Abstract: 【Objective】 The aim of this study was to analyze the potential biological function of Africa swine fever virus (ASFV) D250R protein and prepare polyclonal antibodies against it, and provide materials for the development of ASFV D250R protein function and related diagnostic reagents.【Method】 Bioinformatics softwares were applied to analyze the physicochemical properties, signal peptide, transmembrane structure, phosphorylation site, biological function and protein structure of D250R protein.The recombinant D250R protein was expressed by E.coli expression system, purified using nickel affinity chromatography column and molecular sieve, and the reactogenicity of the purified protein was detected by Western blotting.BALB/c mice were immunized with purified D250R protein to prepare polyclonal antibody.The titer of the polyclonal antibody was determined by indirect ELISA, and the specificity of the polyclonal antibody was detected by indirect immunofluorescence assay (IFA).【Result】 The bioinformatics analysis showed that D250R protein consisted of 250 amino acids with a theoretical molecular mass of 29 822.54 u, the theoretical isoelectric point was 8.96, it was a hydrophilic protein with no signal peptide region and no transmembrane region.The modification site prediction results showed that D250R protein might had 15 phosphorylation modification sites, including 6 serine (Ser), 6 tyrosine (Tyr) and 3 threonine (Thr), and had 2 N-glycosylation modification sites at positions 61 and 197, respectively.Biological function prediction showed that D250R protein contained a Nudix sequence with a dehydrolytic enzyme activity. The structure prediction results showed that the secondary structure of D250R contained 49.20% alpha helices, 7.60% beta turns, 15.20% extended strands and, 28.00% random coils, and more alpha helices existed in the three-dimensional spatial structure, which was basically consistent with the secondary structure prediction results. ASFV D250R gene was cloned into pET-32a(＋) to obtain the pET-32a-D250R recombinant plasmid, which was transformed into E.coli BL21(DE3) receptor cells, and D250R protein was expressed in two forms of supernatant soluble and inclusion body under the induction conditions of 16 ℃ and 1 mmol/L IPTG.The protein supernatant was purified by nickel affinity chromatography column and molecular sieve chromatography to obtain a high purity protein.Western blotting results showed that the recombinant protein had good reactivity.The results of indirect ELISA showed that the antibody titer of D250R protein reached 1:256 000, and the polyclonal antibody was successfully prepared.The IFA test showed that the polyclonal antibody had good specificity.【Conclusion】 In this study, the physical and chemical properties and protein structure of D250R protein were analyzed at the molecular level, and the high-efficiency expression of ASFV D250R protein was realized in the E.coli expression system.The polyclonal antibody prepared by immunizing mice with the purified D250R protein had high specificity, which laid a foundation for further exploring the biological function of ASFV D250R protein and the development of ASFV related diagnostic reagents.

Key words: African swine fever virus (ASFV); D250R protein; bioinformatics analysis; prokaryotic expression; polyclonal antibody