ABSTRACT
Objective To investigate the character and location of a novel gene R049 and its expressed protein in uropathogenic Escherichia coli(UPEC) strain 132 isolated in China. MethodsThe chromosome library of UPEC132 was constructed by a shotgun strategy and the sequence analysis was carried out by a high-throughput pyrophosphate sequencing. Sequence reads were assembled with the Newbler program.The characters of R049-associated specific fragment were analyzed using the bioinformatics methods. Outer and inner membrane proteins of UPEC132 were extracted and then detected by SDS-PAGE and Western blot analysis together with the whole-cell lysates. ResultsThe 169 022 bp contig containing gene R049 was obtained and its sequence was very similar to the chromosome associated sequence of UPEC strain 536. It showed that a 20 773 bp fragment including R049 replaced the pathogenicity island PAI Ⅲ536 of UPEC536 in above 169 022 bp contig. The fragment had a lower GC content (46.97％) and 16 bp direct repeats in two ends. Significantly it also was adjacented to thrW tRNA, insertion element and genes coding integrase. Thus the 20 773 bp fragment was named R049 genome island(R049-GI). There were 25 ORFs in R049-GI, and gene R049 was located in the thirteenth ORF. The results of SDS-PAGE and Western blot revealed gene R049 encoded an outer membrane protein in the size of 47.0× 103. ConclusionGene R049, encoding an outer membrane protein, was a component part of the genome island in UPEC 132 chromosome acquired by horizontal gene transfer.
ABSTRACT
Objective To clone and express the new gene R049 of uropathogenic Escherichia coli 132.and to investigate the immunopmtective effects of the R049 recombinant protein on mice.Methods The pmkaryotic expression system of gene R049 was constructed by directed cloning.Thereafter,the R049 recombinant protein Was expressed and purified by Ni affinity chromatography.Polyclonal antibody was pre-pared by immunizing BALB/c mice with R049 recombinant protein.The R049 recombinant protein and whole bacterial proteins of UPEC132 were analyzed by SDS-PAGE and Western blot.BALB/c mice were im-munized with R049 recombinant protein before challenged by UPECl32 through urinary tract.Then the differences of urine and renal colony counts between immunization group and control group were compared.Results The recombinant strain E coli BL21(DE3)/pET32a-R049 ORF was constructed successfully,and the relative molecular mass of the R049 recombinant protein was 66.9×103 and its purity was up to 95% af-ter purification.The titer of polyclonal antibody wag≥1:102 400 analyzed by indirect ELISA.Both of the R049 recombinant protein and whole bacterial proteins of UPECl 32 were confirmed to show specffic reactions on the antiserunl throughh Western blot.The animal experiments showed the urine and renal colony counts of immunization group were significantly lower than that of the control group(P<0.01,P<0.05).Conclu-sion The new gene R049 of uropathogenic E.coli 132 had immunopmtective effects on mice and the defini-tive mechanism would be needed to further study.