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Molecular cloning of Clostridium difficile toxin A gene fragment in λ gtll
Author(s) -
Muldrow Lycurgus L.,
Ibeanu Gordon C.,
Lee Norveta I.,
Bose Nripendra K.,
Johnson Joe
Publication year - 1987
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(87)81500-4
Subject(s) - microbiology and biotechnology , biology , clone (java method) , clostridium difficile toxin a , toxin , southern blot , genomic library , clostridium difficile , gene , antiserum , cd19 , restriction fragment , molecular cloning , antibody , virology , peptide sequence , genetics , flow cytometry , antibiotics
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.