【摘要】 利用PCR技术从人胎肝扩增并克隆了人VEGF(血管内皮生长因子)的cDNA,构建了其天然蛋白高效表达载体pKPL-3b-VEGF,利用这一载体在大肠杆菌中获得了人VEGF蛋白的高效表达,表达产物占细菌总蛋白的30%以上。更多还原

【Abstract】 VEGF is a recently discovered growth factor specific to vascular endothelial cells. The cDNA for VEGF is isolated and cloned by PCR from human fetal liver, with sense primer 5′-GGG GGA TCC GCC TCC GAA ACC ATG AAC TT-3′ and antisense primer 5′-CCC GAA TTC TCC TGG TGA GAG ATC TGG TT-3′. Sequencing analysis shows that the cloned segment is in accordance with VEGF₁₆₅ reported previously. High-efficiency expression of human VEGF in E. coli is achieved, with the expression products accounting for more than 30% of the total bacterial proteins. The employed expression system includes POP2136 strain of E. coli as engineering host and pKPL-3b as vector, which provides the P_L promoter and two SD sequences. The foreign VEGF cDNA segment is constructed into pKPL-3b between Nco I and EcoR I sites. The triplet ATG in the Nco I site plays the role of translation initiation codon with unchangement of the open reading frame but loses the first two amino acids of the N-terminal of natural mature VEGF. This study provides an easy alternative way for large-scale preparation of VEGF.更多还原