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Current Microbiology

, Volume 20, Issue 6, pp 397–401 | Cite as

Cloning and expression of the toxin B gene ofClostridium difficile

  • John L. Johnson
  • Carol Phelps
  • Lisa Barroso
  • Mary D. Roberts
  • David M. Lyerly
  • Tracy D. Wilkins
Article

Abstract

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5′-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.

Keywords

Peptide Molecular Weight Complete Gene Cloning Study Small Open Reading Frame 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag New York Inc. 1990

Authors and Affiliations

  • John L. Johnson
    • 1
  • Carol Phelps
    • 1
  • Lisa Barroso
    • 1
  • Mary D. Roberts
    • 1
  • David M. Lyerly
    • 1
  • Tracy D. Wilkins
    • 1
  1. 1.Department of Anaerobic MicrobiologyVirginia Polytechnic Institute and State UniversityBlacksburgUSA

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