蓝藻抗病毒蛋白-N衍生物的克隆、发酵与纯化

蓝藻抗病毒蛋白-N( Cyanovirin-N,CVN)能特异性与病毒表面糖蛋白结合,抑制病毒进入宿主细胞及抑制病毒感染细胞与未感染细胞的融合.通过分子设计及优化,在CVN的N-末端连接了5个氨基酸的柔性多肽(GGGGS),构建了SUMO-L5-CVN融合表达系统.SUMO-L5-CVN在大肠杆菌BL21中呈可溶性表达；通过表达条件优化,以0.5 mmol/L IPTG在20℃诱导24h 是最佳的诱导表达条件；SUMO-L5-CVN表达量占菌体总蛋白的30％；经Ni-NTA亲和层析获得融合蛋白SUMO-L5-CVN,SUMO蛋白酶酶切,以及进一步从Ni-NTA亲和层析获得的目的蛋白L5-CVN蛋白纯度＞98％.结果表明,低浓度的L5-CVN与流感病毒表面糖蛋白gp120就有较高的亲和力.

Expression, Fermentation and Purification of Cyanovirin-N Derivative

CVN acts by binding with high affinity to the viral envelope glycoprotein,thus inhibiting viral invasion and fusion of the virus particle to the target cell.Through molecular design and optimization,a 5-amino acids flexible peptide(GGGGS)was linked to the N-terminus of CVN.SUMO-L5-CVN was expressed in the cytoplasm of E.coli BL21 in a soluble form by SUMO fusion expression system.After the optimization of expression conditions,SUMO-L5-CVN was highly expressed in E.coli BL21 under the induction of IPTG(20℃,24 h,0.5 mmol/L). The fusion protein was expressed up to 30% of the total protein.The recombinant L5-CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage.Nanomolar concentration of L5-CVN have a high affinity with the viral envelope glycoprotein gp120.