Screening,Expression,and Purification Of Several African Swine Fever Virus Proteins

African swine fever is an acute,febrile,and highly contagious disease caused by African swine fever virus.The course of disease is short while the mortality rate is high.The mortality rate of domestic pigs is close to 100% within 10 days after infection.The first African swine fever case was discovered in Shenyang,Liaoning Province,China in August 2018.The countrywild spread of African swine fever virus in China has caused serious economic losses in pork product trade,hog breeding and related industries.However no effective vaccine has yet been commercially produced.The genome of ASFV encodes 150-200 proteins.The function of about half of these proteins is still unknown.The analysis of protein structure is of great significance for the study of protein function,binding sites,and the development of related vaccine.However structural information is not available for most of ASFV proteins.As of May 2020,there are 30 entries in the PDB database of eight ASFV proteins(p B119 L,A179L,A238 L,NP419L,p A104 R,p S273 R,E165R,B646L),more than half of which were solved in the past year.In this subject we used Fluorescent Size-Exclusion Chromatography(FSEC)technology to carry out screening of a set of ASFV protein(CP123L,X69 R,EP152R,E199 L,E66L,E183 L,I329L,B475 L,C84L,C217 R,E248R,KR177 R,MGF110-1L,B169 L,O61R,EST-B)using mammalian expression system and found the expression of CP123 L,B475L,KR177 R,E199L,E248 R was good.After optimizing the expression and purification of B475 L and KR177 R proteins,we were able to obtain high purity proteins of MBP-B475 L fusion protein,untaged protein KR177 R,and histidine-tagged protein KR177 R.Crystallization screening was done with histidine tagged KR177 R protein and hexagonal crystals were found,which may lead to way to the structure determination of this protein.The crystal structure of KR177 R protein can be further analyzed by X-ray diffraction.The analysis of the structural protein structure of African swine fever virus KR177 R will have a positive effect on the exploration of related mechanism functions,the determination of infection targets,and the development of related sub-vaccines.The optimization of the expression and purification of B475 L protein and the screening of other ASFV proteins also provide a basis for the study of the functional mechanism of ASFV.