猪圆环病毒3型Rep蛋白的原核表达及酶活性分析OA北大核心CSTPCD

Rep protein plays an important role in porcine circovirus replication.This study aimed to analyze the endonuclease activity,ATPase activity and helicase activity of porcine circovirus type 3(PCV3)Rep protein.The recombinant proteins were purified by Ni2+affinity column chromatography.We established an enzymatic activity assay of Rep protein by optimizing the reaction conditions,and the key active site of Rep protein was explored.Our results confirmed that Rep has endonuclease activity、ATPase activity and helicase activity in vitro.Rep protein exhibited optimal ATPase activity in reaction with 1.6 μmol·L-1 Rep and 1 mmol·L-1 Mg2+at 37 ℃ for 75 min.It exhibited optimal ATPase activity in reaction with 7 μmol·L-1 Rep and 12.5 mmol·L-1 Mg2+at 37 ℃ for 75 min.Y89 of PCV3 Rep was essential for its endonuclease activity.And K173,D210,N250 sites were essential for its ATPase and helicase activities.In this paper,we developed a method for analysis of the enzyme activity and preliminarily revealed the main functional sites of PCV3 Rep protein,which laid a foundation for the functional study of PCV3 Rep protein and the development of antiviral drugs based on the activity of Rep protein.