摘要
以纯化的中华鳖虹彩病毒为抗原免疫Balb／C小鼠，取免疫鼠脾细胞经杂交融合获得了8个能稳定分泌抗中华鳖虹彩病毒特异性单克隆抗体的杂交瘤细胞株。单克隆抗体亚级份分析结果表明，Mab2A4属IgA，Mab8E1是IgG2a，其他的6株单抗Mab1D3、Mab2H1、Mab3A1、MaMB5、Mab5E1和Mab6F2均为IgG1。酶联免疫吸附剂测定（ELISA）分析表明，8株单抗均能特异性地识别中华鳖虹彩病毒，与EPC、CO、FHM等宿主细胞不产生交叉反应，腹水抗体的ELISA效价在10^5～10^6。IFA分析表明，8株单抗中仅Mab5E1没有免疫荧光反应特性，其余7株单抗均能对染毒病灶产生特异性的荧光染色。中和试验结果证实8株单抗均没有中和病毒的特性。应用Western—blotting进行中华鳖虹彩病毒的抗原表位初步分析，结果显示：Mab1D3和Mab2A4分别识别分子量为84ku和35ku中华鳖虹彩病毒结构蛋白，Mab3A1能够同时识别分子量分别为14ku和16ku的两条多肽，说明这3株单抗结合位点是非构象依赖性抗原决定簇，其余单抗不具备Western—blotting反应特性。这些结果提示上述单抗对中华鳖虹彩病毒抗原特异、灵敏，可用于中华鳖虹彩病毒的检测和结构蛋白分析。
Eight stable monoclonal hybridomas were successfully produced by immunization of Balb/C mice with purified soft-shelled turtle iridovirus （STIV） antigen. The Mabs obtained were three kinds of isotype. Mab 2A4 was subclass IgA, Mab8E1 was subclass IgG2a, and the other Mabs 1D3, Mab2H1, Mab3A1, Mab4B5, Mab5E1 and Mab6F2 were subclass IgG1. ELISA（enzyme linked immunosorbent assay） assays showed that eight Mabs could specifically recognize the antigen of STIV, and had no cross reaction with the cell lines EPC, Co, FHM. The ELISA titers of ascites were between 10^5 and 10^6. Immunofluorescent studies showed that all Mabs （except for MabSE1 ） had fluorescence characteristics, and the specific fluorescence signals appeared in the cytoplasm of STIV-infected （EPC） cells. None of the Mabs possessed the ability to neutralize STIV in vitro cell cultures. In this experiment, Western-blot was used to analyze the epitope of monoclonal antibodies against STIV. It demonstrated that Mab1D3 and Mab2A4 reacted specifically to a single linear protein with an approximately molecular weight of 84 ku and 35 ku respectively, Mab3A1 reacted with two STIV proteins at molecular weight of about 14 ku, 16 ku. The results suggested that these three Mabs target conformation-independent determinants within STIV protein. The identity of the target antigen of the other five Mabs could not be determined by Western-blot. These Mabs against STIV might be specific and sensitive, and they might also be used to detect STIV and analyze its structure proteins.
出处
《水产学报》
CAS
CSCD
北大核心
2009年第5期840-846,共7页
Journal of Fisheries of China
基金
国家"八六三"高技术研究发展计划(2006AA100306)
"十一五"国家科技支撑计划重点项目(2006BAK10B06)
国家质量监督检验检疫总局科研项目(2007IK022)
福建省科技计划项目(2009N2003)
关键词
中华鳖
虹彩病毒
单克隆抗体
抗原
Trionyx sinensis
iridovirus
monoclonal antibodies
antigen
