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目的建立CHO细胞表达的重组人白细胞介素-12(rhIL-12)的纯化工艺,并检测纯化的rhIL-12的生物活性。方法取高效表达rhIL-12的CHO工程细胞培养上清液,采用层析结合硫酸铵沉淀的方法进行分离纯化,ELISA法测定目的蛋白的含量,并计算回收率;SDS-PAGE、SEC-HPLC和Westernblot对纯化样品进行鉴定;以PBMCIFNγ诱生法检测纯化rhIL-12的生物活性。结果纯化的rhIL-12的总回收率为56.4%;经SEC-HPLC检测,其纯度达99.2%;SDS-PAGE分析显示,两个亚基的相对分子质量分别与理论值(40000和35000)相符;Westernblot分析显示,rhIL-12可与相应抗体发生特异性结合;rhIL-12诱生IFNγ量与rhIL-12浓度呈典型的S型曲线关系,具有剂量依赖性,其效价为8.3×106IU/mg。结论已建立了CHO细胞表达的rhIL-12纯化工艺,该工艺成本低,操作简便,纯化产物回收率和纯度高,适于工业化生产。
Objective To establish a purification process of recombinant human interleukin-12 (rhIL-12) expressed in CHO cells and to examine the biological activity of purified rhIL-12. Methods The supernatant of CHO engineering cells highly expressing rhIL-12 was isolated and purified by chromatography combined with ammonium sulfate precipitation. The content of target protein was determined by ELISA and the recovery rate was calculated. SDS-PAGE, SEC-HPLC and The purified samples were identified by Western blot and the biological activity of purified rhIL-12 was detected by PBMCIFNγ-induced method. Results The total recovery of purified rhIL-12 was 56.4%. The purity of rhIL-12 was 99.2% by SEC-HPLC. SDS-PAGE analysis showed that the relative molecular mass of the two subunits corresponded to the theoretical values (40000 and 35000) ; Western blot analysis showed that rhIL-12 could specifically bind with the corresponding antibody; rhIL-12-induced IFNγ showed a typical S-curve with rhIL-12 concentration in a dose-dependent manner with a titer of 8.3 × 10 6 IU / mg. Conclusion The purification of rhIL-12 expressed in CHO cells has been established. The method has the advantages of low cost, simple operation, high recovery rate and high purity, and is suitable for industrialized production.