Screening And Identification Of Monoclonal Antibodies Against CD2v Protein Of African Swine Fever Virus And Development Of Blocking-ELISA Detection

African swine fever virus(ASFV),as a newly emerging and highly pathogenic infectious disease,can cause fever and bleeding in domestic pigs.The manifestation is high infection rate and high mortality rate,and the fatality rate can reach 100%.Since the outbreak in 2019,it has dealt a devastating blow to China’s domestic pig breeding industry.CD2 v protein is located in the outer envelope of African swine fever virus.It has good antigenicity,which can enable the host to produce partial protection against the virus and mediate the adhesion between the virus and cells.Therefore,CD2 v protein is an important target for the development of diagnostic reagents and antigens.1.Eukaryotic expression and purification of CD2 v protein of African swine Fever virusFirstly,the constructed p CGS3-CD2 v plasmid was electrically transferred to CHO cells,and the CHO cell lines with stable expression of CD2 v protein were screened by subclonal method.The supernatant of cell line expression was collected and detected by SDS-PAGE,Dot-Blot and Western-Blot.High purity CD2 v protein was purified by anion exchange chromatography and identified by SDS-PAGE and Western-Blot.High purity(90%)and high concentration(1.27 mg/m L)CD2v protein were obtained.2.Preparation and screening of monoclonal antibodies against CD2 v protein of African swine Fever virusBALB/c mice were immunized with CD2 v protein of African swine fever virus,hybridoma cells were fusion,limited dilution,ELISA,IFA,WB and IPMA were used to detect 4positive monoclonal cells,namely 2H10,4C1,7A9 and 11E2.By ELISA,the titers of 2strains were more than 1:51 000,and 1 strain was 1.1 024 000,1 plant was 2 048 000.The high efficient 11E2 monoclonal antibody was coupled to the AGAR bead to prepare the affinity chromatography column with specific binding to CD2 v protein based on monoclonal antibody,which has a good coupling efficiency and establishes a fast and convenient purification method for the purification of CD2 v protein.3.Identification of monoclonal antibody and epitope of CD2 v protein of African swine Fever virusThe four strains of monoclonal antibody were identified by IFA,WB,IPMA and ELISA.ELISA and IFA resuults showed that the monoclonal antibody reacted well with recombinant CD2 v protein.The results of IPMA showed that the four monoclonal antibodies could react well with the virus.WB showed that all four strains recognized linear epitopes.The specific targeting sites and epitopes of the four monoclonal antibodies were identified by the segmented expression of the truncated CD2 v protein gene.The four key amino acids154SILE157 were identified by alanine scanning,and the reactivity of the epitopes was verified by Dot-Blot and ELISA.4.On the basis of the monoclonal antibody blocking antibody ELISA detection method of establishingIt was found that CD2 v protein was relatively simple in the Chinese epidemic strain of African swine fever virus through the establishment of molecular tree,and the epitope sequence was more conservative in the Chinese epidemic strain of African swine fever virus by comparing the identified epitope sequence with the classical strain of African swine fever virus.Again,through the simulation of protein structure,it was determined that the epitopes were located on the surface of CD2 v protein structure,which has the potential to establish antibody detection method.The checkerboard method was used to determine the optimal blocking efficiency with positive serum of African swine fever as high as 82%.The ROC curve was established by the test results of more than 100 groups of positive and negative sera,and the AUC value reached 0.952,which proved the reliability of the system.By testing clinical samples,it was found that the coincidence rate was 91.47%.