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KMID : 0364819990350030205
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Korean Journal of Microbiology
1999 Volume.35 No. 3 p.205 ~ p.210
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Cloning and Nucleotide Sequence Analysis of the Virulence Gene Cassette from Vibrio cholerae KNIH002 Isolated in Korea
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Abstract
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Vibrio cholerae is an important pathogenic organism that causes diarrhea in human beings. V. cholerae KNIH002 was isolated from patients suffering with diarrheal disease in Korea. From Southern hybridization using the amplified PCR product of 307 bp as a probe, which was obtained from PCR reaction using primer detecting cholera toxin gene,
we have found that the ctx gene located in 4.5-kb fragment double digested with PstI and BglII of the chromosome. Therefore, we made mini-libraries of the isolate using PstI and BglII restriction endonuclease and pBluescript SKII(+) vector. As a result, we cloned 4.5-kb PstI-BglII fragment containing the ctx gene encoding a cholera toxin from
the constructed mini-libraries of V. cholerae KNIH002 by colony hybridization using the same probes. This recombinant plasmid was named pCTX75. E. coli XL1-Blue harboring pCTX75 showed the cytotoxicity on Chinese Hamster Ovary cells. From the sequencing of the cloned recombinant plasmid, we confirmed that it has virulence gene cassette
consisting of ace, zot, ctxA and ctxB gene. The ace and zot genes were composed of 291 bp and 1,200 bp with ATG initiation codon and TGA termination codon, respectively. Nucleotide sequence of the ace gene exhibited 100% identity with that of V. cholera E7946 El Tor Ogawa strains. But, nucleotide and amino acid sequence comparison of the zot gene exhibited 99% and 98.8% identity with that of V. cholerae 395 Classical Ogawa strain, respectively. Specially, the Ala-100, Ala-272 and Ala-281 sites of Zot polypeptide presented in V. cholerae 395 Classical Ogawa strain are replaced by Val in V. cholerae KNIH002.
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KEYWORD
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ace gene, cholera toxin, ctxAB gene, Vibrio cholerae KNIH002, zot gene,
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