Development of hyperfimbriated strains of Vibrio cholerae O1

The I:Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR(TM) vector, These clones were sequenced. The fimA sequences were found to be identical between TS cholerae O1 and O139, One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X, The plasmid pVPP thus obtained was transferred into strains of wild-type V. cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation, The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture), All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.