Preparation And Characterization Of Anti-SARS-CoV-2 Papain-like Protease Nanobody

The new coronavirus broke out in Wuhan,China in December 2,019 and has become a global epidemic,posing a serious threat to the global public health and safety.Studies have proven that coronaviruses are critically dependent on protease activity,and two proteases encoded by their genomes,the major protease and papain,are involved in viral replication,packaging,and budding.Due to the rapid mutation iteration of new coronavirus and increased immune escape,the research and development of broadspectrum drugs is particularly urgent.PLpro,a cysteine protease,is located at the C-terminus of NSP3 and is involved in cleaving viral proteins,releasing Nsp1,Nsp2 and NSP3,and PLpro is a deubiquitinating enzyme that recognizes LRLRGG motifs,specifically degrades substrates,hydrolyzes ubiquitin chains and removes interferon stimulated gene 15(ISG15)modifications,PLpro can block the phosphorylation and nuclear translocation of interferon regulatory factor 3(IRF3)to affect the host innate immune response,interact with key regulators in signaling pathways,hijack them to block immunity,and mediate immune escape of viruses.Therefore,targeting PLpro with antiviral drugs may have advantages and could serve as an important target for the treatment of COVID-19.Nanobodies are thought to be the smallest naturally occurring intact antigen binding fragment with a molecular weight of 15 k Da and are distinguished by a long CDR3 loop that can form fingers and can extend into cavity sites on target antigens inaccessible to conventional antibodies;hydrophilic amino acids in the CH2 region,making it more soluble in polar environments,less prone to aggregation,more stable,and tolerable to extreme conditions(p H,temperature).These properties have led to the use of nanobodies in diagnostic therapy and cancer imaging.In this paper,we use PLpro as a research object to understand the mechanism of PLpro action and screen drug candidates by phage display technology for combating new corona viruses.We first constructed a PLpro prokaryotic expression system,performed protein purification,performed seven camelid immunizations,extracted VHHs genes from peripheral blood lymphocytes and constructed an anti-PLpro specific nanobody glycerophore library.Using phage display technology,two consecutive rounds of screening were carried out to make the enrichment reach 117.25 and 14 nanobodies were screened by alignment discovery.The nanobody epitopes were classified by HPLC,their affinity was tested by Octet,the functional nanobodies to PLpro were verified by enzyme activity test,and further explained by structural biology.We have screened the nano antibody NB1A1 and NB1F7 which have the ability to inhibit PLpro.Here we have not obtained the crystal structure which has the ability to inhibit PLpro.We have obtained the crystal structure which has no function to PLpro.The crystal structure shows that NB1F9 has no effect on the active site of PLpro,which is consistent with the results of the enzyme activity test.Through a series of experiments,we finally obtained two nanobodies with obvious inhibitory effects on PLpro,NB1A1,NB1F7,with affinities of 0.059 ± 0.004 n M,2.73 ±0.03 n M,respectively,and IC50 values of 132 n M,68.95 n M,respectively,to provide drug candidates for combating coronaviruses.