【摘要】：阪崎克羅諾桿菌是一種自然界中廣泛存在的,主要污染嬰兒配方奶粉的食源性革蘭氏陰性條件致病菌。由于其明確的致病性,阪崎克羅諾桿菌被列為A類致病菌。它可以引起新生兒腦膜炎、敗血病、菌血癥及壞死性小腸結(jié)膜炎等疾病,死亡率可高達(dá)80%。然而,阪崎克羅諾桿菌的致病機(jī)制還不清楚。阪崎克羅諾桿菌可能通過(guò)在腸道內(nèi)增殖并粘附及入侵腸上皮細(xì)胞而致病,而菌膜的形成可能在這個(gè)過(guò)程中起著重要作用。脂多糖是阪崎克羅諾桿菌細(xì)胞表面的主要致病性大分子,與大腸桿菌、沙門氏菌和空腸彎曲菌等一些革蘭氏陰性菌的菌膜形成密切相關(guān),但是阪崎克羅諾桿菌的脂多糖生物合成途徑中的多數(shù)基因及其與菌膜形成的關(guān)系尚未確定。 本課題對(duì)阪崎克羅諾桿菌的脂多糖生物合成途徑關(guān)鍵基因waaC進(jìn)行了鑒定,并從多方面研究該基因在菌膜形成中的重要作用,從而探索阪崎克羅諾桿菌的脂多糖結(jié)構(gòu)與菌膜形成之間的關(guān)系。主要結(jié)論如下: (1)證實(shí)了ESA_04107為阪崎克羅諾桿菌的脂多糖生物合成途徑中的庚糖基轉(zhuǎn)移酶I的編碼基因waaC。首先,通過(guò)同源比對(duì)發(fā)現(xiàn)阪崎克羅諾桿菌BAA-894中ESA_04107的編碼序列與大腸桿菌W3110的WaaC同源性最高;其次,通過(guò)敲除ESA_04107構(gòu)建了阪崎克羅諾桿菌突變株LWW02;最后,從LWW02中提取脂多糖,用SDS-聚丙烯酰胺凝膠電泳和薄層層析進(jìn)行結(jié)構(gòu)分析,用DEAE-纖維素陰離子交換層析純化,并用ESI/MS確定其分子結(jié)構(gòu)。 (2)研究阪崎克羅諾桿菌脂多糖結(jié)構(gòu)突變株LWW02的菌膜形成能力。與野生型BAA-894相比,LWW02的生長(zhǎng)受到影響,但是在親疏水介質(zhì)表面的菌膜形成能力卻增強(qiáng),表明阪崎克羅諾桿菌的脂多糖結(jié)構(gòu)與菌膜形成能力密切相關(guān)。 (3)探究阪崎克羅諾桿菌脂多糖結(jié)構(gòu)對(duì)細(xì)胞其他特性的影響。與野生型BAA-894相比,LWW02的細(xì)胞表面疏水性、外膜通透性、自凝集性以及胞外物分泌量等都有了顯著提高。這些因素可能是導(dǎo)致LWW02菌膜形成能力增強(qiáng)的主要原因。本課題在鑒定阪崎克羅諾桿菌的脂多糖生物合成途徑關(guān)鍵基因waaC的基礎(chǔ)上,將阪崎克羅諾桿菌的外膜結(jié)構(gòu)組分脂多糖與菌膜形成聯(lián)系起來(lái),從多角度探究了阪崎克羅諾桿菌脂多糖影響菌膜形成能力變化的機(jī)制,對(duì)探究阪崎克羅諾桿菌的致病機(jī)理,檢測(cè)與控制阪崎克羅諾桿菌等都具有重要意義。
[Abstract]:Hanshizaki Crosenia is a foodborne gram-negative opportunistic pathogen that is widely found in nature and mainly contaminates infant formula. Because of its definite pathogenicity, Hanshizaki Crosenia is classified as a class A pathogen. It can cause diseases such as neonatal meningitis, septicaemia, bacteremia and necrotizing small bowel conjunctivitis. However, the pathogenetic mechanism of Hanshizaki Croix is unclear. Hanshizaki Crosenia may cause disease by proliferation, adhesion and invasion of intestinal epithelial cells, and the formation of bacterial membrane may play an important role in this process. Lipopolysaccharide is the major pathogenicity macromolecule on the cell surface of Hanshizaki Crosenia, which is closely related to the membrane formation of some gram-negative bacteria such as Escherichia coli, Salmonella and Campylobacter jejuni. However, most of the genes in the lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia and their relationship with membrane formation have not been determined. In this paper, the key gene waaC of lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia was identified, and the important role of this gene in membrane formation was studied from many aspects. To explore the relationship between lipopolysaccharide structure and membrane formation of Hanshizaki Crosenia. The main conclusions are as follows: (1) it is confirmed that ESA_04107 is the encoding gene waaC. of heptose transferase I in the lipopolysaccharide biosynthesis pathway of Hanshizaki. Firstly, the coding sequence of ESA_04107 in BAA-894 of Hanshizaki Crosenia was found to have the highest homology with the WaaC of Escherichia coli W3110. Secondly, the mutant LWW02; was constructed by knockout ESA_04107. Finally, the lipopolysaccharide was extracted from LWW02. The structure was analyzed by SDS- polyacrylamide gel electrophoresis and thin-layer chromatography. The structure was purified by DEAE- cellulose anion exchange chromatography, and its molecular structure was determined by ESI/MS. (2) to study the membrane forming ability of LWW02. Compared with wild-type BAA-894, the growth of LWW02 was affected, but the membrane formation ability on the surface of hydrophilic medium was enhanced, indicating that the lipopolysaccharide structure of Hanshizaki Croix was closely related to the membrane formation ability. (3) to investigate the effect of lipopolysaccharide structure of Hanshin Crosaki bacillus on other characteristics of cells. Compared with wild-type BAA-894, the surface hydrophobicity, outer membrane permeability, self-agglutination and extracellular secretion of LWW02 were significantly increased. These factors may be the main reasons for the enhancement of membrane formation ability of LWW02 bacteria. On the basis of identifying the key gene of lipopolysaccharide biosynthesis pathway of Hanshizaki, waaC, the lipopolysaccharide of the outer membrane structure of Hanshizaki Crosenia was associated with the formation of the membrane. In this paper, the mechanism of lipopolysaccharide (LPS) from Hanshazaki Crosenia on the change of membrane formation ability is studied, which is of great significance for the study of pathogenicity, detection and control of Hanshizaki Crosenia.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

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本文編號(hào)：2421011