The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain name 110-helix of HA, and the monoclonal antibody realizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide useful insights into H9N2 vaccine design and improvement, as well as Bisacodyl antibody-based therapies for treatment of H9N2 AIV contamination. Keywords: H9N2 avian influenza computer virus, HA protein, neutralization, monoclonal antibody, antigen epitope 1. Introduction The H9N2 subtype of avian influenza is now common Bisacodyl in many eurasian nations and can reduce poultry egg production or co-infect with other infections, resulting in substantial mortality and significant economic losses [1,2,3,4,5]. H9N2 subtype AIV was first isolated in turkeys in 1966 [6], and was first isolated and recognized in 1992 in China [7]. Following this, H9N2 subtype AIV started to appear in additional regions [8,9,10]. The H9N2 subtype of AIV can harm avian physiology and weaken immune systems, leaving them more vulnerable to other harmful pathogenic microorganisms [10,11]. Furthermore, H9N2 AIV has been reported to infect humans [12,13,14,15] and can also pass on gene snippets or even full genomes to other highly fatal AIVs, including H4N6, H5N6, H5N2, H7N2, H10N8, and H7N9 [8,16,17,18,19,20]. AIV is usually a segmented negative-strand RNA computer virus, and eight viral segments encode the following proteins PB2, PB1, PA, Bisacodyl HA, NP, NA, M, and NS. One of the most significant glycoproteins among them is the hemagglutinin HA, whose amino acids are subject to mutation [21,22,23,24,25,26]. As a result, the immune system may develop resistance to the vaccination due to antigenic drift and immunological escape. When HA, which enters cells by attaching to the sialic acid around the cell surface, is targeted, specific neutralizing antibodies (Abs) are generated [5,27]. The recognized neutralizing antibodies primarily target five immunodominant antigenic sites that are extremely changeable around the structural Bisacodyl domain of the HA globular head [28,29,30,31], preventing the adhesion or fusion mediated by HA. However, the neutralizing antibodies located in the head can only produce the neutralizing activity against one or a few strains and are not widely used [32]. Antibodies directed against the stem of the HA protein have also been investigated, which barely have the neutralizing Bisacodyl activity [33]. Recently, a third class of antibodies has been recognized: those directed against the vestigial esterase domain name (VED) [34,35,36,37,38], which is very conserved in the same isotype and varies between isotypes [39,40,41]. Previous studies have reported on H9N2-associated antigenic epitopes. Zhu et al. [42] screened six important antigenic sites by monoclonal antibodies and their acknowledgement of variant sites in different strains: 92, 145, 166, 167, 168, and 197. Wan et al. [43] found nine very important sites, in which 164, 167, 168, 196 and 207 were newly recognized. Wang et al. [44] found rSNHA-200 produced considerably more neutralizing antibodies and provided the highest protection against both homologous and heterologous H9N2 AIVs. Zhu et al. [45] found 53, 274, 83 and 276 were found to be important and neutral sites for the HA protein of H5N1 AIV, all of which are located in the VED region. BSG However, no neutralizing epitopes targeting the VED region have been recognized in H9N2 subtype AIV. In this paper, to investigate the potential epitope of B cell in HA protein, HA gene was cloned and expressed to be used as immunogen to prepare the hybridoma cells. Also, the epitope and neutralization activities of the monoclonal antibodies produced by the hybridmoa cells were performed. Results showed that this monoclonal 1E11 and 2B3 were the neutralizing and targeting epitopes 112NVENLEEL119 and 117EELRSLFS124, which are located in the vestigial esterase domain name (VED). These results suggested that this monoclonal antibodies against this region might take action.