硫氧还蛋白融合表达系统表达小鼠内皮抑素之纯化及生物学活性鉴.doc

硫氧还蛋白融合表达系统表达小鼠内皮抑素之纯化及生物学活性鉴 　 作者：马卫列,刘芳,何承伟,何振辉 【关键词】 蛋白 Purification of mouse endostatin in thioredoxin fusion expression system and identification of its biological activity 【Abstract】 AIM：To purify mouse endostatin by using thioredoxin fusion expression system and to identify its biological activity. METHODS: The pThioHisendo recombinant plasmid was transformed into BL21. After induction with IPTG, thioredoxinendo fusion protein was expressed in BL21 and the product was identified as inclusion body by SDSPAGE. The expression product was purified by affinity chromatography through Nicolumn. The purified protein was detected by chicken chorioallantoic membrane(CAM) angiogenesis inhibitory assay and endothelial cell proliferation inhibitory assay. RESULTS: High purity thioredoxinendostatin fusion protein was obtained by SDSPAGE analysis. The purity of the protein was over 95%. The protein possessed the inhibitory activity of endothelial cell proliferation and inhibited the angiogenesis of CAM ［inhibitory rate: (49.6±4.1)％, (53.2±2.3)％, (55.0±3.6)％ and (67.1±5.7)％］. CONCLUSION: The mouse endostatin recombinant fusion protein expressed in E.coli by using thioredoxin fusion expression system is easy to be purified and possesses high activity. 【Keywords】 endostatin；thioredoxin fusion expression system；fusion protein 【摘要】 目的：将硫氧还蛋白融合表达系统表达的小鼠内皮抑素（endostatin）进行纯化并对其生物学活性进行测定. 方法： 将含有pThioHisendo重组质粒转化大肠杆菌BL21，经IPTG（异丙基βD硫代半乳糖苷）诱导表达重组融合蛋白，SDSPAGE分析表达产物为包涵体，将包涵体纯化后通过镍柱亲和层析得到纯化的、可溶性的小鼠内皮抑素，经SDSPAGE分析鉴定. 将纯化的蛋白质通过鸡胚尿囊膜血管生成抑制实验和内皮细胞抑制实验对纯化的蛋白质进行生物学活性测定. 结果：经SDSPAGE电泳分析获得了高纯度的小鼠内皮抑素重组融合蛋白，纯度可达95%，得率为0.27 g/L. 纯化的重组蛋白在体外能抑制鸡胚囊膜血管生成及具有抑制血管内皮细胞增殖的活性，加入重组蛋白内皮抑素5 μg组与10 μg组血管生成抑制率分别为(22.3±2.0)％和(47.1±4.0)％，Plt;0.05，和对照组经过方差分析,差异都有显著性意义；内皮细胞抑制实验可见不同剂量重组蛋白内皮抑素对内皮细胞的生长具有抑制作用［抑制率分别为(49.6±4.1)％，(53.2±2.3)％，(55.0±3.6)％，(67.1±5.7)％］，经过方差分析和相关性分析，具有剂量依赖效应(r＝0.984，Plt;0.05). 结论： 用硫氧还蛋白融合表达系统在大肠杆菌中表达的小鼠内皮抑素重组融合蛋白易纯化并具有高活性. 【关键词】 内皮抑素；硫氧还蛋白融合表达系统；融合蛋白 0引言 内皮抑素（endostatin）是OReilly等［1］