流产布鲁菌脂多糖单克隆抗体的制备及Asp24蛋白的功能研究
发布时间：2019-06-28 12:09
【摘要】：牛布鲁菌病是一种由流产布鲁菌感染引起的人畜共患细菌性传染病,呈全球性分布。布鲁菌是一种兼性胞内寄生菌,革兰氏染色呈阴性,没有质粒、荚膜,外毒素等经典的毒力因子,布鲁菌的毒力主要体现在其具有入侵宿主细胞并在胞内增殖的能力。布鲁菌致病机制研究为预防和控制该病提供重要基础,目前鉴别诊断和新疫苗研发是控制布鲁菌病面临的两个主要问题。动物感染布鲁菌后产生的针对其脂多糖的抗血清可用于诊断人畜布鲁菌病。本研究采用传统的细胞融合技术,通过流产布鲁菌S2308脂多糖筛选获得一株稳定分泌抗流产布鲁菌脂多糖抗体的单克隆细胞株,命名为51C,并成功制备流产布鲁菌脂多糖单克隆抗体。交叉反应性实验表明,本研究得到的单克隆抗体与流产布鲁菌S2308、猪种布鲁菌1330的脂多糖均有良好的反应性,与马耳他布鲁菌16M、大肠杆菌0157和鼠伤寒沙门菌SL1344的脂多糖不反应,与小肠结肠耶尔森菌0:9型的脂多糖有轻微交叉反应。因此,推测本研究获得的单克隆抗体作用表位为脂多糖的A表位。本研究为进一步建立高特异性的布鲁菌检测方法奠定基础。Asp24是布鲁菌一个重要的毒力相关蛋白,该蛋白在酸性条件下能够诱导表达,asp24基因缺失株作为候选疫苗株具有较大的应用潜力。本实验室在前期研究中发现,脂多糖O-抗原的胞内聚集可以诱导asp24基因上调表达,协助布鲁菌胞内存活。然而,目前对Asp24蛋白的功能研究并不清楚。本研究首先通过定向缺失技术成功构建了Δasp2 缺失株,生长曲线测定发现,Asp24对细菌的生长影响不明显,细胞感染试验发现Asp24对布鲁菌胞内存活无影响,动物感染试验证明Asp24与布鲁菌在小鼠体内早期建立感染无关,而对布鲁菌在小鼠体内建立慢性感染发挥着重要作用。为进一步探究Asp24的功能,后续对Asp24的诱导条件和蛋白活性进行深入分析。在热应激条件下,Asp24的表达出现显著下调。启动子活性分析发现,基因开放阅读框(ORF)前89bp是asp24基因表达所必需的,而前126bp的片段是布鲁菌在酸性环境下诱导表达asp24基因所必需的。生物信息学分析发现Asp24蛋白存在3个典型的钙离子结合型EF手型结构域,为验证Asp24蛋白的钙离子结合活性,本研究表达和纯化了野生型和突变型Asp24蛋白,采用SDS-PAGE分析Asp24蛋白电泳迁移率变化,发现Asp24结合钙离子后电泳速率显著加快,而突变体蛋白电泳速率无变化,表明Asp24确实可以结合钙离子。进一步分析发现,Asp24在布鲁菌内的表达水平与布鲁菌所处环境中钙离子浓度相关,预示细菌可能通过调控Asp24蛋白的表达来维持胞内钙离子稳态。综上所述,本研究成功制备了一株抗流产布鲁菌脂多糖的单克隆抗体。发现Asp24蛋白对布鲁菌在小鼠体内建立慢性感染发挥重要作用,发现Asp24蛋白有结合钙离子活性,为建立布鲁菌特异性的检测方法奠定了基础,为研究布鲁菌致病机制积累了资料。
[Abstract]:The bovine brucellosis is a bacterial and infectious disease caused by the infection of Brucella abortus, and is a global distribution. Brucella is a kind of facultative intracellular parasitic bacteria, Gram-positive staining is negative, no plasmid, membrane, exotoxin and other classical virulence factors, the virulence of Brucella is mainly reflected in its ability to invade host cell and proliferate in the cell. The research of the pathogenesis of Brucella provides an important basis for the prevention and control of the disease, and the present differential diagnosis and new vaccine R & D are two main problems for the control of the brucellosis. The antisera against the lipopolysaccharide produced by the animal after the infection of the Brucella can be used for the diagnosis of the brucellosis of human and animal. In this study, the traditional cell fusion technique was used to obtain a monoclonal antibody to stably secrete an anti-abortion Brucella polysaccharide antibody, named as 51C, and successfully prepared a monoclonal antibody of Brucella abortus. The cross-reactivity experiment shows that the monoclonal antibody obtained in this study has good reactivity with the lipopolysaccharides of Brucella abortus s2308 and the pig breed of Brucella sp., and does not react with the lipopolysaccharide of the strains of Brucella of Malta, Escherichia coli 0157 and S. typhimurium SL1344, There was a slight cross-reaction with the lipopolysaccharide of the type 0:9 of the enterocolon of the small intestine. Therefore, it is presumed that the epitope of the monoclonal antibody obtained in the present study is the A-epitope of the lipopolysaccharide. The present study lays the foundation for the further establishment of a high-specific test method for Brucella. Asp24 is an important virulence-related protein of Brucella, which can induce expression under the condition of acid, and the as24 gene deletion strain has a great potential for application as a candidate vaccine strain. In this lab, it was found that the intracellular aggregation of lipopolysaccharide O-antigen could induce an up-regulated expression of the asp24 gene to assist in the survival of the cell. However, the current functional study of the Asp24 protein is not clear. The results showed that Asp24 had no effect on the growth of bacteria, and the test of cell infection showed that Asp24 had no effect on the survival of Brucella cells. The test of animal infection proves that Asp24 and Brucella are not related to the early establishment of infection in the mouse, and it plays an important role in the development of chronic infection in mice. In order to further explore the function of Asp24, the induction condition and protein activity of Asp24 were further analyzed. The expression of Asp24 was down-regulated under the condition of heat stress. The promoter activity analysis found that the 89 bp before the gene open reading frame (ORF) was necessary for the expression of the asp24 gene, and the first 126 bp fragment was necessary for the expression of the expression of the asp24 gene in an acidic environment. The bioinformatics analysis found that the Asp24 protein has three typical calcium-ion-binding EF-type domains. In order to verify the calcium ion binding activity of the Asp24 protein, the wild type and the mutant Asp24 protein are expressed and purified in the present study, and the change of the electrophoretic mobility of the Asp24 protein is analyzed by SDS-PAGE. It was found that the electrophoresis rate of the Asp24-binding calcium ion was significantly increased, while the electrophoretic rate of the mutant protein was not changed, indicating that the Asp24 could indeed bind to the calcium ion. Further analysis shows that the expression level of Asp24 in the Brucella is related to the concentration of calcium in the environment in which the Brucella is located, which indicates that the bacteria may maintain the homeostasis of intracellular calcium ions by regulating the expression of the Asp24 protein. To sum up, a monoclonal antibody against Brucella abortus was successfully prepared in this study. It is found that Asp24 protein plays an important role in the establishment of chronic infection in mice, and it is found that Asp24 protein has the activity of binding to calcium ions, which lays a foundation for the establishment of the specific detection method of Brucella.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
