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.
出处
《生物工程学报》
CAS
CSCD
北大核心
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 )
