CLONING AND EXPRESSION OF RFB GENES FROM VIBRIO-ANGUILLARUM SEROTYPE O2 IN ESCHERICHIA-COLI - EVIDENCE FOR CROSS-REACTIVE EPITOPES

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (L PS) O antigens containing both specific and cross-reactive epitopes, T he localization of these epitopes on the O antigen is not known. We ha ve cloned and expressed the rfb, gene cluster for O antigen synthesis from V. anguillarum O2 (rfb(VaO2)) in Escherichia coli. E. coli DH5 al pha containing the recombinant plasmid pAM86 expressed O antigens whic h reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protec ted from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfb(VaO2) genes also reacted with MAb A1 6, which is specific for V: ordalii O antigens. Western immunoblot ana lysis revealed that MAb 7B4 reacted with recombinant LPS bearing short er O-antigen repeat units, while MAb A16 reacted with the longer O ant igens. Similar results were obtained when pAM86 was transformed into E . coli CLM4, which has a deletion spanning; the sbcB-rfb region, indic ating that the changes in antigenic profiles of O antigens from the re combinant strains were not due to genes within the E. coli rfb cluster . These data suggest that the epitope recognized by the MAb A16 is exp ressed by V anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfb(Va O2) gene cluster resulted in expression of a novel O antigen. The modi fication(s) which leads to the alterations in antigenic profile of the se recombinant LPS remains to be determined.