高致病性禽流感病毒广谱嵌合抗体的构建表达及活性研究

Construction and Identification of Chimeric Antibody with Broad-spectrum Neutralization to Highly Pathogenic Avian Influenza H5N1 Virus

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 has persistent mutation and is lack of efficient therapeutic drugs.In the early research,a murine broad-spectrum neutralization monoclonal antibody 13D4 had been found that it had broad-spectrum therapeutic effectiveness directly to H5N1 avian Influenza virus in animal protection experiment.In the present study,the genes encoding variable sequences of heavy and light chain were obtained from 13D4 murine hybridoma cells by RT-PCR.Then these genes were combined with human gamma and kappa constant regions separately to construct chimeric antibody（cAbs）.Chimeric antibody was purified from serum-free supernatants of stable CHO cell line.The competitive Elisa results indicated that chimeric antibody could recognize the same epitope as parent antibody with similar affinity.13D4 chimeric antibody was further confirmed that retained broad-spectrum activity to different subtypes of H5N1 viruses in hemoagglutination inhibition assay.It also showed neutralizing activity to two H5N1 virus strains.In summary,13D4 chimeric antibody developed in the research would be valueble in the treatment.