Bacteroides Fragilis OMP A: Utility as a Live Vaccine Vector for Biodefense Agents
Annual rept. 1 Jul 2005-30 Dec 2006
BRENTWOOD BIOMEDICAL RESEARCH INST LOS ANGELES CA
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We are studying the utility of using B.fragilis OmpA as a vehicle on which to put antigenic epitopes of organisms that can be used as bioterror agents, with the aim of eventually constructing a vaccine vehicle vector. OmpA is the major outer membrane protein of B. fragilis, a gram negative anaerobe that normally resides in the gut. There are four homologs for ompA in the genome. The purpose of this study was to construct a B. fragilisompA deletant and to begin to characterize the function of OmpA, as well as the specific functions of the various loops exposed on the surface. We used a PCR-based site-directed mutagenesis technique to insert the FLAG marker into recombinant ompA cloned in the pET-27b expression system. Initial studies indicated difficultyin achievingstableexpression, export to periplasm and integration of B. fragilis OmpA into the E. coli membrane. Thus we will directly express the modified ompAs in B. fragilis. We performed further optimization of the mutagenesis procedure usingthe full-length B. fragilisompA, including upstream and downstream sequences, as template, for use in a two step recombination gene exchange. We will proceed by expressing and detecting the FLAG-tagged OmpAs directly in theB. fragilisompA deletantWAL 186, as well as characterizing the phenotypes of B. fragilis withmodified OmpAs.
- Anatomy and Physiology