A novel gene R049 identified in uropathogenic Escherichia coli provides partial protection in mice from colonization  

作　　者：CHEN JinYing GE Xin ZHANG YuMei LIN Xu ZHANG Wei YANG Xi 

机构地区：[1]Department of Medical Microbiology, Tianjin Medical University, Tianjin 300070, China [2]Research Center of Molecular Medicine, Fujian Medical University, Fuzhou 350004, China [3]Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

出　　处：《Chinese Science Bulletin》2011年第8期755-761,共7页

基　　金：supported by the National Natural Science Foundation of China (30470096);the Specialized Research Fund for the Doctoral Program of Higher Education (20070062010)

摘　　要：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.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.

关 键 词：保护性免疫 免疫小鼠 大肠杆菌 基因组 肾炎 定植 抑制消减杂交技术 GENBANK 

分 类 号：Q78[生物学—分子生物学] S852.659.5[农业科学—基础兽医学]