摘要
Endostatin是一种新发现的血管生成抑制因子 ,具有很强的抗肿瘤活性 .为了研究人 Endostatin的生物学功能和作用机制 ,作者从人胎肝组织中克隆到人 Endostatin c DNA ,然后将其插入到 p SQE表达质粒中 ,转化Escherichia.coli BL 2 1(DE3) ,获得了 p SQE- END/ BL 2 1(DE3)表达工程菌 ,对其诱导表达产物 h Endostatin进行了分离、纯化和复性的研究 .结果表明 ,h Endostatin的表达量可达菌体总蛋白的 30 % ,表达产物主要以包涵体 (Inclusionbodies,IBs)形式存在于菌体中 .菌体经超声破碎、离心、洗涤 ,然后用 Ni2 + - Sepharose 6 B柱一步法亲和层析 ,得到电泳纯度为 95 %的 h Endostatin.通过研究 GSSG浓度、p H值、复性时间等参数 ,获得了 h Endostatin包涵体的最适复性条件 .经细胞毒性试验测定 ,复性产物对 1H11内皮细胞的 ED5 0约为 70 0 ng/ m
Endostatin is a newly found angiogenesis inhibitor, which can inhibit tumor growth. To investigate its biological functions and related mechanisms, we cloned the gene of Endostatin from human fetal liver by RT PCR and constructed the expression plasmid pSQE END. The plasmid was transformed into Escherichia.coli BL21(DE3). Through being induced with IPTG, this strain expressed His6 hEndostatin. SDS PAGE analysis revealed that expression level of the product was up to 30% of the total bacterial protein and was mainly as insoluble inclusion bodies(IBs). After cell lysis, centrifugation, Ni 2+ Sepharose 6B purification, the IBs of 95% purity were obtained. The purified product was refolded perfectly by modulatng the concentration of GSSG, pH, renaturation time. The renaturation product can inhibit 1H11 endothelial cell proli feration with an in vitro ED50 of 700 ng/ml.
出处
《上海大学学报（自然科学版）》
CAS
CSCD
2000年第4期338-342,共5页
Journal of Shanghai University:Natural Science Edition
关键词
基因克隆
原核表达
分离纯化
大肠杆菌
hEndostatin
gene cloning
prokaryotic expression
purification and renaturation
