目的建立重组人凝血因子Ⅶ(rhFⅦ)的纯化方法。方法首先将制备并纯化的单克隆抗体与CNBr-Sepharose 6B Fast Flow介质偶联,制备单克隆抗体亲和层析柱,并用rhFⅦ标准品验证层析柱的层析效果;采用DE-AE-Sephadex A-50吸附细胞表达的上清,对吸附产物用Sephadex G-25脱盐柱做脱盐处理;然后用单克隆抗体亲和层析柱做亲和层析;通过SDS-PAGE、Western Blot等试验测定纯化产物的纯度;通过凝血酶原时间(PT)测定纯化产物的促凝活性。结果制备出rhFⅦ单克隆抗体亲和层析介质;细胞表达产物经DEAE-Sephadex A-50、Sephadex G-25脱盐和亲和层析纯化后,经SDS-PAGE鉴定获得了分子量为50kD的单一蛋白洗脱峰;纯化所获得的重组蛋白具有促凝活性,促凝时间(DT)为52s。结论建立了纯化rhⅦ的亲和层析方法 ,为rhFⅦ的研制奠定了基础。 Objective To establish a method for the purification of recombinant human coagulation factor Ⅶ (rhFⅦ). Methods The monoclonal antibodies were prepared and purified by coupling with CNBr-Sepharose 6B Fast Flow medium to prepare monoclonal antibody affinity chromatography column. The chromatographic results of the column were validated by rhFⅦ standard. The DE-AE-Sephadex A-50 was used to adsorb the supernatant of the cells and the adsorbed product was desalted by Sephadex G-25 desalting column; then affinity chromatography was performed with monoclonal antibody affinity chromatography column; SDS-PAGE, Western Blot and other tests The purity of the purified product; the procoagulant activity of the purified product was determined by prothrombin time (PT). Results The affinity chromatography medium of rhFⅦ monoclonal antibody was prepared. The recombinant protein was purified by DEAE-Sephadex A-50, Sephadex G-25 desalting and affinity chromatography, and then identified by SDS-PAGE. The peaks of the purified recombinant proteins were procoagulant activity and the procoagulant time (DT) was 52s. Conclusion The method of affinity chromatography of purified rhⅦ was established, which lays the foundation for the development of rhFⅦ.