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motA基因在空肠弯曲菌致病性相关趋化和定植中的作用
  • ISSN号:0254-5101
  • 期刊名称:《中华微生物学和免疫学杂志》
  • 分类:R745.43[医药卫生—神经病学与精神病学;医药卫生—临床医学]
  • 作者机构:[1]绍兴文理学院医学院,312000, [2]浙江医学高等专科学校基础医学部, [3]浙江大学医学院病原生物学系,杭州310058
  • 相关基金:基金项目:国家自然科学基金资助项目(30771917)
中文摘要:

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。

英文摘要:

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.

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期刊信息
  • 《中华微生物学和免疫学杂志》
  • 中国科技核心期刊
  • 主管单位:中国科学技术协会
  • 主办单位:中华医学会
  • 主编:
  • 地址:北京市经济技术开发区经海二路38号
  • 邮编:101111
  • 邮箱:cjmia@163.com
  • 电话:010-52245168
  • 国际标准刊号:ISSN:0254-5101
  • 国内统一刊号:ISSN:11-2309/R
  • 邮发代号:2-55
  • 获奖情况:
  • 1995年中华医学会优秀期刊奖,1996年获全国第二届优秀科技期刊奖,中国科协第二届优秀科技期刊奖,中国期刊方阵“双效”期刊
  • 国内外数据库收录:
  • 美国化学文摘(网络版),荷兰文摘与引文数据库,荷兰医学文摘,美国生物科学数据库,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),中国北大核心期刊(2000版)
  • 被引量:16081