Production Of Monoclonal Antibody Against ASFV B602L Protein And Establishment Of Indirect ELISA Method

African Swine Fever（ASF）is an acute hemorrhagic disease of domestic pigs caused by ASF Virus（ASFV）.The disease is characterized by rapid onset,short duration and high mortality.The World Organization for Animal Health（OIE）listed it as a notifiable disease.There is no an effective vaccine by now.The capsid of ASFV has an icosahedral structure,which is assembled by the major capsid protein p72 and other structural proteins.The pB602L of ASFV is the molecular chaperone of p72,which plays a key role in the correct assembly of p72.This protein has good immunogenicity and is believed a potential target for the development of diagnostic tools for ASFV.Given the significance of pB602L in ASFV assembly and clinical diagnosis,the aim of this study was to explore the basis of its antigenicity and develop an indirect ELISA assays based on recombinant pB602L protein.Firstly,ASFV（Pig/HLJ/2018）B602L gene was cloned into vectors p GEX-6P-1,generating plasmids p GEX-6P-1-B602L.The p GEX-6P-1-B602L was transformed into BL21（DE3）E.coil to express a recombinant pB602L protein.The expressed target protein was purified by affinity chromatography and anion-exchange chromatography.The purity of harvested protein was evaluated by SDS-PAGE and Western-blot,which showed that the protein was in high purity and had similar reactivity with the natural pB602L.The purified pB602L protein was used as antigen,eight monoclonal antibodies（m Abs）were obtained and designated 2A1,2F1,2D10,2H10,2B2,2D8,2A3,and 2E12.After antibody subtype identification,the heavy chains of 2A1,2F1,2D10,and 2E12 were identified to belong Ig G1;which of 2H10,2B2,2D8,and 2A3 were Ig G2a;while the light chains of all m Abs were Kappa chains.The epitopes of the m Abs were identified by Western-blot against overlapped polypeptides of pB602L.The shortest peptide recognized by each m Ab was defined as its epitope.Three epitopes were identified and their sequence were ³⁶⁶ANRERYNY³⁷³（recognized by 2A1,2F1,and 2D10）,⁴¹⁵GPDAPGLSi⁴²³（recognized by 2H10,2B2,2D8,and 2A3）,and ⁴⁹⁸EMLNVPDD⁵⁰⁵（recognized by 2E12）,respectively.Western-blot and IFA assays showed that all the 8 m Abs could react specifically with the natural pB602L.Immunohistochemistry and immunoelectron microscopy assays showed that the m Abs detected specifically the antigens in ASFV infected cells and tissues.These results suggest that these m Ab have a broad application promise in ASFV studies.To develop a recombinant pB602L-based i ELISA assay,the established i ELISA was employed to detect positive sera of 15 clinically common pathogens including Classical swine fever virus（CSFV）,Pseudorabies virus（PRV）,Porcine parvovirus（PPV）,etc.All sera were detected to be negative for ASFV,indicating that this assay has no cross reaction with antibodies against these pathogens.The results of intra-batch and inter-batch repeated tests showed that the coefficient of variation was smaller than 10%,suggesting that the reproducibility of this assay is reliable.In the detection of ASFV clinical samples,this assay showed a 95%concordant rate with a commercial p72-based blocking ELISA kit.This assay was further employed to evaluate the immune responses in pigs to the live attenuated ASFV vaccine strain.Results showed that a rise of sera antibodies against pB602L occurred in 30%（3/10）immunized pigs at10 days post immunization（dpi）,and all pigs were detected positive at 15 dpi.The sera antibody levels in immunized pigs increased steadily during the whole observation period of 45 days.The above results suggesting that this assay has the potential of being applied in both clinical diagnosis of ASF and the emulation of humoral responses in pigs immunized with live attenuated vaccine strains.In conclusion,the monoclonal antibody against ASFV Pig/HLJ/2018 strain pB602L protein was successfully prepared in this study,which provides biological materials for the detection of antigens in ASFV infected cells and subcellular localization of pB602L.The i ELISA method based on the purified antigen expressed by the prokaryotic system has good reproducibility,and has no cross-reaction to other common porcine pathogens,having the potential to be used for the clinical diagnosis of ASFV infection,as well as to evaluating the humoral immune response to live attenuated vaccine.