人VEGFcDNA的克隆及其在大肠杆菌中的高效表达  

CLONIGN OF cDNA FOR HUMAN VEGF AND ITS HIGH-EFFICIENCY EXPRESSION IN E.COLI

作　　者：杨亚东[1] 李岱宗[1] 杨和平[1] 王贵春 张晨晖[1] 周爱儒[1] 汤健[1] 马大龙[1] 

机构地区：[1]北京医科大学心血管基础研究所,上海市肿瘤研究所

出　　处：《北京大学学报（医学版）》1994年第S1期85-88,共4页Journal of Peking University:Health Sciences

摘　　要：利用ＰＣＲ技术从人胎肝扩增并克隆人ＶＥＧＦ（血管内皮生长因子）的ｃＤＮＡ，构建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人ＶＥＧＦ蛋白的高效表达，表达产物占细菌总蛋白的３０％以上。EGF is a recently discovered growth factor specific to vascular endothclial cells.The cDNA for VEGF is isolated and cloned by PCR from human fetal liver,with sense primer 5′-GGG GGATCC GCC TCC GAA ACC ATG AAC TT-3′and antisense primer 5′-CCC GAA TTC TCC TGG TGA GAG ATCTGG TT-3′.Sequencing analysi: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 PL promoter and two SD sequences.The foreign VEGF cDNA segment is constructed into pKPL-3b between Nco l 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。

关 键 词：血管内皮生长因子 聚合酶链反应 大肠杆菌 高效表达 血管内皮细胞 

分 类 号：R392.13[医药卫生—免疫学]