Expression And Purification Of CD2v,p30 And PK205R Proteins Of African Swine Fever Virus And Preparation Of Monoclonal Antibodies Against P30 And PK205R Proteins

African swine fever(ASF)is a highly contagious swine infectious disease caused by African swine fever virus(ASFV),and swine animals are susceptible to it.After infection,according to the different clinical symptoms presented by diseased animals,it can be divided into four types: hyperacute,acute,subacute and chronic.Among them,the acute type is one of the types with the highest incidence at present.Loss of appetite,missed fever,and dyspnea are the main symptoms of sick pigs.Sows in gestation may have a miscarriage after infection.Since the first report,African Swine Fever has spread worldwide and has existed for about a century,causing significant losses to the global pig industry.At present,there is no effective treatment method,and vaccines that play a preventive role have not yet been on the market.Therefore,the main way to prevent the spread of ASFV is to identify diseased animals by means of rapid detection and early laboratory diagnosis,and then kill them all.In summary,it is imminent to carry out research on ASFV rapid detection methods and related vaccines.In this study,according to the ASFV(Pig/HLJ/18 strain)gene coding sequence registered in Gen Bank,the ASFV EP402 R,CP204L and K205 R gene codons were optimized and cloned into the p CAG-C vector to construct recombinant expression plasmids p CAG-CD2 v,p CAG-p30,p CAG-p K205 R.The constructed recombinant plasmid was transfected into BHK-21 cells for expression.The antibiotic G418 was used for continuous culture and selection,and at the same time,subcloning was performed.The cell lines expressing CD2 v,p30 and p K205 R proteins were screened by IFA and western blot and named as BASFV-CD2 v,BASFV-p30 and BASFV-p K205 R.The expressed CD2 v,p30 and p K205 R proteins were purified by Ni-NTA affinity chromatography column respectively.Through SDS-PAGE and western blot identification,purified CD2 v,p30 and p K205 R proteins were successfully obtained.The purified p30 protein and Freund's adjuvant were fully emulsified to immunize BALB/c mice.After the immunization,the mouse with the highest serum antibody titer was used for cell fusion,and a p30-specific monoclonal antibody 13G2 was screened by ELISA.The antibody titer of the supernatant of 13G2 cells was determined to be 1:1024 by ELISA.The reactivity of m Ab 13G2 was identified by IFA and western blot,and the results showed that 13G2 had good reactivity to p30 protein.After subtype identification,the heavy chain of m Ab 13G2 is Ig G1,and the light chain is ?.Kunming mice were selected to prepare ascites,and the ascites antibody titer was 1:409600 detected by ELISA.The specificity of m Ab 13G2 was identified by western blot,and the results showed that 13G2 was specific to ASFV and did not react with other common swine pathogens such as CSFV,JEV,PCV and PRRSV.The purified p K205 R protein and Freund's adjuvant were fully emulsified to immunize BALB/c mice.After the immunization,the mice with the highest serum antibody titer were used for cell fusion.Four monoclonal antibodies were screened by ELISA and named separately as 4A5,5D1,7A4 and 7H2.The antibody titer of the cell supernatants of 4A5,5D1,7A4 and 7H2 was detected by ELISA,and the results showed that the antibody titer of 4A5,5D1 and 7A4 were all 1:1024,and the antibody titer of7H2 was 1:256.The reactivity of m Ab 4A5,5D1,7A4 and 7H2 was identified by IFA and western blot.And the results showed that 4A5,5D1,7A4 and 7H2 all had good reactivity to p K205 R protein.After subtype identification,the heavy chains of m Ab 4A5,5D1,7A4 and 7H2 are all Ig G1,and the light chains are all ?.The specificity of m Ab 4A5,5D1,7A4 and 7H2 was identified by western blot.And the results showed that 4A5,5D1,7A4 and 7H2 were specific to ASFV,and did not respond to other common swine pathogens such as CSFV,JEV,PCV and PRRSV.In summary,this study successfully expressed and purified p30,CD2 v,and p K205 R proteins through the mammalian cell expression system,which provided a basis for the preparation of monoclonal antibodies and the preparation of further recombinant subunit vaccines.This study successfully prepared monoclonal antibodies against p30 protein and p K205 R protein,which provided a powerful tool for further research on the function of ASFV p30 and p K205 R protein.This research provides a basis for in-depth research on the prevention and control of ASFV.