摘要
基因工程重组人粒细胞集落刺激因子(rhG-CSF)主要用于癌症患者化疗后的粒细胞减少症.在正确克隆人G-CSFcDNA的基础上,重点对G-CSFcDNA的5′端进行了较为彻底的修饰,修饰后的基因插入pBV220载体组建成功pBV220/G-CSF/2-174高效表达载体.表达后SDS-PAGE分析其表达最高达50%以上.根据G-CSF表达形成包涵体这一特性,建立了一条简便、稳定,适用于大规模生产的分离纯化工艺流程.首先分离纯化包涵体,8mol/L尿素裂解包涵体,稀释复性蛋白,之后一步SP-SepharoseFF柱层析至均质.纯化的G-CSF比活性达3.4×108U/mg蛋白,每升表达菌液回收的G-CSF总活性达1.06×1011U.纯化产物的N-端氨基酸序列分析表明,对甲硫氨酸的去除彻底。
Recombinant human granulocyte colony stimulating factor (rhG CSF) is mainly used in neutropenia induced by cytotoxic chemotherapy in clinical practice.After Chinese human G CSF cDNA was cloned,the 5′ terminal sequence in G CSF cDNA was thoroughly modified in order to raise the expression level.pBV220/G CSF/2 174 was constructed by inserting the modified gene into pBV220 vector.At last,over 50% expression level was achieved as analyzed by SDS PAGE.As G CSF was in inclusion body in E.coli ,a simple and stable purification protocol was established,which was very suitable for large scale purification.Firstly,inclusion body was extracted from E.coli ,and then,8 mol/L urea was used to lysis the inclusion body.G CSF protein was renatured by dilution.At last,the pure G CSF was recovered by one step SP Sepharose FF chromatography.The relative activity of purified G CSF reached to 3 4×10 8 U/mg protein.A total G CSF activity from 1 L fermentation was about 1 06×10 11 U.As demonstrated by N terminal amino acid sequencing,methionine had thoroughly been removed,so this kind of purified G CSF may have a low immunogenicity and toxicity.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1998年第5期479-484,共6页
Chinese Journal of Biochemistry and Molecular Biology