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. Uveathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Serum UPEC virulence factors have been identified, but 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 loca lization 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. novel gene provides a promising new vaccine candidate.