人血管内皮生长因子受体Flt-1胞外区2-3loop在Pichia.pastoris酵母中的表达、纯化及其生物学活性研究

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

目的 :研究人Flt 1胞外区 2 3loopcDNA在Pichia pastoris酵母中的表达 ,获得高效表达的具有生物学活性的重组Flt 1(2 3)。方法 :采用PCR扩增Flt 1(2 3)基因 ,经DNA序列分析后 ,插入含AOX1启动子和α分泌信号肽序列的Pichia pastoris酵母表达载体中 ,构建重组质粒pPIC9K Flt 1(2 3) ,转化酵母宿主菌GS115 ,筛选His+ Muts 表型转化子 ,摇瓶培养 ,1%甲醇诱导表达。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分析显示 ,表达产物以可溶性分子形式存在于上清中 ,诱导 4d的表达量达上清总蛋白的 6 0 %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。经CM SepharoseFF阳离子交换层析和SephacrylS 10 0分子筛层析纯化后 ,Flt 1(2 3)纯度达到 90 %以上。生物学活性检测证实其具有结合hVEGF1 6 5 的能力和抑制hVEGF1 6 5 对HUVEC的促增殖功能。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 2 3loop小片段 ,为进一步的动物实验和临床应用打下了基础。

Objective:To study the expression of human Flt 1 2 3loop cDNA in Pichia pastoris and to obtain high level expressed recombinant human Flt 1(2 3) with good biological activity Methods:Amplifying Flt 1(2 3) cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptides, the recombinant expression plasmids pPIC9K/Flt 1(2 3) was constructed and transformed into GS115 The His +Mut s phenotype transformants were screened,fermented in flasks and induced by 1% methanol Results:After 4 days of methanol induction, the expressed Flt 1(2 3) comes up to 60% of total proteins in supernatant by SDS PAGE ELISA and Western blot assay proved it having good antigenicity and high specificity The recombinant protein was further purified with CM Sepharose Fast Flow and Sephacryl S 100 chromatography, and was proved having good biological activity to bind hVEGF 165 and to inhibit hUVEC proliferation stimulated by hVEGF 165 Conclusion:High level expression of secreted Flt 1(2 3) with good biological activity were successfully achieved in Pichia pastoris expression system and can be applied to further animal and clinical test