motA基因在空肠弯曲菌致病性相关趋化和定植中的作用

Contribution of motA gene in pathogenesis-associated chemotaxis and colonization of Campylobacter jejuni

目的确定鞭毛马达蛋白（flagellarmotorprotein）MotA编码基因在空肠弯曲菌（Campylobacterjejuni）致病性相关趋化和定植中的作用。方法采用PCR扩增motA基因以及用于motA基因敲除的Kan。基因和plus—motA基因片段，目的扩增产物克隆后测序。根据同源重组原理，构建空肠弯曲菌NCTClll68株motA基因自杀质粒（pBlueskrit-Ⅱ-SKmotA-kan）和motA基因敲除突变株（motA-）。采用半固体平板迁移试验、基于硬琼脂平板（hardagarplus，HAP）的脱氧胆酸钠（SDC）体外趋化试验、小鼠空肠定植试验，了解空肠弯曲菌motA-突变株和野生株鞭毛动力、SDC趋化和BALB／c-ByJ小鼠空肠内定植能力差异性。结果所克隆的空肠弯曲菌motA基因核苷酸和氨基酸序列与已报道的相应序列相似性为100％。PCR和测序及含抗生素培养基连续传代培养结果证实，自杀质粒和motA-突变株构建成功。motA-突变株在半固体琼脂平板上的菌斑直径、在HAP上对0．2mol／LSDC趋化聚集环直径、黏附于小鼠空肠黏膜表面以及空肠内容物中motA-突变株数量均明显少于野生株（P〈0．05）。结论本研究成功构建了空肠弯曲菌motA基因敲除突变株。motA基因是空肠弯曲菌鞭毛动力以及致病性相关趋化和定植的必需基因。

Objective To determine the role of flagellar motor protein MotA in the pathogenesisassociated chemotaxis and colonization of Campylobacter jejuni. Methods The motA gene as well as Kan＇ gene and plus-motA gene segments for motA gene knock-out were amplified by PCR and the target amplification fragments were sequenced after cloning. A suicide plasmid （ pBlueskrit-Ⅱ-SKmotA-kan ） and a motA gene knock-out mutant （motA-） were constructed based on homologious recombination. By using semisolid plate migration test, hard agar plus （HAP） -based chemotactic test towards sodium deoxycholate （ SDC ） in vitro, and jejunal colonization test in BALB/c-ByJ mice were performed to determine the differences of flagellar motility, chemotaxis towards SDC and colonization in murine jejunum between motA- mutant and wild-type strain. Results The nucleotide and amino acid sequences of the cloned motA gene were 100% identical to the reported corresponding sequences. The results of PCR, sequencing and continuous passage culture in an- tibiotics-contained medium demonstrated that both suicide plasmid and motA- mutant were successfully generated. The diameters of clonies on semisolid plate and 0.2 mol/L SDC-induced chemotactic rings in HAP as well as the bacterial numbers adhering to the surface of murine jejunal mucosa and in jejunal content of motA - mutant were significantly less than those of wild-type strain（P 〈 0.05 ）. Conclusion A motA gene knock-out mutant of C. jejuni was successfully constructed in this study, motA is an essential gene for flagellar motility, pathogenesis-associated chemotaxis and colonization of C. jejuni.