【摘要】：研究背景:鴨疫里默氏菌(Riemerella anatipestifer,RA)屬黃桿菌科(Flavobacteriaceae)里默氏桿菌屬(Riemerella),為革蘭氏陰性短桿菌,有莢膜、無芽孢、無鞭毛,可引起鴨、鵝、火雞及其它多種鳥類發(fā)生感染。主要感染8周齡以內(nèi)的家鴨,引起鴨傳染性漿膜炎,具有高發(fā)病率、高死亡率的特點。產(chǎn)生以心周炎、肝周炎以及氣囊炎為主的病理變化。血清型多達21種,相互間缺乏有效的交叉免疫保護。不同血清型或即使同一血清型的不同菌株之間也可能存在較大的毒力變化。一旦鴨群感染此菌,便迅速流行,且很難根除。然而,除已被證實的毒力相關(guān)蛋白外膜蛋白A(outer membrane protein A,Omp A)、協(xié)同溶血素(cohemolysin protein,CAMP)、載鐵體蛋白(siderophone protein,SIP)和Ton B-依賴性受體1(Ton B-dependent receptor 1,Tbd R1)外,關(guān)于RA致病的分子機制還知之甚少。本課題組前期從成都某養(yǎng)鴨場分離得到一株RA血清1型SC株,并與在重慶某鴨場分離的RA血清2型AF株進行了比較,研究發(fā)現(xiàn):SC株RA能引起成年產(chǎn)蛋鴨發(fā)病甚至死亡而AF株不能,SC株感染鴨的發(fā)病率和死亡率均顯著高于AF株感染鴨的發(fā)病率和死亡率。目前Gen Bank數(shù)據(jù)庫中已經(jīng)提交了9株RA全基因組序列,其中包括ATCC11845、RA-CH-1、RA-CH-2、DSM 15868、RAGD、RA-SG、CH3、17、153,這些數(shù)據(jù)對深入研究該致病菌的致病機制與免疫保護性抗原有很大的幫助。研究內(nèi)容和結(jié)果:1、SC ideR與AF ideR基因存在差異根據(jù)RA SC和AF全基因組序列,設(shè)計一對鐵吸收調(diào)節(jié)子基因(IRONdependent repressor and activator,ide R)引物,采用PCR擴增目的基因,經(jīng)鑒定正確的產(chǎn)物克隆至p MD18-T載體,序列分析發(fā)現(xiàn),兩株菌的ide R基因的開放閱讀框均為654 bp,編碼217個氨基酸殘基。兩株菌的ide R基因的編碼區(qū)存在41處堿基差異,其中6個堿基差異引起了4個氨基酸殘基改變,導(dǎo)致蛋白質(zhì)三級結(jié)構(gòu)的變化。通過Gen Bank分析,AF株ide R序列與其登載的所有RA菌株完全相同,而SC株成為一個獨立的分支。2、SC株與AF株RA菌體蛋白ELISA抗體存在差異SC株和AF株RA菌體超聲破碎抗原間接ELISA能檢測到同源菌株人工感染耐過鴨血清中的抗RA特異抗體。采取同源菌體吸附去除凝集抗體后,ELISA抗體效價下降1~3個滴度,而異源菌的菌體吸附后則抗體效價未發(fā)生變化。結(jié)果顯示,SC株感染耐過鴨血清中ELISA抗體陽性率低,僅為42.9%,效價也較低,為1:2000~8000,而AF株感染耐過鴨血清中ELISA抗體陽性率高,達到83.3%且效價很高,一般為1:4000~16000。3 SC株與AF株RA菌體蛋白差異SC株和AF株菌體超聲破碎上清,在SDS-PAGE及western blot分析時,共同顯示出2處差異蛋白條帶,即SC株約45 KDa及AF株40 KDa特有蛋白條帶。質(zhì)譜分析結(jié)果顯示,AF差異免疫原性蛋白質(zhì)為肽基脯氨酸順反異構(gòu)酶、假想蛋白、爭光霉素C1B水解肽酶、2-型檸檬酸合成酶和4-羥基苯丙酮酸雙加氧酶;SC差異免疫原性蛋白質(zhì)為延伸因子Tu、谷氨酸脫氫酶和假想蛋白RAYM_00330。
[Abstract]:Background: Riemerella anatipestifer,RA belongs to (Flavobacteriaceae) Riemer's bacilli (Riemerella), is a Gram-negative short bacilli with capsule no spores and no flagella which can cause duck and goose. Turkeys and many other birds are infected. The infection of domestic ducks within 8 weeks of age caused infectious serositis of ducks with high morbidity and mortality. The pathological changes were mainly pericarditis, pericarditis and balloon inflammation. There are as many as 21 serotypes and lack of effective cross-immunological protection between each other. There may be significant virulence changes between different serotypes or even different strains of the same serotype. Once the ducks are infected with the bacterium, it quickly spreads and is difficult to eradicate. However, in addition to the virulence associated protein outer membrane protein A (outer membrane protein Amp A), which is associated with hemolysin (cohemolysin protein,CAMP), ferritin (siderophone protein,SIP) and Ton B- dependent receptor 1 (Ton B-dependent receptor 1 Tbd R1), the virulence associated protein was found to be associated with hemolysin (cohemolysin protein,CAMP), ferritin (siderophone protein,SIP) and Ton B- dependent receptor 1 (Ton B-dependent receptor 1 Tbd R1). Little is known about the molecular mechanism