Establishment Of The IELISA Based On H240R Protein And The Inhibition Of Inflammatory Factors By H240R Protein

African swine fever(ASF)is a highly contagious,hemorrhagic disease caused by African swine fever virus(ASFV)with a mortality rate close to 100%,which has caused huge economic losses to the pig industry worldwide.There is no safe and effective vaccine,and the spread of the disease is mainly controlled by early diagnosis and culling,so it is crucial to establish a rapid and accurate detection method for ASF prevention and control.In addition,ASFV has a complex immune evasion mechanism and antagonizes the host’s natural immunity through a variety of self-encoded proteins,and studying the function of viral proteins is important for further understanding the pathogenesis of the virus and vaccine development.The ASFV H240 R protein is the ASFV capsid five-neighborhood apex protein,which plays a skeletal role in the capsid structure.It has been found that ASFV deletion of H240 R protein substantially reduces the infectivity and replication ability of its deletion strain and induces higher levels of inflammatory cytokine expression,but whether H240 R suppresses the inflammatory response induced by viral infection is unclear.In this study,we conducted correlation studies on ASFV H240 R protein,and the main results are as follows.1.Prokaryotic expression of ASFV H240 R protein and preparation of polyclonal antibodiesThe p ET-28a-H240 R prokaryotic expression vector was constructed,and the H240 R recombinant protein was expressed and purified,and the protein was analyzed by Western blotting for its strong reactivity.Mice were immunized with purified p H240 R protein to prepare mouse-derived anti-H240 R polyclonal antibody,and the potency of the prepared polyclonal antibody reached 1:256000 by indirect ELISA.Western blotting showed that the antibody could effectively recognize the eukaryotic expression of H240 R protein.Mice were immunized with purified p H240 R protein to prepare2.Establishment of the indirect ELISA method for ASFV H240 R protein detectionThe purified H240 R protein was used as the encapsulated antigen and the reaction conditions were optimized.The final determination of the antigen encapsulation concentration was 1 μg/m L and the optimal dilution of the serum to be tested was 1:400.The sensitivity and specificity of the assay were evaluated and compared with the ID-vet African swine fever test kit.The results showed that the method could detect ASFV commercial positive sera at a minimum dilution of 1:3200;and there was no cross-reactivity with five common swine virus positive sera;and the compliance rate with the commercial kit was 98%.3.Inhibition of inflammatory factors by ASFV H240 R proteinThe p CDNA3.1-H240 R eukaryotic expression vector was constructed,transiently transfected into HEK293 T cells,and then poly I:C was used to induce inflammatory factor activation.Fluorescence quantitative PCR results showed that H240 R protein significantly inhibited inflammatory factor activation induced by poly I:C in a dose-dependent manner.The H240 R gene was also truncated for expression and screened for structural domains that inhibit inflammatory factors,and the results showed that the key structural domains were located in 82-121 aa.In summary,this study laid the foundation for further development of an indirect ELISA antibody detection kit for ASFV;and for the first time identified the function of H240 R protein in suppressing host natural immunity,expanding the knowledge of ASFV immune escape and laying the foundation for the development and production of an effective vaccine.