ABSTRACT

H9N2 subtype of avian influenza virus (AIV) is primarily a bird virus, which is widespread in clinical avian disease, and reported in cases of human infection. As one of the surface proteins of AIV, the neuraminidase (NA) protein plays an important role mainly in viral budding. However, vaccine development and detection methods for NA of H9N2 AIVs are in urgent clinical need. In this study, a truncated NA gene (205–900 bp) was cloned from the NA sequence of H9N2 strain, and then expressed using pET-28a (+) vector. This purified recombinant NA protein was used to immunize BALB/c mice, and the monoclonal antibodies were screened through the indirect enzyme-linked immunosorbent assay (ELISA). Next, eight prokaryotic expression vectors were constructed for epitope identification. After cell fusion, three hybridoma cell lines producing the antibodies special to NA protein were screened by ELISA, western blotting, and indirect immunofluorescence; these were named 1B10, 2B6, and 5B2, respectively. Epitope scanning techniques were used to identify three B-cell epitopes recognized by these three monoclonal antibodies, ¹⁹⁶KNATASIIYDGMLVD²¹⁰, ²¹⁰DSIGSWSKNIL²²⁰ and ²²¹RTQESECVCI²³⁰. The subsequent homology analysis revealed the three epitopes were highly conserved in H9N2 AIV strains. The structural predictions of the antigenic epitopes indicated that all three epitopes were located in the catalytic region of NA. These results provide a basis for studying the function of the NA protein of H9N2 AIV and technical support for the development of a universal detection method based on anti-NA monoclonal antibodies.

摘要

H9N2亚型禽流感病毒（AIV）主要是一种禽类病毒，在临床禽类疾病中广泛传播，并在人类感染病例中有报道。神经氨酸酶（NA）蛋白作为AIV的表面蛋白之一，主要在病毒出芽中发挥重要作用。然而，H9N2禽流感病毒NA的疫苗开发和检测方法是临床迫切需要的。本研究从H9N2病毒的NA序列中克隆了一个截短的NA基因（205-900 bp），然后使用pET-28a（+）载体进行表达。利用纯化的重组NA蛋白免疫BALB/c小鼠，通过间接酶联免疫吸附试验（ELISA）筛选单克隆抗体。接下来，构建了八个原核表达载体用于表位鉴定。细胞融合后，通过ELISA、Western blotting、间接免疫荧光等方法筛选出3株产生NA蛋白特异性抗体的杂交瘤细胞系；它们分别被命名为 1B10、2B6 和 5B2。表位扫描技术用于鉴定这三种单克隆抗体识别的三种B细胞表位，¹⁹⁶ KNATASIIYDGMLVD ²¹⁰、²¹⁰ DSIGSWSKNIL ²²⁰和²²¹ RTQESECVCI ²³⁰。随后的同源性分析显示这三个表位在H9N2 AIV毒株中高度保守。抗原表位的结构预测表明，所有三个表位均位于NA的催化区。这些结果为研究H9N2 AIV NA蛋白的功能提供了基础，并为开发基于抗NA单克隆抗体的通用检测方法提供了技术支持。