Cloning and expression of caprine interferon-gamma.
Caprine interferon-gamma (IFN-gamma) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) is 498bp, encoding a putative 166 amino acid (aa) protein (19327Da). The predicted aa sequence homology of caprine IFN-gamma and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN-gamma cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis-encephalitis virus long terminal repeat (CAEV LTR). Recombinant caprine IFN-gamma (rCaIFN-gamma) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN-gamma (rBoIFN-gamma) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN-gamma cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN-gamma produced in E. coli.
Beyer
J C
JC
Department of Veterinary Microbiology, Pathology, Washington State University, Pullman, WA 99164-7040, USA. jcb@vetmed.wsu.edu
Stich
R W
RW
Hoover
D S
DS
Brown
W C
WC
Cheevers
W P
WP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006041:Goats; D007371:Interferon-gamma / Q000737:chemistry* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection / Q000235:genetics; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects; D010948:Viral Plaque Assay
D011994:Recombinant Proteins; D007371:Interferon-gamma