以λRed重组系统突变禽致病性大肠杆菌(APEC)E058株neuC基因，获得中间突变株E058△neuC∷cat和最终突变株E058△neuC，相对于E058△neuC∷cat，E058△neuC在攻毒鸡的肝、肺和肾中的致病作用被显著放大（P<0.05），RT-PCR结果表明，E058△neuC∷cat中的neuC已失活，而E058△neuC却能产生意外转录本（neuC′），推测突变株致病作用的放大与neuC ′的产生有关。申请项目以同样方法突变APEC E058株ompT基因，以证明上述现象的普遍性；以编码意外转录本neuC ′、ompT ′的基因拯救E058△neuC∷cat和E058△ompT∷cat，考察其致病作用的放大；对意外转录本的编码基因进行连续突变，探讨突变株致病作用放大的分子机制。本项目对深入了解λRed系统的工作原理和应用该系统构建突变株及研制活菌苗，均具有重要意义。

The neuC gene of avian pathogenic Escherichia coli (APEC) strain E058 was knocked out by using λRed system, the intermediate mutant E058△neuC∷cat and the final mutant E058△neuC without any antibiotic resistances were obtained. In a colonization and persistence assay, the colonies recovered from the liver, lung and kidney of birds challenged with E058△neuC were increased significantly compared to those from birds challenged with E058△neuC∷cat (P<0.05)..In the reverse transcription-polymerase chain reaction (RT-PCR), an unexpected transcriptome （neuC′）composed of disrupted neuC, primers of the chloramphenicol resistance gene and the FLP recognition target (FRT) sites was generated in the mutant E058△neuC while not in the mutant E058△neuC∷cat. It was postulated that the enhanced colonization and persistence ability of E058△neuC compared to that of E058△neuC∷cat was related to the production of the unexpected transcriptome neuC′. In this project, the ompT gene of APEC E058 strain will be knocked out also using λRed system, and the virulence of E058△ompT and E058△ompT∷cat will be evaluated in the colonization and persistence assay in vivo. It will be proved that the enhanced ability of colonization and persistence exhibited in the mutant E058△neuC is not an accident case, and will be the same in the mutant E058△ompT. The mutant APEC E058△neuC∷cat or E058△ompT∷cat will be complemented with the gene encoding the unexpected transcriptome neuC′or ompT′, respectively. The pathogenicity of the complemented strains will be also assessed in the colonization and persistence assay, and the resuts are going to be the direct evidence of the postulation that the enhanced virulence of E058△neuC or E058△ompT in vivo is related to the production of the unexpected transcriptome neuC′or ompT′, respectively. To probe the molecular mechanism of the enhanced virulence of E058△neuC and E058△ompT, the genomic fragment encoding the unexpected transcriptome neuC′or ompT′, will be knocked out by allelic exchange using the kanamycin or zeocin resistance cassette, and the key sites of the focused region were localized by point mutations. The virulence of a series of mutants mentioned above will be also determined in the colonization and persistence assay. The results of the project help us to understand the working principle of the λ Red system, and to construct mutants and generate live bacterial vaccines of APEC efficiently.