马流感病毒NP单克隆抗体的制备及特性鉴定  

Preparation and identification of monoclonal antibodies against NP of equine influenza virus

作　　者：姬媛媛[1] 郭巍[1] 王征[2] 赵立平[1] 李红梅[1] 相文华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物病研究室,黑龙江哈尔滨150001 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2011年第6期610-613,共4页Chinese Veterinary Science

基　　金：黑龙江省自然科学基金重点项目(ZD200812)

摘　　要：利用原核表达的马流感病毒(EIV)核蛋白(NP)为免疫原,经腹腔接种4周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经4次有限稀释法克隆和间接ELISA法筛选,获得了3株稳定分泌单抗的杂交瘤细胞株2G11、3E10和4A1,并对它们进行了亚类鉴定、间接ELISA检测和特性鉴定。结果显示,单抗2G11为IgG1亚型,3E10为IgG2a亚型,4A1为IgM亚型,轻链均为κ链。单抗2G11、3E10和4A1的细胞上清液及腹水效价分别为1∶640、1∶640、1∶320和1∶409 600、1∶204 800、0。这3株单抗均能特异性识别EIV NP重组蛋白,并且能够与天然EIV结合;获得的3株单抗与马动脉炎病毒、马疱疹病毒1型、马疱疹病毒4型、马乙型脑炎病毒均不发生交叉反应。亲和力试验结果显示,单抗2G11和3E10与EIV的亲和力常数分别为4.39×106 M-1和2.20×106 M-1。Four week-old BALB/c mice were intraperitoneally immunized with the prokaryoticly-expressed NP four times.Three hybridoma cell lines named 2G11,3E10 and 4A1 were obtained by fusing SP2/0 with spleen cells.The first monoclonal antibody（McAb） 2G11 was identified to be IgG1,3E1 to be IgG2a and 4A1 to be IgM,all of them with κ light chain.The antibody titres of their hybridoma supernatant and ascites were 1∶640,1∶640,1∶320 and 1∶409 600,1∶204 800,0,respectively.Western-blot analysis showed that the three McAbs could react with the recombinant NP specifically.IFA showed that they could combine with the natural EIV well.The obtained 3 McAbs had no cross reactions with equine arteritis virus,equine herpes virus-1,equine herpes virus-4 and Japanese encephalitis virus.The affinity test showed that 2G11 and 3E10 were 4.39×106 M-1and 2.20×106M-1 in affinity constant,respectively.

关 键 词：马流感病毒 核蛋白 单克隆抗体 特性鉴定 

分 类 号：S852.659.5[农业科学—基础兽医学]