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

作　　者：沈熹涓 姬晶晶 金慧琴 李宇哲 刘斐[1] 单衍可 SHEN Xijuan;JI Jingjing;JIN Huiqin;LI Yuzhe;LIU Fei;SHAN Yanke(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2024年第1期33-38,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年基金项目(32000028);中国博士后基金面上基金项目(2020M681649);江苏省博士后科研资助项目(2020Z120)。

摘　　要：旨在可溶性表达并纯化猪流行性腹泻病毒(PEDV)解旋酶Nsp13,并验证表达纯化得到的PEDV Nsp13蛋白具有RNA解旋活性。将pET28a(+)-PEDV Nsp13重组质粒转化到大肠杆菌Transetta(DE3)感受态细胞中,利用终浓度0.5 mmol/L的IPTG在18℃条件下诱导16 h,收集诱导后的菌体,超声破碎并收集其上清液,通过镍亲和层析技术获得纯化后的PEDV Nsp13;设计RNA底物,并建立体外解旋反应体系,验证蛋白的RNA解旋活性,并在相同时间内探究不同温度条件下PEDV Nsp13蛋白对RNA的解旋情况,进一步验证其活性。结果表明,在大肠杆菌系统中可溶性表达了PEDV Nsp13蛋白,纯化后蛋白浓度可达0.9 mg/mL;通过体外解旋试验,验证了PEDV Nsp13蛋白具有ATP依赖的RNA解旋酶活性,并发现PEDV Nsp13蛋白的RNA解旋酶活性在一定范围内随解旋温度的升高而提高。本研究结果为进一步深入研究PEDV Nsp13的RNA解旋作用机制奠定了基础。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 transformed 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 concentration 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-dependent 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 

分 类 号：S852[农业科学—基础兽医学]