抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

Gene Construction, Expression, Purification and Activity Analysis of Anti-platelet Aggregation Peptide RGD226

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2

Human proinsulin mutant ([A6-Ser, A11-Ser] proinsulin) gene between B28 and A1 sites was replaced by a gene encoding a peptide of 13 amino acids, SRVARGDWNDDYS, by PCR method. The peptide with an RGD (Arg-Gly-Asp) motif was originated from disintegrin eriststatin. The constructed RGD226 gene was overexpressed in E. coli. RGD226 peptide was purified by Sephadex G-50 chromatography and by Resource TM reverse phase chromatography successively. The RGD226 peptide inhibits ADP-induced human platelet aggregation with an IC 50 of 22.3 μmol/L and keeps 15.1% of immunity of [A6-Ser, A11-Ser] proinsulin, but with 0.1% of receptor binding activity of [A6-Ser, A11-Ser] proinsulin. The in vivo results suggest that the RGD226 peptide can obviously delay the time of bleeding in mice.