人血管生成抑制素基因的克隆、表达载体构建及纯化

Cloning, construction and purification of recombinant human angiostatin gene

目的 克隆人血管生成抑制素基因 (angiostatin) ,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PCR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 2 2b(+) angiostatin 6×His,异丙基硫代 β D 半乳糖苷(IPTG)诱导表达 ,经Ni2 + IDASepharose 6B亲和纯化该表达蛋白 ,并采用血管内皮细胞增殖抑制实验检测其活性。结果 经PCR扩增成功获得 10 5 9bp的人血管生成抑制素基因 ,测序正确 ,在大肠杆菌中表达后 ,该蛋白的表达量占菌体总蛋白的 10 %。SDS PAGE和WesternBlot显示其相对分子质量为 39× 10 3 。经亲和纯化后的angiostatin 6×His纯度可达 90 % ,得率为 0 .2 5mg/ 10 0ml,并且具有抑制血管内皮细胞增殖的活性 ,达到 5 0 %的抑制率 ,angiostatin 6×His浓度为 80 0ng/ml。 结论 人血管生成抑制素基因的克隆、表达及纯化 。

Objective To clone the human angiostatin gene, purify its expression protein and detect its biological activity. Methods Using PCR technique, the gene encoding human angiostatin was cloned from cDNA library of the adult human liver and sequenced and inserted into the expression vector pET 22b(+). The construction was expressed in E.coli and the expression product was purified by affinity chromatography through Ni 2+ IDA Sepharose 6B. The purified protein was tested by endothelial cell proliferation inhibitory assay. Results The acquired angiostatin gene was 1 059 bp and its sequence was verified as correct. The construct was expressed in E. coli at 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 weight of 39×10 3 . The purity of angiostatin 6×His purified by the affinity chromatography reached up to over 90%. The protein possessed the inhibitory activity of endothelial cell proliferation. Conclusion The cloning, expression and purification of human angiostatin protein lay the foundation for the antiangiogenesis therapy in tumor bearing nude mice and tumor patients.