人血管内皮生长因子受体FLT-1胞外区1-3环cDNA的表达、纯化及鉴定

目的　研究人血管内皮生长因子受体FLT 1胞外区 1 3环cDNA在酵母菌中的表达、纯化及生物学活性。方法　将编码 316个氨基酸残基的人FLT 1胞外区 1 3环cDNA插入到含AOX1启动子和d分泌信号肽序列的Pichiapastoris酵母载体中 ,构建了重组表达质粒pPICqk/FLT l(1～ 3) ,转化酵母宿主菌GS115 ,筛选His+ Mut表型转化子 ,经摇瓶培养 ,1%甲醇诱导表达。结果　经SDS -PAGE显示 ,表达产物以可溶性分子形式存在于上清中 ,诱导 4天的表达量达到培养上清总蛋白的 6 0 % ,径Westernblot检测抗原性及特异性良好 ,经CM SepharoseFF阳离子交换层析和Sephacryls 10 0分子筛层析 ,纯度达 90 %以上 ;经生物活性检测具有结合hVEGF16 5的能力和hVEGF16 5促进HUVEC的增殖功能。结论　人血管内皮生长因子受体FLT 1胞外区 1～ 3环cDNA在酵母菌中表达成功。

Expression of Hmnan Vascular Endothelial Growth Factor Receptor FLT-1Extracellular Domain 1-3 Loop cDNA in Pichia. pastoris and Purificationand Biological Activity of Expressed Product

Objective To express human vascular endothelial growth factor receptor FLT 1 extracellular domain 1 3 loop cDNA in Pichia.pastoris ,purify the expressed product and detect the biological activity of it .Methods By inserting human FLT 1(1 3 loop) cDNA coding 316 amino acid residues into Pichia.pastoris expression vector pPIC9K containing AOXI promoter and the sequences of α secreting signal peptides,a recombinant expression plasmid pPIC9K/FLT 1(1 3) was constructed and transformed to GS115,then His +Mut s phenotype transformant was selected and cultured in flasks,and FLT 1(1 3)was expressed under the induction of 1% methanol.Results SDS PAGE showed that,after being induced with 1% methanol for 4 days, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed.Western blot showed good antigenicity and specificity of expressed product.After being purified by CM Sepharose FF and Sephacryl S 100 chromatography,the purity of expressed product reached above 90%.Biological assay proved that the expressed product could combine with hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF 165.Conclusion Human vascular endothelial growth factor receptor FLT 1 extracellular domain (1 3 loop)was successfully expressed.The study laid a foundation of further application of the expressed product in the treatment of vasoformationrelated diseases,such as tumor and diabetic retinopathy.