南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (08): 1124-1127.

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小鼠粒细胞-巨噬细胞集落刺激因子的表达、纯化与鉴定

温茜； 马骊； 苏瑾； 罗微； 王小宁；   

  1. 南方医科大学生物技术学院分子免疫学研究所； 华南理工大学生物科学与工程学院 广东广州510515； 广东广州510515； 广东广州510641；
  • 出版日期:2006-08-20 发布日期:2006-08-20
  • 基金资助:
    国家重点基础研究发展规划(973)资助(2001CB510008)~~

Expression, purification and identification of mouse granulocyte-macrophage colony- stimulating factor

WEN Qian1, MA Li1, SU Jin1, LUO Wei1, WANG Xiao-Ning2 1Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China; 2School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China   

  1. 南方医科大学生物技术学院分子免疫学研究所； 华南理工大学生物科学与工程学院 广东广州510515； 广东广州510515； 广东广州510641；
  • Online:2006-08-20 Published:2006-08-20

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摘要： 目的构建小鼠粒细胞-巨噬细胞集落刺激因子(mGM-CSF)工程菌,通过摸索其变、复性和纯化条件,得到高纯度高比活的重组mGM-CSF蛋白。方法以本实验室构建的hIL-2/mGM-CSF融合蛋白表达载体为模板,PCR扩增mGM-CSF基因,克隆入pET-11c表达载体,转化BL21,构建BL21/pET-11c/mGM-CSF工程菌,用本所专利方法提取包涵体,在含低浓度盐酸胍的复性液中复性,采用镍离子亲和层析纯化。结果工程菌采用TH肉汤培养,32℃、0.1mmol/LIPTG双重诱导,表达量达菌体蛋白总量的60.6%。提取包涵体在含1.5mol/L盐酸胍的谷胱甘肽复性液中复性效果最好,比活达4.2×106U/mg。经过亲和层析一步纯化,目的蛋白纯度达95%,比活与标准品相当。结论构建了高效表达mGM-CSF的工程菌,建立了其复性和纯化工艺,为进一步研究DC、GM-CSF体内抗肿瘤功能奠定了基础,并可为IL-2/GM-CSF双功能分子的生物学功能研究提供对照。 

Abstract: Objective To construct mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) expression vector and express, purify and refold mGM-CSF protein. Method Based on previously constructed fusion protein hIL-2/mGM-CSF expression vector, pET-11c/mGM-CSF expression vector was constructed routinely and transformed into BL21 (DE3). The inclusion body protein was washed with our patented method and refolded with renaturation buffer containing low-concentration guanidinium chloride (Gu·Cl). The refolded protein was purified with affinity chromatography. Results pET-11c/mGM-CSF vector was constructed successfully. The host bacteria was cultured in TH broth and induced with 0.1 mmol/L IPTG at 32 ℃, which resulted in the expression level of 60.6%. The best refolding effect was achieved with the renaturation media containing glutathione and 1.5 mol/L Gu·HCl. After purification with affinity chromatography, the purity of the target mGM-CSF protein reached 95% with activity of 5×106 U/mg. Conclusion Engineered bacteria BL21/pET- 11c/mGM-CSF with efficient mGM-CSF expression and laboratory scale renaturation and purification of mGM-CSF have been established, which facilitates further researches into the anti-tumor function of the dendritic cells and GM-CSF in vivo. 

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