人GM—CSF cDNA的克隆和在大肠杆菌中的表达

从诱导的人胚肺细胞ＨＦＬ株中提取总ＲＮＡ．经ＲＴ－ＰＣＲ反应获取了人ＧＭ－ＣＳＦｃＤＮＡ，ＤＮＡ序列测定表明其顺序与文献报道完全一致。为了获得高效表达，应用ＰＣＲ改造了人ＧＭ－ＣＳＦ的ｃＤＮＡ５’端核苷酸序列，并将改造的人ＧＭ－ＣＳＦ基因插入含Ｔ７启动子的质粒ｐＥＴ－１１ｄ构建成表达质粒ｐＥＴＣ－５，将此质粒转化大肠杆菌株ＢＬ２１（ＤＥ３）得到表达菌株ＢＬＥＣ４。表达菌株用０．５ｍｏｌ／ＬＩＰＴＧ诱导２小时后，产生大量重组蛋白并形成包涵体。ＳＤＳ—ＰＡＧＥ电泳图谱扫描结果表明，ｒｈＧＭ－ＣＳＦ产量占菌体总蛋白量的１６％。ＥＬＩＳＡ和ＴＦ－１细胞培养测定表明，初步纯化和复性的ｒｈＧＭ－ＣＳＦ具有天然的ｈＧＭ－ＣＳＦ生物活性。

Cloning of Human Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) cDNA and Its Expression in E. coli

Human GM-CSF cDNA fragment encoding the mature GM-CSF was obtained by using RT-FCR method with total RNA extracted from induced human fetal lung cells HFL. The sequence of the hGM-CSF cDNA thus obtained was the same as those reported. In order to get expression of a high level in E. coli, the 5'terminal nuclectide sequence of hGM-CSF cDNA was modified by using PCR. the modified hGM-CSF cDNA was insertad into plasmid pET-11d containing T7promoter, with the resulted expression of plasmid pETC-5. E. coli BL21 (DE3)was transformed with pETC-5 and an expressed strain BLEC4 was selectgd. SDSPAGE analysis revealed that rhGM-CSF was produced and accumunlated up to 16% of the t tal cellular protein in the form of inclusion body in BLEC4 cells after induced by 0.5 mM IPTG for 2h. ELISA and TF-1 cell culture assay showed that the biological activity of the partially purified and renatured rhGM-CAF was similar to that of the natural human GM-CSF.