[Construction and identification of nanobody phage display library targeting Middle East respiratory syndrome coronavirus].

He, Lei; Li, Jiangfan; Ren, Shuo; Sun, Shihui; Guo, Yan; Qiu, Hongjie; Liao, Yuanxiang; Ji, Kaiyuan; Fan, Ruiwen; Zhao, Guangyu; Zhou, Yusen

Objective To construct a phage display library of specific nano-antibodies against the Middle East respiratory syndrome coronavirus (MERS-CoV) and apply it to the screening of neutralizing nano-antibodies. Methods MERS-CoV receptor-binding domain (RBD) recombinant protein was used to immunize alpaca. After the last immunization, peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood and total RNA was extracted. The VHH gene was amplified by PCR and used to construct recombinant phages. TG1 Escherichia coli was transformed by these recombinant phages. A phage display library of specific nano-antibodies against the MERS-CoV were obtained and used to screen and characterize the nano-antibodies. Results The volume of this library of nano-antibodies was 1.31×108 and its abundance rate was 5.65×1010/mL. The ratio of VHH insertion in the constructed library reached 96%. There was a rich diversity of nano-antibodies in this library. Nano-antibodies with neutralizing activity were identified and expressed from this library. Conclusion We successfully constructed a library of phages which could be effectively applied to the screening of nano-antibodies against MERS-CoV virus.