新型冠状病毒特异性纳米抗体的筛选和鉴定  

Screening and identification of SARS-CoV-2-specific nanobodies

作　　者：史震中 徐晓驰 吴文惠[1,2] 许剑锋 SHI Zhenzhong;XU Xiaochi;WU Wenhui;XU Jianfeng(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Quality and Safety Risk Assessment Laboratory of Aquatic Products Storage and Preservation,Ministry of Agriculture(Shanghai),Shanghai 201306,China;Qidong Camel Nano-biomedical Co.,Ltd,Qidong 226200,China)

机构地区：[1]上海海洋大学食品学院,201306 [2]农业部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306 [3]启东凯默纳米生物医药有限公司

出　　处：《免疫学杂志》2022年第8期720-725,共6页Immunological Journal

基　　金：国家自然科学基金(82173731)。

摘　　要：目的通过噬菌体展示技术,从骆驼外周血中筛选针对SARS-CoV-2 RBD结构域的特异性纳米抗体,并对纳米抗体的亲和力、结合表位和中和活性进行评估。方法利用重组RBD免疫骆驼,从骆驼外周血中分离淋巴细胞,提取RNA并逆转录为cDNA,接着通过巢式PCR扩增VHH基因,电转化至pMECS噬菌粒载体中,构建噬菌体展示抗体库并进行免疫淘选,鉴定可以与RBD特异性结合的纳米抗体。分别利用Octet Red96e和HPLC测定纳米抗体与重组RBD的亲和力和结合表位,通过SARS-CoV-2假病毒中和实验测定纳米抗体的中和活性。结果从外周血淋巴细胞中成功扩增出VHH基因,构建的噬菌体展示抗体库库容为1.33×10^(8) CFU/ml;经过2轮免疫淘选,鉴定出2株特异性纳米抗体——NB2D11和NB4E9,K_(D)值分别为2.21和0.31 nmol/L;NB2D11和NB4E9具有非竞争性的结合表位,中和SARS-CoV-2假病毒的IC_(50)值分别为340.7和35.5 nmol/L。结论筛选到的2株高亲和力特异性纳米抗体可以有效地地阻断SARS-CoV-2假病毒与ACE2的结合,为COVID-19的诊断和治疗奠定了基础。In response to the growing outbreak of COVID-19,this study was designed to screen and identify specific nanoantibodies against the SARS-CoV-2 RBD domain from the peripheral blood of camels through phage display technology.Camels were immunized with recombinant RBD and lymphocytes were isolated from the camel's peripheral blood;RNA was extracted from the lymphocytes and reverse-transcribed to cDNA for VHH gene amplification by nested PCR,then the VHH gene was electro-transfected into a pMECS bacteriophage vector to construct a phage display antibody library.Nanoantibodies binding specifically to RBD were screened by using the phage display antibody library.The affinity and binding epitopes of the nanobodies to recombinant RBD were determined by Octet Red96e and HPLC,respectively;the neutralizing activity of the nanobodies was determined by SARS-CoV-2 pseudovirus neutralization assay.Data showed that the VHH gene was successfully amplified from peripheral bloodly mphocytes,and the capacity of the constructed phage-display edanti body library was 1.33×10^(8) CFU/ml.After two rounds of immune panning,two specific nanobodies NB2D11 and NB4E9 with K_(D) values of 2.21 nmol/L and 0.31 nmol/L were found.NB2D11 and NB4E9 have non-competitive binding epitopes,and the IC_(50) values for neutralizing SARS-CoV-2 pseudovirus are 340.7 nmol/L and 35.5 nmol/L,respectively.In conclusion,the two highaffinity specific nanobodies obtained in this study can effectively block the binding of SARS-CoV-2 pseudovirus to ACE2,laying a foundation for the diagnosis and treatment of COVID-19.

关 键 词：SARS-CoV-2 RBD 噬菌体展示技术 纳米抗体 

分 类 号：R392-33[医药卫生—免疫学]