H5N8亚型禽流感病毒HA1蛋白的抗原表位鉴定

Epitope identification of HA1 protein of avian influenza virus H5N8 subtype

为了确定H5N8亚型禽流感病毒的新抗原表位,将原核表达的H5N8亚型禽流感QH448毒株HA1蛋白为免疫原免疫小鼠,通过筛选获得5株抗QH448(H5N8) HA1蛋白的单克隆抗体杂交瘤细胞株。经鉴定,1H12B9、8A12D10和9H10F5的重链为Ig G1亚型,2A9D11和7E9G6的重链为Ig G2b亚型,而轻链均为κ链。5株单克隆抗体均能特异性识别H5亚型禽流感病毒HA蛋白。通过截短表达策略确定,1H12B9和7E9G6的识别表位为^(330)ATGLRNSPLREKRRKR^(345),2A9D11、8A12D10和9H10F5的识别表位分别为^(310)IHP LTIGECPKYVKSN^(325)、^(271)KIVKKGDSTIMKSEV^(285)和^(121)LSRINHFEKILIIPKS^(136)。同源性比较结果显示,筛选出的4个抗原表位在H5-HA蛋白序列中均高度保守。综上所述,本研究获得了5株抗H5-HA蛋白不同抗原表位的特异性单克隆抗体细胞株,为H5N8亚型禽流感的诊断提供了新的靶标抗体。

In order to confirm the new epitopes of avian influenza virus H5N8 subtype,the prokaryotic HA1 protein of H5N8 influenza virue QH448 strain was used as immunogen to immunize mice.A total of five stable hybridoma cell lines were eventually obtained by secreting monoclonal antibodies against QH448HA1 protein.The 1H12B9,8A12D10,and 9H10F5 antibodies were Ig G1 type,and the 2A9D11 and 7E9G6 antibodies were Ig G2b type,while the light chains of all antibodies wereκchain.The 5 monoclonal antibodies could specifically recognize the HA protein of avian influenza virus H5 subtype.By using HA1 protein truncations,it was confirmed that the 1H12B9 and 7E9G6 recognized the epitope^(330)ATGLRNSPLREKRRKR^(345)of HA protein of H5N8 avian influenza virus,the 2A9D11 recognized epitope^(310)IHPLTIGECPKYVKSN^(325),the 8A12D10recognizedepitope^(271)KIVKKGDSTIMKSEV^(285),and the 9H10F5 recognized epitope^(121)LSRINHFEKILIIPKS^(136).Homology comparison showed that the four screened antigenic epitopes were highly conserved in the H5-HA proteins.These results indicated that five monoclonal antibodies were developed and characterized,which can recognize four different epitopes of H5-HA protein.The findings provided new antibodies for the diagnosis of H5N8 avian influenza.