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目的建立发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)抗原的超速离心纯化方法。方法将SFTSV工作种子库毒种接种至Vero细胞,病毒培养物经灭活及浓缩后制备病毒浓缩液。病毒浓缩液通过20%蔗糖超速离心富集分离,再经60%、50%、40%、30%、20%、10%蔗糖密度梯度超速离心进一步纯化,将病毒抗原富集的组分进行合并,经SDS-PAGE及Western blot鉴定。结果病毒抗原富集于组分22~26中,合并后经SDS-PAGE分析可见相对分子质量约60 000及28 000的糖蛋白(GP)及核蛋白(NP)条带,纯度约90%,浓度为2.58 mg/ml,且可与兔抗SFTSV全病毒多克隆抗体发生特异性结合。结论成功建立了SFTSV的超速离心纯化方法,纯化制备出了高纯度的病毒抗原,为SFTSV灭活疫苗的研制奠定了基础。
Objective To establish an ultracentrifugation purification method for fever with thrombocytopenia syndrome bunyavirus (SFTSV) antigen. Methods The seeds of SFTSV seed bank were inoculated into Vero cells. The virus culture was inactivated and concentrated to prepare the virus concentrate. The virus concentrate was concentrated by ultracentrifugation in 20% sucrose and further purified by ultracentrifugation at 60%, 50%, 40%, 30%, 20% and 10% sucrose density gradient, and the virus antigen enriched fractions were combined , Identified by SDS-PAGE and Western blot. Results The viral antigens were enriched in fractions 22-26. After SDS-PAGE analysis, the GP and NP bands with molecular weights of about 60 000 and 28 000 were obtained. The purity was about 90% At a concentration of 2.58 mg / ml, and specifically binds to rabbit anti-SFTSV whole virus polyclonal antibody. Conclusion The ultracentrifugation purification method of SFTSV was successfully established and the purified high purity virus antigen was prepared, which laid the foundation for the development of SFTSV inactivated vaccine.