登革病毒NS2B-NS3蛋白酶在大肠埃希菌中表达条件的优化及酶活性测定  

Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity

作　　者：刘晓丽 闫干干 闫浩浩 刘志成 刘晓平[1] 陈云雨 LIU Xiaoli;YAN Gangan;YAN Haohao;LIU Zhicheng;LIU Xiaoping;CHEN Yunyu(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,Anhui Province,China)

机构地区：[1]皖南医学院药物筛选与评价研究所,安徽芜湖241002

出　　处：《中国生物制品学杂志》2023年第11期1306-1312,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81703546);安徽省自然科学基金(1808085QH265);安徽省高等学校自然科学研究项目(KJ2019ZD30,KJ2021-A0839,YJS20210549);安徽省重点研究与开发计划项目(202004a07020041);皖南医学院青年骨干人才资助项目(wyqnyx202104);安徽省研究生学术创新项目(2022xscx129)。

摘　　要：目的在大肠埃希菌(E.coli)中表达登革病毒(dengue virus,DENV)NS2B-NS3蛋白酶,对表达条件进行优化,并测定酶活性,为抗DENV药物先导化合物的筛选与发现奠定基础。方法将密码子优化的NS2B-NS3基因连接到pET-28a载体中,构建重组原核表达质粒pET-28a-NS2B-NS3,转化至E.coli Rosetta(DE3)感受态细胞中,IPTG诱导NS2BNS3蛋白酶表达。通过优化诱导时间、诱导温度和IPTG诱导浓度,确定NS2B-NS3蛋白酶在E.coli中的最佳表达条件。使用HisTrap^(TM)亲和层析柱分离纯化NS2B-NS3蛋白酶,通过荧光共振能量转移(fluorescence resonance energy transfer,FRET)试验测定酶活性。结果重组原核表达质粒pET-28a-NS2B-NS3经双酶切(NheⅠ/XhoⅠ)及测序证明构建正确。NS2B-NS3蛋白酶在E.coli中的最佳表达条件为:诱导温度20℃,诱导时间10 h,IPTG浓度0.2 mmol/L,表达量达20 mg/L。纯化的NS2B-NS3蛋白酶纯度大于90%,可与小鼠抗His-tag单克隆抗体特异性结合,且具有良好的水解活性,其比活力为16111 U/mg、米氏常数(K_(m))值为16.46μmol/L、催化常数(k_(cat))值为0.028/s。结论成功制备了高纯度、高活性的DENV NS2B-NS3蛋白酶,为NS2B-NS3蛋白酶抑制剂高通量筛选模型的建立奠定了实验基础。Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease.The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration.NS2B-NS3 protease was isolated and purified by HisTrap^(TM)affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing.The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20℃,induction length of 10 h and IPTG concentration of 0.2 mmol/L.The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16.111 U/mg,a K_(m) of 16.46μmol/L and a k_(cat)of 0.028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

关 键 词：登革病毒 NS2B-NS3蛋白酶 原核表达 亲和层析 荧光共振能量转移 

分 类 号：Q789[生物学—分子生物学]