發(fā)布時間：2018-03-12 23:00

  本文選題：鴨疫里默氏桿菌　切入點：脂多糖　出處：《中國農業(yè)科學院》2015年碩士論文　論文類型：學位論文

【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RA LPS突變株篩選、鑒定及其生物學特性分析。共篩選獲得2株LPS突變株,并對突變株的免疫效果作了評估。本研究結果表明完整的LPS結構對RA的毒力是必需的。本研究包括以下3部分內容:本試驗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,經過3次免疫后,取免疫小鼠脾細胞與SP2/0骨髓瘤細胞進行細胞融合,將熱酚水法抽提的CH3 LPS作為包被抗原,利用間接ELISA篩選陽性細胞株并進行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效價分別為1:12800和1:6400。應用玻片凝集試驗和懸浮熒光試驗檢測2株單克隆抗體與國內流行血清型1、2和10型鴨疫里默氏桿菌菌株的反應性,結果表明2株單抗與血清1型菌株WJ4發(fā)生特異性反應,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無反應性。本研究成功制備了穩(wěn)定性好、特異性強的LPS單克隆抗體,為篩選RA LPS缺失株和分析RA LPS的結構提供了保障。本研究中,用RA LPS單抗8A9篩選RA轉座子隨機突變庫,獲得突變株CH3?M949_1556,間接ELISA試驗表明該突變株與單抗8A9呈陰性反應。動物試驗表明該突變株半數致死量(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對于體外補體介導的殺菌作用較CH3更敏感。另外,CH3?M949_1556對Vero細胞的粘附、入侵能力增強。CH3?M949_1556油乳劑滅活苗免疫雛鴨后能對RA血清1型(WJ4)、血清2型(Yb2)及血清10型(HXb2)菌株產生較強的保護力,證明突變株CH3?M949_1556能對RA血清1、2、10型產生廣譜保護作用。試驗結果表明M949_1556基因與RA的抗原性和致病性相關。本研究中,用RA LPS單抗8A9篩選RA轉座子隨機突變庫,獲得突變株RA-M1,對其鑒定后發(fā)現突變基因是編碼糖基轉移酶的M949_1603基因�；�因分布檢測表明該基因為RA血清1型菌株所特有。間接ELISA試驗中該突變株不與脂多糖單抗8A9反應。與抗RA CH3兔血清進行Western blotting試驗,結果顯示RA-M1 LPS與CH3 LPS相比,代表LPS O抗原部分的梯狀條帶出現了缺失現象。與野生株CH3相比,RA-M1毒力顯著下降、對血清殺菌更敏感。另外,我們對RA-M1滅活疫苗的交叉保護效果進行了評估。二次免疫后對血清1型(WJ4)、血清2型(Yb2)和血清10型(HXb2)強毒攻擊產生100%的保護率,且心、肝、脾和腦組織均無剖檢病變�？贵w效價檢測表明免疫后針對三種攻毒菌株的ELISA效價均可高達1:12800;細胞因子檢測結果表明免疫鴨血清中白介素2(IL-2)和白介素4(IL-4)的產生水平升高,而γ干擾素(IFN-γ)的水平降低。試驗結果表明M949_1603基因為血清1型型特異性基因,該基因與RA LPS的O-抗原合成相關。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.

【學位授予單位】：中國農業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

【參考文獻】

相關期刊論文 前8條

1 陳昌福,曾妍雄,楠田理一;接種不同種類病原菌脂多糖對翹嘴鱖細菌性敗血癥的免疫保護力比較[J];華中農業(yè)大學學報;2000年04期

2 程安春,汪銘書,陳孝躍,鐘妮娜,賴為民;鴨疫巴氏桿菌的分離和鑒定[J];四川畜牧獸醫(yī);1996年02期

3 郝貴杰;沈錦玉;潘曉義;曹錚;尹文林;;抗哈維氏弧菌脂多糖單克隆抗體的制備及其鑒定[J];上海水產大學學報;2008年03期

4 蘇敬良,黃瑜,呂艷麗,孫艷爭,郭玉璞;鴨傳染性漿膜炎油佐劑滅活疫苗的研究[J];中國獸醫(yī)科技;2000年06期

5 胡清海,張知良,苗晉鋒,劉岳龍,劉曉文,丁鏟;江蘇安徽兩省鴨疫里默氏桿菌病的流行病學調查研究[J];中國獸醫(yī)科技;2001年08期

6 劉紅亮;李克生;陳學忠;杜惠芬;連曉雯;魯學萍;袁明;;大腸桿菌O157 LPS單克隆抗體的制備及ELISA檢測方法的建立[J];中國獸醫(yī)科學;2011年12期

7 張大丙,郭玉璞;不同血清型鴨疫里氏桿菌分離株的部分特性比較[J];中國獸醫(yī)雜志;1999年10期

8 侯灣灣;韓先干;王少輝;王小蘭;岳嘉，

本文編號：1603673