抗创伤弧菌单克隆抗体的制备与鉴定
发布时间：2019-04-26 14:19
【摘要】： 创伤弧菌(Vibrio vulnificus)是一种嗜盐弧菌,广泛存在于海水及海产品中,人体可通过生食海鲜或经过肢体破损创口接触海水、海产品而受其感染。常见于海上劳作的渔民及驻海演习人员从而引起败血症和软组织感染。本菌对于糖尿病、酒精性肝病、肝硬化、肝炎、肝功能障碍患者的危害相当严重,可引起创伤弧菌脓毒血症,起病急,进展迅速,死亡率高[1]。快速准确的早期诊断是指导临床正确用药从而达到理想治疗效果的有力保证;同时,创伤弧菌的治疗手段仍为经验性的抗生素应用,然而,随着细菌对抗生素逐渐出现的不同程度的耐药现象,找到一种非大剂量应用抗生素的治疗也成为亟待解决的问题。单克隆抗体用于疾病的诊断及治疗已在医学领域得到广泛开展,并收到了很好的效果。作为我国海域主要致病菌之一的创伤弧菌,至今未见有诊断与治疗的单克隆抗体制剂问世,为了解决该菌感染的诊断及防治问题,本研究对其单克隆抗体进行了研究。具体试验方法如下: (1)通过动物免疫、细胞融合筛选克隆化及ELISA技术制备并鉴定抗创伤弧菌的单克隆抗体。获得了2株稳定分泌抗创伤弧菌的杂交瘤细胞株,命名为1A2(IgG1)和2D6 (IgG2a)。经测定杂交瘤细胞培养上清及其诱生的腹水mAb效价分别为1×10-2 ,1×10-3和1×10-4 , 1×10-5。经交叉试验,2D6与溶藻弧菌、迟缓爱德华菌、鳗弧菌、温和气单胞菌以及嗜水气单胞菌均不产生交叉反应,可以应用于创伤弧菌感染的快速检测。 (2)以创伤弧菌分别感染乳鼠为模型,对1A2和2D6的治疗保护效果进行研究。结果显示: 1A2和2D6均能提高受创伤弧菌感染的乳鼠的存活率,分别为62.50%和37.50%,1A2具有明显的保护作用。 本实验通过杂交瘤技术共获得2株分泌抗创伤弧菌单克隆抗体的杂交瘤细胞株1A2和2D6。2株杂交瘤细胞的培养上清及腹水能在较长时间内基本保持稳定。其中,1A2的培养上清、腹水效价及亲和力均高于2D6;2D6与溶藻弧菌、迟缓爱德华氏菌、鳗弧菌、温和气单胞菌以及嗜水气单胞菌均不产生交叉反应,可以应用于创伤弧菌感染的快速检测。综上,单克隆抗体2D6在快速检测创伤弧菌感染方面具有重要意义,而1A2有望在治疗方面得以进一步的研究。本实验制备的抗创伤弧菌单克隆抗体为开展海上工作人员遇险后快速准确的检测奠定了实验基础,并且为进一步对于创伤弧菌感染治疗的研究提供了理论依据,不仅有着重要的实验意义,并且具有较高的实用价值。
[Abstract]:Vibrio vulnificus (Vibrio vulnificus) is a halophilic vibrio, which is widely found in sea water and marine products. The human body can be infected by raw seafood or contact with sea water through broken limbs. Fishermen and drills who work at sea often cause septicemia and soft tissue infection. This bacterium is harmful to diabetes mellitus, alcoholic liver disease, liver cirrhosis, hepatitis and hepatic dysfunction. It can cause vibrio vulnificus sepsis, acute onset, rapid progress and high mortality [1]. Rapid and accurate early diagnosis is a powerful guarantee to guide clinical correct use of drugs so as to achieve an ideal therapeutic effect. At the same time, the treatment of Vibrio vulnificus is still an empirical application of antibiotics. However, with the increasing resistance of bacteria to antibiotics, finding a non-large dose treatment of antibiotics has become an urgent problem to be solved. Monoclonal antibodies have been widely used in the diagnosis and treatment of diseases. Vibrio vulnificus, one of the main pathogenic bacteria in the sea area of China, has not been found in the field of diagnosis and treatment. In order to solve the problem of diagnosis and prevention of the infection of Vibrio vulnificus, the monoclonal antibody of Vibrio vulnificus was studied in this study. The specific methods were as follows: (1) Monoclonal antibodies against Vibrio vulnificus were prepared and identified by animal immunization, cell fusion screening and ELISA. Two hybridoma cell lines stably secreting anti-Vibrio vulnificus were obtained and named as 1A2 (IgG1) and 2D6 (IgG2a). The mAb titers of hybridoma cell culture supernatant and ascites were 1 脳 10 ~ (2), 1 脳 10 ~ (- 3) and 1 脳 10 ~ (- 4), and 1 脳 10 ~ (- 5), respectively. 2D6 did not cross-react with Vibrio alginolyticus, Edelia tarda, Vibrio anguillarum, Aeromonas miliformis and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. (2) the therapeutic and protective effects of 1A2 and 2D6 in neonatal rats infected with Vibrio vulnificus were studied. The results showed that both 1A2 and 2D6 could increase the survival rate of newborn mice infected with Vibrio vulnificus by 62.50% and 37.50% respectively. 1A2 had obvious protective effect. In this study, two hybridoma cell lines 1A2 and 2D6.2 secreting monoclonal antibodies against Vibrio vulnificus were obtained by hybridoma technique. The supernatant and ascites of hybridoma cells were stable for a long time. Among them, the titer and affinity of 1A2 in culture supernatant were higher than 2D6; 2D6 did not cross-react with Vibrio alginolyticus, Edwards tarda, Vibrio eel, Aeromonas thermophilus and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. In summary, monoclonal antibody 2D6 plays an important role in rapid detection of Vibrio vulnificus infection, and 1A2 is expected to be further studied in the treatment of Vibrio vulnificus. The monoclonal antibodies against Vibrio vulnificus prepared in this experiment have laid an experimental foundation for rapid and accurate detection of Vibrio vulnificus after distress, and provided theoretical basis for further research on the treatment of Vibrio vulnificus infection. It not only has important experimental significance, but also has high practical value.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2466142
