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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 23loop cDNA in the extracellular region of human Flt1 in Pichia pastoris and to obtain highly expressed recombinant Flt1 (2 3) with high biological activity. Methods: Flt 1 (23) gene was amplified by PCR and inserted into Pichia pastoris yeast expression vector containing AOX1 promoter and alpha secretory signal peptide sequence after DNA sequence analysis. The recombinant plasmid pPIC9K Flt 1 (23) The yeast host strain GS115 was transformed into His + Muts phenotype transformants, shake flask culture, 1% methanol induced expression. The expressed product was purified by CM Sepharose FF cation exchange chromatography and Sephacryl S 10 0 molecular sieve chromatography to determine its biological activity. Results: SDS PAGE analysis showed that the expressed product was soluble in the supernatant. The expression level reached more than 60% of the total supernatant protein after 4 days of induction. ELISA and Western blot showed that the expressed product had good antigenicity and specificity. After purification by CM Sepharose FF cation exchange chromatography and Sephacryl S 10 0 molecular sieve chromatography, the purity of Flt 1 (2 3) reached more than 90%. Biological activity tests confirmed that it has the ability to bind hVEGF1 6 5 and inhibit hVEGF1 6 5 on HUVEC proliferation. CONCLUSION: A small fragment of 2,3loop in the extracellular domain of soluble recombinant Flt 1 receptor was obtained, which laid the foundation for further animal experiments and clinical application.