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| 1 | 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显示文摘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。 | 张晓雷 毛立群 葛新 董小青 | 2013 | 第二军医大学学报2013，34，6： | 3 |
| 2 | 尿道致病性大肠埃希菌感染对人膀胱上皮细胞基因表达谱的影响显示文摘目的 研究尿道致病性大肠埃希菌(UPEC)菌株132与人膀胱上皮EJ细胞的相互作用,分析该菌株感染对EJ细胞基因表达谱的改变.方法 UPEC132感染EJ细胞,用倒置显微镜观察细菌与细胞的黏附,计算黏附率,并通过激光共聚焦显微镜观察UPEC132对细胞的侵袭.感染UPEC132的EJ细胞与未经细菌感染的细胞提取总RNA,用人类全基因组寡核苷酸微阵列芯片分析差异表达基因,并采用RT-PCR对基因芯片数据进行验证.结果 UPEC132能够黏附于EJ细胞表面,黏附率为(73.20±5.26)% 激光共聚焦显微镜观察发现部分细菌位于细胞内部,证实该菌对EJ细胞具有侵袭性.UPEC132感染后的EJ细胞与未经感染的细胞相比,共有28个基因上调,1个基因下调,主要涉及细胞增殖、炎症反应、细胞凋亡等相关基因.结论 UPEC与尿路上皮细胞的相互作用激活宿主细胞内部多种应答反应与信号转导途径,本研究为深入探索UPEC致病机制奠定基础. | 葛新 陈锦英 姚萍 谷超 赵凤玲 | 2010 | 中华微生物学和免疫学杂志2010，30，11： | 2 |
| 3 | A novel gene R049 identified in uropathogenic Escherichia coli provides partial protection in mice from colonization显示文摘Uropathogenic Escherichia coli(UPEC) is the most common causative organism of human urinary tract infection(UTI).Several UPEC virulence factors have been identified,but more are yet to be found.We previously identified a novel 789-bp-long DNA fragment(named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique.In the present study,we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity.Through bioinformatic analysis,we predicted that this gene is a 1311-bp open reading frame(ORF),which we designated ORFR049(GenBank accession No.:EF488001).We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography.Using mouse immunosera generated by the purified protein,we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting.To test the potential of this protein as a vaccine candidate,we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract.The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group.However,the degree of renal pathological damage was not significantly improved in the immunized mice.Our study has identified a novel gene of UPEC which can generate protective immunity against UTI.This novel gene provides a promising new vaccine candidate. | CHEN JinYing GE Xin ZHANG YuMei LIN Xu ZHANG Wei YANG Xi | 2011 | Chinese Science Bulletin2011，56，8： | 0 |
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