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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 murine granulocyte-macrophage colony-stimulating factor plays an extremely important role in antitumor and anti-inflammatory research. The mGM-CSF coding sequence was inserted into pET-28a vector using NcoI and BamH I as restriction sites. After transfection with E. coli host strain BL21, the expressed protein was expressed in inclusion bodies, accounting for 51.6% of the total bacterial protein. The purification conditions of inclusion bodies were further optimized in the experiment and then dissolved in 8 mol / L urea. Dilution method was used to renature protein in 2 mol / L urea buffer. Refolded products were purified by ion-exchange chromatography to obtain high-purity active mGM-CSF. Detection of in vivo leukocyte counts and in vitro myeloid cell proliferation demonstrated that the purified protein possesses all of the biological activity of mGM-CSF. The preparation method can obtain 8.06 mg of protein per liter of fermentation broth, has the characteristics of high protein expression, renaturation and simple and convenient purification method, and is suitable for large-scale production.