畜牧与兽医2024，Vol.56Issue(1)：33-38,6.
猪流行性腹泻病毒解旋酶Nsp13表达纯化及其RNA解旋活性鉴定
Expression and purification of porcine epidemic diarrhea virus helicase Nsp13 and verification of its RNA unwinding activity
摘要
Abstract
The aim of this study was to obtain soluble expressed and purified porcine epidemic diarrhea virus(PEDV)helicase Nsp13,and the RNA unwinding activity of the purified PEDV Nsp13 was verified.The recombinant plasmid pET28a(+)-PEDV Nsp13 was trans-formed into Transetta(DE3),and 0.5 mmol/L IPTG was used to induce the cells at 18℃ for 16 h.The induced bacteria were ultrasonically crushed and the supernatant was collected.The purified PEDV Nsp13 was obtained by nickel affinity chromatography.Next,we established an in vitro unwinding reaction system to verify the RNA unwinding activity of the protein,and its RNA unwinding activity was further verified by exploring the RNA unwinding activity of PEDV nsp13 under the condition of different temperatures.In summary,PEDV Nsp13 at a con-centration of 0.9 mg/mL was solubly expressed and purified in the E.coli system,and it was verified that PEDV Nsp13 protein had ATP-de-pendent RNA helicase activity through in vitro unwinding assay.Besides,it was found that the RNA helicase unwinding activity of PEDV Nsp13 increased when the temperature rose within a certain range.This study laid the foundation for further study of the RNA unwinding mechanism of PEDV Nsp13.关键词
PEDV/Nsp13/可溶性表达/蛋白纯化/RNA解旋活性Key words
PEDV/Nsp13/soluble expression/protein purification/RNA unwinding activity分类
农业科技引用本文复制引用
沈熹涓,姬晶晶,金慧琴,李宇哲,刘斐,单衍可..猪流行性腹泻病毒解旋酶Nsp13表达纯化及其RNA解旋活性鉴定[J].畜牧与兽医,2024,56(1):33-38,6.基金项目
国家自然科学基金青年基金项目(32000028) （32000028）
中国博士后基金面上基金项目(2020M681649) （2020M681649）
江苏省博士后科研资助项目(2020Z120) （2020Z120）
