猪圆环病毒3型Rep蛋白的原核表达及酶活性分析
Prokaryotic Expression and Protein Activity of Porcine Circovirus Type 3 Rep
韩阳 1关帅印 1李振 1周赛赛 1袁红根 1宋云峰1
作者信息
- 1. 华中农业大学动物医学院,武汉 430070
- 折叠
摘要
Rep蛋白对猪圆环病毒(porcine circovirus,PCV)的复制具有重要作用.为分析PCV3 Rep蛋白的核酸内切酶活性、ATPase活性和解旋酶活性,作者使用镍亲和层析法纯化相应的重组蛋白,通过优化反应条件,建立Rep蛋白酶活性的测定方法,并探究Rep蛋白关键活性位点.结果表明,Rep蛋白在体外具有核酸内切酶活性、ATPase活性和解旋酶活性.当蛋白浓度为1.6 μmol·L-1,Mg2+浓度为1.0 mmol·L-1,37 ℃反应75 min时其ATPase活性达到最佳.当蛋白浓度为7μmol·L-1,Mg2+浓度为12.5 mmol·L-1,37 ℃反应60 min时其解旋酶活性达到最佳.Y89为Rep蛋白核酸内切酶活性的关键位点,K173、D210、N250为ATPase活性和解旋酶活性的关键位点.本研究建立了PCV3 Rep蛋白酶活性分析的方法,初步揭示了该蛋白的主要功能位点,为Rep蛋白的功能研究以及基于Rep蛋白活性的抗病毒药物研究奠定了基础.
Abstract
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.
关键词
猪圆环病毒3/Rep蛋白/核酸内切酶活性/ATPase活性/解旋酶活性Key words
porcine circovirus type 3/Rep/endonuclease activity/ATPase activity/helicase activity引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据