Objective To design an effective universal DNA vaccine for influenza A virus and detect its immune efficacy in mice.Methods The DNA sequence containing the M2(3M2e)extracellular domain of H1N1,H3N2 and H9N2 subtypes of influenza A virus was synthesized and cloned into vector pVAX1 to construct eukaryotic expression plasmid pVAX1-3M2e.The plasmid pVAX1-3M2e was mixed with cell penetrating peptides(CPPs)RVG9dR,Protamine,and RVG9dR+Protamine peptide complex(1∶1)at different mass fractions(1∶0.5,1∶1,1∶2,1∶4,and 1∶8),separately,and gel retardation was performed to determine the proper binding of DNA and CPPs.The delivery efficiency of the CPPs was detected by cell fluorescence assay at cellular level(293T cells).Male BALB/c mice were immunized intramuscularly with plasmid pVAX1-3M2e delivered by RVG9dR+Protamine(two doses:25 μg pVAX1-3M2e+500 μg polypeptide complex,50 µg pVAX1-3M2e+1 000 μg polypeptide complex,8 mice in each group)to detect the cross-protection effect of universal influenza A virus DNA vaccine against three subtypes of influenza viruses(H1N1,H3N2 and H9N2).Results The optimum binding ratios of DNA to RVG9dR,Protamine,and RVG9dR+Protamine were 1∶2,1∶2,and 1∶1,respe-ctively.RVG9dR+Protamine had superior delivery efficiency to plasmid pVAX1-3M2e at the mass fraction of 1:20.The protective rates of 50 μg pVAX1-3M2e+1 000 μg polypeptide complex against three subtypes of influenza virus in mice were all 100％.Conclusion The plasmid pVAX1-3M2e delivered by RVG9dR+Protamine polypeptide complex provides cross protection against three subtypes of influenza A virus in mice.
Influenza A virusM2 extracellular domainUniversal DNA vaccineCell penetrating peptides(CPPs)Cross protection
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