人Endostatin cDNA克隆及其在大肠杆菌中的表达

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ＥｎｄｏｓｔａｔｉｎｃＤＮＡ，然后将其插入到ｐＳＱＥ表达质粒中，转化Ｅｓｃｈｅｒｉｃｈｉａ，ｃｏｌｉ ＢＬ２１（ＤＥ３），获得了ｐＳＱＥ－ＥＮＤ／ＢＬ２１（ＤＥ３）表达工程菌，对其诱导表达产物ｈＥｎｄｏｓｔａｔｉｎ进行了分离、纯化和复性的研究，结

Cloning and Expression of Human Endostatin cDNA in E.coli

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.