One-step cloning and expression of Clostridium difficile toxin B gene (tcdB)

Y. J. Tang-Feldman, G. Ackermann, J. P. Henderson, J. Silva, Stuart H Cohen

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments. Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system. The amplified fragment was inserted into the E. coli expression vector pBAD and cloned into competent E. coli TOP 10 cells. tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed. Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts. The recombinant protein expressed in E. coli was identified as toxin B by Western immunoblot analysis using C. sordellii antitoxin. This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C. difficile. Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.

Original languageEnglish (US)
Pages (from-to)179-183
Number of pages5
JournalMolecular and Cellular Probes
Volume16
Issue number3
DOIs
StatePublished - Jun 2002

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Keywords

  • Cloning
  • Clostridium difficile
  • PCR
  • Toxin B gene

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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