RGD修饰多肽EDSM-Y的克隆、表达、纯化和初步活性研究

Cloning,Expression,Purification,and Activity of RGD Modified Peptide EDSM-Y

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。

Synthesized peptide EDSM-Y shows a significant anti-angiogenic activity and anti-tumor activity both in vitro and in vivo. The aim of our study is that EDSM-Y can be obtained by gene engineering,which forms a solid basis for the further study. Exogenous gene for EDSM-Y was obtained by polymerase chain reaction amplification and cloned into prokaryotic expression vector. Induced expression EDSM-Y with IPTG was detected by SDS-PAGE and Western Blot. The expression product was purified via Ni-NTA agarose and its activity was detected by cell proliferation assay, pET-23a（ ＋ ） was first used to construct successfully the expression vector while the subsequent production of interest peptide was low. Then EDSM-Y gene was cloned into pET-32a（ ＋ ） expression vector and a much higher product was obtained. After a first time Ni-NTA column purification, EDSM-Y product was obtained with purity up to 60% and after a second time purification by the same column, the product purity was up to 90%. Experiment in vitro confirmed that EDSM-Y could inhibit cell proliferation. Anti-tumor peptide EDSM-Y can be obtained by gene engineering with activity of inhibiting cell proliferation.