利用真核表达的猪流感病毒NP蛋白制备其特异性的单克隆抗体  

Preparation of the monoclonal antibodies using swine influenza virus NP protein expressed by eukaryotic expression vector

作　　者：郭利敏[1] 乔传玲[1] 陈艳[1] 杨焕良[1] 张健[1] 辛晓光[1] 陈化兰[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/农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2010年第6期460-463,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：哈尔滨市科技攻关计划项目(2009AA6BN078)

摘　　要：为制备猪流感病毒(SIV)核蛋白(NP)单克隆抗体(MAb),本研究将重组质粒pMD-NP中含有的SIVNP基因亚克隆于pCAGGS真核表达质粒中,获得重组质粒pCAGGS-NP,将其转染293T细胞,通过间接免疫荧光(IFA)和western blot检测表明NP蛋白在293T细胞中获得了表达。将pCAGGS-NP以100μg/只剂量免疫4周龄～5周龄BALB/c小鼠,3次免疫后,利用杂交瘤细胞融合技术获得1株稳定分泌抗SIVNP的MAb杂交瘤细胞株(3D7);该MAb经Ig亚类鉴定为IgM,轻链为κ链,其诱导小鼠产生的腹水ELISA效价为1:105;纯化3D7腹水并对其进行亲和力及抗原结合活性的测定,ELISA曲线图显示亲和力常数为1.02×106M-1,IFA结果显示其可与H1N1、H3N2、H9N2亚型SIV发生特异性反应,具有良好的反应活性。该MAb的制备将为进一步建立猪流感的诊断方法以及分析NP蛋白抗原表位等方面的研究奠定基础。To construct the recombinant plasmid pCAGGS-NP, NP gene of swine influenza virus was sub-cloned into a eukaryotic expression vector pCAGGS and transfected into 293T cells. Expression of NP protein was confirmed by IFA and western blot. BALB/c mice were inoculated with 100 ug of the recombinant plasmid and spleen cells used for fusion. Hybridoma was screened and a monoclonal hybridoma cell line （3D7） was successfully obtained. The MAb belonged to IgM subclass, K chain and the ascites titer was 1 ： 105. The relative affinity of the purified MAb against H3 SIV was 1.02 × 10 6 M-1. IFA assay showed that the MAb had good cross-reactivity with H1N1, H3N2 and H9N2 subtype SIVs Vs. The NP protein specific MAb could be a useful reagent for diagnosis of SIV and analyzing the epitope of NP protein.

关 键 词：猪流感病毒 核蛋白基因 真核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]