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OACSTPCD

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.