Cloning and expression of caprine interferon-gamma

• Published in: Gene Vol. 210; no. 1; pp. 103 - 108
• Main Authors: Beyer, Joseph C, Stich, Roger W, Hoover, Debra S, Brown, Wendy C, Cheevers, William P
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 27.03.1998
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Caprine arthritis–encephalitis virus; Cloning, Molecular; COS Cells; Cytokine; Escherichia coli - genetics; Expression plasmid; Goats; Interferon-gamma - chemistry; Interferon-gamma - pharmacology; Leukocytes, Mononuclear - chemistry; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Open Reading Frames - genetics; Plasmid vaccine; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transfection - genetics; Vesicular stomatitis Indiana virus - drug effects; Viral Plaque Assay; aa, amino acid(s); LB, Luria–Bertani (medium); PBMC, peripheral blood mononuclear cells; Ab, antibody(ies); rBoIFN- γ, recombinant bovine interferon- γ; bp, base pair(s); IM, intramuscular; Con A, concanavalin A; Expression plasmid; CAEV LTR, caprine arthritis encephalitis virus long terminal repeat; cDNA, DNA complementary to RNA; Caprine arthritis–encephalitis virus; UTR, untranslated region(s); VSV, vesicular stomatitis virus; Cytokine; ORF, open reading frame; IL-4, interleukin 4; Plasmid vaccine; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; nt, nucleotide(s); CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; IFN- γ, interferon- γ; PMA, phorbol myristate acetate; IPTG, isopropyl β- d-thiogalactopyranoside; rCaIFN- γ, recombinant caprine interferon- γ; RT-PCR, reverse transcription-polymerase chain reaction
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(98)00046-8

Caprine interferon-gamma (IFN- γ) 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 498 bp, encoding a putative 166 amino acid (aa) protein (19 327 Da). The predicted aa sequence homology of caprine IFN- γ and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN- γ 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- γ (rCaIFN- γ) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN- γ (rBoIFN- γ) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN- γ 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- γ produced in E. coli