人白细胞介素7在CHO细胞中的表达及活性鉴定

摘要

Objective: To express human IL-7 in CHO cell and assessment promoting peripheral blood mononu⁃clear cell(PBMC) proliferation function. Methods: The vector pcDNA5/FRT-IL-7 was constructed and co-transfect⁃ed into Flp-In-CHO with pOG44 plasmid using liposome transfection method. The cells expressing IL-7 stably were selected by hygromycin B and the positive cells were monocloned with the method of limited dilution. The IL-7 was detected and quantitative in the supernatant by Wetern blot and ELISA assay. The ability of the protein promoting PBMC differentiation was detected by flow cytometry. Results: Two CHO cells exressing IL-7 stably were 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.%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pcDNA5/FRT-IL-7，用Lipo2000将辅助质粒pOG44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。