白質(zhì)SP0987的三維立體結(jié)構(gòu),在結(jié)構(gòu)的基礎(chǔ)上研究其生物學(xué)功能,并探索sp0987基因影響細(xì)菌毒力的初步機(jī)制。 方法通過(guò)克隆、表達(dá)、純化、蛋白質(zhì)晶體培養(yǎng)、衍射數(shù)據(jù)收集和蛋白質(zhì)三維立體結(jié)構(gòu)解析,得到SP0987的三維立體結(jié)構(gòu);從三維結(jié)構(gòu)所提供的信息,運(yùn)用酶活性分析方法和定點(diǎn)突變技術(shù)進(jìn)行溶菌酶活性的分析和酶活性位點(diǎn)的分析與驗(yàn)證;通過(guò)熒光報(bào)告系統(tǒng)來(lái)確定SP0987的細(xì)胞定位;通過(guò)構(gòu)建肺炎鏈球菌sp0987缺陷菌株研究小鼠生存實(shí)驗(yàn)及體內(nèi)的定植實(shí)驗(yàn)和體外的細(xì)胞粘附和侵襲實(shí)驗(yàn),來(lái)研究sp0987基因缺陷后對(duì)肺炎鏈球菌致病性的影響。 結(jié)果生物信息學(xué)分析顯示,由基因sp0987編碼的假想蛋白質(zhì)SP0987全長(zhǎng)266個(gè)氨基酸,其編碼產(chǎn)物可能為溶菌酶,主要作用是降解細(xì)胞壁中N-乙酰胞壁酸和N-乙酰葡糖胺之間的β-1,4-糖苷鍵,屬于GH25家族溶菌酶;通過(guò)克隆、表達(dá)、純化、蛋白質(zhì)晶體培養(yǎng)、衍射數(shù)據(jù)收集和蛋白質(zhì)三維立體結(jié)構(gòu)解析,首次成功解析SP0987的三維立體結(jié)構(gòu);序列比對(duì)和三維結(jié)構(gòu)疊合分析發(fā)現(xiàn),SP0987是溶菌酶樣蛋白質(zhì),保守性分析及結(jié)構(gòu)特點(diǎn)分析發(fā)現(xiàn)可能的活性位點(diǎn)D33、D131、E133和D222,且都為酸性氨基酸殘基;分子篩實(shí)驗(yàn)提示,SP0987在溶液中以單體形式存在;應(yīng)用綠色熒光蛋白報(bào)告系統(tǒng)分析SP0987的細(xì)胞定位,結(jié)果發(fā)現(xiàn)SP0987定位于細(xì)胞膜,與生物信息學(xué)分析結(jié)果一致;用LFH-PCR技術(shù)構(gòu)建sp0987缺陷菌,小鼠體內(nèi)生存實(shí)驗(yàn)結(jié)果顯示,sp0987缺陷后肺炎鏈球菌毒力明顯減弱;體外細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn),sp0987缺陷菌對(duì)宿主細(xì)胞的侵襲明顯弱于野生菌,而對(duì)宿主細(xì)胞的粘附?jīng)]有明顯差異;體內(nèi)定植實(shí)驗(yàn)發(fā)現(xiàn),肺炎鏈球菌D39野生菌及缺陷菌經(jīng)鼻腔感染后,缺陷菌入血時(shí)間明顯較晚,且在BALB/c小鼠鼻咽部和肺部的細(xì)菌載量有明顯差異,缺陷菌的細(xì)菌載量明顯減少。 結(jié)論用X-射線(xiàn)晶體學(xué)方法首次成功解析肺炎鏈球菌假想蛋白質(zhì)SP0987的三維結(jié)構(gòu);結(jié)構(gòu)分析及功能實(shí)驗(yàn)發(fā)現(xiàn),SP0987是溶菌酶樣膜蛋白,在溶液中以單體形式存在;毒力實(shí)驗(yàn)顯示,sp0987基因缺陷菌在小鼠體內(nèi)毒力顯著降低,并影響肺炎鏈球菌的侵襲能力和定植能力,所以其編碼產(chǎn)物SP0987是一種新的毒力因子。
[Abstract]:The prevention and treatment of Streptococcus pneumoniae is facing great challenge due to the increase of antibiotic resistance and the defects of the vaccine itself. It is necessary for us to further study the pathogenic mechanism of Streptococcus pneumoniae and provide a theoretical basis for the development of antimicrobial agents and vaccines. In previous work, we found that SP0987, a hypothetical protein encoding gene sp0987, is a potential virulence factor of Streptococcus pneumoniae. So far, the structure and function of SP0987 are not clear, so we need further study. Objective to analyze the three-dimensional structure of hypothetical protein SP0987 by X-ray crystallography for the first time, to study its biological function based on the structure, and to explore the mechanism of sp0987 gene affecting bacterial virulence. Methods the three-dimensional structure of SP0987 was obtained by cloning, expression, purification, protein crystal culture, diffraction data collection and protein three-dimensional structure analysis. The lysozyme activity and the site of lysozyme activity were analyzed and verified by enzyme activity analysis and site-directed mutagenesis, and the cellular location of SP0987 was determined by fluorescence report system. In order to study the pathogenicity of streptococcus pneumoniae by constructing a strain of Streptococcus pneumoniae sp0987 deficient in vivo and colonization and in vitro cell adhesion and invasion test the effect of sp0987 gene deficiency on the pathogenicity of Streptococcus pneumoniae was studied. Results Bioinformatics analysis showed that the hypothetical protein SP0987 encoded by gene sp0987 was 266 amino acids in length, and its encoding product might be lysozyme, which was mainly responsible for the degradation of 尾 -1-glucoside bond between N-acetylcytidic acid and N-acetyl glucosamine in cell wall. By cloning, expression, purification, protein crystal culture, diffraction data collection and protein three-dimensional structure analysis, the three-dimensional structure of SP0987 was successfully analyzed for the first time. Sequence alignment and three dimensional structure analysis revealed that SSP0987 was lysozyme like protein. Conserved analysis and structural characteristics analysis showed that the possible active sites were D33OD131E133 and D222, and both were acidic amino acid residues. Molecular sieve experiment indicated that SP0987 existed in the form of monomer in solution, the cell localization of SP0987 was analyzed by green fluorescent protein report system, and the results showed that SP0987 was located on cell membrane, which was consistent with the result of bioinformatics analysis. Sp0987 deficient bacteria were constructed by LFH-PCR technique. In vivo survival test showed that the virulence of Streptococcus pneumoniae was significantly decreased after the deficiency of sp0987, and the invasion of host cells was weaker than that of wild bacteria in vitro, but there was no significant difference in adhesion to host cells. In vivo colonization experiment, it was found that the blood entry time of Streptococcus pneumoniae D39 wild bacteria and defective bacteria was obviously late, and the bacterial load in nasopharynx and lung of BALB/c mice was significantly different, and the bacterial load of defective bacteria was obviously decreased. Conclusion the three-dimensional structure of SP0987 of Streptococcus pneumoniae was successfully elucidated by X-ray crystallography for the first time, and the results of structural analysis and functional experiments showed that SP0987 was a lysozyme-like membrane protein, which existed in the form of monomer in solution. The virulence test showed that the virulence of SNP0987 was significantly decreased in mice, and the invasion and colonization ability of Streptococcus pneumoniae were affected. Therefore, SP0987 was a new virulence factor.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林亞靜;劉志杰;龔為民;;蛋白質(zhì)結(jié)構(gòu)研究[J];生命科學(xué);2007年03期

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本文編號(hào)：1991779