布氏杆菌BP26抗原特异单克隆抗体的制备与初步应用

发布时间：2019-04-02 20:54

【摘要】： 布氏杆菌病是由布氏杆菌引起的一种人畜共患病,给畜牧业的发展和人类的健康带来严重的危害。世界统计资料表明,每年因布氏杆菌病造成的经济损失将近30亿美元。由它引发的人类波浪热、慢性感染以及反刍动物流产和睾丸炎等疾病,至今仍无法根治。此外,布氏杆菌还被用作生物武器。由此可见,布氏杆菌对我国经济建设、人民健康生活、国家安全等存在着严重威胁。布氏杆菌病的发病特点是,一旦发病并转入慢性就无法治愈,因此,控制布氏杆菌病最关键的是预防。传统的血清学诊断存在假阳性血清学反应。假阳性的原因是由于布氏杆菌S-LPS与其他一些革兰氏阴性菌表面的LPS的相似结构引起的交叉反应。常用的布氏杆菌病血清学诊断方法无法区分疫苗免疫和自然感染而引起的血清学反应,研制具有标记性的疫苗以及寻找合适的布氏杆菌诊断性抗原一直是研究者的目标。 BP26蛋白是布氏杆菌的一个周质蛋白,是布氏杆菌非常重要的诊断抗原之一,已有很多用此抗原来进行布氏杆菌诊断的报道。BP26蛋白对布氏杆菌的毒力不起决定性的作用,但却是布氏杆菌的一个优势抗原,能够诱导产生高滴度的抗体。BP26的这个特性,为我们研究基因标记疫苗提供了很好的靶标。如果将该抗原进行缺失标记,一方面可能将标记株的毒力降低,另一方面也为建立鉴别诊断方法提供标识序列。本研究通过原核表达系统表达并纯化两种蛋白pET-30a(+)-bp26和pGEX-6P-1-bp26。用重组蛋白pET-30a(+)-bp26免疫6周龄BALB/c小鼠,加强免疫后取脾细胞与SP2/0细胞进行细胞融合。应用重组蛋白pGEX-6P-1-bp26作为包被抗原,采用间接ELISA方法筛选阳性克隆。经4次克隆,获得5株抗体效价较高的抗BP26蛋白特异性单克隆抗体,分别命名为:3AG5、3AG6、3CE10、4EA2和4EF3。亚型分析得出:重链亚型均为IgG1,轻链均为Kappa链。应用大肠杆菌O:157、小肠结肠炎耶尔森菌O:9、沙门氏菌等菌体裂解液作为抗原检测,均无交叉反应。Western blotting特异性检测这5株MAbs,与B.melitensis M5-90天然菌体反应,但是不与M5-90-△26缺失疫苗株反应。这5株单克隆抗体具有高度特异性,在布氏杆菌的检测方面,具有潜在的应用价值。本研究将制备的单克隆抗体初步应用于竞争ELISA中,发现此单克隆抗体对牛种布氏杆菌的检测效果较为理想,有望将其应用到布氏杆菌的临床检测中。可以根据本研究制备的单克隆抗体来建立血清学鉴别诊断方法,以及区分是自然感染还是疫苗免疫,能够为本研究组构建的两株bp26基因缺失标记疫苗株的应用和诊断提供理论基础。
[Abstract]:Brucellosis is a zoonosis caused by Brucella, which brings serious harm to the development of animal husbandry and human health. World statistics show that economic losses from brucellosis are close to $3 billion a year. It causes human wave fever, chronic infections, ruminant miscarriage and orchitis, and so on. Brucella is also used as a biological weapon. It can be seen that Brucella is a serious threat to China's economic construction, people's healthy life, national security and so on. Brucellosis is characterized by the fact that it is impossible to cure brucellosis once it becomes chronic. Therefore, prevention is the key to control brucellosis. There is false positive serological reaction in traditional serological diagnosis. The reason for false positive is the cross-reaction between Brucella S-LPS and the similar structure of LPS on the surface of some other Gram-negative bacteria. The commonly used methods of serological diagnosis of Brucellosis are unable to distinguish between vaccine immunity and serological reactions caused by natural infection. It is always the goal of researchers to develop marked vaccines and find suitable diagnostic antigens of Brucella. BP26 protein is a periplasmic protein of Brucella and is one of the most important diagnostic antigens of Brucella. There have been many reports of using this antigen to diagnose Brucella. BP26 protein has no decisive effect on the virulence of Brucella brucella. However, BP26 is a dominant antigen of Brucella, which can induce the production of high titer antibodies. This characteristic of BP26 provides a good target for our research on gene-labeled vaccines. If the antigen is deleted, the virulence of the marker strain may be reduced on the one hand, and on the other hand, the identification sequence will be provided for the establishment of the differential diagnosis method. In this study, two proteins, pET-30a ()-bp26 and pGEX-6P-1-bp26., were expressed and purified by prokaryotic expression system. The recombinant protein pET-30a ()-bp26 was used to immunize 6-week-old BALB/c mice. Spleen cells were collected and fused with SP2/0 cells after enhanced immunization. The recombinant protein pGEX-6P-1-bp26 was used as the coating antigen and the positive clones were screened by indirect ELISA. After four clones, five high titer monoclonal antibodies against BP26 protein were obtained. The monoclonal antibodies were named 3AG5, 3AG6, 3CE10, 4EA2 and 4EF3.They were named as 3AG5, 3AG6, 3CE10, 4EA2 and 4EF3. Subtype analysis showed that all the heavy chain subtypes were IgG1, light chain and Kappa chain. No cross-reaction was detected by using E. coli O-157, Yersinia enterocolitica, Salmonella and other lysate as antigen. Western blotting was used to detect the specific reaction between the 5 strains of MAbs, and the natural bacteria of B.melitensis M5-90.The results showed that there was no cross-reaction between the 5 strains of E. coli and E. coli. However, it did not react with M5 / 90-26 deletion vaccine strain. These five monoclonal antibodies are highly specific and have potential application value in the detection of Brucella. In this study, the monoclonal antibody was applied to competitive ELISA. It was found that the monoclonal antibody was effective in the detection of Brucella bovis, and it was expected to be applied to clinical detection of Brucella bovis. The method of serological differential diagnosis can be established according to the monoclonal antibody prepared in this study, and the distinction between natural infection and vaccine immunization can be made. It can provide a theoretical basis for the application and diagnosis of two bp26 gene deletion marker vaccine strains constructed by our study group.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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