本文選題：鴨疫里默氏桿菌　切入點(diǎn)：脂多糖　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：鴨疫里默氏桿菌(Riemerella anatipestifer,RA)感染能造成雛鴨高死亡率,給世界各國(guó)養(yǎng)鴨業(yè)造成巨大損失。本研究成功制備了RA脂多糖(Lipopolysaccharide,LPS)單克隆抗體,并用于對(duì)RA LPS突變株篩選、鑒定及其生物學(xué)特性分析。共篩選獲得2株LPS突變株,并對(duì)突變株的免疫效果作了評(píng)估。本研究結(jié)果表明完整的LPS結(jié)構(gòu)對(duì)RA的毒力是必需的。本研究包括以下3部分內(nèi)容:本試驗(yàn)旨在研制鴨疫里默氏桿菌脂多糖(LPS)單克隆抗體。將鴨疫里默氏桿菌CH3作為免疫原免疫BALB/c小鼠,經(jīng)過(guò)3次免疫后,取免疫小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行細(xì)胞融合,將熱酚水法抽提的CH3 LPS作為包被抗原,利用間接ELISA篩選陽(yáng)性細(xì)胞株并進(jìn)行3次亞克隆后制備小鼠腹水單克隆抗體。共獲得2株穩(wěn)定分泌鴨疫里默氏桿菌CH3 LPS單克隆抗體的雜交瘤細(xì)胞株,分別命名為7H1和8A9。2株單克隆抗體均為Ig G1亞類。腹水單克隆抗體ELISA效價(jià)分別為1:12800和1:6400。應(yīng)用玻片凝集試驗(yàn)和懸浮熒光試驗(yàn)檢測(cè)2株單克隆抗體與國(guó)內(nèi)流行血清型1、2和10型鴨疫里默氏桿菌菌株的反應(yīng)性,結(jié)果表明2株單抗與血清1型菌株WJ4發(fā)生特異性反應(yīng),而與鴨疫里默氏桿菌血清2型菌株NJ-3及血清10型菌株HXb2無(wú)反應(yīng)性。本研究成功制備了穩(wěn)定性好、特異性強(qiáng)的LPS單克隆抗體,為篩選RA LPS缺失株和分析RA LPS的結(jié)構(gòu)提供了保障。本研究中,用RA LPS單抗8A9篩選RA轉(zhuǎn)座子隨機(jī)突變庫(kù),獲得突變株CH3?M949_1556,間接ELISA試驗(yàn)表明該突變株與單抗8A9呈陰性反應(yīng)。動(dòng)物試驗(yàn)表明該突變株半數(shù)致死量(LD50)大于1010CFU,較野生株CH3(LD50=2×108)減毒50倍以上。突變株CH3?M949_1556感染雛鴨血液載菌量與野生株感染雛鴨相比顯著下降。突變株CH3?M949_1556對(duì)于體外補(bǔ)體介導(dǎo)的殺菌作用較CH3更敏感。另外,CH3?M949_1556對(duì)Vero細(xì)胞的粘附、入侵能力增強(qiáng)。CH3?M949_1556油乳劑滅活苗免疫雛鴨后能對(duì)RA血清1型(WJ4)、血清2型(Yb2)及血清10型(HXb2)菌株產(chǎn)生較強(qiáng)的保護(hù)力,證明突變株CH3?M949_1556能對(duì)RA血清1、2、10型產(chǎn)生廣譜保護(hù)作用。試驗(yàn)結(jié)果表明M949_1556基因與RA的抗原性和致病性相關(guān)。本研究中,用RA LPS單抗8A9篩選RA轉(zhuǎn)座子隨機(jī)突變庫(kù),獲得突變株RA-M1,對(duì)其鑒定后發(fā)現(xiàn)突變基因是編碼糖基轉(zhuǎn)移酶的M949_1603基因�；蚍植紮z測(cè)表明該基因?yàn)镽A血清1型菌株所特有。間接ELISA試驗(yàn)中該突變株不與脂多糖單抗8A9反應(yīng)。與抗RA CH3兔血清進(jìn)行Western blotting試驗(yàn),結(jié)果顯示RA-M1 LPS與CH3 LPS相比,代表LPS O抗原部分的梯狀條帶出現(xiàn)了缺失現(xiàn)象。與野生株CH3相比,RA-M1毒力顯著下降、對(duì)血清殺菌更敏感。另外,我們對(duì)RA-M1滅活疫苗的交叉保護(hù)效果進(jìn)行了評(píng)估。二次免疫后對(duì)血清1型(WJ4)、血清2型(Yb2)和血清10型(HXb2)強(qiáng)毒攻擊產(chǎn)生100%的保護(hù)率,且心、肝、脾和腦組織均無(wú)剖檢病變。抗體效價(jià)檢測(cè)表明免疫后針對(duì)三種攻毒菌株的ELISA效價(jià)均可高達(dá)1:12800;細(xì)胞因子檢測(cè)結(jié)果表明免疫鴨血清中白介素2(IL-2)和白介素4(IL-4)的產(chǎn)生水平升高,而γ干擾素(IFN-γ)的水平降低。試驗(yàn)結(jié)果表明M949_1603基因?yàn)檠?型型特異性基因,該基因與RA LPS的O-抗原合成相關(guān)。RA-M1可用作新型疫苗候選株。
[Abstract]:Riemerella anatipestifer (Riemerella anatipestifer, RA) infection can cause high mortality in ducklings, causing huge losses to the world breeding. RA prepared in this study were successfully lipopolysaccharide (Lipopolysaccharide, LPS) monoclonal antibody, and used for screening of mutants of RA LPS analysis, identification and biological characteristics of 2 strains were obtained. The LPS mutation strain, and the immune effect of mutant strains were evaluated. The results of this study show that the complete structure of the LPS of RA virulence is required. This study includes the following 3 parts: the development of Riemerella Bacillus lipopolysaccharide (LPS) to test the monoclonal antibody. The Riemerella anatipestifer CH3 as immunogen BALB/c mice, after 3 times of immunization, cell fusion of immunized mouse spleen cells and myeloma cells SP2/0, the hot phenol water extracted CH3 LPS as antigen screening positive cells by indirect ELISA Cell lines and 3 subclones after preparation of mouse ascites monoclonal antibody. There were 2 strains secreting antibody of Riemerella anatipestifer CH3 LPS monoclonal hybridoma cell lines, named 7H1 and 8A9.2 monoclonal