粒-巨噬系集落刺激因子基因在大肠杆菌中的高效表达

EXPRESSION OF HUMAN GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR GENE (hGM-CSF) IN E COLI

粗-巨噬系集落刺激因子(GM-CSF)是一种重要的造血生长因子,主要是刺激粒细胞和巨噬细胞大量增殖。其临床疗效显著而且副作用小。本文先从正常人外周血细胞中提取总RNA,经逆转录——PCR扩增克隆了GM-CSF之cDNA。以DNA序列分析证实与文献一致。再设计合成一对引物定向改造编码成熟GM-CSF基因的5′端:换成大肠杆菌偏性密码,并避免起始点周围形成强二级结构,把终止密码子换成TAA。将改造后的基因克隆到载体pBV220中,转化受体菌HB101,经42℃诱导高效表达出GM-CSF。表达产物约占全菌蛋白的20％,初步纯化和复性后比活性】1×10~7U/mg。

Total RNA was isolated from human peripheral blood mononuclear cells stimulated by PHA in vitro, and used as templates in reverse transcription reaction to synthesize first strand cDNA. The cDNA of hGM-CSF was then obtained through PCR amplification. After recombination of the cDNA into pUC19 plasmid, we determined the sequence of it. The result of sequencing amplified cDNA was identical with the sequence of hGM-CSF reported. In order to enhancing expression of hGM-CSF in E coli, a recombinant hGM-CSF gene which is avoid of strong secondary structure at 5'- terminal and changed the codon to E coli like was cloned with PCR. It was constructed into pBV220 vector, then transferred to E coli HB101. After thermal induction, the expression product of hGM-CSF gene accounted as much as 20%of total bacterial soluble proteins. GM CSF inclusion bodies were separated from the soluble part and solubilized with 8 mol/L urea, then purifed through a Sepharyl S-200. The purity of the GM-CSF was 90% as analyzed on SDS-PAGE and the specific activity was about 1 × 107U / mg.