Preliminary Study On Expression,Purification And Crystallization Conditions Of Four Proteins Of African Swine Fever Virus

African swine fever(ASF)is an acute,febrile,widespread and highly contagious hemorrhagic disease caused by African swine fever virus(ASFV),a member of the African swine fever related virus family,African swine fever virus(ASFV).ASFV encodes 54 structural proteins and more than 100 non-structural proteins,which are involved in viral genome replication,DNA repair,transcription,virus assembly,and immune escape.This thesis mainly studies the four proteins K196 R,A240L,K205 R and CP312 R in the ASFV protein.Among them,K196 R and A240 L are two thymidine kinase proteins,and K205 R and CP312 R are two strong immunogenic antigen proteins expressed in the early stage of the virus.The four proteins were fusion-expressed in a prokaryotic expression system by adding a solubilizing MBP tag to their N-terminus.After TEV digestion,the target protein was purified by affinity chromatography.In view of the fact that K196 R and A240 L proteins are very uns Tablein the case of monomers,experiments were mainly carried out to improve protein stability.For example: retain the solubilizing label MBP protein to form a fusion protein;predict the secondary structure through bioinformatics technology,and cut the N-terminus or C-terminus of the protein sequence according to the prediction results;compare with the amino acid sequence of the homologous protein with known structure for hydrophilicity and hydrophobicity,and mutate carry out amino acid;co-expression of K196 R and A240 L proteins;add detergents and arginine of different concentrations in the solvent.Only the K205 R protein is s Tableafter removing the MBP tag.The K205 R protein was screened for the crystallization condition solution of a commercial kit,and filamentous crystals were found in two of the condition solutions.However,in the subsequent optimization condition liquid experiment,no more optimized conditions can be found to make the crystal larger.Attempts to crystallize the CP312 R protein in the form of the MBP-CP312 R fusion protein,but no crystals have been found yet.The preliminary exploration of the four protein crystallization conditions in this experiment will be helpful to the analysis of the protein structure in the future.