Screening And Identification Of The B Cell Epitopes In P54 Protein Of African Swine Fever Virus

African Swine Fever(ASF)is an acute,febrile and highly fatal viral disease of pigs caused by the African Swine Fever Virus(ASFV),which causes great economic losses to the breeding industry.The World Organization for Animal Health(OIE)listed African swine fever(ASF)as an animal disease in the statutory report,and it was also listed as a class I animal disease in the animal pathogenic microorganisms in China.Due to the lack of a safe and effective vaccine for ASFV,it is extremely important to establish a rapid and accurate diagnostic method for ASFV prevention and control.P54protein is an important structural protein of ASFV,which can induce obvious immune response in the body,and is an ideal target for serological detection of ASFV.Therefore,the preparation of monoclonal antibody against ASFV p54 protein and the identification of the B cell epitope of p54 protein are of great significance for the detection and monitoring of ASF.In this study,the p54 gene of ASFV-China 2018 Anhuix CGQ strain was inserted into lentiviral vectors pLVX-IRES-Zs Green1 and pTrip-IRES-puro to construct lentiviral expression vectors pLVX-p54-IRES-Zs Green1(pLVX-p54)and pTrip-p54-IRES-Puro(pTrip-p54).pLVX-p54 vector was co-transfected with lentivirus packaging plasmids p SPAX2 and pMD2.G into HEK 293T cells to package lentivirus,then the lentivirus was transfected into H22 cells.After the transfected H22 cells were screened by flow cytometry for high expression clones,and a stable H22 cell line expressing p54 protein was obtained,named p54-H22.The pTrip-p54 vector was used to package lentivirus in the same way and transduce HEK 293T cells.After transfected HEK 293T cells were screened with purinomycin and subcloned,a stable HEK 293T cell line expressing p54 protein was obtained,which was named p54-293T.Balb/c mices were immunized with p54-H22 cells as immunogen,and p54-293T cells as detection.Positive hybridioma cells were detected and screened by immunoperoxidase monollayer assay(IPMA).The IPMA titer of mice serum reached1:25600 after one week of the third immunization.Ten hybridioma cell lines were prepared by hybridioma technology and IPMA screening.Western blot assay was used to verify the reactivity of these monoclonal antibodies to denatured p54 protein,and 8monoclonal antibodies specifically reacted with denatured p54 protein were identified.They were named 4C2,4C6,4E3,5E6,5G11,10C2,16B11,and 21A2.Bioinformatics tools were used to predict the B cell epitope region of p54 protein.According to the predicted results,10 overlapping peptides(P1 to P10)spanning the full length of p54 protein were synthesized.The ELISA results of peptides with ASFV-positive pig serum and immunized mouse serum showed that peptides P3(54-75 aa),P4(70-90 aa),P6(93-115 aa)and P10(162-184 aa)were the immunodominant regions of p54 protein.The ELISA and Dot-blot results of peptides with anti-p54 m Abs showed that polypeptide P3(54-75 aa)was recognized by monoclonal antibodies 4C2,4C6,4E3,5E6,5G11 and 10C2,and polypeptide P4(70-90 aa)was recognized by monoclonal antibody 21A2.Peptide P6(93-115 aa)is recognized by monoclonal antibody 16B11.By further truncated P3,P4 and P6 peptides,the epitopes were mapped to ⁶⁴EEDIQFI⁷⁰?⁷⁵DQQWVEVTPQP⁸⁵ and ¹⁰³TGRPATNRPATNK¹¹⁵ by reacting with monoclonal antibodies.Among them,⁷⁵DQQWVEVTPQP⁸⁵ was the first reported B cell epitope.In this study,mice were immunized with H22 cells expressing ASFV p54 protein,and 8 monoclonal antibodies against p54 protein were prepared and the linear B cell epitope of p54 protein was identified.The monoclonal antibody prepared and epitopes identified in this study provide a basis for the establishment of ASFV detection methods.