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高致病性禽流感病毒高度变异且缺乏有效的治疗药物.在前期研究工作中,本课题组发现一株鼠源广谱中和单抗13D4,在动物实验中显示出对H5N1禽流感病毒具有广谱治疗效果.在本研究中,从分泌13D4单克隆抗体的杂交瘤细胞株中抽提总RNA,经RT-PCR扩增出轻重链可变区DNA序列,并分别与人IgG1的轻重链恒定区基因序列拼接,构建人-鼠嵌合抗体.筛选出稳定表达嵌合抗体的CHO细胞株,从培养上清中纯化出嵌合抗体.竞争Elisa结果表明,嵌合抗体与鼠源单抗能够识别同一个抗原表位并具有相似的亲和力.血凝抑制反应和中和活性测定结果证明,13D4嵌合抗体保留了对不同亚型H5N1病毒的广谱反应性,并且对两株H5N1病毒具有中和活性.本研究获得的13D4嵌合抗体将具有潜在的治疗价值.
Highly pathogenic avian influenza virus highly variable and the lack of effective treatment drugs.In the previous work, our group found a murine broad-spectrum neutralizing mAb 13D4 in animal experiments showed that the H5N1 bird flu virus has Broad-spectrum therapeutic effect.In this study, total RNA was extracted from the hybridoma cell line secreting 13D4 monoclonal antibody, and the light and heavy chain variable region DNA sequences were amplified by RT-PCR and compared with the light and heavy chain The constant region gene was spliced to construct the human-mouse chimeric antibody.The CHO cell line stably expressing the chimeric antibody was screened and the chimeric antibody was purified from the culture supernatant.Competitive Elisa results showed that the chimeric antibody and the mouse monoclonal antibody Can recognize the same epitope and have similar affinity.Hepaglutination inhibition and neutralization activity assay results show that 13D4 chimeric antibody retains broad-spectrum reactivity to different subtypes of H5N1 virus, and has two strains of H5N1 virus Neutralizing activity.The 13D4 chimeric antibody obtained in this study will have potential therapeutic value.