Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus.
Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus.
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NP蛋白特异性B细胞表位的鉴定，用于鸟类流感病毒的H9N2亚型。
DOI：
10.3390/v14061172
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发表时间：
2022-05-28
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由H9 N2亚型禽流感病毒（AIV）引起的禽流感（Avian Influenza，AI）对养禽业和公共卫生安全构成严重威胁。NP是流感病毒的主要结构蛋白之一。位于NP蛋白上的B细胞决定簇引起了越来越多的关注。本研究根据H9 N2（A/chicken/Shandong/LY 1/2017）毒株NP基因序列，克隆了NP基因截短片段（71 AA-243 AA），并在pET-28 a（+）载体中进行原核表达。用纯化的NP蛋白的重组体免疫BALB/c小鼠，以制备针对NP蛋白的单克隆抗体。利用原核表达的4个重叠片段NP-N-96、NP-C-103、NP-C-54和NP-C-49识别NP蛋白的抗原表位。结果表明，经细胞融合后，经ELISA和间接免疫荧光筛选，获得1株分泌NP蛋白特异性抗体的杂交瘤细胞克隆，命名为4F 5单克隆抗体（mAb）。Western blotting结果显示，230 bp TAAQRA 237基序为4F 5单抗识别的最小基序，为NP蛋白的线性B细胞表位。同源性分析表明该表位在本研究分析的18株AIV中高度保守，表位预测结果表明该表位可能位于NP蛋白的表面。这些结果为研究H9 N2亚型禽流感病毒NP蛋白的功能提供了有力的实验基础，也为建立基于NP蛋白单克隆抗体的通用检测方法提供了有力的技术支持。
Avian Influenza (AI) caused by the H9N2 subtype of the avian influenza virus (AIV) poses a serious threat to both the poultry industry and to public health safety. NP is one of the major structural proteins in influenza viruses. B-cell determinants located on NP proteins have attracted increasing attention. In this study, based on the NP sequence of the H9N2 (A/chicken/Shandong/LY1/2017) strain, the truncated NP gene (71 AA–243 AA) was cloned and prokaryotically expressed in a pET-28a (+) vector. BALB/c mice were immunized with a purified recombinant of an NP protein to prepare a monoclonal antibody against NP proteins. The prokaryotic expression of four overlapping fragments, NP-N-96, NP-C-103, NP-C-54 and NP-C-49, were used to recognize an antigenic epitope of the NP protein. The results show that, after cell fusion, one hybridoma cell clone secreted the antibody specific to the NP protein, following screening with ELISA and indirect immunofluorescence, which is named the 4F5 monoclonal antibody (mAb). Western blotting on the overlapping fragments showed that the 230FQTAAQRA237 motif was identified as the minimal motif recognized by 4F5mAb, which was represented as the linear B-cell epitope of the NP protein. Homology analysis of this epitope shows that it was highly conserved in 18 AIVs analyzed in this study, and the epitope prediction results indicate that the epitope may be located on the surface of the NP protein. These results provide a strong experimental basis for studying the function of the NP protein of the H9N2 AIV and also strong technical support for the development of a universal assay based on an anti-NP monoclonal antibody.
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