Development of hyperfimbriated strains of Vibrio cholerae O1.
The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V 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.
Ehara
M
M
Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan. ehara@net.nagasaki-u.ac.jp
Iwami
M
M
Ichinose
Y
Y
Hirayama
T
T
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial; D014734:Vibrio cholerae / Q000235:genetics* / Q000648:ultrastructure
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C063055:fimbrillin; D036781:Fimbriae Proteins