Expression Of African Swine Fever Virus PS273R Recombinant Protease And Preliminary Evaluation Of The Effect Of Small Molecule Inhibitor E-64 On Its Enzyme Activity

African swine fever is a major animal disease caused by the infection of the African swine fever virus that harms the pig industry.The disease shows different clinical symptoms in the acute phase,with a case fatality rate of 100%.There is still no effective vaccine and treatment plan.ASFV pS273 R protein is a special SUMO-1-like cysteine protease with a high degree of conservation.It can decompose ASFV p P220 and p P62 proteins into multiple mature capsid proteins,which are involved in the assembly of viral particle nucleocapsid.Play an important role in the ASFV replication process.Therefore,screening drugs that can inhibit the protease activity of ASFV pS273 R,inhibit the assembly of ASFV nucleocapsid,and form non-infectious virus particles,which is of great significance for the comprehensive prevention and treatment of African swine fever.In this study,the prokaryotic and eukaryotic expression vectors of the ASFV pS273 R protein were first constructed,and the crystal structure of the ASFV Georgia 2007/1 strain pS273 R protein was predicted and analyzed by Modeller 9.20 software,and then combined with the data of the Inter Pro website to screen out small molecular targets for the pS273 R protein inhibitor.Through molecular docking,covalent docking and in vitro verification experiments with small molecule inhibitors and pS273 R protein,the characteristics of targeted inhibition of pS273 R protease active center by small molecule inhibitors were comprehensively evaluated.The identification results of SDS-PAGE and Western Blot experiments showed that the ASFV pS273 R recombinant protein was successfully expressed.The selected small molecule inhibitor E-64 carbon atom No.14 can form a stable C-S covalent bond with the pS273 R protein residue CYS 232,and also form 7 hydrogen bonds as an auxiliary binding.The recombinant vector Flag-pS273 R and the recombinant vector HA-p P62 were co-transfected into 293 T cells.Under the action of different concentrations of E-64 inhibitors,Western Blot technology was used to detect the inhibition of the small molecule inhibitor E-64 on the protease activity of ASFV pS273 R The results show that 4 m M E-64 inhibitor can inhibit pS273 R protease activity,and the cell morphology after adding 4 m M E-64 inhibitor is regular without excessive dead cells,and the cell viability is 101.86%,indicating that 4 m M E-64 The inhibitor does not cause cytotoxicity to 293 T cells.Fluorescence quantitative PCR detection results showed that ASFV pS273 R protein can down-regulate the m RNA transcription levels of cellular immune factors IFN-? and IL-12 after transfection of 293 T cells,which is significantly different from the transfection blank vector(P<0.01);add 4 m M E-64 inhibitor can up-regulate the m RNA transcription levels of cellular immune factors IL-12,IFN-? and IFN-?,and IL-12 is significantly different from transfected ASFV pS273 R protein(P<0.01),IFN-? and The difference of IFN-? was significant(P<0.05).In summary,the small molecule inhibitor E-64 can inhibit the activity of ASFV pS273 R at the cellular level without affecting the biological activity of the cells.This study provides new ideas for the development of African swine fever drugs,and provides a theoretical basis for a comprehensive understanding of the structure and function of the ASFV pS273 R protein and the screening of drug targets.