登革热病毒(DENV)包膜蛋白特异性人源抗体筛选与鉴定

Screening and identification of human antibodies against dengue virus(DENV)envelope protein

目的建立自外周血快速筛选识别登革热病毒衣壳的人源抗体的方法,从病毒感染者外周血记忆B细胞中筛选出特异性抗体并进行性质分析。方法通过流式细胞术分选得到外周血中登革热病毒包膜蛋白特异性的单个记忆B细胞,结合高效单细胞PCR技术获得抗体基因序列,对重组表达的单克隆抗体进行结合活性及中和活性检测。结果成功筛选到识别DENV-1包膜蛋白的6株人源单克隆抗体,其中4株抗体均具有很强的结合活性以及中和活性。结论成功的通过流式细胞术对DENV特异性记忆B细胞进行分选,并结合单细胞PCR技术,实现了对DENV特异性人源单克隆抗体的高效筛选。通过该平台有希望筛选得到DENV的广谱中和抗体,用于登革热的预防和治疗。

We amied to establish a method for rapid screening of human antibodies against dengue virus(DENV)capsids from peripheral blood,and to screen specific antibodies from peripheral blood memory B cells of virus-infected patients for qualitative analysis.By flow cytometry,DENV envelope protein-specific memory B cells in peripheral blood were single-cell sorted.The gene sequence of antibody was amplified using efficient single-cell PCR.We determined the binding activity and neutralization potency of recombinant full-length antibodies.Six human monoclonal antibodies recognizing DENV-1 envelope protein were successfully screened,and four of them had strong binding activity and neutralizing activity.DENV-specific memory B cells were successfully sorted by flow cytometry,and DENV-specific human monoclonal antibodies were efficiently obtained using single-cell PCR.The results revealed that it is hopeful to obtain DENV-specific antibody with broadly neutralizing activity for the prevention and treatment of dengue fever.