以MMP酶切位点为连接臂构建Vasostatin和TRAIL的融合蛋白及其生物学活性分析

Expression and Characterization of Vasostatin-TRAIL Fusions with a MMP Restriction Site Linker

以基质金属蛋白酶(MMP)酶切位点(PLGLWA)为连接臂,采用重组PCR技术获得血管形成抑制素(vasostatin,V)和肿瘤坏死因子相关凋亡诱导配体(TRAIL,T)的融合蛋白质编码序列,将该融合编码序列插入原核表达载体pMAL-c2中,重组质粒转化大肠杆菌BL21,IPTG诱导表达,分别得到MBP-VT和MBP-TV融合蛋白,Amylose Resin亲和层析柱纯化.初步纯化的融合蛋白MBP-VT在体外内皮细胞增殖抑制实验、肿瘤细胞凋亡诱导实验中显示了明显的活性,而MBP-TV的作用不明显;体外酶切实验和培养肿瘤细胞上清酶切融合蛋白的免疫印迹分析,证实融合蛋白MBP-VT皆能被正确酶切.上述结果表明成功表达了融合蛋白VT,该融合蛋白具有双重抗肿瘤活性,并可在肿瘤高表达的MMP作用下裂解为V和T.

The sequences of vasostatin （V） and TRAIL （T） were amplified by recombinant PCR and fused with linker coding for PLGLWA of a restriction site of matrix metalloproteinases（MMPs） , then inserted into a pMAL-c2 vector. The partially purified pMAL-VT and pMAL-TV fused proteins were obtained by transfecting the recombinant plasmids into the E. coli BL21 with IPTG induction, and amylose resin chromatography. The results indicated that MBP-VT substantially inhibited the proliferation of endothelial cells and exhibited a potent apoptosis-inducing effect in tumor cells, whereas MBP-TV showed none of these effect. As expected, bifunclional MBP-VT fusion proteins could be cleaved into MBP-V and T when treated with the supernatant of cultured tumor cells as demonstrated by Western blotting.