Preparation And Epitope Identification Of Monoclonal Antibody Against African Swine Fever Virus PA137R Expressed In Escherichia Coli

African Swine Fever(ASF)is an acute,thermal and highly contagious disease caused by the infection of domestic pigs or wild boars by the African Swine Fever virus(ASF virus).The disease was first reported in the 1990s.The disease was first detected in Shenyang,Liaoning province in China,in August 2018,and quickly spread throughout the country,causing huge economic losses for the pig farming industry.ASFV,the only member of the African Swine Fever Viridae family,is a encapsulated double stranded DNA virus.The ASFV genome encodes 150 to 200 proteins,of which more than 50 are structural proteins of the virus.These proteins are not only involved in viral assembly,DNA replication and gene expression,but also encode many proteins involved in evasive host defences.The pA137R protein is expressed late in viral replication and is localized in the viral replication factory,but it's not clear how it functions.In this study,genomic DNA from ASFV HLJ/18 isolate was used as template to amplify ASFV A137R gene,and then cloned into prokaryotic expression vector p ET-21a.The recombinant expression plasmid was successfully constructed and named p ET-21a-A137R.Then the recombinant protein was expressed and purified.Rabbit polyclonal antibodies were prepared by immunizing rabbits with the recombinant protein as immunogens.Two mouse monoclonal antibodies(named 3D1 and 2C3,respectively)were obtained by immunizing mice.Western blot and indirect immunofluorescence(IFA)results showed that the prepared antibody could specifically recognize the transient expression of FLAG-pA137R in HEK293T cells and the pA137R protein in ASFV-infected porcine alveolar macrophages(PAMS).The results of laser confocal assay showed that the pA137R protein was mainly localized in the cytoplasm of infected PAMS.The results of co-immunoprecipitation assay(Co-IP)showed that the prepared antibody could be used in the Co-IP assay.The epitopes of 3D1and 2C3 were identified by Western blot and enzyme-linked immunosorbent assay(ELISA),and the amino acid sequence of the epitope recognized by monoclonal antibody 3D1 was ¹⁸NFHRCAWEE²⁶.The amino acid sequence of the epitope recognized by the monoclonal antibody 2C3 was ⁶⁴AWHEVPECREFI⁷⁵.In this study,polyclonal and monoclonal antibodies against ASFV pA137R protein were successfully prepared,and the antigenic epitopes of pA137R monoclonal antibodies were identified,which laid a theoretical foundation for exploring the function of ASFV pA137R protein,developing new diagnostic reagents for ASFV and further studying ASFV.