Unique characteristics of the C. perfringens enterotoxin (cpe) promoter and the biology of the C. perfringens type A has made it possible to deliver a large amount of foreign protein to the terminal ileum (a major location of the mucosal immune inductive sites in gastro-intestinal tract) and therefore, could be used as a general oral delivery vector of antigens. However, there are two potential problems with this delivery vector. First, C. perfringens type A produces two extracellular toxins, alpha toxin (pic) and theta toxin (pfoA) which could cause gas gangrene and pose a potential danger when used as a vaccine vector. Secondly, the C. perfringens expressed foreign antigen from a plasmid has potential problems related to instability and transferring a plasmid-encoded antibiotic resistant gene in the environment. Our hypothesis is that a pic /pfoA' mutant of cpe-negative C. perfringens, with the capacity to produce a high level of foreign proteins from its chromosome without antibiotic selection, will be an ideal vector to deliver the antigen to the immune inductive PPs in the GALT. The overall objective of this proposal is to construct a safe recombinant C. perfringens type A that is incapable of producing toxins and stably expresses high levels of foreign protein gene without involvement of antibiotic resistant gene. This will be accomplished by a) knocking out the chromosomal pfoA gene of a cpe negative ATCC 3624 C. perfringens type A using a mobile group II target-tron method; b) inserting a foreign gene cassette into the chromosomal pic gene of the pfoA' mutant by the target-tron methods to create a pIc'/pfoA' mutant and measure expression of the foreign protein from the chromosomal DNA in vitro and in vivo. Successful completion of this study will create a safe, low-cost and efficient oral delivery vector for vaccine and therapeutic agents. ? ?
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