title: Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11.
abstract: 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 lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 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 lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification; D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins; D007159:Immunologic Tests; D009693:Nucleic Acid Hybridization
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D004262:DNA Restriction Enzymes