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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，用于人体时可能具有较小的免疫原性和毒性
Genetically engineered recombinant human granulocyte-colony stimulating factor (rhG-CSF) is mainly used for neutropenia in cancer patients after chemotherapy. Based on the correct cloning of human G-CSF cDNA, the 5 ’end of G-CSF cDNA was modified thoroughly. The modified gene was inserted into pBV220 vector to construct pBV220 / G-CSF / 2-174 high expression vector. After the expression of SDS-PAGE analysis of its expression up to 50% or more. According to the characteristic of inclusion body formed by G-CSF expression, a simple and stable separation and purification process suitable for large-scale production was established. First of all, the inclusion bodies were isolated and purified, then 8mol / L urea was used to lyse the inclusion bodies and dilute the renaturation protein, followed by one-step SP-Sepharose FF column chromatography to homogeneity. The specific activity of purified G-CSF was 3.4 × 108U / mg protein, and the total activity of G-CSF recovered per liter of bacterial solution reached 1.06 × 1011U. N-terminal amino acid sequence analysis of the purified product showed that the removal of methionine was completely intact and could have less immunogenicity and toxicity when used on humans