两株鸭疫里默氏菌免疫原和ideR基因差异研究
发布时间：2018-11-06 11:55
【摘要】：研究背景:鸭疫里默氏菌(Riemerella anatipestifer,RA)属黄杆菌科(Flavobacteriaceae)里默氏杆菌属(Riemerella),为革兰氏阴性短杆菌,有荚膜、无芽孢、无鞭毛,可引起鸭、鹅、火鸡及其它多种鸟类发生感染。主要感染8周龄以内的家鸭,引起鸭传染性浆膜炎,具有高发病率、高死亡率的特点。产生以心周炎、肝周炎以及气囊炎为主的病理变化。血清型多达21种,相互间缺乏有效的交叉免疫保护。不同血清型或即使同一血清型的不同菌株之间也可能存在较大的毒力变化。一旦鸭群感染此菌,便迅速流行,且很难根除。然而,除已被证实的毒力相关蛋白外膜蛋白A(outer membrane protein A,Omp A)、协同溶血素(cohemolysin protein,CAMP)、载铁体蛋白(siderophone protein,SIP)和Ton B-依赖性受体1(Ton B-dependent receptor 1,Tbd R1)外,关于RA致病的分子机制还知之甚少。本课题组前期从成都某养鸭场分离得到一株RA血清1型SC株,并与在重庆某鸭场分离的RA血清2型AF株进行了比较,研究发现:SC株RA能引起成年产蛋鸭发病甚至死亡而AF株不能,SC株感染鸭的发病率和死亡率均显著高于AF株感染鸭的发病率和死亡率。目前Gen Bank数据库中已经提交了9株RA全基因组序列,其中包括ATCC11845、RA-CH-1、RA-CH-2、DSM 15868、RAGD、RA-SG、CH3、17、153,这些数据对深入研究该致病菌的致病机制与免疫保护性抗原有很大的帮助。研究内容和结果:1、SC ideR与AF ideR基因存在差异根据RA SC和AF全基因组序列,设计一对铁吸收调节子基因(IRONdependent repressor and activator,ide R)引物,采用PCR扩增目的基因,经鉴定正确的产物克隆至p MD18-T载体,序列分析发现,两株菌的ide R基因的开放阅读框均为654 bp,编码217个氨基酸残基。两株菌的ide R基因的编码区存在41处碱基差异,其中6个碱基差异引起了4个氨基酸残基改变,导致蛋白质三级结构的变化。通过Gen Bank分析,AF株ide R序列与其登载的所有RA菌株完全相同,而SC株成为一个独立的分支。2、SC株与AF株RA菌体蛋白ELISA抗体存在差异SC株和AF株RA菌体超声破碎抗原间接ELISA能检测到同源菌株人工感染耐过鸭血清中的抗RA特异抗体。采取同源菌体吸附去除凝集抗体后,ELISA抗体效价下降1~3个滴度,而异源菌的菌体吸附后则抗体效价未发生变化。结果显示,SC株感染耐过鸭血清中ELISA抗体阳性率低,仅为42.9%,效价也较低,为1:2000~8000,而AF株感染耐过鸭血清中ELISA抗体阳性率高,达到83.3%且效价很高,一般为1:4000~16000。3 SC株与AF株RA菌体蛋白差异SC株和AF株菌体超声破碎上清,在SDS-PAGE及western blot分析时,共同显示出2处差异蛋白条带,即SC株约45 KDa及AF株40 KDa特有蛋白条带。质谱分析结果显示,AF差异免疫原性蛋白质为肽基脯氨酸顺反异构酶、假想蛋白、争光霉素C1B水解肽酶、2-型柠檬酸合成酶和4-羟基苯丙酮酸双加氧酶;SC差异免疫原性蛋白质为延伸因子Tu、谷氨酸脱氢酶和假想蛋白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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61
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相关期刊论文 前10条
1 万春和;程龙飞;陈红梅;傅光华;施少华;黄瑜;;鸭疫里默氏菌铁依赖抑制子基因的克隆及序列分析[J];中国畜牧兽医;2014年12期
2 侯湾湾;韩先干;王少辉;王小兰;岳嘉，
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