鸡白痢沙门氏菌flgK、fliI基因的必需性及致病作用机理研究

项目名称： 鸡白痢沙门氏菌flgK、fliI基因的必需性及致病作用机理研究

项目编号： No.31272582

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 余旭平

作者单位： 浙江大学

项目金额： 80万元

中文摘要： 前期工作发现与鞭毛及其III型分泌器相关的flgK、fliI基因在鸡白痢沙门氏菌527菌株为必需基因。为确定是其本身必需性还是下游基因极性作用的结果，我们拟对其及同一操纵子的下游基因进行无极性插入倍增突变敲除和必需性测定。进一步，选定flgK、fliI或其极性影响基因，应用RED重组和反向筛选系统，通过构建基因双拷贝敲入中间菌株，获得鸡白痢沙门氏菌527二选定基因的敲除菌株；在摸索敲除菌株体外培养和诱导条件的基础上，测定敲除菌株鞭毛、动力、形态等表型及Biolog生化表型，应用芯片和定量PCR测定敲除菌株全基因组基因表达调控变化，应用细胞和动物试验测定敲除菌株的粘附、侵入、胞内存活等致病性参数，通过定量PCR和组织病理学测定炎性细胞浸润和组织细胞炎性因子表达状况等免疫特征，最终获得flgK、fliI或其极性影响基因的必需性和致病性数据，为鸡白痢沙门氏菌控制药物筛选和免疫制剂研究奠定基础。

中文关键词： 鸡白痢沙门氏菌；flgK；fliI；flhDC；致死性

英文摘要： Previous work revealed that the flgK gene and fliI gene, encoding proteins of the flagellar hook and the flagellar type III secretion apparatus respectively, were showed to be essential in Salmonella enterica serovar Pullorum CVCC527 strain. To determine whether this was because of their own essentiality or of the polar effect of the downstream genes, fragments of the flgK and fliI genes and the downstream genes in the same operon will be cloned into pIDM-T vector and be subjected to insertion-duplication mutagenesis. The essentiality will be determined via the calculation of the integration rate per cell. Next, two knockout strains of the flgK and fliI genes or their polar effected downstream genes of the S. Pullorum CVCC527 strain will be constructed respectively, using RED recombination and counter-selection cassette, after another copy of the selected gene is knocked into the strain firstly. The knockout strains will be subjected to phenotype the fla, mot, che and growth character etc. in the established liquid medium and on the plate as well as to be analyzed with Biolog PM plates. The regulation of gene expression of the knockout strains will be determined with DNA chip and real-time PCR analysis. The pathogenicity of the knockout strains, such as adhesion, invasion and the ability to survive in the cell,

英文关键词： Salmonella enterica serovar Pullorum；flgK；fliI；flhDC；lethal