EV71两种结构形式的免疫学特性比较  

Immune characteristics of two enterovirus 71 virion fractions

作　　者：程晨[1] 冯敏[1] 杨二霞[1] 普晶[1] 崔萍芳[1] 张莹[1] 李建平[1] 陈姝樾 王丽春[1] 赵树栋[1] 王晶晶[1] 李剑兰[1] 刘龙丁[1] 李琦涵[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2014年第11期1388-1393,共6页Chinese Journal of Biologicals

基　　金：国家"973"计划项目(2011CB504903);国家"863"计划项目(2012AA02A404);云南省应用基础研究计划面上项目(2011FB116)

摘　　要：目的分析肠道病毒71型（Enterovirus 71,EV71）在病毒增殖的动力学过程中不同时间点的病毒样品所具有的两种结构形式及免疫学特性,为疫苗的研发及质控提供参考。方法利用Opti Prep密度梯度离心获得EV71两种结构形式,电镜扫描观察两种结构形式的颗粒形态;采用病毒微量滴定法测定病毒两种结构形式的感染性滴度;EV71抗原生物素-亲和素酶联免疫（BA-ELISA）方法测定病毒两种结构形式的抗原含量;QRT-PCR法测定两种结构形式的病毒RNA含量;SDS-PAGE及Western blot鉴定病毒两种结构形式。将分离得到的空壳及实心病毒颗粒成分用甲醛灭活后,与Al（OH）3佐剂混合,对小鼠进行皮下多点注射,分别于初免28 d及二免7、14 d,经小鼠尾静脉采血,分离血清,采用微量中和试验法检测抗EV71中和抗体;将二免14 d后的雄、雌鼠合笼,生产后24 h内,用EV71FY-23对乳鼠进行颅内注射,10 d内观察乳鼠存活率。结果经Opti Prep密度梯度离心后,可获得两条沉降系数不同的病毒条带,位于上层的病毒结构成份基本以空心衣壳颗粒形式存在;下层病毒结构成分在感染前期主要为以实心颗粒形式存在,感染后期则以空壳和实心颗粒同时存在,两种结构形式均能被抗EV71特异血清识别。在病毒感染过程中,下层毒粒感染性滴度均比上层毒粒高2~3个log值;两种结构形式的抗原滴度均呈增加的趋势,但下层毒粒抗原滴度增加更为明显;病毒两种结构形式两层的RNA含量不同,下层毒粒中VP0均已裂解形成VP2和VP4,而上层毒粒中以VP0为主要成分。初免后28 d及二免后7、14 d,两种结构形式的EV71均诱导小鼠抗体阳转,但上层毒粒诱导的中和抗体效价及增长速度均明显低于下层毒粒（P均〈0.001）。病毒两种结构形式免疫小鼠产生的乳鼠免疫保护效率均为100%。结论 EV71的两种结构形式虽然具有差异,但作为病毒疫苗抗原用于免疫时,均Objective To analyze the two entervirus 71（EV71）virion fractions and their immune characteristics at various time points in dynamic process of viral proliferation and provide a reference for development and quality control of vaccine.Methods Two EV71 virion fractions were obtained by Opti Prep density gradient centrifugation and observed for morphology by electron microscopy,determined for infectious titer by microtitration,for antigen content by BA-ELISA,for viral RNA content by QRT-PCR,and identified by SDS-PAGE and Western blot. The obtained capsid and solid virus particles were inactivated with formaldehyde,mixed with aluminium hydroxide as an adjuvant,and injected s.c. to mice in several sites.Intravenous blood samples of mice were collected 28 d after the first immunization and 7 and 14 d after the second immunization,from which sera were separated and determined for neutralizing antibody against EV71 by microneutralizat-ion test. The male mice 14 d after the second immunization were mated with the female ones,of which the suckling mice24 h after birth were injected i.c. with EV71 FY-23 and observed for survival rate within 10 d. Results Two virus bands with different sedimentation coefficients were obtained by Opti Prep density gradient centrifugation. The virion fraction in upper layer was mainly consisted of empty particles,while that in lower layer with solid particles. However,the virion fraction in lower layer consisted of empty and solid particles,both of which were recognized by specific antisera against EV71. The infectious titer of virion fraction in lower layer during virus infection was 2 ~ 3 log higher than that in upper layer. Both the titers of two virion fractions showed increasing tendencies,while that in lower layer showed a significantly increasing tendency. The RNA contents of two virion fractions in upper and lower layers were different. The VP0 in lower layer was cleaved into VP2 and VP4,while the virion fraction in upper layer was mainly consisted of VP0. Both the virion fractions

关 键 词：肠道病毒71型 结构形式 免疫学特性 

分 类 号：R373.2[医药卫生—病原生物学] R392-33[医药卫生—基础医学]