Protein Expression And Establishment Of Detection Method Of African Swine Fever Virus

African swine Fever（ASF）is an acute,highly contagious disease of domestic pigs and wild boars caused by African swine Fever virus（ASFV）.The incidence of infection in domestic pigs and wild boars can reach 100%,with a mortality rate of 0-100%depending on the virulence of different strains.The first case of ASF was reported in Shenyang,Liaoning province,in August 2018,and it quickly spread across the country,causing huge losses to China’s pig industry.However,the function of ASFV protein remains largely unknown.In view of the lack of effective vaccines against ASFV,it is important to characterize the function of ASFV protein and provide new insights for the development of ASFV vaccine.At the same time,in the absence of an effective vaccine,the control of ASF relies on accurate,efficient and low-cost testing methods.Objective:The purpose of this study was to establish the foundation for the functional study of ASF virus protein by verifying its expression,to establish an ELISA method for the preparation of African swine fever by screening proteins with good immunogenicity and to provide necessary biological materials for specific diagnosis of ASFV by preparing monoclonal antibodies to related proteins.Methods:（1）In this study,87 selected Eukaryotic plasmids of African swine fever protein were transfected into 293T cells.Flag antibody was used as the primary antibody,and the expression of 87 plasmids was verified by indirect immunofluorescence（IFA）and Western-blot experiments.（2）The positive prokaryotic expression plasmids p ET-32a-P30,p ET-30a-p54 and p ET-28a-K205R were transformed into BL21 competent state,after induced expression,protein solubility was verified by SDS-PAGE analysis.The p30 and K205R proteins expressed in inclusion body form were sent to the company for purification,and the p54 expressed in supernatant was purified with the kit,and the concentration of p54 was determined with the BCA Protein Quantification kit.（3）Using Bac-to-Bac insect baculovirus expression system,CP204L in ASFV was inserted downstream of p Fast Bac1 shuttle plasmid vector,and the resulting recombinant plasmid p Fast Bac-P30 was inserted into DH10Bac bacterium as transposon.After screening the recombinant baculovirus plasmid DNA Bacmid-p30,the recombinant baculovirus was transfected into Sf9 cells and passed for 3 times.The recombinant baculovirus was named r Bac-p30.Indirect immunofluorescence（IFA）and Western-blot were used to identify its expression.（4）The 4 expressed proteins were titrated by square array.The optimal coating concentration was used to coat the plate,and the same ASFV positive serum was diluted at different concentrations as primary antibody,and indirect enzyme-linked immunosorbent assay（ELISA）was performed.According to the results,the optimum protein was selected for indirect ELISA of African swine fever virus.The ELISA method was optimized,and 35ASFV negative serum samples were used to determine the criterion of negative and positive.Finally,14 samples of pig serum were collected for clinical detection,and the results were compared with those of commercial kits.（5）After immunizing mice with p30 protein,cells were fused.The positive hybridoma cells were screened by Western-blot,IFA and ELISA,and subcloned after expansion,and the subcloned cell lines were verified.Results:（1）Western blot results showed that 33 of 87 ASFV eukaryotic plasmids could express proteins in 293T cells.IFA results showed that green fluorescence could be seen in 293T cells transfected with 34 plasmids,indicating that these 34 plasmids could express proteins in293T cells.Among them,23 proteins could be recognized by both Western-blot and IFA.（2）ASFV prokaryotic proteins p ET-32a-p30、p ET-30a-p54 and p ET-28a-K205R were successfully expressed,in which p ET-32a-p30 and p ET-28a-K205R were expressed as inclusion bodies,and p ET-30a-p54 was expressed in supernatant.p54 protein was successfully purified and its concentration was 1.39mg/m L.（3）PCR identification showed that p Fast Bac-p30 was successfully constructed and recombinant Bacmid-p30 was successfully screened.Indirect immunofluorescence（IFA）and Western-blot were used to identify the supernatants obtained after transfection and passage.p30 positive serum specificity recognized the protein expressed in Sf9 cells,demonstrating that the baculovirus expression system was used to successfully express p30protein.（4）The optimal encapsulation concentration of prokaryotic expression proteins p30,p54,K205R and p30 baculovirus expression system was 100ng/well,1000ng/well,100ng/well and 10000ng/well,respectively.This concentration was coated with ELISA plate,and the same serum was used as primary antibody for indirect ELISA detection.After comparing the results,p30 was selected for indirect ELISA detection.The optimal dilution of serum was1:3200,and OD₄₅₀>PC（0.745）was determined as ASFV positive serum.OD₄₅₀<PC（0.281）is considered as negative.If the OD₄₅₀ value of the sample is between 0.745 and 0.590,it is considered as suspicious.The results of clinical testing were consistent with those of commercial kits.（5）Eight p30 positive hybridoma polyclonal cells were screened,and two stable monoclonal cells secreting p30 specific antibodies were obtained after subcloning,which were named 2D6C5 and 2D6D6,respectively.