人粒细胞巨噬细胞集落刺激因子的基因修饰及其在大肠杆菌中的高效表达

Modification of N Terminal cDNA of hGM CSF and High Expression in E. coli.

目的对人粒细胞巨噬细胞集落刺激因子（ｇｒａｎｕｌｏｃｙｔｅ－ｍａｃｒｏｐｈａｇｅｃｏｌｏｎｙｓｔｉｍｕｌａｔｉｎｇｆａｃｔｏｒ，ＧＭ－ＣＳＦ）ｃＤＮＡ５′端进行修饰以实现在大肠杆菌中的高效表达。方法采用ＰＣＲ方法进行插入和定点突变，转化大肠杆菌实现原核系统表达。通过细胞和生物学方法对表达产物进行鉴定。结果修饰后基因的氨基酸序列与文献报道完全一致，重组蛋白表达量占菌体总蛋白的２５％；重组蛋白可刺激体外培养的ＴＦ－１细胞生长和细胞集落形成；其蛋白比活达到１．５×１０７／ｍｇ。氨基酸Ｎ末端序列分析表明，前１６位氨基酸序列与天然序列完全一致。结论对ＧＭ－ＣＳＦｃＤＮＡ５′端进行修饰可有效地提高Ｎ末端启始区的翻译效率，从而实现在大肠杆菌中的高效表达。获得具有生物学活性的重组蛋白。

Objective N terminal cDNA of human granulococyte macrophage colony stimulating factor(GM CSF) was designed to be modified and highly expressed in E.coli. Methods A pair of oligo nucleotide primers were used to modify the mature N terminal cDNA sequence of hGM CSF with the method of PCR. the modified cDNA of hGM CSF was cloned into E.coli. expressive vector PBV220 and expressed in E.coli DH5α strain, the biological activities of the recombinant protein was identified by means of cell colony formation and TF 1 cell growth assay in vitro. Results The expression level of modified hGM CSF cDNA was higher than that of unmodified native type. SDS PAGE revealed that expressed protein of hGM CSF accounted for about 25% of total bacterial cell protein. The biological activity of recombinant protein was about 1 5×10 7 U/mg protein. The sequence of 1￣16 amino acid of N terminal of the recombinant protein was same with native hGM CSF.Conclusions Modification of the N terminal cDNA of hGM CSF could dramatically enhance the expression level in E. coli system.