Cloning and expression of an immunogenic membrane associated protein of Helicobacter hepaticus for use in an enzyme-linked immunosorbent assay

Helicobacter hepaticus is a bacterial pathogen that causes chronic active h epatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assa y (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hep aticus was constructed and was screened with sera from H. hepaticus-infecte d mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes codi ng for outer membrane proteins of other bacteria, and the predicted amino a cid sequence of MAP18 had a putative membrane-tracking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was e xpressed in Escherichia coli as a glutathione S-transferase (GST) fusion pr otein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusio n protein,vas detected on Western blots probed with sera from H. hepaticus- infected mice but was not detected on blots probed with sera from mice infe cted with Helicobacter muridarum or Helicobacter bilis or with sera from mi ce free of Helicobacter infection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA t hat uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bills, or H. m uridarum and sera from mice free of Helicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed,vith a higher sensitivity (89%) than the rec ombinant protein-based ELISA (sensitivity, 66%). These data indicate that H . hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-asso ciated protein; however, antibodies to this protein are not detected in all infected mice.