antibodies were Ig subtype G1. Reaction ascites monoclonal antibody ELISA titer was 1:12800 and 1:6400. respectively. The application of slide agglutination test and suspension immunofluorescence assay of 2 monoclonal antibodies and popular serotype 1,2 and 10 riemerellaanatipestifer strains, the results showed that 2 strains of monoclonal antibody and serotype 1 strain WJ4 had specific reaction with duck disease in silent Salmonella serum NJ-3 2 strain and serotype 10 strain HXb2 no response. This study successfully prepared good stability and specificity of LPS monoclonal antibody, provide a guarantee for the screening of RA structure of LPS mutant and RA LPS analysis. In this study, using RA LPS RA to screening of monoclonal antibody 8A9 Transposon random mutation library, the mutant strain of CH3? M949_1556, indirect ELISA test showed that the mutant and 8A9 antibody negative. Animal experiments showed that the mutant median lethal dose (LD50) than 1010CFU, compared with wild strain CH3 (LD50=2 * 108) more than 50 times less toxic. The mutant CH3 M949_1556 infection of young duck? Liquid microbial load and wild strains of Ducklings Infected markedly decreased. The mutant CH3? M949_1556 is more sensitive to the bactericidal effect of in vitro complement mediated with CH3. In addition, CH3? M949_1556 on Vero cell adhesion, invasion ability of.CH3? M949_1556 oil emulsion inactivated vaccine to ducklings RA serotype 1 (WJ4), serotype 2 (Yb2) and serotype 10 (HXb2) strains produce strong force protection, the mutant CH3? M949_1556 can produce broad-spectrum protective effect on serum RA 1,2,10. Experimental results show that the antigen and pathogenicity of M949_1556 gene associated with RA in this study. Screening, RA transposon random mutation library using RA LPS monoclonal antibody 8A9, the mutant strain RA-M1, the mutant gene was identified encoding glycosyltransferase enzyme M949_1603 gene. Gene distribution showed that the gene for RA specific serotype 1 strain. The mutant with lipopolysaccharide monoclonal antibody 8A9 reaction of the indirect ELISA test Western. Blotting test and anti RA rabbit serum CH3, LPS and CH3 results showed that compared to RA-M1 LPS, a representative of LPS O antigen ladder shaped part of the band have lost. Compared with wild-type CH3, RA-M1 virulence significantly more sensitive to serum bactericidal. In addition, we cross protection effect of inactivated vaccine of RA-M1 were evaluated. After the two immunization against serotype 1 (WJ4), serotype 2 (Yb2) and serotype 10 (HXb2) rate, the protection of virulent attack 100% and the heart, liver, spleen and brain tissue showed no pathological changes showed that the antibody titer after immunization. For the three kinds of virulent strains of the ELISA titer can be up to 1:12800; the detection results show that the cytokine interleukin 2 immunity in duck serum (IL-2) and interleukin 4 (IL-4) increased production levels, and interferon gamma (IFN- gamma) levels decreased. The test results show that the M949_1603 gene as serotype 1 type specific the gene, RA gene and LPS O- antigen associated with the synthesis of.RA-M1 can be used as a new vaccine candidate strains.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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本文編號(hào)：1603673