重组人Endostatin/MBP融合蛋白片段的构建及体外抑制内皮细胞增殖活性

CONSTRUCTION OF RECOMBINANT HUMAN ENDOSTATIN/MBP FUSION PROTEIN AND ITS SEGMENTS AND THEIR INHIBITION ON ENDOTHELIA PROLIFERATION IN VITRO

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 active segments of human endostatin gene, human endostatin gene were obtained by PCR technology from human liver cDNA library. Using restriction enzymes, five segments of endostatin were obtained and cloned to E. coli expression vector pMAL-c2. Recombinant vectors were transformed to E. coli DH5a. Six kinds of Endostatin/MBP fusion protein were purified by Amylose Resin beads, and were applied in experiments of inhibition of bovine adrenal capillary en-dothelial cell (BCE) proliferation in vitro. Each segment of Endostatin/MBP fusion protein can inhibit proliferation of the en-dothelial cell stimulated by bFGF in different level. The active segment of endostatin is located in the N terminal No. 55 to 96 amino acid.