Preparation Of Monoclonal Antibody Against P72 Protein Of African Swine Fever Virus

African Swine Fever(ASF)is an acute,hemorrhagic and severe infectious disease of pigs caused by African Swine Fever Virus(ASFV).The main clinical symptoms are high fever,short course of disease,high mortality,extensive bleeding of internal organs and dysfunction of respiratory system and nervous system.The incidence rate and mortality of virulent strain infection are as high as 100%,which has caused huge economic losses to the global pig industry.Because the infection and immune evasion mechanism of ASFV is extremely complex,no effective vaccine or drug has been developed for prevention and treatment.Therefore,the establishment of a rapid and specific serological diagnosis method of ASF is the key to early diagnosis,regular monitoring and control of the epidemic of ASF.At the same time,it also laid a good foundation for the development and production of vaccines.Studies have shown that ASFV p72 is an important structural protein encoded by B646 L gene,which constitutes the viral capsid,and its content is about 32% of the total viral protein.Because p72 gene is highly conserved in different strains,the protein encoded by p72 gene has good immunogenicity,which provides a good research target for establishing a rapid and effective serological diagnosis of ASFV.The accuracy of serological diagnosis is inseparable from highly specific monoclonal antibodies.Monoclonal antibodies have the advantages of strong specificity,high sensitivity and good specificity.Since the advent of this technology,they have been widely used as a means of detecting a variety of virus infections.In this study,p72 protein was expressed and purified by prokaryotic expression system,and used as antigen to immunize BALB/c mice to prepare monoclonal antibody against p72 protein.Hybridoma cell lines that can stably secrete p72 antibody were screened by the established indirect ELISA method,and monoclonal cell lines that can stably secrete p72 antibody were established after three subclones.The chromosome number of the established monoclonal cells was analyzed and their antibody subclasses were identified.At the same time,the established monoclonal cells were taken to prepare ascites,and the ascites was purified to obtain p72 monoclonal antibody.The specificity of the purified antibody was identified by indirect immunofluorescence assay(IFA),Western blot and cross reactivity test.In addition,a double antibody sandwich ELISA for p72 was established by coating the ELISA plate with purified antibody,and the sensitivity and repeatability of the method were tested.Four monoclonal hybridoma cell lines,named 5F11-2,16E2-1,16E2-2 and19B7-2,were successfully obtained.The antibodies were identified as Ig G1,Ig G2 a,Ig G2 a and Ig G2 b.The results showed that the average chromosome number of the four monoclonal cells was 108,which was about the sum of the chromosome numbers of the two parent cells.The purified antibodies were tested by IFA,Western blot and cross reactivity test.The results showed that these antibodies could react specifically with p72 protein,and there was no cross reaction with other common porcine pathogens,indicating that these antibodies had good specificity.The established double antibody sandwich ELISA for p72 was used to detect p72 protein,and the minimum detection concentration was 0.031 μg.The coefficients of variation between batches and within batches were less than 10%,indicating that the method has high sensitivity and good repeatability.In conclusion,p72 monoclonal antibody with good reactivity was successfully prepared in this study,and the detection method of p72 double antibody sandwich ELISA was preliminarily established,which provides a diagnostic technology for the prevention,control and purification of ASF.