RESUMEN
ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.
Asunto(s)
Toxinas Bacterianas , Escherichia coli Enterotoxigénica , Enterotoxinas , Epítopos de Linfocito B , Proteínas de Escherichia coli , Vacunas contra Escherichia coli , Proteínas Fimbrias , Proteínas Recombinantes de Fusión , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Línea Celular Tumoral , Simulación por Computador , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/inmunología , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/química , Vacunas contra Escherichia coli/genética , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Vibrio cholerae is considered one of the major health threats in developing countries. Lack of efficient vaccine, short incubating time of the disease, and bacterium ability to survive in aquatic environment have made cholera one of the most epidemic diseases yet known. The lipopolysaccharide is one of the bacterium key antigens used to classify V. cholerae into 206 serogroups. V. cholerae serogroup O1 is a causative agent of all cholera pandemics. Research has shown that anti-lipopolysaccharide (LPS) antibodies could provide protective immunity in cholera cases. In this research, we used N-terminal fragments of the camel's heavy-chain antibodies called VHH or nanobodies and produced a phagemid library. The obtained library was panned against V. cholerae O1 LPS, and four monoclonal nanobodies were isolated. Isolated nanobodies were tested in LPS ELISA and bacterial ELISA. The nanobody with the highest affinity toward the bacterium was used in an in vivo challenge and successfully neutralized the bacterium infection. The isolated nanobody showed high thermostability and proteolytic resistance in characterization tests.