重组人白细胞介素-18的高浓度复性、纯化和活性检测

The high-concentration refolding, purification and bioactivity assay of recombinanthuman interleukin-18

目的摸索大规模生产重组人白细胞介素 18(rhIL 18)高效简便、成本低廉的生产工艺。优化rhIL 18高浓度下最优复性、纯化方案 ,建立快速准确的活性检测方法。方法采用液相色谱和光谱学方法检测复性效率 ,筛选确定rhIL 18最优复性条件 ,再用SepheroseFFANX阴离子交换柱结合分子筛HiPrepSephacrylS 10 0进行纯化。采用3 H 掺入法检测rhIL 18产品对人外周血单个核细胞的促增殖作用 ,用流式细胞术检测其促γ 干扰素生成能力。结果建立了一套rhIL 18复性、纯化、活性检测方案。结论液相色谱和光谱学方法相结合 ,可用于检测rhIL 18的复性效率 ,流式细胞术也可用于检测rhIL 18的生物活性。

PurposeTo set up production techniques with high efficiency and low cost in the large-scale production of recombinant human interleukin(rhIL-18), to optimize the refolding and purification strategies and to establish fast and accurate bioactivity assay methods.MethodsUsing liquid chromatography and spectroscopy to examine the refolding efficiency in the evaluation of refolding conditions,and combining Sepherose FFANX anions exchange chromatography with size exclusive chromatography to purify the recombinant protein. Using 3 H-TdR incorporation methods to assay the proliferating activity of rhIL-18 on PBMC and using flow cytometry to detect the intracellular IFN-γ secreted from PBMC after the stimulation of rhIL-18.ResultsThe refolding,purification and bioactivity assay strategies of rhIL-18 were established.ConclusionLiquid chromatography combining spectroscopy can be used in the determination of refolding efficiency and flow cytometry can be adopted in the bioactivity assay of rhIL-18.