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Development of hyperfimbriated strains of Vibrio cholerae O1
1Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan. ehara@net.nagasaki-u.ac.jp
Abstract:
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.
Insights
Researchers developed an efficient method to produce large quantities of Vibrio cholerae fimbriae. This breakthrough facilitates the rapid and cost-effective development of cholera vaccines.
Area of Science:
- Microbiology
- Molecular Biology
- Vaccine Development
Background:
- Vibrio cholerae causes cholera, a severe diarrheal disease.
- Fimbriae are crucial virulence factors for V. cholerae.
- Efficient production of fimbriae is needed for vaccine development.
Purpose of the Study:
- To develop an overexpression system for V. cholerae fimbriae.
- To characterize recombinant fimbriae produced by the system.
- To assess the utility of the system for vaccine design.
Main Methods:
- Polymerase chain reaction amplification and cloning of fimbrillin genes (fimA or mshA) from V. cholerae O1 and O139 into an E. coli vector.
- Construction of the pVPP plasmid for overexpression.
- Electroporation of pVPP into V. cholerae strains.
- Purification of recombinant fimbriae via sucrose-gradient centrifugation.
Main Results:
- Sequencing confirmed identical fimA sequences between V. cholerae O1 and O139.
- The pVPP plasmid successfully overexpressed mature fimbriae in V. cholerae.
- Recombinant fimbriae exhibited identical properties (subunit structure, hydrophobicity, hemagglutination, immunogenicity) to wild-type fimbriae.
- High yield of purified fimbriae (7.8 mg/L-culture) was achieved.
Conclusions:
- A robust overexpression system for V. cholerae fimbriae has been established.
- This system enables rapid, large-scale, and cost-effective production of fimbriae.
- The system is highly valuable for advancing cholera vaccine design and development.