title: Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.
abstract: The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12.
authors.affiliation: Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Hsing Chieh
authors.initials: HC
authors.lastname: Wu
authors.affiliation: Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Yu San
authors.initials: YS
authors.lastname: Chen
authors.affiliation: Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Pin Chun
authors.initials: PC
authors.lastname: Shen
authors.affiliation: Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Jui Hung
authors.initials: JH
authors.lastname: Shien
authors.affiliation: Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Long Huw
authors.initials: LH
authors.lastname: Lee
authors.affiliation: Dept. of Biotechnology, College of Environmental and Life Science, Foo Yin University, Kaohsiung, 831, Taiwan.
authors.forename: Hua Hsien
authors.initials: HH
authors.lastname: Chiu
mesh_terms: D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D007371:Interferon-gamma; D004121:Dimethyl Sulfoxide
keywords: DMSO; chIL-12 expression; chicken IL-12; interferon-γ; recombinant DF1 cell