RESUMO
Anti-idiotype monoclonal antibody 1E10 can mimic the protective epitope of Vibrio anguillarum and be used as vaccine to prevent fish infection of V. anguillarum. In this study, the variable heavy (V(H)) domain and variable light (V(L)) domain of mAb1E10 were cloned by RT-PCR and were linked to each other by a disulfide bond engineered at position 44 of V(H) and position 105 of V(L) that lie between structurally conserved framework positions. Mutated V(H) 44 and V(L) 105 were inserted into phagemid pCANTAB5E. When co-transfected by recombinant pCANTAB5E and helper phage M13KO7, the host Escherichia coli cells secreted disulfide-stabilized Fv fragment (dsFv) which displayed on the surface of filamentous phage. The binding specificity of the phage-displayed dsFv was proved by ELISA method. Protection experiment showed that Japanese flounders can develop high titer of antibody against the dsFv and survival ratio of vaccinated group was significantly different from control groups. Thus, this phage-displayed dsFv may be used as vaccine against V. anguillarum in fishery.
Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Linfocinas/imunologia , Biblioteca de Peptídeos , Sialoglicoproteínas/imunologia , Vibrioses/prevenção & controle , Vibrioses/veterinária , Vibrio/imunologia , Sequência de Aminoácidos , Animais , Vacinas Bacterianas/química , Clonagem Molecular , Dissulfetos/química , Ensaio de Imunoadsorção Enzimática , Linfocinas/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/química , Análise de Sobrevida , Vacinas Sintéticas/imunologiaRESUMO
AIM: To clone and sequence V(H) and V(L) genes of anti-idiotype monoclonal antibody (mAb) against vibrio alginolyticus. METHODS: Total RNA was extracted from hybridoma cell AL1 secreting mAb against vibrio alginolyticus and cDNA was amplified by RT-PCR. Then the cDNA was inserted into PMD18-T vector and its sequence was analyzed. RESULTS: The V(H) gene contained 369 bp and encoded 123 amino acid residues; the V(L) gene contained 339 bp and encoded 113 amino acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) and V(L) genes, respectively. CONCLUSION: The successful cloning of the V(H) and V(L) genes of anti-idiotype mAb against vibrio alginolyticus provides a sound basis for construction of gene-engineering vaccine of the anti-idiotype mAb against vibrio alginolyticus.
