title: One-step cloning and expression of Clostridium difficile toxin B gene (tcdB).
abstract: The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments. Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system. The amplified fragment was inserted into the E. coli expression vector pBAD and cloned into competent E. coli TOP 10 cells. tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed. Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts. The recombinant protein expressed in E. coli was identified as toxin B by Western immunoblot analysis using C. sordellii antitoxin. This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C. difficile. Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.
authors.affiliation: Department of Internal Medicine, Division of Infectious and Immunologic Diseases, University of California, Davis, CA 95817, USA.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Tang-Feldman
authors.forename: G
authors.initials: G
authors.lastname: Ackermann
authors.forename: J P
authors.initials: JP
authors.lastname: Henderson
authors.forename: J
authors.initials: J
authors.lastname: Silva
authors.forename: S H
authors.initials: SH
authors.lastname: Cohen
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics / Q000472:pathogenicity; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000166:cytology; D016133:Polymerase Chain Reaction; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile