Wu Hsing Chieh, Chen Yu San, Shen Pin Chun, Shien Jui Hung, Lee Long Huw, Chiu Hua Hsien
Dept. of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 402, Taiwan.
Dept. of Biotechnology, College of Environmental and Life Science, Foo Yin University, Kaohsiung, 831, Taiwan.
Biotechnol Prog. 2015 May-Jun;31(3):641-9. doi: 10.1002/btpr.2052. Epub 2015 Feb 2.
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.
鸡白细胞介素-12（chIL-12）蛋白的佐剂活性已被描述为与哺乳动物IL-12的佐剂活性相似。重组chIL-12可以通过几种方法生产，但真核细胞中chIL-12的产量低于原核细胞。可以向动物细胞培养物中添加刺激化合物，如二甲基亚砜（DMSO），以克服这一缺点。在本研究中，我们构建了一种细胞系DF1/chIL-12，其稳定表达一种融合蛋白，即通过（G4 S）3接头序列连接的chIL-12和增强型绿色荧光蛋白（eGFP）。当细胞在DMSO存在下培养时，融合蛋白产量增加。当将1×10(6)个DF1/chIL-12细胞接种到含有30 mL培养基的T-175培养瓶中，孵育15 h，然后在4% DMSO存在下进一步培养48 h时，与使用其他浓度DMSO诱导的细胞裂解物产生的总融合蛋白产量相比，总融合蛋白产量大多得到提高。细胞裂解物中未纯化和纯化的总融合蛋白浓度分别为2,781±2.72 ng mL(-1)和2,207±3.28 ng mL(-1)。回收率为79%。融合蛋白刺激鸡脾细胞产生IFN-γ，使用酶联免疫吸附测定法在培养上清液中进行测量，这表明用DMSO处理DF1/chIL-12细胞或以融合蛋白形式生产chIL-12对chIL-12的生物活性没有不利影响。
