人内皮抑素基因的克隆以及在大肠杆菌中的可溶性表达研究

Research about Soluble Expression of Human Endostatin in E.coli

用PCR的方法从人胎肝cDNA文库中得到人内皮抑素基因 ,克隆测序正确后连接到硫氧还蛋白融合表达载体上 ,转化大肠杆菌BL2 1 (DE3)得到表达人内皮抑素的工程菌。用IPTG诱导表达 ,表达量达到全菌蛋白的 64%。经分析硫氧还蛋白可以辅助内皮抑素可溶性表达 ,表达的融合蛋白保持了天然蛋白的免疫学特性。而且表面带有多聚组氨酸的突变的硫氧还蛋白还简化了蛋白纯化的步骤 ,使融合蛋白可以通过固相金属螯和层析 (IMAC)的方法纯化。纯化后的融合蛋白经IgA蛋白酶的切割可得到大小正确的重组人内皮抑素 ,用此方法获得的重组人内皮抑素可以在CAM试验中抑制新生血管的形成。高效可溶型表达内皮抑素的工程菌的构建成功 。

Endostatin is a potent,endogenous inhibitor of angiogenesis,which corresponds to the C-terminal fragment of collagen XVIII.cDNA coding for human endostatin was obtained from a human fetal liver cDNA library using PCR ,After subcloned into the express vector pETTrxHisL and subsequenced to prove its correctness, pETTrxHisL-endostatin was transformed to E.coli BL21(DE3).The fusion protein Trx-Endostatin was expressed as soluble form,and can be purified by IMCA (Immobilized metal affinity chromatography) method.The SDS-PAGE thin layer scanning analysis found expression level was more than 64% total protein,and the Mol.Weight was about 31.5kDa.Western blot analysis indicate the immunoreactive was identical with natural hEndostatin.Biological activity was confirmed by CAM angiogenesis assay,the soluble human endostatin expressed by E.coli is potent to inhibit the angiogenesis in CAM.;