人血管内皮抑素的基因克隆和表达载体构建及纯化

Cloning, Construction and Purification of Recombinant Human Endostatin Gene

目的 克隆人血管内皮抑素基因(endostatin),纯化其表达蛋白并初步检测纯化蛋白的生物学活性。方法 用PCH技术从正常成人肝cDNA库扩增出人血管内皮抑素基因,将其克隆进pBluescript中测定其核苷酸序列。构建大肠杆菌分泌性表达载体pET-22b(+)-endostatin-6xHis,IPTG诱导表达,经Ni^(2+)-IDA Sepharose 6B亲和纯化该表达蛋白,并采用血管内皮细胞增殖抑制试验检测其活性。结果 经PCR扩增成功获得55lbp的人血管内皮抑素基因,测序正确,在大肠杆菌中表达后,该表达蛋白的表达量占菌体总蛋白的10％。SDS-PAGE和Western blot显示其分子最为21kd。经亲和纯化后的endostatin-6xHis纯度可达90％,得率为0.3mg/100ml,并且具有抑制血管内皮细胞增殖的活性,达到50％的抑制率需endostatin-6xHis浓度为600ng/ml。结论 endostatin基因克隆、表达和纯化的成功,为采用抗血管生成方法治疗裸鼠肿瘤模型和肿瘤病人的研究奠定了基础。

Objective To clone human endostatin gene, purify its expression protein and detect its biological activity. Methods By using PCR technique, the gene encoding human endostatin was cloned from the cDNA library of the adult human liver and sequenced. Then this gene was inserted into expression vector pET -22b( + ). The construct was expressed in E. coli and the expression product was purified by affinity chromatography through Ni + - IDA Sepharose 6B. The purified protein was tested by en-dothelial cell proliferation inhibitory assay. Results The acquired endostatin gene was 551 bp and its sequence was correct. The construct was expressed in E. coli with a high level as soluble protein, accounting for 10% of the total bacterial proteins. This gene product, characterized by SDS - PAGE and Western blot appeared to be a protein with molecular mass of 21kd. The purity of endostatin -6xHis purified by affinity chromatography reached more than 90% . The protein possesses the inhibitory activity of endothelial cell proliferation. Conclusion The cloning, expression and purification of human endostatin protein lay the foundation for antiangiogenesis therapy in tumor bearing nude mice and tumor patients.