本文编号：2507292
[Abstract]:The bovine brucellosis is a bacterial and infectious disease caused by the infection of Brucella abortus, and is a global distribution. Brucella is a kind of facultative intracellular parasitic bacteria, Gram-positive staining is negative, no plasmid, membrane, exotoxin and other classical virulence factors, the virulence of Brucella is mainly reflected in its ability to invade host cell and proliferate in the cell. The research of the pathogenesis of Brucella provides an important basis for the prevention and control of the disease, and the present differential diagnosis and new vaccine R & D are two main problems for the control of the brucellosis. The antisera against the lipopolysaccharide produced by the animal after the infection of the Brucella can be used for the diagnosis of the brucellosis of human and animal. In this study, the traditional cell fusion technique was used to obtain a monoclonal antibody to stably secrete an anti-abortion Brucella polysaccharide antibody, named as 51C, and successfully prepared a monoclonal antibody of Brucella abortus. The cross-reactivity experiment shows that the monoclonal antibody obtained in this study has good reactivity with the lipopolysaccharides of Brucella abortus s2308 and the pig breed of Brucella sp., and does not react with the lipopolysaccharide of the strains of Brucella of Malta, Escherichia coli 0157 and S. typhimurium SL1344, There was a slight cross-reaction with the lipopolysaccharide of the type 0:9 of the enterocolon of the small intestine. Therefore, it is presumed that the epitope of the monoclonal antibody obtained in the present study is the A-epitope of the lipopolysaccharide. The present study lays the foundation for the further establishment of a high-specific test method for Brucella. Asp24 is an important virulence-related protein of Brucella, which can induce expression under the condition of acid, and the as24 gene deletion strain has a great potential for application as a candidate vaccine strain. In this lab, it was found that the intracellular aggregation of lipopolysaccharide O-antigen could induce an up-regulated expression of the asp24 gene to assist in the survival of the cell. However, the current functional study of the Asp24 protein is not clear. The results showed that Asp24 had no effect on the growth of bacteria, and the test of cell infection showed that Asp24 had no effect on the survival of Brucella cells. The test of animal infection proves that Asp24 and Brucella are not related to the early establishment of infection in the mouse, and it plays an important role in the development of chronic infection in mice. In order to further explore the function of Asp24, the induction condition and protein activity of Asp24 were further analyzed. The expression of Asp24 was down-regulated under the condition of heat stress. The promoter activity analysis found that the 89 bp before the gene open reading frame (ORF) was necessary for the expression of the asp24 gene, and the first 126 bp fragment was necessary for the expression of the expression of the asp24 gene in an acidic environment. The bioinformatics analysis found that the Asp24 protein has three typical calcium-ion-binding EF-type domains. In order to verify the calcium ion binding activity of the Asp24 protein, the wild type and the mutant Asp24 protein are expressed and purified in the present study, and the change of the electrophoretic mobility of the Asp24 protein is analyzed by SDS-PAGE. It was found that the electrophoresis rate of the Asp24-binding calcium ion was significantly increased, while the electrophoretic rate of the mutant protein was not changed, indicating that the Asp24 could indeed bind to the calcium ion. Further analysis shows that the expression level of Asp24 in the Brucella is related to the concentration of calcium in the environment in which the Brucella is located, which indicates that the bacteria may maintain the homeostasis of intracellular calcium ions by regulating the expression of the Asp24 protein. To sum up, a monoclonal antibody against Brucella abortus was successfully prepared in this study. It is found that Asp24 protein plays an important role in the establishment of chronic infection in mice, and it is found that Asp24 protein has the activity of binding to calcium ions, which lays a foundation for the establishment of the specific detection method of Brucella.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
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