Production And Characterisation Of Monoclonal Antibodies Against African Swine Fever Virus PH240R

African swine fever(ASF)is a hemorrhagic disease of pigs caused by African swine fever virus(ASFV)and virulent strains can cause nearly 100% mortality in domestic pigs.The disease is highly contagious and poses a huge threat not only to the pig industry but also to global trade.China,which supplies about 50% of the world’s pork,has been particularly affected by ASF.Given there is no effective vaccines and therapeutic drugs,disease control relies mainly on rapid diagnosis,culling of infected pigs,and enhancing biosecurity measures.p H240 R,a minor capsid protein of ASFV,is essential for ASFV icosahedral capsid formation and infectious particle production.At present,there are few related studies on p H240 R,and further research is needed on the specific mechanism in ASFV assembly and other biological functions.In this study,two forms of p H240 R were obtained by prokaryotic expression and purification to immunize mice,so as to produce monoclonal antibodies(m Abs)against p H240 R and further characterize.The contents and results are as follows:1.Expression and purification of monomeric and granulated p H240 R in E.coli.The recombinant plasmid p ET-29b-H240 R was constructed and expressed in E.coli.It was purified by affinity chromatography and gel filtration chromatography to obtain monomeric p H240 R with a high purity.The results of mass spectrometry showed that there was disulfide bond structure between p H240 R.To improve the immunogenicity of p H240 R and maintain its pentameric structure,the pentameric 52 B protein was selected to be fused and expressed with p H240 R,and then mixed with dimeric 52 A protein to form nanoparticle.Therefore,the H240R-52 B,52A and 52 B genes were inserted into the p ET-29 b vector to synthesize the whole gene,and then transformed into BL21(DE3)competent cells for prokaryotic expression,and purified by a nickel column.The purified H240R-52 B and 52 A proteins were mixed in an equimolar ratio and DLS analysis showed that the p H240 R assembled into complexes with an average particle size of 34.4 nm.52 B protein was used for screening of m Abs against granulated p H240 R.2.Production and screening of m Abs against p H240 R.BALB/c mice were immunized subcutaneously with equal amounts of monomeric and granulated p H240 R at an interval of twenty-one days.After the fourth immunization,indirect ELISA(i ELISA)method was used to determine the serum titers of mice,and mice No.4 and No.6 with the highest serum titer were given the final booster immunization before the cell fusion.Finally,Ten hybridoma cell lines,namely 13E11,2H7,13D2,21G3,14E16,17F12,18F10,11B9,8G7 and 12D6,were generated by i ELISA method screening and three times subcloning.3.Characterisation of m Abs against ASFV p H240 R.The affinity of m Abs for purified p H240 R protein was tested by i ELISA,and the titration of m Abs ranged from 80 000 to 1 280 000.After resuscitating ten hybridoma cell lines frozen for more than three months,and the stability of the antibody secreted by the cell lines was tested.The results showed that seven m Abs could stably secrete antibodies.Isotypes of these m Abs were characterized and were found to be Ig G2 b or Ig G1 with kappa light chain.Western blot and Dot blot showed that eight m Abs recognized the linear epitopes of the p H240 R,while the other two m Abs recognized the conformational epitopes.To characterize the reactivity of these m Abs with the eukaryotic expressed p H240 R,the p CAGGS-HA-H240 R recombinant vector was constructed and transfected into HEK293 T cells.Results from IFA demonstrated the specific binding of these m Abs to p H240 R.Results from IPMA showed that screened m Abs against p H240 R could specifically bind to ASFV HLJ/18 strain.The cross reactivity test of five other unrelated swine virus antigens showed that m Abs had good specificity.In addition,to evaluate the potential of m Abs for ASFV diagnosis,ten m Abs were evaluated by competition ELISA.m Ab 17F12 has a good performance which showed the highest PI of 77%.To characterize the linear epitopes recognized by the m Abs,H240 R gene was truncated and expressed.Results from western blot showed that m Ab 18F10 was able to recognize only the N-terminal domain of p H240R(1-155aa),while m Abs 13E11 and 13D2 identified the C-terminus part(127-240aa).In summary,monomeric and granulated ASFV p H240 R were successfully expressed and purified in this study,and the immunogenicity of granulated p H240 R was significantly higher than that of monomeric protein.A total of ten high-affinity m Abs that can specifically react with p H240 R were produced and seven of them are able to stably secrete antibodies.MAb 17F12 showed better ability to compete with ASFV positive serum for binding to p H240 R.In addition,we found that there are different epitopes at the N-terminal and Cterminal of p H240 R.These findings not only enrich the knowledge of p H240 R,but also provide new target for the development of subunit vaccines and the diagnosis of ASFV.