| ObjectiveUrinary tract infection (UTI) is a common clinical disease. Escherichia coli are the main causative agents of UTI and account for>80% of cases. UPEC clinical isolates showed multiple drug resistance to antibiotics.It is difficult to treat the infections. The construction of vaccine is very important.The objective is to construct and identify an uropathogenic Escherichia coli fimH eukaryotic expression vector pcDNA3.0-fimH. And investigate the immunoreaction of the fimH gene vaccine of type 1 pilus of Uropathogenic Escherichia coli and its immune protection to mice and makes comparisons to the immune efficacy through different immune approaches.Methods1. Genomic DNA of uropathogenic Escherichia coli was extracted from uropathogenic Escherichia coli 6704. The fimH gene fragment was amplified from the genomic DNA of uropathogenic Escherichia coli by PCR. The purified PCR fragment was ligated into the pMD19-T simple vector, the positive clones were screened and identified by PCR from bacteria directly, and the PCR products were digested via double enzymes following sequencing of the recombinant plasmid. The fimH gene fragments from TA clones were cloned into the eukaryotic expression vector pcDNA3.0 using restriction enzymes. The recombinant plasmid pcDNA3.0-fimH was identified through PCR, enzyme digestion and sequencing.2. We performed a large number of these plasmids and immunized BABL/c mice. A total of 40 BALB/c mice were selected and divided into 4 groups randomly: a PBS group, a pcDNA3.0 group, an experimental group of intramuscular injection, and an experimental group of intranasal immunization. It uses recombinant pcDNA3. 0-FimH to immume BALB/c mice by means of intramuscular injection and intranasal immunization. At the same time, the PBS group and the pcDNA3.0 group were intramuscularly immunized with PBSor pcDNA3.0. All m ice w ere immunized three times successively at 2 weeks interval. The specific IgG antibody in the blood-serum of the mice was tested. In the last time of immune after 10 days, all mice were challenged by UPEC through urinary tract.Finally,5 days after attacting,we made an experiment of germiculture and counting to the urine (10μl per mouse urine seeded on MacConkey agar for 24 hours at 37℃. Observed and counted the bacteria colonies after one day.).Results:1. The fimH gene fragment was amplified correctly, as the size of gene was about 910bp.After being digested by BamH and XhoⅠ, the recombinant plasmid produced one little fragment which is similar with the PCR products of fimH gene, and one big fragment which is different with the recombinant plasmid of pcDNA3.0-fimH. It proved that the fimH gene connected with the pcDNA3.0.The sequences of the recombinant plasmid were similar with ones of the J96 in GenBank. The test proved that the uropathogenic Escherichia coli fimH eukaryotic expression vector pcDNA3.0- fimH was successfully constructed.2. The mice immunized with the plasmids expressed in muscle in mice and produced anti—fimH antibodies. After the immunization, the level of specific IgG antibody in the blood-serum of the mice of the experimental group of intramuscular injection and intranasal immunization is increasing apparently, which is higher than control group and pcDNA3.0 group (p< 0.05). 3. After UPEC attack, the urine colony counts of the experimental group of intermuscular injection or intranasal immunization were significantly lower than the control group and pcDNA3.0 group (p< 0.05).Conclusion:1. Type I pilus of pathogenic E. coli fimH gene eukaryotic expression vector pcDNA3. 0-f imH was successfully constructed.2. As an immune vaccine, recombinant pcDNA3.0-filmH can lead BALB/c mice to produce specific humoral response and have an immune protection to a degree for the ascending infection of the mice' urethra; different immune approaches has different immune efficacy. |
PDF Full Text Request