一种具有抗肿瘤活性的新型尿激酶原Kringle结构域变体蛋白的研究

利用PCR技术获得人尿激酶原Kringle结构域基因,将其克隆至具有T7启动子的表达质粒pET29a中,并进而插入plasminogen Kringle 5一段16肽片段构建了其变体,重组质粒转化至大肠杆菌BL21中,经IPTG诱导表达,其产物以包涵体形式存在．通过体外复性,得到可溶性的prourokinase Kringle及其变体．所得蛋白质经过动物肿瘤模型测活,确定变体蛋白具有抑制肿瘤生长作用．

A Novel Kringle Mutant of Prourokinase Suppressing Tumor Growth

Kringles of plasminogen and other proteins, obtained by proteolytic fragments, have been reported to display the anti tumor activity, which represent potent anti cancer candidates. However, there remains controversy on whether it is the sequence or the tertiary structure that renders Kringle the anti tumor activity. In order to address such an issue, we cloned the genes of Kringle of prourokinase and obtained its mutant by inserting a previously demonstrated fragment of 16 amino acids from Kringle 5 of plasminogen that manifested anti tumor activity. The constructed recombinant vectors pET29a were expressed in E.coli BL21 (DE3), induced by IPTG. Prourokinase Kringle and the mutant were first purified by Ni NTA affinity chromatography and then subjected to renaturation. Finally, the folding solutions were applied to CM ion exchange chromatography for further purification and concentration. As a result, appropriately folded proteins with high purity were obtained, which were confirmed by SDS?PAGE analysis. To compare the in vivo anti tumor activities of prourokinase Kringle and its mutant, male 6 week C57/BL6 mice were used for tumor study. Lewis lung carcinoma cells were subcutaneously injected and the anti tumor efficacy was evaluated on the basis of tumor volume. Here, prourokinase Kringle almost displayed no anti tumor activity while its mutant comparatively stifled the growth of subcutaneous tumor, illustrating that equipping proteins with certain anti tumor fragment will inhibit tumor growth and it is the amino acid sequence rather than the tertiary structure of protein that enables several Kringle structures to prevent tumor from growing.