Structural And Functional Investigation On The African Swine Fever Virus P15 Protein

African swine fever?African swine fever ASF?is a highly contagious disease that can be transmitted between stocked pigs,wild boars and soft ticks.It is caused by African swine fever virus?African swine fever virus ASFV?and the mortality rate is close to 100%.ASFV is a large double-stranded DNA virus and the only member of the Asfarviridae family.The virus particles have a diameter of 200 to 250nm,a genome length of about 170 to 193kbp,and contain 150 to 167 open reading frames,the entire genome encodes more than 150 proteins.The pp62 is an ASFV multi-protein precursor,encoded by the gene CP530R,Processed by p S273R protease to mature virion structural proteins p15 and p35,and the p15 and p35 proteins are important components of ASFV Core shell.Because the three-dimensional structure,specific location and functional role of the p15 and p35 proteins on the Core shell are unknown,it has prevented humans from further exploring the composition of the ASFV Core shell,the mechanism of viral infection,and the antiviral vaccine manufacture.In this dissertation,the virion structural protein p15 of ASFV is used as the research object.Using S2 Drosophila cells expression system,the structural protein p15 stably transfected cell line is selected,and a large amount of recombinant protein with stable-nature is obtained.First,the orthogonal optimization of crystallization conditions is used to obtain high-quality p15 protein crystals with X-ray diffraction resolution of 2.6?.Finally,the three-dimensional structure of p15 was analyzed by preparing selenomethionine derivative crystals and single-wavelength anomalous scattering.The space group of p15 crystal is P2₁3,one asymmetry unit contains two p15 monomer molecules,and each molecule is composed by five?-helices,one 3₁₀-helix and five?-strands,adopted as a wedge-like structure.The protein has three aggregate forms of monomer,dimer and hexamer,the hexamer is shaped like a"three-bladed propeller"with two sides that are named"side A"and"side B".Based on the analysis of the related amino acid residues in the structure of ASFV p15 polymer,the site-directed mutations are designed to verify the forming mechanism of p15 dimer and hexamer.The position of p15 protein on the Core shell is further clarified by liposome floating test and gel electrophoresis migration test?EMSA?,where the“side A”of the p15 hexamer faces the inner membrane and the“side B”faces the Nucleoid,as well as reveals its dual role for membrane-association and DNA binding.Based on the research results of the structure and function of ASFV p15 protein,we propose a possible model to illustrate the position and function of p15 hexamer in the structure of ASFV Core shell.In order to provide structural biological basis for the development of defensive vaccines,and provide potential targets for the design of antiviral specific therapeutic drugs.