[Abstract]:Vibrio vulnificus (Vibrio vulnificus) is a halophilic vibrio, which is widely found in sea water and marine products. The human body can be infected by raw seafood or contact with sea water through broken limbs. Fishermen and drills who work at sea often cause septicemia and soft tissue infection. This bacterium is harmful to diabetes mellitus, alcoholic liver disease, liver cirrhosis, hepatitis and hepatic dysfunction. It can cause vibrio vulnificus sepsis, acute onset, rapid progress and high mortality [1]. Rapid and accurate early diagnosis is a powerful guarantee to guide clinical correct use of drugs so as to achieve an ideal therapeutic effect. At the same time, the treatment of Vibrio vulnificus is still an empirical application of antibiotics. However, with the increasing resistance of bacteria to antibiotics, finding a non-large dose treatment of antibiotics has become an urgent problem to be solved. Monoclonal antibodies have been widely used in the diagnosis and treatment of diseases. Vibrio vulnificus, one of the main pathogenic bacteria in the sea area of China, has not been found in the field of diagnosis and treatment. In order to solve the problem of diagnosis and prevention of the infection of Vibrio vulnificus, the monoclonal antibody of Vibrio vulnificus was studied in this study. The specific methods were as follows: (1) Monoclonal antibodies against Vibrio vulnificus were prepared and identified by animal immunization, cell fusion screening and ELISA. Two hybridoma cell lines stably secreting anti-Vibrio vulnificus were obtained and named as 1A2 (IgG1) and 2D6 (IgG2a). The mAb titers of hybridoma cell culture supernatant and ascites were 1 脳 10 ~ (2), 1 脳 10 ~ (- 3) and 1 脳 10 ~ (- 4), and 1 脳 10 ~ (- 5), respectively. 2D6 did not cross-react with Vibrio alginolyticus, Edelia tarda, Vibrio anguillarum, Aeromonas miliformis and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. (2) the therapeutic and protective effects of 1A2 and 2D6 in neonatal rats infected with Vibrio vulnificus were studied. The results showed that both 1A2 and 2D6 could increase the survival rate of newborn mice infected with Vibrio vulnificus by 62.50% and 37.50% respectively. 1A2 had obvious protective effect. In this study, two hybridoma cell lines 1A2 and 2D6.2 secreting monoclonal antibodies against Vibrio vulnificus were obtained by hybridoma technique. The supernatant and ascites of hybridoma cells were stable for a long time. Among them, the titer and affinity of 1A2 in culture supernatant were higher than 2D6; 2D6 did not cross-react with Vibrio alginolyticus, Edwards tarda, Vibrio eel, Aeromonas thermophilus and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. In summary, monoclonal antibody 2D6 plays an important role in rapid detection of Vibrio vulnificus infection, and 1A2 is expected to be further studied in the treatment of Vibrio vulnificus. The monoclonal antibodies against Vibrio vulnificus prepared in this experiment have laid an experimental foundation for rapid and accurate detection of Vibrio vulnificus after distress, and provided theoretical basis for further research on the treatment of Vibrio vulnificus infection. It not only has important experimental significance, but also has high practical value.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 李晓莉;张以芳;曾令兵;许映芳;肖晶;童亮;;单克隆抗体在水产养殖中的应用[J];动物医学进展;2009年09期
，本文编号：2466142
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