Development of Hyperfimbriated Strains of Vibrio cholerne O1

Abstract

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCRTm 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.

Minor pilin subunits are conserved inVibrio choleraetype IV pili

“…A prepilin‐expression vector was constructed to use for further transformation into V. cholerae strain 88UDT119 as described by Ehara et al [19]. Primers EXS14 and HV12 (Table 1) were used to amplify the vcfA gene by PCR, and the PCR product was ligated to pCR R 2.1 vector to obtain plasmid pNG14.…”

“…Except for a few modifications, NAGV14 pili were purified essentially as described by Ehara et al [19] from V. cholerae 88UDT119 carrying plasmid pGV3. Briefly, bacteria were grown in LB broth supplemented with 100 μg ampicillin ml −1 and induced with IPTG for 4 h. A heavy suspension of the harvest cells in 50 mM Tris–HCl buffer (pH 8.0) was agitated in a biomixer and centrifuged at 12,000× g for 30 min.…”

“…The pellet was resuspended and applied to sucrose density gradient (10–60% (w/v) in Tris buffer) and centrifuged at 150,000× g for 3 h. Fractions rich in pili were pooled and regarded as purified pili. MSHA pili were purified by the same procedure from V. cholerae Bgd17 carrying plasmid pVPP [19] with the exception that bacteria were grown in LB broth supplemented with 50 μg ampicillin ml −1 .…”