Cloning and expression of the toxin B gene ofClostridium difficile
- 46 Downloads
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.
KeywordsPeptide Molecular Weight Complete Gene Cloning Study Small Open Reading Frame
Unable to display preview. Download preview PDF.
- 2.Borriello SP, Barclay F, Welch A, Ketley J, Mitchell T, Stephen J, Griffin G (1985) Host and microbial determinants of the spectrum ofClostridium difficile mediated gastrointestinal disorders. Microecol Ther 15:231–236Google Scholar
- 4.Dower WJ (1988) Transformation ofE. coli to extremely high efficiency by electroporation. Mol Biol Rep No. 6:3–4. Richmond, CA: Bio-Rad LaboratoriesGoogle Scholar
- 10.Lyerly D, Roberts M, Phelps C, Wilkins T (1986) Purification and properties of toxins A and B ofClostridium difficile. FEMS Microbiol Lett 33:31–35Google Scholar
- 11.Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryGoogle Scholar