Development Of Monoclonal Antibody Against Classical Swine Fever Virus And Identification Of A Novel Epitope On E2 Protein Of CSFV

Classical swine fever(CSF)is a highly contagious,fatal infectious disease caused by classical swine fever virus(CSFV).In the CSFV-encoded structural protein,E2 envelope protein as the main immunogenicity protein of CSFV,can induce the body to produce neutralizing antibodies,is not only the main research object of new vaccines,but also the establishment of CSFV serological detection method of the preferred antigen.Most of the monoclonal antibodies prepared with CSFV E2 protein have the ability of virus neutralization,and have important application value in the biological characteristics,antigenicity and epitope research,immunological diagnosis and epidemiological study of CSFV.Studies have shown that there are multiple epitopes on the E2 protein,including linear epitopes and conformational epitopes.In order to further locate and analyze the E2 epitope,we used baculovirus to express the swine fever virus C strain E2 protein as the immunogen,and immunized BABL / c mice.By cell fusion,screening and subcloning,four strains were obtained Secreted anti-CSFV E2 protein monoclonal antibody,named E2-1,E2-2,E2-3,E2-4,respectively.Four monoclonal antibodies were found to belong to IgG1 subtype and the light chain was ? chain.Preparation of ascites and purification of monoclonal antibody,identification results show that the potency and are very high,and can react with the swine fever C strain,and BVDV no cross reaction,and have a virus neutralization activity.In order to further identify the antigenic epitopes identified by monoclonal antibody,the monoclonal antibody with the highest neutralizing activity and titer was selected from the four monoclonal antibodies selected for epitope identification.In this study,PEPperMAP? epitope pattern technique was used to synthesize 15 amino acid peptides according to the full length of E2(except transmembrane region)of CSFV strain E,and 14 amino acids were prepared per peptide to prepare polypeptide chip.E2-3,and found that a 9-amino acid-specific polypeptide sequence(which is a national patent)is specifically associated with monoclonal antibody E2-3 and is associated with monoclonal antibody WH303 epitope(TAVSPTTLR).In order to further determine the key amino acid sites of this epitope,the amino acid substitutions of the epitope were replaced by amino acid substitutions,and the peptide chips were prepared and analyzed with monoclonal antibody E2-3.The results showed that the affinity of monoclonal antibody E2-3 with acidic amino acid(D,E)and aromatic amino acid(W,Y)was strong,and there were 3 aspartic acid(D)in antigen epitope The Thus,the initial formulation of the three aspartic acid in the epitope may be a critical binding site.Based on the identification of the level of epitope of peptides,in order to further study the mechanism of action of three Ds,the key amino acid mutation expression test was carried out by using baculovirus expression system,and three D were respectively on the bovine virus CSFV E2 transfer vector Mutations,double mutations,three mutations,and identification of the expressed product.The results showed that the binding capacity of monoclonal antibody E2-3 and mutant expression product did not show a significant decrease,suggesting that the three D sites may not be the key sites.In order to determine the reactivity between monoclonal antibody E2-3 and CSFV strains of different genotypes,22 strains of CSFV were isolated and identified,and the main coding region of E2 gene and E2 were sequenced.The results showed that 22 strains were subordinate to 1.1,2.1 and 2.2 subtypes,and 2.1 subtypes were mainly subtypes.The response of CSFV to monoclonal antibody E2-3 was analyzed.The monoclonal antibody E2-3 reacted with three subtypes of the virus.The full length sequences of 14 strains were determined.The results showed that there were two key difference sites in the epitope(the 4th position D And the fifth position G),while the monoclonal antibody E2-3 was compared with the untreated strain.The mechanism of its need for further amino acid mutation research.In conclusion,in this study four CSFV E2-specific monoclonal antibodies were developed.The virus neutralized activity and the highest titer of monoclonal antibody E2-3,the use of the monoclonal antibody to achieve antigen epitope positioning,the successful identification of a linear epitope(patent pending);The key amino acid sites in the linear epitope were not accurately mapped.The specificity of E2-3 was analyzed and its broad spectrum was verified.For the obtained epitope,The differences between the isolates and the reference strains were compared,and two key differences were found in the epitopes.The above study laid the foundation for further understanding of the structure and function of CSFV E2 protein and the establishment of differential diagnosis of CSFV.