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 hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera from H. hepaticus-infected 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 coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice 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 that uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum 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 with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.
Livingston
R S
RS
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA. livingstonr@missouri.edu
Riley
L K
LK
Hook
R R
RR
Besch-Williford
C L
CL
Franklin
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics; D016998:Helicobacter / Q000235:genetics* / Q000276:immunology*; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; C121959:MAP18 protein, Helicobacter hepaticus; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase