重组人凝血因子Ⅶ的纯化与鉴定  

Purification and identification of recombinant coagulation factor Ⅶ using monoclonal antibody immunoaffinity chromatography

作　　者：吕茂民[1] 王卓[1] 冯晶晶[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中国输血杂志》2010年第4期252-255,共4页Chinese Journal of Blood Transfusion

基　　金：军事医学科学院创新基金/输血保障工程项目资助(9009)

摘　　要：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 purify and character the recombinant human coagulation factor Ⅶ （rhFⅦ） from the cells expression supernatant fluid. Methods The monoclonal antibody was coupled with the CNBr-Sepharose 6B Fast Flow media and formed FⅦ immunoaffinity chromatography gel, which was used to prepare the column. The column was validated by using standard recombinant human coagulation factor Ⅶ. Then the rhFⅦ was adsorbed by DEAE-Sephadex A-50 gel from the cells expression supernatant, removed salts by Sephadex G-25 gel column and followed by FⅦ immunoaffinity chromatography column. The total and purify protein were monitored at each stage using SDS-PAGE and Western Blot. The biology activity of purified rhFⅦ was confirmed by prothrombin time （PT） assay. Results The FⅦ immunoaffinity chromatography column showed a good absorption to standard and expressed rhFⅦ protein; non-special proteins were removed after three steps purification; the coagulation time of purified protein was 52s, which indicated the protein was fully biological activity. Conclusion Recombinant human coagulation factor Ⅶ monoclonal antibody immunoaffinity chromatography method was constructed successfully and provided a useful system for future study.

关 键 词：亲和层析 重组人凝血因子Ⅶ(rhFⅦ) 纯化 单克隆抗体 

分 类 号：R392.33[医药卫生—免疫学] Q784[医药卫生—基础医学]