KMID : 0613820040140020362
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Journal of Life Science 2004 Volume.14 No. 2 p.362 ~ p.367
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Construction and Characterization of the Vibrio parahaemolyticus Collagenase Inactivated Mutant
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Lee Jae-Won
Kang Ho-Young Jun In-Joon Cha Jae-Ho
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Abstract
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For better understanding of the host infection mechanism of Vibrio, a Vibrio parahaemolyticus collagenase mutant was generated by insertional inactivation of a vppC gene encoding extracellular collagenase. A recombinant DNA containing vppC::nptII was cloned into a suicide plasmid pDMS197, resulted in pVCM03. The recombinant suicide plasmid pVCM03 contained in E. coli ¥ö7213 was transferred to a wild-type V. parahaemolyticus 04 through conjugation. The recombinant vppC::nptII DNA in pVCM03 was exchanged with wild-type allele by homologous recombination resulting vppC mutant, V. parahaemolyticus CM. The mutant was selected and screened on TCBS media containing 10% sucrose and kanamycin. The mutation by allele exchange was confirmed with the comparison of the size of DNAs amplified by PCR. V. parahaemolyticus CM showed at least 4-fold less collagen-degrading activity than those of wild-type, and the mutant exhibited less cytotoxicity than that of wild-type in MTT assay.
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KEYWORD
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V. parahaemolyticus, vppC, conjugation, allelic exchange
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