Molecular cloning of Clostridium difficile toxin A gene fragment in λ gtll

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in λ gtll. Approx. 35000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, λ cd 19. Verification of the immunological identity of the isolated toxin A gene fragment in λ cd 19 was determined by affinity purifying toxin A antibodies specific for λ cd 19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in λ cd 19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in λ cd 19 was not cytotoxic for 3T3 mammalian tissue culture cells.