【作者】 王涛；

【导师】 毕丁仁； 周祖涛；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2016， 硕士

【摘要】 鸭疫里氏杆菌病是由鸭疫里氏杆菌引起的鸭、鹅、火鸡等多种禽类的一种细菌性传染病。血清型众多、交叉保护力低、易产生耐药性使该病难以防控。因此,加强对鸭疫里氏杆菌致病机制的研究对防制该病具有重要意义。Por分泌系统（Por secretion system,Por SS）,也称为Ⅸ型分泌系统（T9SS）。广泛分布于拟杆菌门成员中,介导拟杆菌门细菌的运动性及致病机制。生物信息学分析发现RA-YM株基因组存在完整的Por SS系统编码基因。sprT基因是Por SS系统关键组件。因此,本研究构建了RA-YM株sprT基因（RAYM₀3924）缺失株,对缺失株生物学特性开展了研究,具体研究内容如下:1、鸭疫里氏杆菌RA-YM株sprT基因缺失株的构建与鉴定以RA-YM株基因组为模板,扩增sprT基因上、下游同源臂,利用重叠PCR方法将上、下游同源臂与壮观霉素基因融合,构建壮观霉素抗性基因表达盒,经双酶切后与p RE-112连接,构建自杀性质粒p RE-LSR。将构建成功的自杀性质粒p RE-LSR转化E.coli X7213,以含有p RE-LSR的E.coli X7213为供体菌,以RA-YM株为受体菌,采取双亲本接合转移的方法筛选sprT基因缺失株。挑取在含有壮观霉素的TSA平板上生长的单菌落,利用双重PCR鉴定sprT基因缺失突变株,成功构建了RA-YM株sprT基因缺失株ΔsprT。2、鸭疫里氏杆菌RA-YM株sprT基因缺失株生物学特性研究比较了突变株ΔsprT与亲本株RA-YM的生长特性和生化特性,结果显示ΔsprT在对数早期的生长速度快于RA-YM,ΔsprT和RA-YM主要生化特性一致,但ΔsprT不能液化明胶。突变株ΔsprT和RA-YM对高盐耐受浓度一致,均为1.88%。ΔsprT对胆盐的耐受浓度为23.4μg/m L,RA-YM对胆盐的耐受浓度为46.9μg/m L。血清抗性试验结果显示,突变株ΔsprT在12.5%正常鸭阴性血请的存活率显著高于RA-YM。对突变株ΔsprT和亲本株胞外分泌蛋白的明胶酶谱分析发现,ΔsprT不能够分泌明胶酶,表明sprT基因缺失导致某些蛋白分泌障碍。3、鸭疫里氏杆菌RA-YM株sprT基因缺失株对雏鸭致病性的研究比较了ΔsprT与RA-YM对10日龄雏鸭的毒力,经腿部肌肉注射感染,RA-YM株LD50为3.82×104CFU,ΔsprT株LD50为1.61×109CFU,毒力较亲本株下降约4.21×104倍;雏鸭感染试验结果显示,在感染后24h,ΔsprT在血液、肝脏、和脾脏的组织载菌量显著低于RA-YM;在感染后48h,ΔsprT在心脏、肝脏、脾脏和脑的组织载菌量显著低于RA-YM,表明ΔsprT对组织的侵袭力比亲本株下降;病理切片观察发现,ΔsprT对心脏,肝脏、脾脏和脑组织的病变程度显著比亲本株轻微,表明ΔsprT的致病性较RA-YM显著下降。本研究发现sprT基因缺失突变后致其毒力显著下降,表明通过Por SS系统分泌的因子是鸭疫里氏杆菌重要的毒力因子,值得进一步深入研究。更多还原

【Abstract】 Riemerella anatipestifersis is a bacterial disease of domestic ducks, geese, turkey and other poultry caused by Riemerella anatipestifer. It owns series of serotypes but lacks cross-protection between serotypes. In addition, the drug resistance makes it difficult to control. So, to strengthen the research of pathogenic mechanism of the Riemerella anatipestifer will be of great significance to control the the disease.The Por secretion system is named T9 SS, which distributes in the bacteroidetes members mediates the motility of bacteroidetes bacteria and pathogenic mechanism. Bioinformatics analysis shows that RA-YM genome owns complete Por secretion system coding genes. The sprT gene is critical component of the Por secretion system. So, our research constructed sprT gene（RAYM₀3924）deletion mutant strain and made a series of studies on its biological characteristics as follows.1 Construction and identification of sprT gene deletion mutant strain Amplified upstream and downstream homology arms of sprT gene using RA-YM genome. Connect upstream and downstream homology arms and spectinomycin gene together by using overlapping PCR method, and then connected it to plasmid p RE112. The recombination plasmid was transformed into the E.coli X7213. In E.coli X7213, which contains p RE-LSR as donor strain, RA-YM as receptor strain. After transconjugation, select spectacular resistant colonies on the plate, and then using the PCR method to identify whether there has been exchange of homology arms. We constructed sprT gene deletion strain successfully in the end.2 The biological characteristics of sprT gene deletion mutant strain Comparison growth characteristics and biochemical characteristics of RA-YM sprT gene deletion mutant strain with the parent strain RA-YM, we found that the growth rate of sprT gene deletion mutant strain was faster than that of the parent RA-YM strain. Biochemical experiments found that the main biochemical characteristics of the parent strain and the sprT gene deletion mutant strain did not change, while the sprT gene deletion mutant strain did not degrad gelatin. In respect of hypertonic saline, the hypertonic saline tolerance concentration of the sprT gene deletion mutant strain was 1.88% as well as the parent RA-YM strain. The Choline salts tolerance concentration of the sprT gene deletion mutant strain was 23.4 μg/m L, while the parent RA-YM strain was 46.9 μg/m L. Serum survival rate of sprT gene deletion mutant strain in 12.5% normal duck serum was higher than that of the parent RA-YM strain. Analysis of gelatin zymography proved that the sprT gene deletion mutant strain can not secret gelatinase, which showed that some proteins can not be secreted because of the deleted sprT gene.3 The pathogenicity of sprT gene deletion mutant strain and the parent RA-YM strain Comparison the virulence of ducklings aged 10 days between the sprT gene deletion mutant strain and the parent RA-YM strain. All the ducklings were infected through muscle. The LD50 value of parent RA-YM strain was 3.82×104CFU, while the LD50 value of the sprT gene deletion mutant strain was 1.61×109CFU. It showed that the virulence of the sprT gene deletion mutant strain fell about 4.21 × 104 times. It showed that 24 h after infected, the blood, liver and spleen tissue bacterial content of the sprT gene deletion mutant strain was significantly lower than that of the parent RA-YM strain; 48 h after infected, the heart, liver, spleen and brain tissue bacterial content of the sprT gene deletion mutant strain was significantly lower than that of the parent RA-YM strain. It showed that the tissue invasiveness of the sprT gene deletion mutant strain was lower than that of the parent RA-YM strain.The research found that the virulence of the sprT gene deletion mutant strain was significantly decreased. It showed that the secreted factors through Por SS were important virulence factors which deserves further investigation.更多还原