Preparation Of Monoclonal Antibody Against African Swine Fever Virus H240R Protein

African swine fever(ASF)is an animal infectious disease caused by African swine fever virus(ASFV),which causes high fever,severe bleeding and extremely high fatality rate.The first ASF case broke out in China on August 3,2018 in Liaoning and rapidly spread across the entire country,causing serious economic losses to our country swine industry.It’s been reported that H240R is one of the main structural proteins of ASF V and is involved in virus assembly,but no specific functional studies have been repored.The study of H240R protein will contributed to understand ASFV.Preparation of specific monoclonal antibody against H240R protein provides an important material for the establishment of immunological detection methods for ASFV,and also provides a good tool for the function study of H240R protein.Monoclonal antibody targeting only one epitope and is widely used in detecting of diseases due to the high sensitivity and specificity.In the present study,we prepared five monoclonal antibodies against H240R of ASFV with high stability and specificity.The main results of our study were shown as follows:1.Prokaryotic expression and purification of H240R protein of ASFVThe H240R gene was synthesised according to the genetic sequence published on NCBI website and cloned in pET28a vector to construct prokaryotic expression plasmid pET28aH240R.Then the plasmid pET28a-H240R was transformed into the expression competent cells.After being induced with IPTG,H240R protein was expressed in the form of inclusion body.H240R protein was purified and analysed with SDS-PAGE and Western Blot methods.The results showed that H240R protein was stably expressed and the purification effect was good.The concentration of purified H240R protein was 1.4 mg/mL,which could be used as antigen to immunize Balb/c mice.2.Eukaryotic expression and analysis of H240R protein of ASFVH240R gene was cloned in pcDNA3.1 vector to construct eukaryotic expression plasmid pcDNA3.1-H240R.Then the plasmid pcDNA3.1-H240R was transfected into the HEK293T cells and analysed with Western Blot and IFA methods.Results showed that H240R protein was expressed in HEK293T cells,which could be used for the detection of H240R specific monoclonal antibody in the following experiments.3.Preparation of H240R protein specific monoclonal antibodyBalb/c mice were immunized with purified H240R protein to prepare H240R protein specific monoclonal antibody.After the fusion of splenocytes and SP2/0 cells,the supernatants of hybridoma were screened by ELISA,IFA and Western Blot.Then the positive hybridoma were subcloned twice.Results showed that five monoclonal antibodies against H240R of ASFV were prepared with high stability and specificity.These antibodies subtypes were identified with commercial kit.Results showed that one monoclonal antibody subtype was IgG1,and others were IgG2b.The specificity test of these five monoclonal antibodies showed that there was no cross reaction with other common swine diseases.