Preparation Of Monoclonal Antibody Against P30 Protein Of African Swine Fever Virus

African swine fever(ASF)caused by African swine fever virus(ASFV)is an acute,hemorrhagic and virulent infectious disease.Clinical symptoms include multiple bleeding cyanosis on the skin surface,high fever,blood in stool or diarrhea,which can lead to abortion of pregnant sows.ASFV can be transmitted through contact between pigs,and it spreads rapidly.The outbreak of ASF has caused huge economic losses to the global aquaculture industry.Despite much research since the outbreak,there is still no vaccine or specific drug on the market that can induce effective immunity.Until an effective vaccine is developed,rapid diagnosis and timely containment are effective means to prevent and control the disease.The preparation of monoclonal antibodies against key proteins of ASFV and the establishment of accurate,simple and efficient diagnostic techniques can provide effective help for the prevention and control of ASF.Studies have shown that p30 protein is one of the main structural proteins of ASFV,an important inner capsule protein encoded by CP204 L gene,and one of the most immunogenic proteins.It can mediate the adsorption and internalization of ASFV to macrophages,and interfere with transcription and translation of host cells.It can be detected in cytoplasm 4 h after infection with abundant content and high antigenicity,which is an early diagnostic target of ASFV.Since monoclonal antibodies have strong specificity,and the accuracy of serological diagnosis depends on this advantage,the application and development of this technique is of great importance for detection after viral infection.In order to prepare monoclonal antibody against p30 protein,in this study,the prokaryotic expression plasmid p ET30a-His-p30 was synthesized to induce the expression of p30 protein,and the soluble p30 protein was purified.After immunizing BALB/c mice,two strains of monoclonal antibody against ASFV p30 were prepared.They were named 2G10-2 and 6D2-1.An indirect ELISA method was established to analyze the number of chromosomes in the selected monoclonal hybridoma cell lines.The results showed that the average number of chromosomes in the two monoclonal cells was about the sum of the number of chromosomes in the two parent cells.The subtypes were identified as Ig G2 b and Ig G1.The titers of the antibody were detected by indirect ELISA,and the titers were 1:12 800 and 1:3 200,respectively.Immunofluorescence,western blotting and cross-reactivity tests were used to identify the specificity of the obtained antibodies.The results showed that the antibodies could bind to p30 protein specifically and did not cross-react with other common pathogenic microorganisms.In addition,by predicting the secondary and tertiary structure of ASFV p30,we identified the epitope of ASFV.It was found that the epitope region of the two monoclonal antibodies was aa.170 ~ aa.194,and the amino acid sequence was as follows: IYGTPLKEEEKEVVRLMVIKLLKKK,and in predicting the tertiary structures of the p30 monoclonal antibodies to identify antigen epitope regions.This study prepared p30 monoclonal antibody,which laid a foundation for the study of the structure and function of ASFV p30 protein and the development of serological detection methods.