易通过冷冻食品传播的新冠病毒解旋酶的表达纯化及酶活研究  

Expression and Purification of SARS-CoV-2 helicase NSP13 and Preliminary Study on its Enzymatic Activity

作　　者：李海红[1] 郑亚婷 高博 韩晓睿 何怡宁 侯锡苗[1] LI Haihong;ZHENG Yating;GAO Bo;HAN Xiaorui;HE Yining;HOU Ximiao(Department of Biochemistry and Molecular Biology,College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,生物化学与分子生物学教研室,陕西杨凌712100

出　　处：《农产品加工》2021年第24期68-71,74,共5页Farm Products Processing

基　　金：国家自然基金项目(32071225)。

摘　　要：通过原核表达方式纯化得到新冠病毒解旋酶(SARS-CoV-2-Nsp13),并利用荧光各向异性和凝胶阻滞试验探究了其体外结合DNA、RNA底物的亲和力及解旋双链DNA的最佳解旋条件。结果表明,SARS-CoV-2-Nsp13解旋酶具有结合单链DNA(ssDNA)、带有5'尾链的双链DNA(5'Oh-dsDNA)及RNA发卡结构(RNA-HP)的活力,亲和性依次为5'Oh-dsDNA≈RNA-HP>ssDNA,结合曲线呈S型。SARS-CoV-2-Nsp13解旋酶与DNA、RNA底物的结合具有协同性。SARS-CoV-2-Nsp13解旋酶可以解旋带有5'尾链的双链DNA,解旋的方向为5'-3',发挥完全解旋活性的酶浓度为3μmol/L;而该解旋酶解旋DNA双链的活性相对较弱,解旋活性在NaCl浓度达到80 mmol/L时受到明显的抑制。研究结论可为深入理解冠状病毒解旋酶的生化活性及工作机理提供参考和帮助。In this study,SARS-CoV-2-encoded nonstructural protein(SARS-CoV-2-Nsp13)was expressed and purified in Escherichia coli.The characteristics of SARS-CoV-2-Nsp13 binding with different substrates single-stranded DNA(ssDNA),double-stranded DNA with 5'ssDNA tails(5'Oh-dsDNA),and RNA hairpin structure(RNA-HP)and the effects of helicase concentration and NaCl concentration on DNA unwinding activities of SARS-CoV-2-Nsp13 were analyzed by fluorescence anisotropy and Electrophoretic Mobility Shift Assay(EMSA).The results showed that SARS-CoV-2-Nsp13 helicase can bind with different DNA and RNA substrates with the binding activity in decreasing order of 5'Oh-dsDNA≈RNA-HP>ssDNA,and the binding curve is S-shaped,indicating that the binding of SARS-CoV-2-Nsp13 helicase with DNA and RNA substrates are collaborative.Its unwinding activity to 5'Oh-dsDNA was suppressed by the 80 mmol/L NaCl,and the concentration of SARS-CoV-2-Nsp13 helicase was 3μmol/L,indicating that the activity of the helicase to dsDNA is relatively weak.This study can provide reference and help for further understanding the biochemical activity and working mechanism of coronavirus helicase.

关 键 词：冠状病毒 Nsp13解旋酶 蛋白表达纯化 DNA结合活性 DNA解旋活性 

分 类 号：Q556[生物学—生物化学]