肠病毒71型分离株的鉴定及免疫原性研究  

Identification and immunogenicity of enterovirus 71 isolates

作　　者：李秀玲[1] 张中洋[1] 王潇潇[1] 杨永娟[1] 郝春生[1] 赵敏[1] 何薇薇[1] 张晨[1] 沈心亮[1] 

机构地区：[1]北京生物制品研究所,100024

出　　处：《中华微生物学和免疫学杂志》2009年第10期944-948,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家科技支撑计划（2008BA169D00）

摘　　要：目的在对肠病毒71型（enterovirus 71，EV71）分离株进行鉴定的基础上，对其诱导小鼠产生的免疫应答水平进行研究，拟为进一步的EV71候选疫苗研究奠定基础。方法超速离心纯化病毒后，采用磷钨酸负染法通过电镜观察病毒形态、大小；采用特异性EV71单抗通过间接免疫荧光法（IFA）检测病毒的特异性；合成EV71特异性引物，通过RT—PCR对病毒进行分子生物学鉴定；病毒的VP1基因序列测定后，与其他EV71序列进行比较，绘制种系发育树，确定病毒基因型；通过腹腔注射途径免疫小鼠，免疫后分离血清通过微量细胞病变抑制法测定血清中和抗体效价，ELISA法检测血清抗体水平和抗体亚型水平。结果电镜下可观察到典型的圆形病毒颗粒，直径为20～30nm，呈典型的肠病毒形态；085和087株病毒感染细胞中均能观察到黄绿色荧光，提示该两株病毒可与EV71单抗特异性结合；RT—PCR可以从病毒感染细胞中扩增出226bp大小的特异性产物带，而采用CA16特异性引物则不能扩增出相应大小的产物带；基于VP1基因序列的种系发育分析显示，087和085株均为c4基因型；085和087株E、EV71免疫小鼠后可诱导产生能中和包括其本身在内的多个病毒株的中和抗体，针对同一株中和病毒，实验组间差异无统计学意义；针对不同中和病毒株，各实验组中和本株、523-07T株的能力明显高于FY-02T株（P〈0．05）；ELISA法检测结果显示，接种灭活前后的EV71病毒085和087株后均能诱导小鼠产生抗EV71特异性IgG；IgG亚型为IgG1／IgG2a混合型，灭活病毒免疫组小鼠血清EV71特异性IgG、IgG1、IgG2a水平明显高于活病毒免疫组（P〈0．05）。两株病毒之间差异无统计学意义。结论本研究分离到的085和087株病毒均为EV71CA基因亚型，并具有一定的免疫原性。Objective To identify two enterovirus 71 （ EV71 ） isolates and evaluate their immunogenicity for the purpose of EV71 vaccine development. Methods The morphology of EV71 isolates was determined by electronic microscopy, both of the 087 and 085 isolates were identified by indirect fluorescence assay （IFA） using anti-EV71 monoclonal antibody and by RT-PCR with EV71 specific primers, the genotype was determined by phylogenetic analysis based on VP1 gene sequence. The immunogenicity was evaluated on BALB/c mice by the detection of neutralization antibody and anti-EV71 IgG in the serum with neutralization test and ELISA. Results The round virus particles with 20-30 nm in diameter can be objected by electronic microscopy in the virus infected cells culture which is typical for enterovirus. Both 087 and 085 strains can react with anti-EVT1 monoclonal antibody, the positive virus infected cells stained with FITC can be detected by IFA. The 226 bp products were amplified from the total RNA extracted from virus infected cells by RT-PCR with EV71 special primers, not CA16 special primers. The genotype of both strains is C4, which is identical to the other epidemiological EV71 strains in mainland, China in the latest decade. Both strains can induced high level of neutralization antibody before or after inactivation which can neutralize more EV71 strains than themselves, for the same neutralized virus strain, no difference was found between 085 and 087 strains and between live and inactivated group for each strain either, but the ability to neutralize FY- 02T strain for the serum from all the immunized groups is lower than other strains significantly（ P 〈 0.05 ）. Compared with control, anti-EV71 IgG can be detected in the sera from immunized mice with the subtype IgG1/IgG2a by ELISA, which are not different between 087 and 085 strains but higher in inactivated virus group. Conclusion Both of the two EV71 isolates, 085 and 087 strains were identified to be EV71 with genotype CA and are highly immunogenic.

关 键 词：肠病毒71型 鉴定 免疫原性 

分 类 号：R3[医药卫生—基础医学]