of RA pathogenesis. A RA serotype 1 SC strain was isolated from a duck farm in Chengdu and compared with RA serotype 2 AF strain isolated from a duck farm in Chongqing. The results showed that the incidence and mortality of SC strain RA was significantly higher than that of AF strain in adult laying ducks, but AF strain could not, and the incidence and mortality of SC strain were significantly higher than that of AF strain. Up to now, 9 RA complete genome sequences have been submitted in Gen Bank database, including ATCC11845,RA-CH-1,RA-CH-2,DSM 15868 RAGDX RA-SGN CH3H17153. These data are helpful to further study the pathogenic mechanism and immune protective antigen of the pathogen. Results: 1 the difference between SC ideR and AF ideR gene. According to the whole genome sequence of RA SC and AF, a pair of primers of iron absorption regulator gene (IRONdependent repressor and activator,ide R were designed, and the target gene was amplified by PCR. The correct product was cloned into p MD18-T vector. Sequence analysis showed that the open reading frame of ide R gene of the two strains was 654 bp, encoding 217 amino acid residues. There were 41 nucleotide differences in the coding region of the ide R gene of the two strains, of which 6 nucleotide differences led to the change of 4 amino acid residues and the change of protein tertiary structure. Gen Bank analysis showed that the ide R sequence of the AF strain was identical to that of all the RA strains it carried, while the SC strain became an independent branch. There was difference between SC strain and AF strain in RA protein ELISA antibody. Indirect ELISA could detect anti-RA specific antibody in serum of duck infected with homologous strain SC strain and AF strain. The titer of ELISA antibody decreased by 1 ~ 3 titers after removal of agglutination antibody by homologous bacterial adsorption, but the antibody titer did not change after bacterial adsorption. The results showed that the positive rate of ELISA antibody was only 42.9 and the titer was 1: 2000 ~ 8000 in the serum of the duck infected with SC strain, while the positive rate of ELISA antibody in the serum of the duck infected with AF strain was 83.3% and the titer was very high. The protein difference between 1: 4 000 SC strain and AF strain was generally 1: 4000,16000.3. In the analysis of SDS-PAGE and western blot, there were two differential protein bands, that is, about 45 KDa of SC strain and 40 KDa band of AF strain. The results of mass spectrometry showed that AF differential immunogenicity proteins were peptide proline cis-trans isomerase, hypothetical protein, Lim C 1B hydrolytic peptidase, 2-type citric acid synthase and 4-hydroxyphenylpyruvate dioxygenase. SC differential immunogenicity proteins are extension factors Tu, glutamate dehydrogenase and hypothetical protein RAYM_00330.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S852.61

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本文編號：2314186