Expression of a recombinant sheep IgE gene.
An ovine cosmid library was screened with a bovine C epsilon gene probe. IgE and IgA constant region genes (C epsilon and C alpha, respectively) were isolated within a single 26 kb cosmid insert. The C epsilon gene was cloned into an expression vector which contained the rearranged VDJ genomic segment encoding an anti-(4-hydroxy-3-iodo-5-nitrophenyl) acetic acid (anti-NIP) VH chain. This chimeric construct was transfected into murine hybridoma cells producing L chain with anti-NIP antibody specificity and stable transfectomas secreting chimeric ovine-murine IgE anti-NIP antibodies were obtained. Chimeric antibody, affinity purified from culture supernatants of transfected cells on a NIP-sepharose column, was characterised by SDS-PAGE and shown to contain H and L chains of expected size. These experiments demonstrated the power of advanced molecular genetic techniques to generate immunological reagents against low abundance immunoglobulin isotypes thus facilitating monoclonal antibody production for further immunological studies.
Clarke
R A
RA
CSIRO Division of Animal Health, McMaster Laboratory, NSW, Australia.
Beh
K J
KJ
D000818:Animals; D015139:Blotting, Southern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015723:Gene Library; D007073:Immunoglobulin E / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D008297:Male; D051379:Mice; D009592:Nitrohydroxyiodophenylacetate / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012756:Sheep / Q000276:immunology*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009592:Nitrohydroxyiodophenylacetate; D007073:Immunoglobulin E; D004247:DNA