RESUMO
Background and Aim: Enterohemorrhagic Escherichia coli [EHEC] cause a wide spectrum of infections, such as diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Considering the risks associated with antibiotic therapy against EHEC infection, vaccines can be a promising method for prevention of infections. Recombinant chimeric proteins containing multiple immunogens could induce immunity against bacterial infections. The aim of this study was to evaluate immunogenecity of trivalent chimeric antigen EspA -Stx2b - Intimin against E.coli O157 H7 infection
Material and Methods: In this descriptive-laboratory study, recombinant chimeric protein was expressed in E.coli BL21 DE3 by use of IPTG. The protein expression was evaluated by SDS-PAGE and western blotting analysis. The recombinant protein was purified using Ni- NTA affinity chromatography. The immunization was conducted in mice with purified protein and antibody titers were determined by ELISA. Following immunization, mice were infected with E.coli O157:H7 and evaluated for bacterial shedding and mortality. Using SPSS software, statistical analysis was performed by Duncan's test and T-test
Results: The protein was expressed in E.coli BL21 [DE3] and SDS-PAGE analysis showed expression of recombinant protein with molecular weight of 63kD. Western blot analysis confirmed presence of chimeric protein. ELISA results showed that immunogenic properties of chimeric protein induced humoral response to EspA, intimin and Stx2b. Bacterial shedding in immunized mice decreased to 102 cfu/ml and mortality rate was reduced to 60%
Conclusion: The results showed that the chimeric protein induced humoral response and protected the mice against E.coli O157:H7
RESUMO
Background and aims: Enterotoxigenic Escherichia coli [ETEC] is the main cause of diarrhea in children in developing countries and also in travelers to these areas. ETEC attaches to host cells via filamentous bacterial surface structures, known as colonization factors. Epidemiological studies suggest that the prevalence of CFA/I is higher than other colonization factors. CFA expressed by ETEC and so represents an important component of any vaccine. Investigation supposed that CfaB as a major subunit of fimbriae is an appropriate candidate for vaccine preparation. In the present study we investigated cloning and expression of CfaB
Methods: In this descriptive study, information about CfaB gene was obtained from gene bank and appropriate primers were designed accordingly. Genomic PCR reaction was performed and its product [CfaB gene] was cloned into pTZ57R/T cloning vector and then subcloned pET28a expression vector. CfaB gene expression was evaluated
Results: Cloning was confirmed by using restriction enzyme and sequencing. Expression of recombinant protein was determined in different conditions [time, media, and host], but native gene inserted in pET28a was not expressed
Conclusion: Native gene employs tandem rare codon which can reduce the efficiency of expression