尿路致病性大肠杆菌iha基因敲除及其对细菌生物膜形成的影响

发布时间：2018-07-21 08:45

【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(P0.01)。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。
[Abstract]:Objective to construct the iha deficient strain of urinary tract pathogenic Escherichia coli (UPECW140) and analyze the effect of iha gene deletion on biofilm formation. Methods three kinds of plasmids (pKD46, pKD3, pCP20) of Red recombination system were used to knockout the iha gene of W140 strain. PKD46 expressed three recombinant proteins of 位 phage. The chloramphenicol resistance gene with reverse enzyme binding site (FRT) on both sides of pKD3 was used to replace the target gene iha. then the recombinant plasmid pCP20 expressing reverse enzyme was used to delete the chloramphenicol resistance gene between FRTs and obtain the knockout strain of iha gene. The difference of bacterial biofilm formation between wild strain and gene knockout strain was compared by crystal violet staining. Results PCR and DNA sequencing showed that the iha gene on the chromosomes of UPEC W140 was successfully knocked out and the iha gene deficient strain UPEC W140 螖 IIA was obtained. The ability of biofilm formation of gene deficient strain was lower than that of wild strain (P0.01). Conclusion iha gene and its encoding products are involved in the formation of biofilm, which may provide a new target for the control of urinary tract infection by inhibiting the expression of this gene.
【作者单位】：武警后勤学院病原生物与免疫学教研室;
【基金】：武警后勤学院科研基金面上项目(WHM201202)~~
【分类号】：R378

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