人血管内皮细胞生长因子cDNA克隆和在大肠杆菌的高效表达

CLONING cDNA OF HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR AND HIGH LEVEL EXPRESSION IN ESCHERICHIA COLI

目的 在大肠杆菌高效表达人血管内皮生长因子蛋白质。方法 用PCR从人胎儿脑cDNA文库扩增血管内皮细胞生长因子 (VEGF )cDNA ,得到 5 17bp的DNA片段。扩增片段重组到M 13mP18中 ,经测序证实为VEGF165cDNA。将该片段重组到PRL62 1温控表达质粒中 ,在大肠杆菌表达 2 0kd的重组蛋白。结果 该表达产物占菌体总蛋白的 3 5 % ,其N 端 15个氨基酸序列与天然VEGF165蛋白相应序列一致。结论 工程菌TG1/PRL62 1/VEGF高效表达人VEGF165蛋白质。

Objective To be expressed human vascular endothelial growth factor (VEGF) recombinant protein in Escherichia Coli in high level. Methods VEGF was amplified from human fetal brain cDNA library, the amplified fragment was inserted into M13mP18 and confirmed to be VEGF165cDNA by restriction mapping and DNA sequencing, then it was combined with an expression vector PRL621. This recombinant plasmid overexpressed a 20kd recombinant protein in E.Coli(TG1), the protein was isolated and purifed from E.Coli, and initially renatured. Results The overexpressed recombinant protein was 35% of the total cell protein, the sequence of its first 15-N terminal amino acid was identrical to that of the human natural VEGF protein, Chorioallantoic membrane(CAM) assay showed that the rhVEGF promated new capillary vessels formation. Conclusion The genetic engineering Escherichia Coli can express human vascular endothelial growth factor in high level.