Preparation Of Monoclonal Antibody Against P72 Protein Of African Swine Fever Virus And Immune Characteristic Analysis

African swine fever（ASF）is an acute,febrile and highly fatal infectious disease caused by African swine fever virus（ASFV）infective domestic pigs and wild boars,with a fatality rate of up to 100%.ASFV has a complex structure that include a large genome,a large number of viral proteins leaded to immune evasion.It is a key factor to develop vaccines unsuccessfully.ASFV virions was composed of an outer membrane（CD2v）,a nucleocapsid（p72,B602L,M1249L,etc.）,an inner membrane（p54,E248L,etc.）,a nucleocapsid（PP220,PP62 and their hydrolyzed products）,and a nucleocapsid.p72 encoded by B646L gene,which presented on capsid with 8280 copies.It is one of the important antigenic proteins of ASFV virions and target for the detection of ASFV serology and molecular biology.In this study,monoclonal antibody against ASFV p72 was prepared to provide a research tool for ASFV studies.Specific research contents are as follows:1.Expression and purification of African Swine Fever Virus p72 protein:In this study,the ASFV B646L gene was amplified by polymerase chain reaction（PCR）and subcloned into the pET-32a（+）vector to construct the prokaryotic expression vector pET-32a-p72.The vector was transformed into BL21（DE3）cells,which were induced by 1 mM IPTG.Supernatant and precipitation were obtained by highly speed centrifugation and analyzed by SDS-PAGE electrophoresis.The results showed that p72 protein mainly existed in precipitation by the form of inclusion bodies.Then,p72 was purified in prokaryotic expression protein.In addition,the Bac-to-Bac baculovirus expression system was used to construct the p72 eukaryotic expression vector.After transposition,blue-white spot was screened and identified.The bacmid plasmid DNA was extracted and transfected into Sf9 cells.After three passes,them were verified by IFA and Western Blot.The results showed that the p72 recombinant baculovirus expression system was constructed,successfully.2.Preparation and epitope identification of African swine Fever Virus p72 protein monoclonal antibody:The purified p72 protein was quantified and each mouse was immunized by intraperitoneal injection with 100μg.On the 5th day after three immunizations,orbital blood was collected to prepare poly-anti-serum.The serum titer was more than 1:100,000 by ELISA detection.The recognition specificity of p72 expressed by baculovirus was verified by IFA.Spleen cells of mice were selected for fusion with the highest antibody titer and hybridoma cells were screened.Then,three strains monoclonal antibodies were obtained for against ASFV p72.That named as 1A3,2B5 and 4A5,respectively.The results showed that three strains monoclonal antibody was lgG1 by detecting the heavy chain.The light chain was Kappa for three.The 4A5 clones were selected to prepare ascites for the best specificity.The antibody titer up to 1.6×10⁶ by ELISA detection.The monoclonal antibody showed a good specific response to ascites by IFA and Western Blot verification using ASFV-infected BMDM cells as antigen.Next,4A5 monoclonal antibody recognition region was further clarified in this study.Then,p72 gene were respectively connected to the pET-32a vector for expression.The antigen recognition epitopes of monoclonal antibody were identified by Western Blot using protein expressed.The result showed that 4A5 recognized antigen epitope located at pp.245～285 amino acid residues for linear table(²⁴⁵IHDLHKPHQSKP-ILTDENDTQRTCSHTNPKFLSQHFPENSH²⁸⁵),and that located at p72 trimer surface and conformation with the top of the table.The sequence is conserved relatively during ASFV strains.In this study,a monoclonal antibody 4A5 recognized ASFV p72 protein specifically was prepared successfully using p72 protein expressed in prokaryota as immune source and expressed in baculovirus as screening source.Meanwhile,the antigen recognition epitope of the monoclonal antibody was determined successfully.This laid a foundation for the prevention or control of ASF,and serological diagnosis and vaccine development.