重组血小板糖蛋白Ibα活性片段在CHO细胞中表达及其功能研究  

Study of expression in Chinese hamster ovary cells and function of recombinant active fragment of glycoprotein IbαH1-V289

作　　者：赵益明[1] 邵波静[1] 董宁征[1] 沈飞[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一人民医院江苏省血液研究所,苏州215006

出　　处：《中国免疫学杂志》2007年第2期108-111,共4页Chinese Journal of Immunology

基　　金：苏大附一院血液病学"135工程"重点学科开放课题基金资助项目(编号:135XY0405);苏州市科技基金项目(S2D0607)

摘　　要：目的:建立表达重组糖蛋白Ibα活性片段(rGPIbαH1-V289)细胞株,并且纯化其表达产物,研究其生物学功能。方法:利用脂质体将含有编码GPIbαH1-V289的DNA质粒pCMV3转染CHO细胞。用亲和层析法纯化重组片段。用SDS-PAGE和Western blot鉴定纯化产品的纯度和免疫学活性。用血小板聚集法检测纯化产品对瑞斯托霉素诱导血小板聚集功能的影响。用ELISA测定纯化产品对vWF与血小板结合能力的影响。结果:每1L培养上清液可纯化到6.15mgrGPIbαH1-V289重组产品。SDS-PAGE显示,纯化的重组片段主要在42kD处显一蛋白区带,Western blot显示,抗GPIbα单抗SZ-2能与重组片段在42kD处显单一蛋白区带。纯化重组片段能抑制瑞斯托霉素诱导的血小板聚集功能。ELISA测定显示纯化产品抑制血浆vWF与血小板的结合能力。结论:CHO细胞株A1能恒定表达rGPIbαH1-V289,重组片段具有较高的纯度、免疫学活性和生物学活性,有可能开发为有效的抗血栓药物。Objective：To study the biological function of glycoprotein protein Ibα, a cell line expressing recombinant active fragment rGPIbαH1-V289 was constructed and purified fragment was acquired to research its function. Methods：The CHO cells were transfected with plasmid pCMV3 including GPIbαH1-V289 DNA by Lipofectamine. The recombinant fragment was purified with affinity chromatography column. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. The effect of rGPIbαH1-V289 on the platelet aggregation function was detected with platelet aggregation assay. The effect of purified product on binding activity between plasma vWF and platelet was measured with ELISA. Results：6. 15 mg recombinant fragment in culture supernatants per liter could be purified with affinity chromatography. The recombinant fragment formed a main lane at the position of 42 kD with SDS-PAGE and could react with monoclonal antibody SZ-2 by Western blot. It could inhibit the platelet aggregation induced by ristocetin and inhibit binding activity between plasma vWF and platelets. Conclusion： CHO cell line A1 can express rGPIbαH1-V28 stably and the recombinant fragment which has high purity, immune activity and biology activity, can be a potential anti-thrombosis drug.

关 键 词：CHO细胞 重组糖蛋白Ibα 表达 纯化 

分 类 号：R392[医药卫生—免疫学] Q78[医药卫生—基础医学]