摘要
目的 获得具有抑制血管内皮细胞生长活性的人重组内皮生长抑素 (endostatin)蛋白 ,以研究其在治疗肿瘤及血管性疾病中的潜在应用价值。方法 从人的胎盘中分离总RNA经反转录聚合酶链反应 (RT PCR) ,得到en dostatin的全基因 ,并将其与质粒pUCm T重组 ,测序证明获及的人内皮生长抑素基因的序列与先前报道一致后 ,用限制性内切酶将其切下与表达载体pET 1 1a重组 ,转化受体菌BL 2 1 (DE3)中 ,用IPTG诱导以包涵体形式表达 ,经层析纯化 ,鸡绒毛尿囊膜血管 (CAM)检测活性。结果 经SDS PAGE检测 ,重组人内皮生长抑素蛋白的分子量为2 0KD ,表达量约占蛋白总量的 40 %。纯化后的内皮生长抑素对鸡的绒毛尿囊膜血管的生长有明显的抑制作用。结论 得到了具有生物活性的重组人内皮生长抑素蛋白 。
Objective To construct an engineering bacteria which can express with human endostatin protein to observe a latent valuation in curing cancer and vascular disease.Methods Human endostatin gene fragment was acquired from human placenta total RNA by means of RT PCR.The gene was inserted into prokaryotic vector pUCm T and transformed into E.coli JM109.Gene analysis showed that acquired endostatin gene was the same as the published one in Genebank. Then using restriction enzyme and DNA ligase,endostatin gene was recombined with expression vector pET 11a,transformed into BL21(DE3),induced by IPTG,endostatin protein was purified by sephadex G 75 gel filtration chromatography and its biological activity was analyzed by means of CAM.Results SDS PAGE analysis showed that endostatin was about 20KD and took up about 40% in bacterial total protein.Chick embryo allantoid membrane with nine days injected this purified protein was found that CAM was decreasing after 48 hours.Conclusion The recombinant human endostatin protein can inhibit proliferation of vascular endothelial cell.This method gives us a new thought for treatment of cancer and vascular disease.
出处
《哈尔滨医科大学学报》
CAS
2002年第2期105-108,共4页
Journal of Harbin Medical University
基金
黑龙江省教育厅科研项目( 9551071)
黑龙江省卫生厅科研课题(2 0 0 1- 0 13)
