摘要
粒细胞-巨噬细胞集落刺激因子(GM-CSF)是一种造血生长因子和促炎细胞因子。重组鼠源粒细胞-巨噬细胞集落刺激因子在抗肿瘤及抗炎研究中有着极其重要的作用。以NcoI和BamH I为酶切位点将mGM-CSF编码序列插入到pET-28a载体中。转染E.coli宿主菌BL21后得到以包涵体形式表达的蛋白,表达量占菌体总蛋白量51.6%。包涵体的纯化条件在实验中进一步优化,然后溶解于8 mol/L尿素中。采用稀释法在2 mol/L尿素缓冲液中添加PEG对蛋白进行复性。复性后的产物经过离子交换层析纯化后得到高纯度的有活性的mGM-CSF。通过体内白细胞计数和体外髓样细胞增殖检测表明纯化的蛋白具有mGM-CSF全部的生物活性。这种制备方法能够从每升发酵液中获得8.06 mg蛋白,具有蛋白表达量高,复性和纯化方法简便易得的特点,适用于规模生产。
Granulocyte macrophage-colony stimulating factor （GM-CSF） is a hematopoietic growth factor and a proinflammatory cytokine. Recombinant mouse GM-CSF （rmGM-CSF） is necessary in antineoplastic and anti-inflammatory research. To obtain rmGM- CSF, the encoding region for mGM-CSF was cloned between NcoI and BamHI in pET28a- The protein was expressed in the form of inclusion bodies in E. coli host strain BL21 （DE3）. The expression level was more than 51.6% of total cell lysate. Inclusion bodies were washed under optimized conditions, and dissolved in a buffer containing 8 mol/L urea. Dilution method combined with polyethy- lene glycol （PEG） as a co-solvent was employed to refold proteins in 2 raoL/L urea. After ion-exchange chromatography, rmGM-CSF with a high purity was obtained. Purified rmGM-CSF has a good fidelity, which makes it possible to promote proliferation of myeloid cells in vitro and to increase the number of leukocyte and neutrophil in vivo. A high yield of 8. 06 mg rmGM-CSF per litre of cell culture was achieved by using this method. Owing to its high yield and simple operation, this protocol is suitable for large-scale production.
出处
《药物生物技术》
CAS
2013年第5期386-391,共6页
Pharmaceutical Biotechnology
基金
supported by NFSC grant 81172973/H3004,31270985
College Students Innovation Project for the R&D of Novel Drugs J1030830
关键词
重组鼠粒细胞巨噬细胞集落刺激因子
包涵体
聚乙二醇
纯化
复性
生物活性
Recombinant mouse granulocyte - macrophage colony-stimulating factor, Inclusion bodies, Polyethylene glycol, Purifi-cation, Refolding, Bioactivity
