PRRSV-GP5 Antigen Structure Analysis And Nanobody Phage Display Library Construction

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by reproductive and respiratory disorders in sows.Its incidence rate and mortality rate are high,which has caused huge economic losses to the global pig industry,and has also done great harm to the mental health of the pig farmers.As the main protective antigen of PRRSV,glycoprotein 5(GP5)plays an important role in the invasion of cells and the clearance of PRRSV by immune system.However,there are decoy non neutralizing epitopes in GP5 protein,which greatly reduces the recognition and clearance efficiency of PRRSV by immune system.Nanobody(Nb)has small molecular weight and simple structure.It has a convex antigen binding epitope in space,and can be embedded in the depression of the surface of the antigen to recognize potential epitopes that traditional tetramer antibody can not recognize.In this study,the PRRSV-GP5 nanobody display library was constructed by phage display technology,and the structural characteristics of PRRSV-GP5 were analyzed by bioinformatics software to predict the dominant B cell antigen epitopes,so as to lay a foundation for panning GP5 specific Nb phage immune library.The research content of this paper includes two parts.1.The structural characteristics of GP5 antigen were analyzed by bioinformatics software.The analysis of protparam online server showed that GP5 was a hydrophilic protein.The online analysis by sopma software showed that alpha helix was the most in the whole amino acid chain,and its distribution was relatively concentrated;extended strand and random coil were alternately distributed on both sides;beta turn was scattered on the whole chain,which preliminarily inferred that there was a greater possibility of dominant antigen epitopes on both sides of the protein.Netphos 3.1 online server analysis showed that GP5 antigen had 10 serine phosphorylation sites at 12,16,34,37,78,109,110,133,157 and 193 amino acid sites,9 threonine phosphorylation sites at 46,53,83,90,95,98,139,142 and 187 amino acid sites,and one tyrosine phosphorylation site at 141amino acid sites.The transmembrane region,flexibility,hydrophilicity,antigen index and surface accessibility of GP5 antigen were analyzed by dnaman and tmhmm 2.0 online server.The results showed that there might be dominant B cell epitopes in 32-37,149-151,164-165 and 196-198 regions of GP5 antigen.2.Construct PRRSV-GP5 nanobody phage display library,immunize adult healthy male Alpaca with GP5 antigen,and detect the immune effect by ELISA.The results show that strong positive reaction can still be detected after immunized Alpaca serum dilution 10⁵,which indicates that the immune effect is good and can be used for the subsequent construction of immune Library.After 4 times of immunization,peripheral venous blood of alpaca was collected,lymphocytes were isolated,and total RNA was extracted by Trizol method.VHH fragment was amplified by nested PCR,and then transformed into TG1cells by ligating plasmid vector to construct the library.The results showed that:the phage immune Library of PRRSV-GP5 was constructed successfully,the insertion rate of the library was 95%,the volume of the library was 5.52×10¹⁰,and the abundance was 4.2×10¹⁰ cells/ml.In conclusion,this study predicted that there might be dominant B cell epitopes of PRRSV-GP5 in 32-37,149-151,164-165 and 196-198 regions,and successfully established a good quality phage immune Library of PRRSV-GP5 nanobody.