【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。更多还原

【Abstract】 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 rmGMCSF,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 polyethylene glycol( PEG) as a co-solvent was employed to refold proteins in 2 mol / 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.更多还原