番茄溃疡病菌多克隆抗体和单克隆抗体的制备与鉴定

发布时间：2018-04-10 04:37

本文选题：番茄溃疡病菌　切入点：多克隆抗体　出处：《兰州大学》2011年硕士论文

【摘要】：目的：番茄溃疡病(Bacterial canker of tomato)是一种严重危害番茄生产的细菌性病害,普遍发生在世界各大番茄主要产区。番茄溃疡病的致病菌是密执安棒杆菌(Clavibacter michiganensis subsp.michiganensis, Cmm)。Cmm主要是通过浸染番茄植株维管束系统从而导致植株干枯致死,由于Cmm具有致病性强、潜伏期长、易于传播等特点,对番茄溃疡病的产量和质量造成了严重损失带来了巨大的经济损失。Cmm病菌的检测对于番茄溃疡病的防治是至关重要的,番茄溃疡病菌的检测方法主要有生物学、血清学、分子生物学等方法,由于血清学检测如酶联免疫方法特异性好、灵敏度高且操作简单而被广泛应用。但是目前国内的番茄溃疡病菌抗体全部依赖国外进口,检测成本较高,使得免疫学方法无法在国内普及运用。为此,我们制备了效价高、特异性好的多克隆抗体和单克隆抗体,其目的是逐渐解决番茄溃疡病菌抗体和酶联免疫试剂盒的国产化问题,打破国外公司在该领域的技术和产品垄断,降低国内该病菌检测成本,促进该类试剂盒在我国的普及运用。方法：用番茄溃疡病菌全菌皮下免疫新西兰兔, ELISA测抗体效价,颈动脉采血,收集血清,对抗体特异性鉴定,最后对制备的多克隆抗体进行纯化。用番茄溃疡病菌全菌皮下免疫BALB/c小鼠制备单克隆抗体,采用B细胞杂交瘤技术,经免疫、融合、间接ELISA筛选和克隆等,获得稳定分泌抗体的阳性杂交瘤细胞株,最后对得到的番茄溃疡病菌的单抗进行效价、特异性、亲和力等鉴定。结果：制备了针对番茄溃疡菌的多克隆抗血清,抗体效价为1：256000,Western Blot分析表明该抗体能特异性识别番茄溃疡病菌；经免疫BALB/c小鼠后获得3株番茄溃疡病菌单抗分别为1A4、1C3和1B7,经亚类鉴定分别是IgM、IgG1、IgG1;纯化腹水间接ELISA效价分别为1：25600、1：12800、1：12800；与其同属不同亚种无明显交叉反应。结论：制备的番茄溃疡病菌多克隆抗体和3株单克隆抗体均具有较高特异性和敏感性,可作为番茄溃疡病菌的检测抗体,其中,1A4的特异性最好。番茄溃疡病菌多克隆抗体和单克隆抗体的获得为进一步研发番茄溃疡病检测试剂盒奠定了基础。
[Abstract]:Objective: the bacterial canker of tomato canker is a kind of bacterial disease which seriously endangers tomato production.The pathogen of tomato canker is Clavibacter michiganensis subsp. michiganensis. Cmm).Cmm is mainly caused by soaking the vascular bundle system of tomato plant to cause the plant to dry to death. Because Cmm has the characteristics of strong pathogenicity, long incubation period, easy to spread, etc.It is very important for the prevention and cure of tomato canker to detect the pathogen of Cmm, which has caused serious loss to the yield and quality of tomato canker. The detection methods of tomato ulcer pathogen mainly include biology and serology.Molecular biology and other methods have been widely used for serological detection such as enzyme linked immunosorbent assay (Elisa) because of its high specificity high sensitivity and simple operation.However, at present, the domestic antibodies against tomato ulcer bacteria all rely on foreign imports, and the cost of detection is high, which makes immunological methods can not be widely used in China.Therefore, we have prepared high titer and good specificity polyclonal antibodies and monoclonal antibodies, the purpose of which is to gradually solve the problem of localization of tomato ulcer pathogen antibodies and enzyme linked immunosorbent kits.To break the monopoly of technology and products of foreign companies in this field, to reduce the cost of detection of this pathogen in China, and to promote the popularization and application of this kind of kit in our country.Methods: new Zealand rabbits were immunized subcutaneously with tomato ulcer bacteria. The antibody titers were measured by ELISA, blood samples were collected from carotid artery, serum was collected, antibody specificity was identified, and the polyclonal antibody was purified.BALB/c mice were immunized subcutaneously with tomato ulcer bacteria to prepare monoclonal antibody. By using B cell hybridoma technique, immunizing, fusion, indirect ELISA screening and cloning, positive hybridoma cell lines secreting antibodies stably were obtained.Finally, the titer, specificity and affinity of the McAb of tomato ulcer pathogen were identified.Results: the polyclonal antiserum against tomato ulcer bacteria was prepared. The titer of the antibody was 1: 256 000. Western Blot analysis showed that the antibody could specifically identify tomato ulcer bacteria.After immunizing BALB/c mice, three McAbs of tomato ulcer pathogen were obtained, which were 1A4C3 and 1B7, respectively, and the titers of purified indirect ELISA from ascites were 1: 25600, 1: 12800 and 112800, respectively, and there was no obvious cross reaction with the same subspecies.Conclusion: the polyclonal antibody and three monoclonal antibodies of tomato ulcer bacteria have high specificity and sensitivity, and can be used as the detection antibody of tomato ulcer bacteria, among which the specificity of 1 A4 is the best.The acquisition of polyclonal antibodies and monoclonal antibodies against tomato ulcer bacteria laid a foundation for further research and development of tomato canker detection kit.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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