肠道病毒71型的快速纯化及其鉴定  

Rapid purification and identification of Eenterovirus 71

作　　者：胡小云[1] 朱冰[1] 王长兵[1] 邝璐[1] 肖密丝[1] 

机构地区：[1]广州医学院附属广州市妇女儿童医疗中心实验室,510120

出　　处：《国际检验医学杂志》2012年第8期897-898,905,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金项目(30972630);广东省自然科学基金项目(10151012001000002)

摘　　要：目的建立一种简单、快速有效的从细胞培养物中纯化肠道病毒71(EV71)的方法。方法 EV71病毒在恒河猴肾细胞(LLC-MK2)中增殖后,将获得的细胞培养物经反复冻融、聚乙二醇6000沉淀、超速离心、氯化铯垫层超速离心的方法纯化病毒。用聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹(Western blot)和透射电镜(TEM)的方法对纯化病毒进行鉴定,并测定其感染性的滴度及回收率。结果 SDS-PAGE显示出3个条带,相对分子质量分别为3.6×103、3×103和2.6×103与EV71的VP1、VP2和VP3相符合。Westernblot证实为EV71特异性条带。经磷钨酸负染后电镜观察,能看到典型病毒颗粒。采用终浓度为10%的聚乙二醇6000沉淀后的病毒的感染性回收率为82.0%,再经氯化铯垫层超速离心后浓缩病毒的感染性回性率为29.0%。结论聚乙二醇6000沉淀结合氯化铯垫层超速离心的方法比氯化铯密度梯度区带离心方法更简便,易于操作,并且比单独聚乙二醇6000沉淀有更高的纯度,是一种简便、有效的病毒纯化方法。Objective To establish a simple,rapid and effective method to purify Enterovirus 71（EV71） from cell culture.Methods EV71,propagated in rhesus renal cells（LLC-MK2 cells） were purified by precipitating with polyethylene glycol（PEG） 6000,differential centrifugation and ultracentrifugation with cesium chloride（CsCl） cushion.The purified virus samples were further identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis（SDS-PAGE） ,Western blot and transmission electron microscope（TEM） .Results SDS-PAGE showed three protein straps with relative molecular weights of 3.6×103,3×103 and 2.6×103 respective1y,which were according with the VP1,VP2 and VP3 of EV71 and could be confirmed by Western blot.The typical virus particle could be observed with TEM.Infectivity recovery of virus samples,precipitated by PEG-6000 with final concentration of 10% was 82.0%,and that of the final purified virus samples was 29.0%.Conclusion New method,constructed in this research,could be more convenient and easier than CsCl density gradient centrifugation and might get higher purity of virus than PEG-6000 precipitation alone.

关 键 词：肠道病毒71型 病毒纯化 超速离心 

分 类 号：R446.5[医药卫生—诊断学]