基础医学与临床 ›› 2018, Vol. 38 ›› Issue (5): 674-678.

• 研究论文 • 上一篇    下一篇

Zika病毒NS3具有依赖于ATP水解的dsDNA解链活性

徐杉,王蕾杰,石磊   

  1. 中国医学科学院基础医学研究所
  • 收稿日期:2018-01-11 修回日期:2018-03-20 出版日期:2018-05-05 发布日期:2018-04-28
  • 通讯作者: 石磊 E-mail:shilei@ibms.pumc.edu.cn
  • 基金资助:
    协和青年基金

Zika virus NS3 can unwind dsDNA depending on ATP hydrolysis

  • Received:2018-01-11 Revised:2018-03-20 Online:2018-05-05 Published:2018-04-28
  • Contact: Lei -SHI E-mail:shilei@ibms.pumc.edu.cn

摘要: 目的 研究Zika 病毒NS3蛋白是否具有依赖于ATP水解的dsDNA解链活性。方法 构建原核表达载体,在大肠杆菌BL21菌株中表达带有His标签的Zika NS3蛋白,并用Ni琼脂糖珠进行纯化;利用ATPase检测试剂盒对NS3的ATP水解活性进行检测;利用FRET(荧光共振能量转移)原理,对NS3的dsDNA解链活性进行实时动力学检测;利用定点突变方法将NS3的第198位甘氨酸(G)突变为丙氨酸(A),得到G198A突变体,检测其是否影响NS3解旋酶活性。结果 Zika NS3蛋白在体外可以水解ATP,酶解最大反应速率为2.76μmol /L?min,Km值为0.11 mmol/L;Zika NS3蛋白在体外可以解链dsDNA;G198A突变体与野生型相比,ATP水解速率和dsDNA的解链速率均明显减弱。结论Zika NS3蛋白在体外具有ATP水解活性以及dsDNA解链活性,且198位甘氨酸对于NS3酶活性至关重要。

Abstract: Objective To investigate whether Zika virus NS3 have ATP hydrolase and DNA helicase activity. Methods Zika NS3 with His tag was expressed in E.coli BL21 and purified by Nickelagarose beads. ATP hydrolysis assay was performed to examinethe ATPase activity of NS3. The dsDNA unwinding activity of NS3 was detected according to the principle of FRET(Fluorescence Resonance Energy Transfer). NS3 G198A mutant was generated by site-directed mutagenesis and its enzymatic activity was measured subsequently. Results Zika NS3 was capable of hydrolyzing ATP in vitro(Vmax=2.76 μmol /(L?min),Km =0.11 mmol/L).Moreover, Zika NS3 could unwind dsDNA in vitro as the fluorescence was enhanced in FRET system. G198A mutation impaired NS3 enzymatic activities, including ATP hydrolysis and dsDNA unwinding activities. Conclusions Zika NS3 has DNA helicase activity in vitro depending on ATP hydrolysis, and the 198 glycine is important for its enzymatic activity.

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