人血管内皮细胞生长因子受体Flt-1胞外区cDNA在毕赤酵母中的表达和鉴定  

Expression and Characterization of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain in Pichia pastoris

作　　者：马骊[1] 张智清[1] 周小明[1] 曾革非[1] 陈爱君[1] 姚立红[1] 王小宁[1] 

机构地区：[1]中国预防医学科学院病毒学研究所,北京100052

出　　处：《生物工程学报》2000年第4期447-450,共4页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划资助项目!( 10 2 0 8 0 1 0 3 )

摘　　要：将编码血管内皮细胞生长因子受体Flt 1胞外区 1 3loop 316个氨基酸残基的cDNA插入到含AOX1启动子和α分泌信号肽序列的Pichiapastoris酵母载体中 ,构建了重组表达质粒 pPIC9K/Flt 1(1 3) ,转化酵母宿主菌GS115 ,筛选His+ Muts 表型转化子 ,经摇瓶培养 ,1%甲醇诱导表达 4d后 ,SDS PAGE结果显示 ,培养上清中Flt 1(1 3)表达量达总蛋白的 30 %以上。ELISA及Westernblot实验表明 ,表达产物具有良好的抗原性和特异性。生物学活性检测证实其具有结合VEGF的能力和抑制VEGF对HUVEC细胞的促增殖功能。By inserting VEGF receptor Flt\|1(1\|3 loop) cDNA into \%Pichia pastoris\% expression vector pPIC9K containing AOX 1 promotor and the sequences of α secreting signal peptides,the expression plasmid pPIC9K/Flt\|1(1\|3) was constructed and transformed into GS115.The multi\|copy insert transformants were selected and cultivated in flasks.After 4 days of 1% methanol induction,the expressed Flt\|1(1\|3) accumulated up to 30% of total proteins in supernatant.The expressed Flt\|1(1\|3) was further proved with good antigenicity and high specificity by ELISA and Western blot.They can bind to VEGF and inhibit HUVEC proliferation stimulated by VEGF.

关 键 词：血管内生长因子 FIt-1 酵母表达系统 鉴定 

分 类 号：Q786[生物学—分子生物学]