Screening And Structure Analysis Of Novel Coronavirus RBD Neutralizing Nanobody

COVID-19 is a severe respiratory disease caused by novel coronavirus(SARS-COV-2),which is spreading rapidly around the world and seriously affecting human health.SARS-Co V-2 is an enveloped virus with forward single-stranded RNA.Structurally,it has a double lipid envelope containing S protein,E protein,M protein and N protein,in which S protein forms the spiky-glycoprotein on the surface of the virus in the form of trimer.SARS-Co V-2 mainly enters cells through the upper respiratory tract or the facial mucosal surface.The receptor binding domain(RBD)of the S1 subunit on the surface of the S protein binds to the receptor binding unit(RBM)of ACE2,and the S2 subunit of the S protein mediates the fusion of the virus with the cell membrane.After SARS-Co V-2 infects cells,viral genetic material and proteins are uncoated.The virus releases RNA into the host cell and translates proteins,including RNA-dependent polymerase,within the host cell,which is then transcribed and assembled,followed by viral shedding to complete the replication cycle.Nanobody is a single domain antibody containing only the heavy chain variable region(VHH).It was obtained by molecular cloning from the heavy chain antibody containing only the heavy chain naturally occurring in camels.Nanobodies have the function of traditional antibodies.Compared with the traditional antibodies,nanobodies have smaller molecular weight,higher stability and stronger tissue permeability.In addition,nanobodies also have the characteristics of high solubility and easy preparation.Due to their unique structure and function,nanobodies are widely used in the diagnosis and treatment of tumors.In this paper,SARS-Co V-2 was associated with nano antibody,aiming to obtain several different neutralizing nanobodies in the RBD domain of SARS-Co V-2,and elucidate their mechanism of action from the perspective of structure.The RBD domain of S protein on the surface of SARS-Co V-2 was constructed by molecular cloning.After expression in HEK-293-F cells,RBD with high purity was obtained by isolation and purification.Bactrian camels were immunized with RBD for seven times.RNA was extracted from their peripheral blood and c DNA was synthesized.Finally,an antibody library with a capacity of 1.33×10~8CFU/m L was constructed,including anti-RBD nanobody.Through the phage display technology,two rounds of immunoscreening were performed on the constructed antibody library using RBD,and the phage enrichment of displaying the anti-RBD nanobody gene reached 116.7.Subsequently,after TG1 infection with phages,950 single colonies were selected to express nanobodies in small quantities.By ELISA identification and sequence alignment,32 anti-RBD nanobodies with different CDR3 regions were obtained.The epitopes of 32 nanobodies were analyzed by HPLC and the affinity of the nanobodies was determined by Octet.The nanobodies termed 1A7 and 1B11 were identified and selected due to high binding affinity and different binding epitopes of RBD.The KD value of 1A7 and 1B11 are 0.74±0.003 n M and 6.76±0.027 n M,respectively.After1A7 and 1B11 were formed into complexes with RBD,the structure of the two complexes was analyzed by X-ray diffraction,and the different binding epitopes of 1A7and 1B11 with RBD were determined,and the two nanobodies could block the binding of RBD with ACE2 in the spatial position.Finally,the activity of 1A7 and 1B11 was verified by SARS-Co V-2 pseudovirus assay,and it was confirmed that the two nanobodies could inhibit the binding between RBD and ACE2.In this paper,a neutralizing nanobody against the RBD domain of SARS-Co V-2 was obtained by screening.The interaction epitopes of 1A7 and 1B11with RBD were analyzed from the perspective of structural biology,and the ways in which the binding between RBD and ACE2 was blocked by the two different nanobodies were explained,which laid an important foundation for the subsequent diagnosis and treatment of SARS-Co V-2 and drug development.