基因工程人干细胞因子原核表达、纯化及生物学活性研究  

Prokaryotic Expression, Purification and Bioactivity Determination of Recom-binant Human SCF

作　　者：陈国友[1] 厉永建[1] 蒋应明[1] 张意[1] 黄欣[1] 姜波[2] 施群英[1] 卢琳[1] 陆君燕 

机构地区：[1]第二军医大学免疫学研究所,上海200433 [2]第二军医大学长海医院放射科

出　　处：《中国肿瘤生物治疗杂志》2002年第2期126-128,共3页Chinese Journal of Cancer Biotherapy

摘　　要：目的：探索一种适合于中试生产的重组人干细胞因子（recombinant human stem cell factor,rhSCF）的发酵表达与纯化工艺。方法：研究不同培养基和热诱导时间对rhSCF表达量影响；探索最佳纯化工艺和各因素对rhSCF得率和生物学活性的影响。结果：rhSCF工程菌在含甘油、酪蛋白水解物、酵母浸出粉、胰蛋白胨及微量元素的培养基中表达量最高。灰度扫描显示热诱导后2h开始表达，6h表达量最高，目的蛋白约可达菌体总蛋白30％。高表达蛋白经复性后，可用酸沉淀作粗分离纯化，随后用阳离子交换层析分离，纯度达80％，经反相层析去除二聚体后，最后用DEAE柱浓缩后并经S200转换缓冲液，最终获得纯度＞95％和经活性＞6×10^5U/mg的rhSCF制品。结论：成功地建立了注射用rhSCF的中试生产工艺，为随后进行的临床前研究及临床试验奠定基础。Objective: To set up an optimal method of fermentation and purification of recombinant human stem cell factor (rhSCF). Methods; The effect of culture medium on the expression of rhSCF and the contents of rhSCF after different induction time were observed. The optimal condition for purification of rhSCF was also studied by changing pH, concentration of protein and chromatography procedure. The bioactivity of rhSCF was determined by UT-7 proliferation. Results: The expression level of rhSCF increased significantly in M9 culture medium containing glycerol, casein hydroly-sate, yeast extract, peptone, and trace element. The optimal induction time for rhSCF expression was 6 h, approximately 30% of total protein. The insoluble inclusion body of rhSCF was denatured by 8M urea. After refolding for 24 h, the protein was firstly purified by acid precipitation and the supernatant was applied to an Source SOS column. The disulfide-linked dimeric rhSCF was excluded by reverse phase chromatography. The buffer was converted to PBS by S200. The purity of final product was over 95% with the biological activity more than 6 × 105 U/mg. Conclusion: The optimal condition for rhSCF preparation was successfully established, which can be used for scale-up production in the future.

关 键 词：基因工程 干细胞因子 蛋白纯化 蛋白表达 生物学活性 重组人干细胞因子 rhSCF 

分 类 号：TQ937[化学工程]