摘要
为研究荚膜在禽多杀性巴氏杆菌(Pasteurella multocida)致病过程中的作用,本研究利用同源重组基因敲除技术构建禽P.multocida C48-3株荚膜多糖输出蛋白基因hexABC的缺失突变株,并以小鼠为动物模型检测荚膜缺陷对细菌毒力的影响。PCR、RT-PCR和DNA测序结果均表明253 bp的hexC基因下游序列、798 bp的hexB基因全序列和290 bp的hexA基因上游序列完全被四环素抗性基因替代,表明构建了基因敲除突变株C48-3ΔhexABC。电镜观察结果表明突变株荚膜合成能力缺失,对小鼠的致病性试验结果显示突变株毒力基本丧失。本研究获得的无荚膜突变株为进一步研究禽P.multocida的致病机理奠定基础。
To further investigate the role of capsule involved in virulence of avian Pasteurella multocida, a mutant of hexABC gene deletion of P. multocida （C48-3AhexABC） was generated by homologous recombination. First of all, a recombinant knockout plasmid of pWSK29AhexABC was constructed consisting of tetracycline resistance （TetR） cassette with the flanking homology regions of hexABC genes based on pWSK29 vector, which was transformed into competent P. multocida C48-3 accordingly. The hexABC genes replaced with TetR cassette in C48-3AhexABC was confirmed by PCR, RT-PCR of the target gene. Furthmore, the acapsular phenotype of the mutant was observed under electron microscopy. Eventually, the pathogenicity of the wild-type strain and the mutant strain were tested in mice, and the results demonstrated that the C48-3AhexABC was significantly attenuated and no deaths occurred following intraperitoneal injection of 345 cfu which was equivalent to 50 LD50 of the wild-type strain. In conclusion, the construction of C48-3AhexABC would facilitate further study on pathogenesis of the bacteria.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2012年第8期597-601,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30972206)
湖南省研究生创新项目(CX2011B397)
