Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector

G. Ackermann, B. Löffler, Y. J. Tang-Feldman, Stuart H Cohen, J. Silva, A. C. Rodloff

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.

Original languageEnglish (US)
Pages (from-to)271-274
Number of pages4
JournalMolecular and Cellular Probes
Volume18
Issue number4
DOIs
StatePublished - Aug 2004

Fingerprint

Organism Cloning
Polymerase Chain Reaction
Clostridium difficile
Genes
Virulence
Clone Cells
Cell Culture Techniques
Bacterial Toxins
Virulence Factors
Diarrhea
Western Blotting
Enzyme-Linked Immunosorbent Assay
Clostridium difficile tcdA protein
Escherichia coli

Keywords

  • Cloning
  • Clostridium difficile
  • Toxin A

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector. / Ackermann, G.; Löffler, B.; Tang-Feldman, Y. J.; Cohen, Stuart H; Silva, J.; Rodloff, A. C.

In: Molecular and Cellular Probes, Vol. 18, No. 4, 08.2004, p. 271-274.

Research output: Contribution to journalArticle

Ackermann, G. ; Löffler, B. ; Tang-Feldman, Y. J. ; Cohen, Stuart H ; Silva, J. ; Rodloff, A. C. / Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector. In: Molecular and Cellular Probes. 2004 ; Vol. 18, No. 4. pp. 271-274.
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AU - Silva, J.

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