乙型脑炎病毒非结构蛋白1单克隆抗体的制备  

Preparation and Characterization of Monoclonal Antibody against Nonstructural Protein 1 of Japanese Encephalitis Virus

作　　者：周丹娜[1] 夏菲[1] 段正赢[1] 刘学[1] 郭锐[1] 杨克礼[1] 袁芳艳[1] 刘泽文[1] 孟丽[1] 蔡行[1] 田永祥[1] 

机构地区：[1]湖北省农业科学院畜牧兽医研究所/动物胚胎工程及分子育种湖北省重点实验室,武汉430064

出　　处：《湖北农业科学》2014年第23期5809-5812,共4页Hubei Agricultural Sciences

基　　金：湖北省农业科学院青年基金项目(2013NKYJJ12);湖北省科技支撑计划项目(2013BEC0002);湖北省科技支撑计划公益性课题项目(2014BBB010);湖北省农业科技创新中心资助项目(2011-620-001-003)

摘　　要：日本乙型脑炎(JE)是由乙型脑炎病毒(Japanese encephalitis virus,JEV)引起的蚊媒性人畜共患病,造成人畜中枢神经系统损害,威胁人畜健康。JEV的非结构蛋白1(Nonstructural protein 1,NS1)是其重要的非结构蛋白,一方面可以诱导机体产生保护性免疫,另一方面参与病毒复制等多种生物学过程。试验采用化学融合法获得分泌乙型脑炎NS1蛋白单克隆抗体(Mc Ab)的杂交瘤细胞系5D5,经检测获得的杂交瘤细胞系平均染色体数目约为90条,分泌的单抗为Ig G1亚型,细胞上清效价为27。接种小鼠后,收取腹水,经辛酸-硫酸铵法进行纯化后,测得腹水效价为105。Western blot鉴定结果表明所制备单抗能够与JEV NS1蛋白特异性结合,具有良好的免疫反应性,可为JE诊断方法的建立以及NS1蛋白生物学功能的研究提供物质基础。Japanese Encephalitis （JE） caused by Japanese Encephalitis virus （JEV） is a mosquito-born disease mainly causing central nervous system damage to human and animals. NS1 protein of JEV is a very important nonstrnctural protein of JEV inducing the body to produce protectie immune and participating in many biological processes including viral replication. A hybridoma cell line （5D5） secreting Monoclonal Antibody （McAb） against NS1 protein was selected and obtained by chemical fusion method. The results showed that the average chromosome number of hybridoma cell line obtained was 90. The sub-type of its secreting McAb was IgG1 and the titers of cell supernatant was 2^7. The hybridoma cell line was used to pro- duce ascites containing monoclonal antibody. The ascites was purfied by using octylic acid ammonium sulfate method. The result of NS1-ELISA showed that purified ascites titer was 10^5. The result of western blot showed that the monoclonal antibodies bound with JEV NS1 protein specifically and had good immunoreactivity. It is indicated that the MeAb obtained against NS1 will provide the basis for establishing JE diagnostic methods, and further study of the NS1 protein.

关 键 词：日本乙型脑炎 非结构蛋白1 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]