登革2型病毒NS3蛋白NTPase/RNA解旋酶结构域的原核表达及活性研究

目的 表达登革病毒非结构蛋白NS3 NTPase／RNA解旋酶结构域（dNS3），纯化表达产物并对其活性作初步研究。方法提取感染登革2型病毒的BHK-21细胞总RNA，RT-PCR扩增目的基因，经NcoⅠ、HindⅢ双酶切后连入表达载体pET28a，转化宿主菌BL21（DE3），优化诱导表达条件，SDS-PAGE和免疫印迹鉴定表达蛋白。表达产物经亲和层析纯化，超滤浓缩脱盐，孔雀绿钼酸铵法检测NTPase活性。结果成功构建重组表达载体pET28a-dNS3并在大肠杆菌中获得可溶性表达，纯化产物经测定具有良好的NTease活性及抗原性。体外条件下证实登革2型病毒非结构蛋白NS5（RDRP）对dNS3的ATPase活性有促进作用，提示在病毒复制时NS3与NS5间的相互作用对NS3的生物活性及对病毒复制过程可能具有调控作用。结论本研究为基于抑制NS3 NTPase／RNA解旋酶活性的抗病毒药物的筛选提供了实验依据。

Expression, purification and characterization of NTPase/RNA helicase domain of dengue virus type 2 nonstructural protein 3

Objective To express NTPase/RNA helicase domain of dengue virus type 2 nonstructural protein 3(dNS3) in Escherichia coli BL21(DE3) and to characterize the NTPase activity and immunoreactivity of the recombinant dNS3. Methods DEN2 dNS3 gene was cloned from DEN2 infected cells by RT-PCR and then inserted into pET28a vector, and transformed into E.coli. The recombinant protein was identified by SDS-PAGE and Western blot. The colorimeric assay for inorganic phosphate was used to measure the NTPase activity of DEN2 dNS3. Results DEN2 dNS3 was expressed with C-terminal histidine tag in E.coli and purified in a soluble form. The purified protein has NTPase activity and good antigenicity. NTPase activity was stimulated by the viral NS5 protein. Conclusion NTPase activity of NS3 protein may be regulated by the viral NS5 protein during virus replication.