| Background:Influenza virus often infected human and animal.And influenza widespread pandemics can bring significant health and socio-economic damage to humans.The most effective means to prevent the spread of influenza virus is through vaccination.The current influenza vaccines have better prevention effect on influenza virus infection of the same subtype,but have weaker cross protection activity on influenza virus of different subtypes.Influenza virus has high variability.The World Health Organization(WHO)predicts influenza candidates strains based on influenza epidemic pandemic strains monitored by the global influenza surveillance response system.Correct prediction affect the efficacy of the current season vaccine Therefore,it is necessary to develop an effective influenza vaccine that has protective effects against different subtypes of influenza virus.DNA vaccine,as a new vaccine production method,can mode of production the genes which encoding the target antigen directly into the body,it can induce stronger cellular and humoral immunity when combined with adjuvant.DNA vaccines have many advantages,such as simple operation,high safety,long-lasting immune response,easy storage and transportation,so it can be used as a platform for research and development of universal influenza vaccine.Objective:The universal influenza DNA vaccine was constructed by the HA,M2e and NP,and to screen a universal influenza vaccine with broad-spectrum protective activity.Method:1.In this study,three construction strategies of mHA,cHA and sHA were desigend base on the short conserved region peptide genes of HA,M2e and NP of H5N1 influenza virus A/me-erkat/Shanghai/SH-1/2012(clade 2.3.2.1).And the genes were cloned it into pcDNA3.1(+)vector,three recombinant DNA vaccines,pcDNA3.1-mHA,pcDNA3.1-cHA and pcDNA3.1-sHA were cons-tructed.2.After transfection of 293T cells,Westren blot and indirect immunofluorescence were used to verify the correct expression of the target protein.After identification,a large number of amplification and extraction were carried out.Then we immunized mice by intramuscular at 0,3,6 and 9 weeks with a bacterial CPG Oligonucleotide(CPG ODN)and monophosphoryl lipid A(monophosphoryl pid A)to evaluate the immunogenicity and protective efficacy against H1N1,H3N2,and H5N1 influenza viruses.Results:1.Three recombinant DNA vaccines,pcDNA 3.1-mHA,pcDNA 3.1-cHA,pcDNA 3.1-sHA were successfully constructed by molecular biological techniques2.The correct expression of the above DNA vaccines was verified by transfection of 293T cells with Western blot analysis and indirect immunofluorescence3.After the third immunization,pcDNA3.1-sHA serum antibody titers reached 1:32000 detected by ELISA in H5N1 influenza virus attack group.Micro neutralization experiments showed that the titer of neutralizing antibody in pcDNA3.1-cHA group and pcDNA3.1-sHA group was 2 to 3 times higher than that in HA control group.pcDNA 3.1-cHA and pcDNA 3.1-sHA were able to induce stronger humoral immunity.4.Spleen lymphocytes were isolated from immunized mice,the immune group of universal DNA vaccine could secrete high levels of IFN-y and IL-4 when stimulated by antigens,which showed that DNA vaccine could induce antigen-specific cellular immunity,and Thl type immune response was the main immune response.5.When mice were immunized with the combination of adjuvant CpG and MPLA,both the pcDNA 3.1-cHA and pcDNA 3.1-sHA immune groups had a survival rate of 80%in the challenge groups of H5N1 and H1N1 homologous viruses,significantly protecting the mice from lethal challenge of both homologous and heterologous influenza virusesConclusion:This study showed that the DNA vaccine constructed with conserved short peptide genes could induce strong cellular and humoral immunity when combined with adjuvants,which could improve the protection rate against influenza viruses of different subtypes and provide a reference for the development of universal influenza vaccine with broad-spectrum protection. |
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