Cloning and sequencing of badger (Meles meles) interferon gamma and its detection in badger lymphocytes.
The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.
Dalley
D J
DJ
TB Research Group, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK.
Hogarth
P J
PJ
Hughes
S
S
Hewinson
R G
RG
Chambers
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002335:Carnivora / Q000097:blood / Q000235:genetics / Q000276:immunology* / Q000382:microbiology*; D002417:Cattle; D003001:Cloning, Molecular; D004197:Disease Reservoirs / Q000662:veterinary*; D004285:Dogs; D005434:Flow Cytometry; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008214:Lymphocytes / Q000276:immunology*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000302:isolation & purification*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000382:microbiology; D019444:Vaccines, DNA / Q000592:standards
D019444:Vaccines, DNA; D012313:RNA; D007371:Interferon-gamma