Preparation Of Nanobodies Against Porcine Circovirus Type 2 And Identification Of Antigentic Epitope It Recognizes

China is the world’s largest pig-raising country,pig stock and pork output in the world’s first.At present,the pig industry in China is facing many bottleneck problems,and epidemic disease is the main problem that restricts the healthy development of the pig industry in China.Porcine circovirus type 2 is the main pathogen of porcine circovirusassociated disease(PCVAD).The epidemic of PCVAD has caused serious economic losses to the pig industry in China.Antigen epitope as antigen-antibody interaction identification,binding sites,is one of the most important nature of the antigen,through identification of virus antigen epitope can understand the pathogenic mechanism of pathogenic microorganisms and antibodies play a role of protection mechanism,at the same time,the antigen epitope identification also has paved the way for the research and development new epitope-based vaccine.As an immune protein of PCV2,Cap protein has been identified in some immune segments on its surface,but there are still many linear and structural epitopes that have not been identified.For epitopes identification of PCV2 antigen,monoclonal antibodies are mostly used at present.However,due to the large size of traditional monoclonal antibodies,they can only bind to epitopes located on the surface of intact antigen molecules,but cannot recognize some hidden epitopes.Appears to overcome these shortcomings of traditional monoclonal antibodies very well,Its small relative molecular mass enables it to recognize hidden epitopes located inside antigen molecules that are difficult for traditional antibodiesto recognize.In this study,six anti-PCV2 Cap nanobody gene sequences were obtained through four rounds of bio-panning on the phage library of PCV2 Cap nanobody established in the laboratory.Six recombinant prokaryotic expression vectors were obtained by cloning the gene sequence of the screened nanobody into the pCold-SUMO expression vector.They were named PCV2-Cap-Nb-50,PCV2-Cap-Nb-55,PCV2-Cap-Nb-56,PCV2-Cap-Nb-57,PCV2-Cap-Nb-85,and PCV2-Cap-Nb-86,Subsequently,the recombinant vector was transformed into Escherichia coli BL21(DE3)for induced expression,and the recombinant protein was purified by nickel column.SDS-PAGE and Western blot results showed that the six nanobody were successfully expressed in soluble form in Escherichia coli.The titer of nanobody was determined by ELISA experiments,and it was found that all the six nanobody specifically bound to PCV2 Cap protein,among which the ELISA titer of PCV2Cap-Nb-56 was the highest,which could reach 1:104,THE ELISA of PCV2-Cap-Nb-50,PCV2-Cap-Nb-55,PCV2-Cap-Nb-57,and PCV2-Cap-Nb-86 was 1:103,and the ELISA potency of PCV2-Cap-Nb-85 was only 1:102.The results of virus neutralization experiment showed that PCV2-Cap-Nb-55 and PCV2-Cap-Nb-56 could neutralize PCV2 virus,and the titer was 24,while the other nabs had no virus-neutralizing activity.To further confirm whether PCV2-Cap-Nb-55 and PCV2-Cap-Nb-56 bind to different antigen sites,we used PCV2 Cap protein as the coated antigen and identified the binding sites of the two natant antibodies by competitive ELISA.The results showed that PCV2-Cap-Nb-55 and PCV2Cap-Nb-56 competed for the same epitope binding Cap protein.The experimental results of PCV2-Cap-Nb-55 are relatively ideal and stable.After comprehensive consideration,this study selected PCV2-Cap-Nb-55 for subsequent experiments.In order to further analyze the mechanism of the neutralizing Nanobody blocking PCV2 infection,PK-15 cells were infected after incubation with PCV2 and PCV2-Cap-Nb-55,and the infection was observed by IFA experiment.PCV2 binds to prevent the virus from adsorbing on the surface of the cell membrane,thereby achieving the effect of blocking PCV2 infection of PK-15 cells.In this study,we also identified the epitope of PCV2-Cap-Nb-55 binding to Cap protein by using the truncated fragment of Cap protein.Firstly,molecular biology software was used to predict the epitopes of Cap protein.According to the prediction results,Cap protein was divided into 9 truncated fragments,namely(Cap-A,Cap1-122),(Cap-A1,Cap178),(Cap-A2,Cap79-122),(Cap-B,Cap123-203),(Cap-B1,Cap123-163),(Cap-B2,Cap163-203),(Cap-B3,Cap123-159),(Cap-B4,Cap148-184),and(Cap-B5,Cap185-203),Primer5.0 software was used to design specific primers.After PCR amplification,the truncated fragment was cloned into pET-32a expression vector,which was transformed into BL21(DE3)expression strain for induction expression and purification.Western blot and ELISA were used to analyze the specific binding reaction between PCV2-Cap-Nb-55 and truncated protein,and the antigenic epitopes recognized by PCV2-Cap-Nb-55 were initially identified in the aa148-aa159 region of PCV2 Cap protein.In this study,six PCV2-specific antibody sequences were screened by biological panning of phage library of PCV2 Cap protein nanobody constructed earlier in the laboratory and successfully expressed in Escherichia coli.The neutralization test showed that two of the six nanobody had neutralization activity and were bound to the same antigen site.By identifying the epitope recognized by PCV2-Cap-Nb-55,a new epitope of PCV2 Cap protein was identified,which laid a foundation for the study of PCV2 immune mechanism and the development of PCV2 epitope vaccine.