重组人干扰素β1a的纯化及其理化性质

Purification and physico-chemical property of recombinant human interferon β1a

目的纯化重组人干扰素β1a(recombinant human interferonβ1a,rhIFNβ1a),并分析其理化性质。方法应用15 L生物反应器悬浮微载体培养表达rhIFNβ1a的重组CHO细胞,细胞培养收获液经超滤浓缩、蓝胶亲和层析、脱盐和CM离子交换层析纯化后,采用水泡性口炎病毒抑制法测定rhIFNβ1a的效价;Lowry法测定蛋白含量;等电聚焦电泳法测定其等电点;SDS-PAGE和高效液相色谱法测定其纯度;Western blot法分析其免疫活性;SDS-PAGE和质谱仪测定其相对分子质量;圆二色谱法分析其二级结构。结果共纯化了3批样品,纯化的rhIFNβ1a平均蛋白浓度为0.284 0 mg/ml,平均比活可达1.06×108IU/mg,纯度达95%以上,蛋白质平均回收率为58.36%,蛋白质相对分子质量为20 445.0,等电点约为6.6,免疫活性良好,二级结构与国家标准品基本一致。结论成功纯化了rhIFNβ1a,并检测了其理化性质,为建立大规模制备rhIFNβ1a的生产工艺和制造检定规程,实现产业化奠定了基础。

Objective To purify recombinant human interferon β1a（rhIFNβ1a）and analyze its physico-chemical property.Methods Recombinant CHO cells for expression of rhIFNβ1a were cultured on suspended micro-carriers in a 15 L bioreactor,of which the harvest liquid was concentrated by ultrafiltration and purified by Blue Sepharose 6 Fast Flow,Sephadex G-25 and CM ion exchange chromatography,then determined for rhIFNβ1a titer by vesicular stomatitis virus（VSV）inhibition test,for protein content by Lowry method,for isoelectric point by isoelectric focusing electrophoresis,for purity by SDS-PAGE and HPLC,for immunological activity by Western blot,for relative molecular mass by SDSPAGE and mass spectrometry,and for secondary structure by circular dichroism spectrum.Results Three batches of rhIFN-β1a samples were purified,of which the mean protein content was 0.284 0 mg/ml,the mean specific activity was 1.06 × 108 IU/mg,the purity was more than 95%,the mean recovery of protein was 58.36%,the relative molecular mass was 20 445.0,the isoelectric point was about 6.6,the immunological activity was high,and the secondary structure was basically consistent with that of national standard.Conclusion The rhIFNβ1a was successfully purified,and its physic-chemical property was determined,which laid a foundation of development of requirement for large-scale preparation,control test and industrialization of rhIFNβ1a.