关键词: 非洲猪瘟病毒, MGF505-5R蛋白, 大肠杆菌表达系统, 可溶性表达, 纯化

Abstract: In order to obtain African swine fever virus (ASFV) MGF505-5R protein with biological activity and its specific polyclonal antibodies,the His-SUMO,His-TrxA,His-GST,His-MBP and His-NusA gene fragments were recombinanted with MGF505-5R gene respectively,and then the pET28a-SUMO-MGF505-5R,pET28a-TrxA-MGF505-5R,pET28a-GST-MGF505-5R,pET28a-MBP-MGF505-5R and pET28a-NusAMGF505-5R were transformed into E.coli BL21(DE3) competent cells,and induced by IPTG,respectively.The optimal expression conditions were explored by optimizing the concentration of IPTG,induction temperature and induction time.The target protein was purified by MBP affinity chromatography and Ni2+affinity chromatography.The BALB/c mice were immunized with purified MGF505-5R protein,and the specificity of the anti-MGF-505-5R serum was identified by Western blot. The results showed that the MBP-MGF505-5R recombinant protein was successfully expressed in E.coli,and the expression quantity of soluble MBP-MGF505-5R recombinant protein was the highest after induced by 0.5 mmol/L IPTG at 20℃ for 12 h.Purified protein was obtained by MBP and Ni²⁺affinity chromatography,and its purity reached 90%.The polyclonal antibody could specifically recognize the MGF-505-5R protein.In summary,this study successfully expressed ASFV MGF-505-5R protein using E.coli expression system,and the purified recombinant protein had high purity and good biological activity.

Key words: ASFV, MGF505-5R protein, E.coli expression system, Soluble expression, Purification