鸭疫里默氏杆菌脂多糖单克隆抗体制备、突变株筛选及生物学特性研究

发布时间：2018-03-12 23:00

本文选题：鸭疫里默氏杆菌　切入点：脂多糖　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：鸭疫里默氏杆菌(Riemerella anatipestifer,RA)感染能造成雏鸭高死亡率,给世界各国养鸭业造成巨大损失。本研究成功制备了RA脂多糖(Lipopolysaccharide,LPS)单克隆抗体,并用于对RA LPS突变株筛选、鉴定及其生物学特性分析。共筛选获得2株LPS突变株,并对突变株的免疫效果作了评估。本研究结果表明完整的LPS结构对RA的毒力是必需的。本研究包括以下3部分内容:本试验旨在研制鸭疫里默氏杆菌脂多糖(LPS)单克隆抗体。将鸭疫里默氏杆菌CH3作为免疫原免疫BALB/c小鼠,经过3次免疫后,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,将热酚水法抽提的CH3 LPS作为包被抗原,利用间接ELISA筛选阳性细胞株并进行3次亚克隆后制备小鼠腹水单克隆抗体。共获得2株稳定分泌鸭疫里默氏杆菌CH3 LPS单克隆抗体的杂交瘤细胞株,分别命名为7H1和8A9。2株单克隆抗体均为Ig G1亚类。腹水单克隆抗体ELISA效价分别为1:12800和1:6400。应用玻片凝集试验和悬浮荧光试验检测2株单克隆抗体与国内流行血清型1、2和10型鸭疫里默氏杆菌菌株的反应性,结果表明2株单抗与血清1型菌株WJ4发生特异性反应,而与鸭疫里默氏杆菌血清2型菌株NJ-3及血清10型菌株HXb2无反应性。本研究成功制备了稳定性好、特异性强的LPS单克隆抗体,为筛选RA LPS缺失株和分析RA LPS的结构提供了保障。本研究中,用RA LPS单抗8A9筛选RA转座子随机突变库,获得突变株CH3?M949_1556,间接ELISA试验表明该突变株与单抗8A9呈阴性反应。动物试验表明该突变株半数致死量(LD50)大于1010CFU,较野生株CH3(LD50=2×108)减毒50倍以上。突变株CH3?M949_1556感染雏鸭血液载菌量与野生株感染雏鸭相比显著下降。突变株CH3?M949_1556对于体外补体介导的杀菌作用较CH3更敏感。另外,CH3?M949_1556对Vero细胞的粘附、入侵能力增强。CH3?M949_1556油乳剂灭活苗免疫雏鸭后能对RA血清1型(WJ4)、血清2型(Yb2)及血清10型(HXb2)菌株产生较强的保护力,证明突变株CH3?M949_1556能对RA血清1、2、10型产生广谱保护作用。试验结果表明M949_1556基因与RA的抗原性和致病性相关。本研究中,用RA LPS单抗8A9筛选RA转座子随机突变库,获得突变株RA-M1,对其鉴定后发现突变基因是编码糖基转移酶的M949_1603基因。基因分布检测表明该基因为RA血清1型菌株所特有。间接ELISA试验中该突变株不与脂多糖单抗8A9反应。与抗RA CH3兔血清进行Western blotting试验,结果显示RA-M1 LPS与CH3 LPS相比,代表LPS O抗原部分的梯状条带出现了缺失现象。与野生株CH3相比,RA-M1毒力显著下降、对血清杀菌更敏感。另外,我们对RA-M1灭活疫苗的交叉保护效果进行了评估。二次免疫后对血清1型(WJ4)、血清2型(Yb2)和血清10型(HXb2)强毒攻击产生100%的保护率,且心、肝、脾和脑组织均无剖检病变。抗体效价检测表明免疫后针对三种攻毒菌株的ELISA效价均可高达1:12800;细胞因子检测结果表明免疫鸭血清中白介素2(IL-2)和白介素4(IL-4)的产生水平升高,而γ干扰素(IFN-γ)的水平降低。试验结果表明M949_1603基因为血清1型型特异性基因,该基因与RA LPS的O-抗原合成相关。RA-M1可用作新型疫苗候选株。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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