重组人粒细胞集落刺激因子在Escherichia coli中的表达研究

Recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli

根据人天然粒细胞集落刺激因子 (G -CSF)基因序列设计出引物 ,通过RT -PCR从人外周血单个核细胞mRNA获得G -CSFcDNA片段 .将该片段与原核表达载体 pT 7构建成重组体 ,导入大肠杆菌后发现天然G -CSFcD NA表达量并不理想 .这可能是由于天然hG -CSF 5’端G +C的比例过高 ,使转录后的mRNA很容易形成二级结构而影响翻译起始 ,因此在大肠杆菌中很难获得高效表达 .根据密码子简并性原则 ,在不改变编码氨基酸顺序的前提下 ,通过重新设计PCR引物 ,将hG -CSF的前 3个氨基酸密码子中的几个GC碱基作了改动 ,获得了新的cDNA突变体 ,将其与pT 7的重组体导入大肠杆菌 。

The primers are designed according to gene sequence of natural granulocyte colony-stimulating factor (G-CSF). G-CSF cDNA has been fished from the mRNA of mononuclear cells from human peripheral blood through RT-PCR. The cDNA was inserted into a prokaryotic expression vector pT 7 to make the recombinant plasmid. Finding that the expression level of the E.coli containing natural human G-CSF cDNA was not satisfied. The reason why the high expression level is not easily reached in E.coli may be that the G/C ratio at 5' terminus of human G-CSF cDNA is too high, and so the transcribed mRNA is liable to form secondary structure, which affects the beginning of transcription. On the principle of codon degeneracy, altered the codons of the first three amino acids of G-CSF cDNA by redesigning PCR primers and gained a new rhG-CSF cDNA mutant. A remarkable increase in the expression of rhG-CSF was obtained in the E.coli transformed with the recombinant plasmid of the mutant.