人血管内皮生长因子受体胞外1-3、2-3 loop cDNA在酵母中的表达及其生物学活性研究

Expression and biological activity analysis of human vascular endothelial growth factor receptor FLT-1 1-3 and 2-3 loop within extracellular domain in Pichia pastoris

目的 在酵母中实现人血管内皮生长因子 (hVEGF165)受体FLT 1胞外 1 3及 2 3loopcDNA的高效分泌性表达 ,并比较两种表达产物的生物学活性。方法 构建酵母重组表达质粒pPIC9K/FLT 1(1 3)及pPIC9K/FLT 1(2 3) ,并分别转化酵母宿主菌GS115。表达产物经CM SepharoseFF阳离子交换层析和SephacrylS 10 0分子筛层析纯化后测定其生物学活性。结果 SDS PAGE显示 ,表达产物以可溶性分子形式存在于上清中 ,诱导 4d的表达量占上清总蛋白的 6 0 %以上。表达的sFLT 1(1 3)及sFLT 1(2 3)都具有结合hVEGF165的能力和抑制hVEGF165对人脐静脉内皮细胞 (HUVEC)的促增殖功能 ,其中前者的作用虽较强 ,但二者相差不大。结论 获得了具有生物学活性的可溶性重组FLT 1受体胞外区 1 3及 2 3loop小片段 。

Objective To study the biological activity of soluble recombinant peptides represent the FLT 1 1 3 and 2 3 loops of human VEGF receptor. Methods Two recombinant expression plasmids, pPIC9K/FLT 1(1 3) and pPIC9K/FLT 1(2 3), were constructed and then separately transformed into Pichia pastoris GS115. Soluble recombinant protein was purified with CM Sepharose fast flow chromatography and Sephacryl S 100 chromatography. Biological activities were analyzed with indirect ELISA, competition binding experiment, cell based binding experiment, HUVEC MTT assay and [ 3H] thymidine incorporation assay. Results After 4 days of 1% methanol induction, the expressed FLT 1(1 3) was up to 60% of total proteins in supernatant. Compared with FLT 1(1 3), the expressed FLT 1(2 3) was proved having almost the same biological activity to bind hVEGF 165 and to inhibit HUVEC proliferation stimulated by hVEGF 165 . Conclusion High level expression of soluble FLT 1(1 3) and FLT 1(2 3) with good biological activity was achieved in Pichia pastoris .