| 摘要: |
| 目的 构建尿路致病性大肠杆菌(UPEC)W140菌株的黏附素基因iha缺陷株,分析iha基因缺失对UPEC生物膜形成的影响。方法 采用Red重组系统的3种质粒(pKD46、pKD3、pCP20)敲除W140菌株的iha基因。pKD46表达λ噬菌体的3个重组蛋白,转入W140菌株使其具有同源重组能力。以pKD3携带的两侧带有翻转酶结合位点(FRT)的氯霉素抗性基因替换目的基因iha,再利用表达翻转酶重组酶的质粒pCP20将FRT之间的氯霉素抗性基因删除,从而获得iha基因敲除菌株。采用结晶紫染色法比较野生菌株与基因敲除菌株的细菌生物膜形成差异。结果 PCR验证和DNA测序表明，UPEC W140菌株染色体上的iha基因被成功敲除,得到iha基因缺陷株UPEC W140Δiha。基因缺陷菌株与野生菌株相比生物膜形成能力降低(P<0.01)。 结论 iha基因及其编码产物参与UPEC生物膜的形成,通过抑制该基因的表达有望为控制尿路感染提供新的靶点。 |
| 关键词: 大肠杆菌 生物膜 尿路感染 基因敲除 |
| DOI：10.3724/SP.J.1008.2013.00671 |
| 投稿时间：2012-12-01修订日期：2013-03-19 |
| 基金项目:武警后勤学院科研基金面上项目(WHM201202). |
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| Knockout of the iha gene in uropathogenic Escherichia coli and its influence on biofilm formation |
| ZHANG Xiao-lei,MAO Li-qun,GE Xin*,DONG Xiao-qing |
(Department of Pathogenic Biology and Immunology, Logistics College of Chinese People’s Armed Police Force, Tianjin 300162, China
*Corresponding author.) |
| Abstract: |
| Objective To construct the adhesin gene iha mutant of uropathogenic Escherichia coli (UPEC) strain W140, and analyze the effects of iha deletion on UPEC biofilm formation. Methods Knockout of the iha gene was done with plasmid pKD46, pKD3, and pCP20 of Red recombinant system. pKD46 was transformed into W140 strain to express the three recombination proteins of λ phage. The chloramphenicol resistance gene flanked by flipase recognition target (FRT) of pKD3 was amplified by PCR and transformed into W140 strain to replace the iha gene. Finally pCP20 was transformed into W140 strain and expressed flipase recombinase, which deleted the chloramphenicol resistance gene between FRT sites. The differences of biofilm formation ability between the wild strain and the mutant were measured by crystal violet staining. Results PCR verification and DNA sequencing indicated that the iha gene was successfully knocked out and UPEC W140Δiha was constructed. The biofilm growth level of the mutant was significantly lower than that of wild strain (P<0.01). Conclusion The iha gene and its encoding product are involved in the process of UPEC biofilm formation. Suppressing iha gene may provide a new target for controlling urinary tract infection. |
| Key words: Escherichia coli biofilms urinary tract infection gene knockout |
