[1]李敏华,王 倩,田志军*,等.一株PRRSV N蛋白单克隆抗体制备及其抗原表位鉴定[J].中国预防兽医学报,2019,(03):279-283.[doi:10.3969/j.issn.1008-0589.201805026]
 LI Min-hua,WANG Qian,TIAN Zhi-jun*,et al.Preparation of the monoclonal antibody against the PRRSVN protein and identification of the B-cell epitope[J].Chinese journal of preventive veterinary medicine,2019,(03):279-283.[doi:10.3969/j.issn.1008-0589.201805026]
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一株PRRSV N蛋白单克隆抗体制备及其抗原表位鉴定()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年03
页码:
279-283
栏目:
免疫学
出版日期:
2019-04-25

文章信息/Info

Title:
Preparation of the monoclonal antibody against the PRRSV
N protein and identification of the B-cell epitope
文章编号:
1008-0589(2019)03-0279-05
作者:
 

李敏华1王 倩2田志军2*金 涛1*

 (1. 黑龙江大学 生命科学技术学院,黑龙江 哈尔滨 150080;2. 中国农业科学院 哈尔滨兽医研究所,黑龙江 哈尔滨 150069)
Author(s):
 

LI Min-hua1 WANG Qian2 TIAN Zhi-jun2* JIN Tao1*

 (1. College of Life Science and Technology, Heilongjiang University, Harbin 150080, China;
2. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
猪繁殖与呼吸综合征病毒单克隆抗体细胞融合N蛋白表位鉴定
Keywords:
porcine reproductive and respiratory syndrome virus  monoclonal antibody  cell fusion  N protein  identify epitope
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201805026
文献标志码:
A
摘要:
为了制备具有阻断能力的抗猪繁殖与呼吸综合征病毒(PRRSV)的单克隆抗体(MAb),本研究以PRRSV SD53株感染Marc-145细胞后的上清作为免疫原免疫BALB/c雌鼠,筛选获得一株稳定分泌抗PRRSV MAb的杂交瘤细胞株(1H4)。鉴定结果显示,1H4 MAb是IgG1亚类,轻链为κ链。1H4细胞上清经间接免疫荧光(IFA)检测其效价是1∶40,腹水的IFA效价是1∶3 200。阻断ELISA试验显示1H4 MAb与病毒的结合能够被PRRSV阳性血清阻断。Western blot试验显示1H4 MAb可以特异性识别PRRSV的N蛋白,经截短表达N蛋白鉴定该MAb的抗原识别序列为N蛋白末端aa 107~aa 123。该抗原表位在美洲型PRRSV中高度保守,该MAb的制备为进一步建立特异性强、灵敏度高的PRRSV阻断ELISA检测方法奠定了基础。
  
Abstract:
In order to prepare the monoclonal antibody (MAb) against porcine reproductive and respiratory syndrome virus (PRRSV), the splenic cells of immunized mice with PRRSV were fused with SP2/0 myeloma cells, and a hybridoma cell line (1H4) stably secreting the MAb against PRRSV was screened. The identification results indicated the 1H4 MAb was IgG1 subclass with κ light chain. The MAb titer in cell culture medium and ascites were 1∶40 and 1∶3,200, respectively. The blocking ELISA detection showed that the binding of the MAb to the virus was blocked by PRRSV positive serum. The MAb was able to specifically recognize the N protein of PRRSV detected by western blot. The antigen sequence of the epitope recognized by the MAb was identified within aa107-aa123 of N protein (107PTQHTVRLIRATASPSA123) by pepscan technique, which is the highly conservative epitope sequences among the N protein of American (Type 2) PRRSV strains. The preparation of MAb provided foundation for further development of detecting technique of blocking ELISA against PRRSV with high specificity and sensitivity.

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                    (本文编辑:李 娜)

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更新日期/Last Update: 2019-04-26