布氏桿菌BP26抗原特異單克隆抗體的制備與初步應(yīng)用
發(fā)布時(shí)間：2019-04-02 20:54
【摘要】： 布氏桿菌病是由布氏桿菌引起的一種人畜共患病,給畜牧業(yè)的發(fā)展和人類(lèi)的健康帶來(lái)嚴(yán)重的危害。世界統(tǒng)計(jì)資料表明,每年因布氏桿菌病造成的經(jīng)濟(jì)損失將近30億美元。由它引發(fā)的人類(lèi)波浪熱、慢性感染以及反芻動(dòng)物流產(chǎn)和睪丸炎等疾病,至今仍無(wú)法根治。此外,布氏桿菌還被用作生物武器。由此可見(jiàn),布氏桿菌對(duì)我國(guó)經(jīng)濟(jì)建設(shè)、人民健康生活、國(guó)家安全等存在著嚴(yán)重威脅。布氏桿菌病的發(fā)病特點(diǎn)是,一旦發(fā)病并轉(zhuǎn)入慢性就無(wú)法治愈,因此,控制布氏桿菌病最關(guān)鍵的是預(yù)防。傳統(tǒng)的血清學(xué)診斷存在假陽(yáng)性血清學(xué)反應(yīng)。假陽(yáng)性的原因是由于布氏桿菌S-LPS與其他一些革蘭氏陰性菌表面的LPS的相似結(jié)構(gòu)引起的交叉反應(yīng)。常用的布氏桿菌病血清學(xué)診斷方法無(wú)法區(qū)分疫苗免疫和自然感染而引起的血清學(xué)反應(yīng),研制具有標(biāo)記性的疫苗以及尋找合適的布氏桿菌診斷性抗原一直是研究者的目標(biāo)。 BP26蛋白是布氏桿菌的一個(gè)周質(zhì)蛋白,是布氏桿菌非常重要的診斷抗原之一,已有很多用此抗原來(lái)進(jìn)行布氏桿菌診斷的報(bào)道。BP26蛋白對(duì)布氏桿菌的毒力不起決定性的作用,但卻是布氏桿菌的一個(gè)優(yōu)勢(shì)抗原,能夠誘導(dǎo)產(chǎn)生高滴度的抗體。BP26的這個(gè)特性,為我們研究基因標(biāo)記疫苗提供了很好的靶標(biāo)。如果將該抗原進(jìn)行缺失標(biāo)記,一方面可能將標(biāo)記株的毒力降低,另一方面也為建立鑒別診斷方法提供標(biāo)識(shí)序列。 本研究通過(guò)原核表達(dá)系統(tǒng)表達(dá)并純化兩種蛋白pET-30a(+)-bp26和pGEX-6P-1-bp26。用重組蛋白pET-30a(+)-bp26免疫6周齡BALB/c小鼠,加強(qiáng)免疫后取脾細(xì)胞與SP2/0細(xì)胞進(jìn)行細(xì)胞融合。應(yīng)用重組蛋白pGEX-6P-1-bp26作為包被抗原,采用間接ELISA方法篩選陽(yáng)性克隆。經(jīng)4次克隆,獲得5株抗體效價(jià)較高的抗BP26蛋白特異性單克隆抗體,分別命名為:3AG5、3AG6、3CE10、4EA2和4EF3。亞型分析得出:重鏈亞型均為IgG1,輕鏈均為Kappa鏈。應(yīng)用大腸桿菌O:157、小腸結(jié)腸炎耶爾森菌O:9、沙門(mén)氏菌等菌體裂解液作為抗原檢測(cè),均無(wú)交叉反應(yīng)。Western blotting特異性檢測(cè)這5株MAbs,與B.melitensis M5-90天然菌體反應(yīng),但是不與M5-90-△26缺失疫苗株反應(yīng)。這5株單克隆抗體具有高度特異性,在布氏桿菌的檢測(cè)方面,具有潛在的應(yīng)用價(jià)值。 本研究將制備的單克隆抗體初步應(yīng)用于競(jìng)爭(zhēng)ELISA中,發(fā)現(xiàn)此單克隆抗體對(duì)牛種布氏桿菌的檢測(cè)效果較為理想,有望將其應(yīng)用到布氏桿菌的臨床檢測(cè)中�？梢愿鶕�(jù)本研究制備的單克隆抗體來(lái)建立血清學(xué)鑒別診斷方法,以及區(qū)分是自然感染還是疫苗免疫,能夠?yàn)楸狙芯拷M構(gòu)建的兩株bp26基因缺失標(biāo)記疫苗株的應(yīng)用和診斷提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
本文編號(hào)：2452912
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
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