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

Abstract

Toxin A of Clastr~dium d~~cile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. dl~ci~e chromosomal DNA was developed which allowed for the construction of a genomic library in d gtll. Approx. 35 000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, d cd19. Verification of the immunological identity of the isolated toxin A gene fragment in A cdl9 was determined by affinity purifying toxin A antibodies specific for 1 cdl9 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in 1 cdl9 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. d@cile. The peptide coded for by the toxin A gene fragment in 1 cdl9 was not cytotoxic for 3T3 mammalian tissue culture cells.

Genetic Engineering of Clostridium Difficile Toxin A Vaccine

“…The sequence data demonstrates that there is only one 300 bp jTa.1 fragment within the toxin A gene, which is located within a 4.5 Pstl fragment, and a large HindlII fragment. Hybridization data published by our laboratory (32) demoustrates that [alpha-32P] labeled ýxd19 hybridized to a 4.5 kb fragment in a Pstl chromosomal digestion of C. difficile, and a 16 kb fragment in a HindIII digestion ( fig. 3.…”

“…Studies directed toward the molecular genetics or DNA of C. difficile toxin A have been appearing in the literature in increasing numbers in the last several years. The trust paper pubhshed on cloning part of C. difficile toxin A was reported by our group (32). Since this publication, several other studies have followed on cloning, expression of E. coli, and sequencing of toxin A and B (33,34,35,36,37,38).…”

Genetic Engineering of Clostridium Difficile Toxin a Vaccine

“…The sequence data demonstrates that there is only one 300 bp Tadf fragment within the toxin A gene, which is located within a 4.5 Pstl fragment, and a large Hindlll fragment. Hybridization data published by our laboratory (32) fig. 2).…”

“…Based on the data published by this group, and data published by Von Ichil-Streiber's group (35) on overlapping toxin A DNA fragments which express antigenic sites, the DNA sequence of an 800 bp fragment has been identified which is thcugh to have a major antigenic site. This 800 bp fragment has also been identified to contain the 300 bp fragment that we earlier reported on cloning (32). Using the above information epitopes were predicted using the Antigenic Index of the IBi Pustell software program.…”

“…Studies directed toward the molecular genetics or DNA of C. difficile toxin A have been appearing in the literature in increasing numbers in the last several years. The first paper published on cloning part of C. difficile toxin A was reported by our group (32). Since this publication several other studies have followed on cloning, exoression in E. coli, and sequencing of toxin A (33, 34,35,36,37).…”