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？…

将编码血管内皮细胞生长因子受体ＦＩｔ－１胞外区１－３ｌｏｏｐ３１６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Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ酵母载体中，构建了重组表达质粒ｐＰＩＣ９Ｋ／ＦＩｔ＝１（１－３），转化酵母景菌ＧＳ１１５，筛选Ｈｉｓ＾＋Ｍｕｔ＾ｓ表型转化子，经插瓶培养，１％甲醇诱导表达４ｄ后，ＳＤＳ－ＰＡＧＥ结果显示，培养上清中ＦＩｔ－１（１－３）表达这总蛋白的３０－％

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

By inserting VEGF receptor Flt-1(1-3 loop) cDNA into Pichia pastoris expression vector pPIC9K containing AOX1 promotor and the sequences of alpha 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.