Bacteroides Fragilis OMP A: Utility as a Live Vaccine Vector for Biodefense Agents
Abstract
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 function(s) 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 deletant(WAL 186), as well as characterizing the phenotypes of B. fragilis withmodified OmpAs.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2007
- Accession Number
- ADA467965
Entities
People
- Hannah M. Wexler