【摘要】 目的　利用大肠杆菌原核表达系统表达重组人内皮抑素Endostatin ,并对其进行生物学活性鉴定。方法　采用RT PCR方法从人胚肝中钓取人内皮抑素cDNA ,将其克隆入pGEM TEasy克隆载体中 ;经全自动序列分析仪测序确证后 ,将人内皮抑素cDNA亚克隆入原核表达载体pWR450 1 ,构建含人内皮抑素cDNA的重组质粒 ;将该重组质粒转化入大肠杆菌TG1 中进行诱导表达 ,表达产物经SDS PAGE鉴定 ;表达产物初步纯化复性后以鸡胚尿囊膜血管生成抑制实验和小鼠伤口愈合实验对其进行生物学活性鉴定。结果　成功获得 549bp人endostastin基因 ,测序证实序列正确。经IPTG诱导的重组原核表达质粒表达出重组人内皮抑素的融合蛋白 ,此蛋白在SDS PAGE凝胶上显示出一条约 75KD的阳性条带。重组人内皮抑素融合蛋白的初纯物在体外能抑制鸡胚尿囊膜血管生成及延长小鼠伤口愈合时间。结论　人内皮抑素融合蛋白在大肠杆菌原核表达系统中高水平表达 ,并具有抗血管生成活性 ,为采用抗血管生成方法治疗恶性实体肿瘤的研究奠定了基础更多还原

【Abstract】 OBJECTIVE To express recombinant human endostatin in E.coli and identify its biological activity. METHOD Endostatin cDNA was amplificated by RT PCR from human fresh fetal liver. The synthesized cDNA fragment was cloned into pGEM T Easy vector. After identifying the sequence of the insert, the fragment was subcloned into pWR450 1 prokaryotic expression vector. The recombinant human endostatin was expressed in E.coli TG 1 and the product was identified by SDS PAGE. After the expression product was purified and renatured, its protein biological activity was detected by the chicken chorio allantoic membrane(CAM) angiogenesis inhibition assay and the experiment of rat wound healing.RESULTS The acquired fragment of 549bp encoding human endostatin 183 amino acids was correct, and an induced fusion protein by IPTG with a relative molecular mass of 75KD was expressed in E.coli TG 1. The recombinant protein possessed the anti angiogenesis activity and could inhibit the angiogenesis of CAM and extend the time of rat wound healing. CONCLUSION Recombinant human endostatin protein was expressed in E.coli in a high level and showed the anti angiogenesis activity. The expression and application of human endostatin gene lay the foundation of the anti angiogenesis therapy of tumors.更多还原