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固相合成多肽EDSM-Y在体内外具有良好的抗血管生成和抗肿瘤的活性,该研究希望通过基因工程重组手段获得目的蛋白EDSM-Y,为进一步研究开发奠定基础。PCR扩增得到目的基因,连接入原核表达载体;以IPTG诱导实现外源基因表达,SDS-PAGE和Western Blot验证;摸索发酵表达纯化条件,分离目的蛋白;细胞增殖实验检测EDSM-Y的活性。成功构建了pET-23a(+)-EDSM-Y重组表达载体,但目的蛋白表达量较低。后将EDSM-Y克隆入pET-32a(+)重组表达载体进行融合表达,改变表达载体后目的蛋白表达量显著增加。目的蛋白经过先后两次亲和层析,纯度分别可到达60%和85%,活性实验表明EDSM-Y对血管内皮细胞具有增殖抑制作用。通过基因工程重组能获得具有抑制细胞增殖活性的抗肿瘤多肽EDSM-Y。
The solid-phase synthetic peptide EDSM-Y has good antiangiogenic and anti-tumor activity both in vitro and in vivo. The study hopes to obtain the target protein EDSM-Y by genetic engineering recombination, which lays the foundation for further research and development. The target gene was amplified by PCR and inserted into the prokaryotic expression vector. The expression of foreign gene was induced by IPTG. SDS-PAGE and Western Blot were used to verify the expression. Purification conditions of the fermentation were explored to isolate the target protein. Cell proliferation assay was used to detect EDSM-Y activity. The pET-23a (+) - EDSM-Y recombinant expression vector was successfully constructed, but the expression of the target protein was low. After EDSM-Y was cloned into pET-32a (+) recombinant expression vector for fusion expression, the expression of the target protein expression was significantly increased. After two consecutive affinity chromatography, the target protein can reach 60% and 85% purity, respectively. The activity test shows that EDSM-Y can inhibit the proliferation of vascular endothelial cells. The recombinant antitumor polypeptide EDSM-Y with inhibiting cell proliferation activity can be obtained through genetic engineering recombination.