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为了寻找人,endostatin基因的核心作用片段,先用PCR方法从人的肝脏cDNA文库克隆出人endostatin基因。然后,利用限制性内切酶酶切得到5个不同的endostatin基因片段,并将它们构建到大肠杆菌表达载体pMAL-c2上。经转化并由大肠杆菌表达得到rh Endo-statin/MBP 6个不同片段的融合蛋白,用亲和层析技术分离纯化,并分别作用于体外培养的牛肾上腺毛细血管内皮细胞(BCE),检测它们 对内皮细胞增殖的影响。rh Endostatin/MBP不同片段的融合蛋白对经bFGF刺激引起的BCE细胞的快速增殖有不同程度的抑制作用。Endostatin作用的活性片段位于Endostatin蛋白N端的第55-96氨基酸位置内。
In order to find the core functional fragment of human endostatin gene, human endostatin gene was cloned from human liver cDNA library by PCR. Then, five different endostatin gene fragments were obtained by restriction enzyme digestion and constructed into E. coli expression vector pMAL-c2. The fusion protein of 6 different fragments of rh Endo-statin / MBP was transformed and expressed in E. coli. The fusion protein was purified by affinity chromatography and applied to cultured bovine adrenal capillary endothelial cells (BCE) On the proliferation of endothelial cells. The fusion protein of different fragments of rh Endostatin / MBP inhibited the proliferation of BCE cells stimulated by bFGF to a certain extent. The active fragment of Endostatin is located at the amino acid position 55-96 of the N-terminal endostatin protein.