流產布魯菌脂多糖單克隆抗體的制備及Asp24蛋白的功能研究
發(fā)布時間：2019-06-28 12:09
【摘要】：牛布魯菌病是一種由流產布魯菌感染引起的人畜共患細菌性傳染病,呈全球性分布。布魯菌是一種兼性胞內寄生菌,革蘭氏染色呈陰性,沒有質粒、莢膜,外毒素等經典的毒力因子,布魯菌的毒力主要體現在其具有入侵宿主細胞并在胞內增殖的能力。布魯菌致病機制研究為預防和控制該病提供重要基礎,目前鑒別診斷和新疫苗研發(fā)是控制布魯菌病面臨的兩個主要問題。動物感染布魯菌后產生的針對其脂多糖的抗血清可用于診斷人畜布魯菌病。本研究采用傳統(tǒng)的細胞融合技術,通過流產布魯菌S2308脂多糖篩選獲得一株穩(wěn)定分泌抗流產布魯菌脂多糖抗體的單克隆細胞株,命名為51C,并成功制備流產布魯菌脂多糖單克隆抗體。交叉反應性實驗表明,本研究得到的單克隆抗體與流產布魯菌S2308、豬種布魯菌1330的脂多糖均有良好的反應性,與馬耳他布魯菌16M、大腸桿菌0157和鼠傷寒沙門菌SL1344的脂多糖不反應,與小腸結腸耶爾森菌0:9型的脂多糖有輕微交叉反應。因此,推測本研究獲得的單克隆抗體作用表位為脂多糖的A表位。本研究為進一步建立高特異性的布魯菌檢測方法奠定基礎。Asp24是布魯菌一個重要的毒力相關蛋白,該蛋白在酸性條件下能夠誘導表達,asp24基因缺失株作為候選疫苗株具有較大的應用潛力。本實驗室在前期研究中發(fā)現,脂多糖O-抗原的胞內聚集可以誘導asp24基因上調表達,協(xié)助布魯菌胞內存活。然而,目前對Asp24蛋白的功能研究并不清楚。本研究首先通過定向缺失技術成功構建了Δasp2 缺失株,生長曲線測定發(fā)現,Asp24對細菌的生長影響不明顯,細胞感染試驗發(fā)現Asp24對布魯菌胞內存活無影響,動物感染試驗證明Asp24與布魯菌在小鼠體內早期建立感染無關,而對布魯菌在小鼠體內建立慢性感染發(fā)揮著重要作用。為進一步探究Asp24的功能,后續(xù)對Asp24的誘導條件和蛋白活性進行深入分析。在熱應激條件下,Asp24的表達出現顯著下調。啟動子活性分析發(fā)現,基因開放閱讀框(ORF)前89bp是asp24基因表達所必需的,而前126bp的片段是布魯菌在酸性環(huán)境下誘導表達asp24基因所必需的。生物信息學分析發(fā)現Asp24蛋白存在3個典型的鈣離子結合型EF手型結構域,為驗證Asp24蛋白的鈣離子結合活性,本研究表達和純化了野生型和突變型Asp24蛋白,采用SDS-PAGE分析Asp24蛋白電泳遷移率變化,發(fā)現Asp24結合鈣離子后電泳速率顯著加快,而突變體蛋白電泳速率無變化,表明Asp24確實可以結合鈣離子。進一步分析發(fā)現,Asp24在布魯菌內的表達水平與布魯菌所處環(huán)境中鈣離子濃度相關,預示細菌可能通過調控Asp24蛋白的表達來維持胞內鈣離子穩(wěn)態(tài)。綜上所述,本研究成功制備了一株抗流產布魯菌脂多糖的單克隆抗體。發(fā)現Asp24蛋白對布魯菌在小鼠體內建立慢性感染發(fā)揮重要作用,發(fā)現Asp24蛋白有結合鈣離子活性,為建立布魯菌特異性的檢測方法奠定了基礎,為研究布魯菌致病機制積累了資料。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61
本文編號：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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61
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