摘要
目的:用CHO细胞表达人白细胞介素7(IL-7),并验证其促细胞增殖功能。方法:构建表达质粒pc DNA5/FRT-IL-7,用Lipo2000将辅助质粒p OG44和表达质粒共转染Flp-In-CHO细胞,潮霉素B筛选并单克隆化稳定表达目的蛋白的细胞;通过Western印迹、ELISA方法验证上清中的目的蛋白,并对其定量;用流式细胞仪检测该蛋白促外周血单核细胞增殖的能力。结果:获得了2株稳定表达IL-7的细胞,IL-7的表达量均为3 mg/(L·d);流式细胞术检测表明,所表达的IL-7的促外周血单核细胞增殖能力强于R&D原核系统表达的IL-7。结论:获得了稳定表达人IL-7的CHO细胞系,且表达的蛋白具有促外周血单核细胞增殖的功能。
Objective：To express human IL-7 in CHO cell and assessment promoting peripheral blood mononu-clear cell（PBMC） proliferation function.Methods：The vector pc DNA5/FRT-IL-7 was constructed and co-transfect-ed into Flp-In-CHO with p OG44 plasmid using liposome transfection method.The cells expressing IL-7 stablywere selected by hygromycin B and the positive cells were monocloned with the method of limited dilution.TheIL-7 was detected and quantitative in the supernatant by Wetern blot and ELISA assay.The ability of the proteinpromoting PBMC differentiation was detected by flow cytometry.Results：Two CHO cells exressing IL-7 stablywere selected and the expression quantity was about 3 mg/（L·d）.Conclusion：The CHO cell lines stable express-ing IL-7 were established and the function of recombinant IL-7 on promoting PBMC proliferation was like the nat-ural IL-7.
出处
《生物技术通讯》
CAS
2015年第4期477-480,共4页
Letters in Biotechnology
基金
国家科技重大专项(2014ZX10004002-004-001)
