λ Red系统引入抗性的去除与APEC突变株致病作用的放大

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.

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。

以λ&nbsp;Red重组系统构建ompT基因突变株，对其定居、持续能力进行了评价。结果表明，与E058ΔneuC相似，即无抗性突变株E058ΔompT的致病作用被放大了，阐明了同一现象在不同基因中的重现率。构建含意外转录本编码基因的重组表达质粒，对有抗性“中间体”突变株E058ΔneuC∷cat、E058ΔompT∷cat进行拯救，证实部分含1/4 neuC基因、1/4质粒ompT基因的意外转录本确实导致了E058ΔneuC、E058ΔompT致病作用的放大。通过克隆、表达，证明neuC’意外转录子可成功表达相应的蛋白。对能放大无抗突变株致病作用的1/4质粒ompT基因中的酶活性位点关键氨基酸进行点突变，证明单个点突变株的致病性未见明显下降。申请项目无论对λRed重组系统，工作原理深入了解，还是对应用该系统构建突变株和研制活菌苗，均具有重要意义。

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