Establishment And Application Of CELISA For Detection Of African Swine Fever Based On P54 Protein Epitopes

African swine fever(ASF)is an infectious disease of domestic and wild pigs of all breeds and ages,caused by a virus that produces a range of syndromes.Acute disease is characterised by high fever,haemorrhages in the reticuloendothelial system,and a high mortality rate.After 2007 it was become to a local endemic disease in Eastern Europe and the Caucasus region.Currently widespread concern due to the increase in international trade activities as well as the increase in the number of breeding pigs,African swine fever may spread across Europe to Asia,especially to East Asia.It must be notified a catalogue of animal disease OIE regulations.It's classified in the first animal epidemics in the catalogue of our country animal pathogenic microorganisms.In this study,a c ELISA method for the detection of ASFV was established by preparation of the antigen and the monoclonal antibody.Hoped effectively apply to the African swine fever virus antibody detection timely,ASF is prevented into China in the international trade.Recombinant antigen targets for serodiagnosis of African swine fever.ASFV P54 gene was cloned into the p ET-52b(+)3C/LIC of prokaryotic expression vector.The resultant plasmid was transformed into Escherichia coli BL21(DE3)and induced expression.By making use of the Ni column purification kits,African swine fever virus P54 protein was successfully purified.The expressed protein P54 was identified by SDS-PAGE and Western-blot analysis.The expressed protein could react with antiserum against African swine fever virus,indicating the expressed protein had good reactogenicity.African swine fever virus P54 gene was successfully introduced into baculovirus which would infect Sf9 cells.P54 protein was less expressed by sds-page electrophoresis test.The recombinant protein P54 has good response to the original by western blot test,and can be used as a screening antigen in this study.To identification of the B cell epitopes on the p54 of African swine fever virus,a series of short peptides were synthesized according to the amino acid sequence of p54 based on the hydrophilic and antigenic indexes for the epitope prediction by the software analysis.And their immunological characteristics were verified by indirect ELISA and blocking ELISA.The experimental results show that the five short peptides of 23-29,36-45,72-94,114-120,137-150 have antigenicity by indirect ELISA.And the short peptides of 36-45 and 72-94 can react with two monoclonal antibodies specifically.This will make the foundation for the establishment of the clinical immunology detection method.In order to preparation for monoclonal antibody(MAb)against known specific antigen epitope of the p54 protein of African swine fever virus,the mice were immunized with the recombinant protein p54 that is expressed in prokaryotic system.Antigens used for screening the prepared MAb include the p54 protein of prokaryotic expression,the p54 protein of eukaryotic expression protein and the synthetic specific antigen epitope polypeptide of p54.A hybridoma secreting MAb against specific antigenic epitope of p54 was generated by fusion of the SP2/0 cells with the splenic cells from the recombinant protein p54 immunized BALB/c mice.The monoclonal antibody affinity was analyzed by the Protein On XPR36 interaction array system.And the monoclonal antibody belongs to the Ig G1 subspecies,which shows a good specificity and affinity.It laid a foundation for the establishment of immunological detection method.The minimal number and sequences of amino acids were identified at a certain antigen site of ASFV p54 protein.Compared with all the ASFV p54 protein sequences available in the National Center for Biotechnology Information database,the amino acid sequences of the antigenic site were very conservative.The antigenic site corresponded to the monoclonal antibody was analyzed.A conservative antigenic site of ASFV p54 protein and its amino acid sequence was found and confirmed in this study.The competitive ELISA method for the detection ASFV antibodies was established in this study,and it had advantages such as specificity,repeatability,and high compliance.The protein sequence alignment test verified that this ELISA method could theoretically detect the anti-serums produced by pigs after being affected by almost all ASFVs around the world.