摘要
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 develop a ultracentrifugation method for purification of severe fever with thrombocytopenia syndrome bunyavirus（SFTSV）antigen. Methods The working seeds of SFTSV were inoculated into Vero cells, and the viral culture was inactivated and concentrated, in which the viral particles were enriched by ultracentrifugation with 20%sucrose, followed by a polish process with a sucrose density gradient of 60%, 50%, 40%, 30%, 20% and 10%. The viral antigen fractions were pooled, and identified by SDS-PAGE and Western blot. Results Viral antigen was enriched in components 22 ~ 26. The pooled viral antigen showed glycoprotein（GP） and nucleoprotein（NP） bands, with relative molecular masses of about 60 000 and about 28 000 respectively, on SDS-PAGE profile, which reached a purity of about90% and a concentration of 2. 58 mg/ml, and showed specific binding to rabbit polyclonal antibody against whole SFTSV. Conclusion The ultracentrifugation method for purification of SFTSV was developed, and viral antigen with high purity was prepared, which laid a foundation of preparation of inactivated SFTSV vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第11期1191-1195,共5页
Chinese Journal of Biologicals
基金
十二五重大新药创制项目(2013ZX09102029)
关键词
发热伴血小板减少综合征布尼亚病毒
病毒纯化
蔗糖密度梯度超速离心
Fever with thrombocytopenia syndrome virus （SFTSV）
Virus purification
Sucrose density gradient ultracentrifugation
