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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。
Objective To study the expression, purification and biological activity of cDNA encoding human vascular endothelial growth factor receptor 1 (FLT 1) extracellular domain in yeast. Methods The cDNA of FLT 1 extracellular domain of 316 amino acid residues was inserted into the Pichia pastoris yeast vector containing the AOX1 promoter and the signal peptide sequence of d secretion. The recombinant plasmid pPICqk / FLT1 (1 ~ 3 ) Was transformed into yeast host strain GS115, His + Mut phenotype transformants were screened and expressed in 1% methanol after shake flask culture. Results SDS-PAGE showed that the expressed product was soluble in the supernatant, and the expression reached to 60% of the total protein in the supernatant after 4 days of induction. The antigenicity and specificity of the expressed protein were determined by Western blotting. The CM Sepharose FF cation Exchange chromatography and Sephacryls 10 0 molecular sieve chromatography, purity of more than 90%; bioactivity detection with the ability to bind hVEGF16 5 and hVEGF16 5 promote HUVEC proliferation. Conclusion The 1 ~ 3 loop cDNA of human vascular endothelial growth factor receptor FLT 1 extracellular domain was successfully expressed in yeast.