重组人可溶性gp130的纯化及生物学活性鉴定  

Purification and identification of recombinant human sobluble gp130

作　　者：邱玉华[1] 马泓冰[1] 程钢[1] 孙玮丽[1] 孙中文[1] 周立峰[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,苏州215007

出　　处：《现代免疫学》2006年第4期302-305,共4页Current Immunology

基　　金：江苏省自然科学基金资助项目(BK2004203);江苏省高校高新产业发展基金资助项目(JHB05-45)

摘　　要：采用转瓶培养可溶性gp130基因转染细胞。运用免疫亲和层析法，将抗人可溶性gp130的单克隆抗体偶联于CNBr活化的Sepharose 4B。收获基因转染细胞的培养上清，经抗gp130单抗／Sepharose 4B亲和层析柱吸附，用pH2．8、0．1mol／1．甘氨酸溶液洗脱，获取可溶性gp130重组蛋白纯品。基因转染细胞培养上清中可溶性gp130的表达量为100～120μg／ml，纯化后的回收率为70％～75％，SDS-PAGE电泳后出现一条蛋白曲带。经Western blot分析，纯化的可溶性gp130能与特异性抗体结合。将可溶性gp130（终浓度为5μg／m1）与IL-6依赖性生长细胞株XG2共同培养，细胞的生长与增殖出现抑制。提示经免疫亲和层析法纯化的可溶性gp130具有良好的生物学活性。The cells transfected with human soluble gp130 gene were cultivated in DMEM medium supplemented with 10% heated-inactivated fetal hovine serum and 800μg G418, and the content of soluble gp130 in the culturalsupernatants was about 100～120μg/mlasdemontrated by ELISA hy using theimmuno affinity chromatography, the anti human solublegp130 mono clonal antibody purified from ascitic fluids with G protein was conjugated to CNBr-activated Sepharose 4B , and the superna rants of cultural cells transfeeted with human soluble gp130 gene were harvested, pre-treated and passed through the antigp130 mAh/Sepharose 4B column. Then, the soluhle gp130 protein was eluated from column by pH 2.8, 0. 1 mol/l, glycine or 3 mol/l. KCNS. Finally, the purified recomhinant human soluhlegpl30 was thusohtained. It was found that the rate of re covery of soluhle gp130 protein was 70%～75%, and only one hand of purified suluhle gp130 protein was demonstrated after SDS-PAGE analysis. This purified protein hound with specific antihodies as demonmstrated by Western blot analysis. When this protein（ final concentration 5 μg/ml） was co cultivated with IL-6-dependent cell line XG2, cell growth and proliferation were definitely inhibited, indicating that this purified protein possess good purity with excellent hiological activities.

关 键 词：重组 可溶性GP130 单克隆抗体 亲和层析 纯度 生物学活性 

分 类 号：R392.33[医药卫生—免疫学]