GII.P16-GII.2型诺如病毒VLPs的纯化研究

Study on purification of GII.P16-GII.2 norovirus virus-like particles

目的诺如病毒(Norovirus)是一种重要的人畜共患病病毒,可感染人、猪、牛、犬等多种动物,引起诺如病毒病,每年给全球造成巨大的经济损伤。本研究基于AKTA纯化系统探索诺如病毒VLPs的纯化工艺。方法将诺如病毒重组杆状病毒接种到SF9细胞中培养,通过Western blot、间接免疫荧光试验以及SDS-PAGE进行鉴定,对鉴定正确的细胞培养产物采用阴、阳离子柱进行病毒的层析纯化,并对层析柱的选择、层析缓冲液的pH值、洗脱液NaCI浓度等进行优化。基于优化后的参数或条件通过AKTA纯化系统纯化诺如病毒VLPs,分析纯化效果。结果经Western blot、间接免疫荧光、以及SDS-PAGE等鉴定,诺如病毒重组杆状病毒转染SF9细胞后表达诺如病毒VLPs。采用阴、阳离子柱进行病毒的层析纯化,优化的纯化参数或条件为:阴离子纯化柱,层析缓冲液pH值为6.0,洗脱液NaCI浓度为0.2 mol/L。结论诺如病毒重组杆状病毒转染SF9细胞后表达诺如病毒VLPs。优化的AKTA纯化系统可用于诺如病毒VLPs的纯化。

Objective Norovirus is an important zoonotic virus that can infect humans,pigs,cattle,dogs and other animals,causing huge economic damage to the world every year.In this study,the purification of norovirus virus-like particles was explored based on the AKTA purification system.Methods Norovirus recombinant baculovirus was inoculated into SF9 cells and identified by Western blot,indirect immunofluorescence,and SDS-PAGE.After the identification is correct,the obtained cell culture products are optimized for the selection of anion and cation columns,pH value,eluent concentration and other parameters.Purification of norovirus virus-like particles was performed by the AKTA purification system based on the optimized parameters.Results Identification methods such as Western blot,indirect immunofluorescence,and SDS-PAGE proved that the recombinant baculovirus successfully expressed norovirus virus-like particles.The results of purification parameters showed that norovirus virus-like particles could be successfully purified by using an anion purification column,pH value of 6.0,and eluent concentration of 0.2 mol/L.Conclusion The study successfully purified norovirus virus-like particles using the AKTA purification system,which can be used for the purification of norovirus virus-like particles.