重组人可溶性gp130的纯化及生物学活性鉴定

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pH 2.8、0.1 mol/L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/ml)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。

Purification and identification of recombinant human sobluble gp130

The cells transfected with human soluble gp130 gene were cultivated in DMEM medium supplemented with 10% heated-inactivated fetal bovine serum and 800 μg G418,and the content of soluble gp130 in the cultural supernatants was about 100~120 μg/ml as demontrated by ELISA by using the immuno-affinity chromatography,the anti-human soluble gp130 monoclonal antibody purified from ascitic fluids with G protein was conjugated to CNBr-activated Sepharose 4B,and the supernatants of cultural cells transfected with human soluble gp130 gene were harvested,pre-treated and passed through the anti-gp130 mAb/Sepharose 4B column.Then,the soluble gp130 protein was eluated from column by pH 2.8,0.1 mol/L glycine or 3 mol/L KCNS.Finally,the purified recombinant human soluble gp130 was thus obtained.It was found that the rate of recovery of soluble gp130 protein was 70%~75%,and only one band of purified suluble gp130 protein was demonstrated after SDS-PAGE analysis.This purified protein bound with specific antibodies 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 biological activities.