乙脑病毒猪源分离株CSF.XZ-2D感染BHK-21细胞毒的浓缩及纯化  

Concentration and Purification of Japanese Encephalitis Virus( JEV)Isolate CSF. XZ-2D Propagated in BHK-21 Cells

作　　者：滕蔓[1] 冯丽丽[2] 罗俊[1] 樊剑鸣[3] 王方雨[1] 郅玉宝[1] 李秀杰[1] 邓瑞广[1] 张改平[1,4] 

机构地区：[1]河南省农业科学院动物免疫学重点实验室/农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,河南郑州450002 [2]河南省农业科学院农业经济与信息研究所,河南郑州450002 [3]郑州大学公共卫生学院,河南郑州450001 [4]河南农业大学牧医工程学院,河南郑州450002

出　　处：《河南农业科学》2015年第12期117-120,139,共5页Journal of Henan Agricultural Sciences

基　　金：河南省农业科学院科研发展专项资金项目(20157817);河南省科技攻关计划项目(152102110127);河南省农业技术推广财政补助资金(YCN201519111);国家自然科学基金项目(31372453);国家公益性行业(农业)科研专项经费项目(201203082-5)

摘　　要：研究了日本流行性乙型脑炎病毒（Japanese encephalitis virus,JEV）猪源分离株CSF.XZ-2D感染的BHK-21细胞培养物中JEV的浓缩及纯化方法,对浓缩及纯化后的病毒TCID_（50）效价分别进行测定。结果表明,经差速离心后病毒液在4℃条件下分别以30 000 r/min、40 000 r/min、50 000r/min、60 000 r/min超速离心3 h,其中50 000 r/min超速离心可将绝大部分JEV病毒粒子充分沉淀至离心管底部,浓缩效果最佳;病毒沉淀用PBS重悬后进行不连续蔗糖密度梯度离心,病毒粒子主要集中在35%~50%的区带蔗糖层。In thepresent work,the method for concentration and purification of Japanese encephalitis virus（ JEV） isolate CSF. XZ-2D propagated in BHK-21 cells was studied. The BHK-21 cell cultures infected with CSF. XZ-2D were first ultracentrifugated with 30 000 r/min,40 000 r/min,50 000 r/min or 60 000 r/min for 3 h at 4 ℃,respectively. Then,the virus titers of TCID_（50） were determined and showed that most of the virus particles can be fully deposited to the bottom of centrifuge tube when they were ultracentrifugated with 50 000 r/min for 3 h,showing the best concentration effect. The virus precipitation was suspended with PBS and used for further sucrose density gradient centrifugation. We found that JEV particles were mainly concentrated in the sucrose layers of concentrations of 35% —50%.

关 键 词：乙脑病毒 BHK-21细胞 蔗糖密度梯度离心 浓缩 纯化 

分 类 号：S855.3[农业科学—临床兽医学]