[1]郭利敏,乔传玲*,陈 艳,等.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[J].中国预防兽医学报,2010,(06):460-463.
 GUO Li-min,QIAO Chuan-ling*,CHEN Yan,et al.Preparation of the monoclonal antibodies using swine influenza virus NP protein expressed by eukaryotic expression vector[J].Chinese journal of preventive veterinary medicine,2010,(06):460-463.
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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
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2010年06期
页码:
460-463
栏目:
免疫学
出版日期:
2010-06-01

文章信息/Info

Title:
Preparation of the monoclonal antibodies using swine influenza virus NP protein expressed by eukaryotic expression vector
文章编号:
1008-0589(2010)06-0460-04
作者:
郭利敏乔传玲*陈 艳杨焕良张 健辛晓光陈化兰*
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
Author(s):
GUO Li-min QIAO Chuan-ling* CHEN Yan YANG Huan-liang ZHANG Jian XIN Xiao-guang CHEN Hua-lan*
Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology,  Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China
关键词:
猪流感病毒核蛋白基因真核表达单克隆抗体
Keywords:
Swine influenza virus NP gene eukaryotic expression monoclonal antibody
分类号:
S852.65
文献标志码:
A
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SIV NP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100 μg/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SIV NP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1.02×106 M-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。
Abstract:
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 μg 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, κ chain and the ascites titer was 1∶105. The relative affinity of the purified MAb against H3 SIV was 1.02×106 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.

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备注/Memo

备注/Memo:
收稿日期:2010-02-01
基金项目:哈尔滨市科技攻关计划项目(2009AA6BN078)
作者简介:郭利敏(1982-),女,山西浑源人,硕士研究生,主要从事猪流感诊断方法的研究.
更新日期/Last Update: 2010-07-08