As a major zoonotic pathogen,influenza A virus(IAV)poses serious threats to global public health security.The cap-binding domain(CBD)of PB2 protein plays an important role in IAV transcription process,and is therefore one of the important targets for anti-influenza drugs.To obtain high-purity CBD protein by prokaryotic expression system,the CBD gene sequence of a highly pathogenic IAV H5N1 and an 8×His tag were constructed into the prokaryotic expression vector pET28a by homologous recombination strategy.The constructed recombinant plasmid pET28a-8×His-CBD was identified by PCR and sequen-cing,and then transformed into Escherichia coli receptor cells BL21(DE3).The expression of CBD protein was detected by SDS-PAGE after induction with different concentrations of IPTG for different hours,and then purified by Ni-NTA.The results showed that a distinct band at 21ku was visible in the cell lysate supernatant of the bacterium,which was consistent with the expected size of 8×His-CBD protein.The highest expression of the protein was obtained under the condition of 0.5mmol/L IPTG,cultured for 6 hours at 37℃,and the purity of the 8×His-CBD protein was more than 95%after Ni-NTA purification.To determine whether the 8×His-CBD protein in this study was biologically active,it was subjected to an isothermal titration calorimetry(ITC)assay with m7GTP,the cap structure analogue.Apparent thermal peaks were observed when the two were combined,indicating that the 8×His-CBD protein was biologically active,and the Kd value was calculated to be 1.71×10-5(±2.92×10-6)mol/L.In this study,the recombinant plasmid pET28a-8×His-CBD was correctly constructed,and the optimal induction conditions were determined.The high-purity 8×His-CBD protein was obtained with biological activity.This study could have reference significance for the basic research of influenza virus and for the development of anti-influenza virus drugs.
influenza A virusPB2 proteincap-binding domainprokaryotic expressionpurification
NETL
NSTL
万方数据
