Genetic Engineering of Clostridium difficile Toxin A Vaccine
Abstract
Recombinant DNA technology allows for an approach to subunit vaccine production that should provide advantages over existing techniques. Improvement of vaccine biotechnology in the area of recombinant DNA studies using Clostridium difficile toxin A as the model, is the major long range objective of this project. However, prior to accomplishing this goal C. difficile toxin genes must be cloned, sequenced and characterized, which was the objective of the first year of work, and is reported in part in this document. A genomic library in lambda gt11 of c. difficile chromosomal DNA was 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. Sequence analysis of DNA encoding for toxin A was determined using the Sanger chain-termination sequence procedure. The amino acid sequence was deduced for the DNA sequence and a hydropathy plot of the open reading frame given.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 14, 1988
- Accession Number
- ADA201097
Entities
People
- Joe Johnson
- Lycurgus L. Muldrow
Organizations
- Spelman College