Identification of a novel linear epitope on the NS1 protein of avian influenza virus.
BACKGROUND
The NS1 protein of avian influenza virus (AIV) is an important virulent factor of AIV. It has been shown to counteract host type I interferon response, to mediate host cell apoptosis, and to regulate the process of protein synthesis. The identification of AIV epitopes on NS1 protein is important for understanding influenza virus pathogenesis.


RESULTS
In this paper, we describe the generation, identification, and epitope mapping of a NS1 protein-specific monoclonal antibody (MAb) D9. First, to induce the production of MAbs, BALB/c mice were immunized with a purified recombinant NS1 expressed in E. coli. The spleen cells from the immunized mice were fused with myeloma cells SP2/0, and through screening via indirect ELISAs, a MAb, named D9, was identified. Western blot assay results showed that MAb D9 reacted strongly with the recombinant NS1. Confocal laser scanning microscopy showed that this MAb also reacts with NS1 expressed in 293T cells that had been transfected with eukaryotic recombinant plasmids. Results from screening a phage display random 7-mer peptide library with MAb D9 demonstrated that it recognizes phages displaying peptides with the consensus peptide WNLNTV--VS, which closely matches the (182)WNDNTVRVS(190) of AIV NS1. Further identification of the displayed epitope was performed with a set of truncated polypeptides expressed as glutathione S-transferase fusion proteins, and the motif (182)WNDNT(186) was defined as the minimal unit of the linear B cell epitope recognized by MAb D9 in western blot assays. Moreover, homology analysis showed that this epitope is a conserved motif among AIV.


CONCLUSIONS
We identified a conserved linear epitope, WNDNT, on the AIV NS1 protein that is recognized by MAb D9. This MAb and its epitope may facilitate future studies on NS1 function and aid the development of new diagnostic methods for AIV detection.
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. wenxuexia0420@163.com.|College of Veterinary Medicine, China Agricultural University, Beijing, People's Republic of China. wenxuexia0420@163.com.
Xuexia
X
Wen
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. conansun6812@163.com.
Jiashan
J
Sun
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. wxr@hvri.ac.cn.
Xiurong
X
Wang
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. 772152723@qq.com.
Hongmei
H
Bao
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. 859829800@qq.com.
Yuhui
Y
Zhao
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. 395457707@qq.com.
Xianying
X
Zeng
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. 781198447@qq.com.
Xiaolong
X
Xu
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. mayong135166@163.com.
Yong
Y
Ma
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. 18854801560@163.com.
Linlin
L
Gu
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. chenhualan@caas.cn.
Hualan
H
Chen
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification; D001717:Birds; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005585:Influenza in Birds / Q000821:virology; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D017361:Viral Nonstructural Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; C085820:INS1 protein, influenza virus; D019151:Peptide Library; D017361:Viral Nonstructural Proteins