发热伴血小板减少综合征病毒表面糖蛋白Gc的真核表达、纯化和结晶

Expression,Purification and Crystallization of Severe Fever with Thrombocytopenia Syndrome Virus Envelope Glycoprotein Gc

用真核表达系统表达纯化发热伴血小板减少综合征病毒的表面糖蛋白Gc,通过研究其结构与功能,了解病毒组装出核和粘附宿主细胞的机制,并且通过对病毒表面结构的研究为进一步的抗病毒药物研发提供了重要的理论依据.克隆Gc蛋白的分泌表达基因,利用sf9昆虫细胞表达系统进行表达.SDS-PAGE蛋白纯化结果分析显示,通过镍亲和纯化和Superdex200分子筛纯化后得到了高纯度的Gc蛋白.以气相扩散-悬滴法对Gc蛋白晶体进行晶体筛选.置于16℃恒温室中进行蛋白质晶体生长.在对晶体初筛条件pH浓度和沉淀剂浓度优化后,在两种结晶条件下生长出蛋白质晶体,分别为2 mol/L硫酸铵和0.1 mol/L Hepes,pH值7.5,0.8 mol/L的一水磷酸钠,0.8 mol/L磷酸二氢钾.实验结果得到了纯度较高的Gc蛋白,并生长得到了蛋白质晶体.

To understand the mechanism of virus assembly and adhesion to host cells,this study prepared glycoprotein Gc of severe fever with thrombocytopenia syndrome virus(SFTSV)by insect cells expression system,and provides an important theoretical basis for further antiviral drug development.The secretory expressive construct of recombined Gc ectodomain protein was designed.The purified protein was expressed by insect cells expression system.The results of SDS-PAGE analysis after purified by nickel affinity chromatography and size-exclusive chromatography shows the high purity of Gc ectodomain protein.Crystal screening was carried out by hanging drop vapor diffusion method at a constant temperature of 16℃.After optimization of the pH and precipitant concentration,crystals were grown under two crystallization conditions:2 mol/L ammonium sulfate;0.1 mol/L Hepes,pH 7.5,0.8 mol/L Sodium phosphate monobasic monohydrate,and 0.8 mol/L potassium dihydrogen phosphate.The Results showed that the Gc ectodomain protein with high purity was obtained.The protein crystals were grown.