Generation Of Monoclonal Antibody Against ASFV P72 Protein And Preliminary Establishment Of DAS-ELISA For Detection

African Swine Fever(ASF) is a severe and highly contagious disease of pigs caused by African Swine Fever Virus(ASFV),which poses huge threaten to pig industry.African Swine Fever(ASF)first spread into China in August 2018,thereafter national-wide spread,causing enormous economic losses to the pig industry.Efficient,convenient and sensitive pathogen detection methods are of great significance for the diagnosis,prevetion and control of ASF.At present,quantitative PCR(q PCR)methods for ASFV have been widely used,but q PCR methods have some shortcomings,such as high false positive rate,excessive dependence on professional laboratory and professional operators,which cannot be detected on the farm site.The double-antibody sandwich ELISA method for antigen detection is simple and quick to operate,does not need expensive equipment,and can be used for rapid detection of pathogens in clinical field,which contributes to clinical diagnosis and prevention of ASF substantially.At present,there are still few researches in this area in China,and no commercial kits available,leaving a huge gap to fill.African swine cholera virus(ASF) is a large double-stranded DNA virus with capsule,encoding 151-200 viral proteins,among which P72 protein is the main component of the outer capsid of the virus.It is the dominant protein in virion and highly conservative,which is suitable for the construction of double-antibody sandwich ELISA method.In this study,BALB/c mice were immunized with purified P72 protein expressed by Escherichia coli prokaryotic expression system,and 5 monoclonal antibodies against ASFV P72 protein were generated.Combined with rabbit polyclonal antibody against ASFV P72 protein prepared by our laboratory,a sandwich ELISA method was preliminarily established for ASFV dual antibody detection,and the sensitivity of the detection method was tested.The specific research contents are as follows:1.Expression of African swine Fever virus P72 protein and generation of monoclonal antibodyAccording to the sequence of Auhui XCGQ strain of ASFV published in Gen Bank,the synthesized P72 gene was constructed into prokaryotic expression vector p ET-28 a and eukaryotic expression vector p CAGGS-Myc,and named p ET-28a-P72 and p CAGGS-Myc-P72,respectively.The recombinant protein P72 was induced and purified in Escherichia coli BL21.BALB/c mice aged 6-8 weeks were immunized with prokaryotic recombinant His-P72 protein as antigen.Five monoclonal antibodies against P72 protein were obtained by three subclonal screening,which were named 2C4B8,2C4C8,5C11D12,5C11F11 and 7D10C9.Western blot and IFA experiments confirmed that the prepared monoclonal antibody had good reactivity and specificity with the eukaryotic expression of P72 protein,which laid a foundation for the establishment of P72 double-antibody sandwich ELISA detection method.2.Preliminary establishment of double-antibody sandwich ELISA method for detection of African swine fever virusUsing purified mouse monoclonal antibodies as detection antibody,rabbit anti-ASFV P72 polyclonal antibody stored in our laboratory as capture antibody,and goat anti-mouse Ig G(Ig G-HRP)as labeled antibody,a sandwich double-antibody ELISA method for the detection of ASFV was preliminarily established.When onoclonal antibodies 2C4C8 and5C11D12 were used to detect the recombinant P72 protein,the lower limit of detection was0.25 μg/m L.When using 7D10C9 as the detection antibody,the lower limit of detection for recombinant P72 protein was 0.05 μg/m L.The ELISA method established by the three monoclonal antibodies showed a good linear relationship with different concentrations of P72 antigen.The establishment of P72 double-antibody sandwich ELISA method provides a literature and reagent basis for the development of ASFV antigen detection assay.Conclusion: The prokaryotic expression plasmid and eukaryotic expression plasmid of ASFV P72 gene were successfully constructed,and the prokaryotic recombinant protein of P72 gene was expressed and purified.Five monoclonal antibodies against P72 protein were successfully prepared,and all the five monoclonal antibodies showed good specificity and reactivity.A double-antibody sandwich ELISA method was established for the detection of ASFV,and the validation showed that the double-antibody sandwich ELISA method had good sensitivity.The double-antibody sandwich ELISA method established in this study provides important material for the development of ASFV antigen ELISA kit and lays the experimental foundation.