Preparation Of Monoclonal Antibody Against African Swine Fever Virus P72 Protein

African swine fever(ASF)is a severe infectious disease of swine caused by African swine fever virus(ASFV),The main symptoms are high fever,dyspnea and severe bleeding of tissues and organs.The mortality rate of swine infected with the virulent strain can be as high as 100%.Since the disease entered China in 2018,it has caused a serious blow to pig industry.At present,Chinese scholars provide important theoretical basis for vaccine research and development by analyzing the fine three-dimensional structure of ASFV.It is of great significance to find an efficient and accurate detection method for the disease control.The accuracy of detection results depends on highly specific antigens or antibodies,since the advent of monoclonal antibody technology,it has been widely used as a means of disease detection.This kind of antibody only targets at one epitope,which has high sensitivity and specificity,and can accurately detect diseases.In this study,p72 protein was used as the research object,with the purpose of obtaining stable and specific monoclonal antibodies,related studies were carried out.The main contents are as follows:1.Prokaryotic expression and purification of ASFV p72 protein.In this study,p72 gene sequence was synthesized according to the published sequence of Georgia 2007/1 strain in NCBI,and the prokaryotic expression recombinant plasmid pet-30a-p72 was successfully constructed.The p72 protein expressed in the form of inclusion body was successfully obtained by using the prokaryotic expression system.After denaturation,renaturation and dialysis,the purified p72 protein was detected by SDS-PAGE.The concentration of p72 was 1.13mg/ml The subsequent preparation of monoclonal antibody laid a foundation.2.Eukaryotic expression and analysis of ASFV p72 protein.In order to obtain eukaryotic expressed p72 protein for the validation of monoclonal antibodies.the eukaryotic expression recombinant plasmid pCAGGS-HA-p72 was constructed in this study and expressed by transfecting 293 T cells.After 24 h,samples were detected by Western Blot.The results showed that the eukaryotic expressed p72 protein had good specificity and could be used for the validation of monoclonal antibodies in the later stage.3.Preparation of monoclonal antibody against ASFV p72 proteinThe purified p72 prokaryotic protein was used to immunize Balb/c mice to prepare monoclonal antibodies.The positive hybridoma cells were screened by indirect ELISA method,after three times of subcloning,combined with IFA and Western Blot verification,a hybridoma cell line with stable secretion of monoclonal antibody against P72 protein was obtained,and it was named 3E5.The type was identified as the IgM subtype,and the specificity test showed that there was no cross-reaction with other common diseases and has strong specificity.