应用抑制性消减杂交和信号标签诱变技术筛选鸭疫里默氏杆菌的毒力基因

Riemerella anatipestifer infection is one of the most serious infectious diseases confronting the duck industry throughout the world. However, so far little is known about the virulence factors and the molecular basis of the pathogenesis of Riemerella anatipestifer. The full genomes of two strains of Riemerella anatipestifer have been published on GenBank, and these data would be of benefit to screen the genes involved in the virulence from genome-wide analysis. In our preliminary studies, strong variations of virulence as assessed by mortality and morbidity rates in duck infection experiment were observed for the different serotypes or within a given serotype of Riemerella anatipestifer strains. In this study, we would screen the candidate genes involved in the virulence of Riemerella anatipestifer by suppression subtractive hybridization and signature-tagged transposon mutagenesis using highly pathogenic strain Yb2 （serotype 2）. Then 4～6 candidate genes were selected and would be deleted by allelic exchange through a recombinant suicide plasmid, and moreover, a recombinant plasmid based on Riemerella anatipestifer- Escherichia coli shuttle vector was constructed to complement the mutant respectively. Finally,the genes involved in virulence were identified by comparing the biological characteristics of the mutant, the complemented strain and wild type strain Yb2, including the median lethal dose (LD50), bacteria loading in the infected ducks, adherence and invasion capacity to Vero cells. This study may help us to uncover the molecular pathogenesis of Riemerella anatipestifer infection and to develop novel attenuated vaccine candidates.

鸭疫里默氏杆菌感染是当前危害养鸭业最为严重的传染病之一，但目前人们对鸭疫里默氏杆菌的毒力相关基因及分子致病机制等仍知之甚少。鸭疫里默氏杆菌的全基因组序列已经发表，这为从全基因组范围内筛选其毒力相关基因提供了有利条件。在前期的研究中我们发现不同的鸭疫里默氏杆菌菌株对雏鸭的致病性存在着较大的差异。本研究将选择强致病性的血清2型Yb2株为研究对象，先采用抑制性消减杂交和转座子信号标签突变技术相结合的方法来筛选潜在的毒力基因；然后选取4～6个基因用自杀质粒同源重组的方法进行定向缺失，再向突变株中导入基于鸭疫里默氏杆菌-大肠杆菌穿梭载体构建的互补质粒，通过比较突变株、突变株的互补菌株和野生株对雏鸭的半数致死量、感染鸭体内的细菌载量以及对Vero细胞的粘附和入侵能力等生物学特性，对相关的毒力基因进行鉴定。本项目的研究对于解析鸭疫里默氏杆菌的分子致病机制以及研制新型弱毒菌苗等具有重要意义。

鸭疫里默氏杆菌感染是当前危害养鸭业的主要传染病之一。但人们对其毒力相关基因和分子致病机制还知之甚少。本研究采用抑制性消减杂交和转座子信号标签突变技术相结合的方法成功地筛选了一些潜在的毒力相关基因，并对其中的5个基因进行了生物学特性分析。研究结果表明：（1）应用抑制性消减杂交筛选到了35个强毒株HXb2携带而弱毒株NJ-1无的强弱毒株基因组间差异片段。其中的差异基因Riean_1915缺失后，缺失株对8日龄雏鸭的半数致死量比野生株HXb2株升高了约137倍；（2）基因组测序后分析，强毒株HXb2株与弱毒株NJ-1株基因组间有1837个共有基因，有548个HXb2株特异性基因和410个NJ-1株特异性基因；有1523个基因是HXb2株与GenBank中已公开全基因组序列的DSM15868株、CH3株、CH-1株、CH-2株和RA-GD株共有的基因；（3）应用构建WJ4株携带不同信号标签转座子的随机突变库，通过雏鸭感染试验负筛选到了20个与WJ4株在鸭体内存活或致病性相关基因；通过构建鸭疫里默氏杆菌转座子随机突变库和雏鸭感染试验，分别从YZb1和CH3株中筛选到了29个和8个毒力相关的基因；（4）Riean_0060、Riean_1537、sip、tonB1和tonB2与鸭疫里默氏杆菌的致病性相关，且后3个基因与鸭疫里默氏杆菌的获铁、生物被膜的形成、及对Vero细胞的粘附和入侵相关；（5）除了sip和Riean_1915外，目前已鉴定的其他10个毒力相关基因在所检测的不同致病性、不同血清型的鸭疫里默氏杆菌菌株中均携带。本研究的结果为进一步解析鸭疫里默氏杆菌的分子致病机制以及研制新型弱毒菌苗等奠定了基础。