Development of hyperfimbriated strains of Vibrio cholerae O1

The I:Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were ampl ified by polymerase chain reaction and cloned into an Escherichia coli pCR( TM) vector, These clones were sequenced. The fimA sequences were found to b e identical between TS cholerae O1 and O139, One of the plasmids was digest ed with EcoR I and inserted into the EcoR I site of pGEX-3X, The plasmid pV PP thus obtained was transferred into strains of wild-type V. cholerae O1 B gd17 (classical in biotype) and its fimbriated strain by electroporation, T he recombinant plasmid pVPP overexpressed mature fimbriae following inducti on of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The clo ned gene product was purified to homogeneity by sucrose-linear gradient cen trifugation (7.8 mg of fimbriae/L-culture), All the properties of the recom binant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were iden tical to those of the wild-type fimbriae. This overexpression system will b e extremely useful for rapid, inexpensive preparation of large amounts of f imbriae for vaccine design and development.