多杀性巴氏杆菌aroA基因缺失突变株的构建及鉴定

【目的】构建多杀性巴氏杆菌aroA基因缺失突变株,并验证其致病性。【方法】采用正向筛选同源重组技术构建多杀性巴氏杆菌aroA基因缺失突变株,利用PCR对突变株进行鉴定,分析其遗传稳定性、生长特性和致病性。【结果】成功构建多杀性巴氏杆菌aroA基因缺失突变株,连续传代20代,遗传稳定;突变株体外生长曲线表明,在前6h生长速度稍慢于亲本菌,随后两者生长速度一致。对小鼠的致病性试验表明:经腹腔注射aroA基因缺失突变株在1.0×106 CFU对小鼠无致死性,而亲本菌株在1.0×102 CFU对小鼠是致死性的。【结论】本研究获得多杀性巴氏杆菌aroA基因缺失突变株,对小鼠的致病性是减弱的。多杀性巴氏杆菌突变株的构建有助于研究其致病机理。

Construction and characterization of aroA deletion mutant of Pasteurella multocida strain C51-17

[Objective]We constructed Pasteurella multocida strain C51-17 aroA mutant and characterized its virulence.[Methods] Suicide recombinant plasmid of pBC-aroA-Tn903 was constructed with kanamycin gene and transformed into Pasteurella multocida by electroporation.aroA mutant strain of P.multocida was screened by kanamycin resistance and chloromycetin sensitivity and identified by PCR.Virulence of aroA mutant strain was tested in mice by intraperitoneal injection.[Results] The aroA mutant strain of P.multocida C51-17 was successfully constructed.Growth rate of aroA mutant strain was slower within the first 6 hour and similar to the wild-type strain in vitro.The virulence test indicated that aroA mutant strain was significant attenuated and no death was found in mice even inoculated at a dosage of 1.0×106 CFU.[Conclusion] The virulence of aroA mutant strain of P.multocida was highly attenuated in mice.The results of this study will contribute to a better understanding of pathogenesis of P.multocida.