重组人白细胞介素-12(CHO细胞)的纯化及其活性

Purification and Activity of Recombinant Human IL-12(CHO Cells)

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 develop a purification procedure for recombinant human IL-12 （rhIL-12） expressed in CHO cells and determine the biological activity of purified rhlL-12. Methods The culture supernatant of CHO cells highly expressing rhlL-12 was purified by chromatography combined with ammonium sulfate precipitation. The content of target protein was determined by ELISA, and the recovery, rate was calculated. The purified product was identified by SDS-PAGE, SEC-HPLC and Western blot. The biologieal activity of purified rhlL-12 was determined by induction of IFNγ in PBMCs. Results The total recovery rate of purified rhlL-12 was 56. 4%. SEC-HPLC showed that the purified rhIL-2 reached a purity of 99. 2%. SDS-PAGE proved that the relative molecular masses of two subunits were consistent with their theoretical values （40 000 and 35 000） respectively. Western blot revealed specific binding of rhIL-12 to the corresponding antibody. The dose-response curve of rhIL-12 for induction of IFNγwas in a typical S shape, indicating the dose-dependent biological activity of rhlL-12. The potency of rhlL-12 was 8. 3 × 10^6 IU/mg. Con- clusion The purification procedure of rhIL-12 expressed in CHO cells was developed, which was low-cost and simple to handle, and both the recovery rate and purity of purified product were high, indicating that the developed procedure was suitable for industrial production.