Study On Construction And Experimental Immunity Of DNA Vaccine Against Influenza Virus Of H3,H5,H7 Subtype

Influenza (Influenza) referred to as influenza, is an acute respiratory infectious diseases caused by the influenza virus. Among them, A-type influenza virus mutates very rapidly, and some subtypes of virulence is very strong in the past hundred years which has caused many times the global pandemic, however, cause great economic losses, it also seriously endanger the health of humans and livestock, today the global influenza viruses still constitute a very serious public health threat. For decades, the prevalence has been a human influenza H3, H1 subtypes and influenza B dominated, while the avian flu epidemic in order to H5, H7, H9 subtypes based. In recent years, several subtypes of avian influenza virus (H5/H7/H9/H10) begins to break the barrier between species and infect humans, and has caused the world's pandemic potential.This study based on the current status of pandemic influenza and a number of highly pathogenic avian influenza virus subtype cross-species transmission of the status quo, the application of molecular biological information theory, against influenza in different species and the purpose designed and successfully constructed three kinds of lead-based multivalent DNA vaccine, against influenza virus H5 and H3 subtypes pVAX1-H5-H3 DNA vaccine,against influenza virus H5 and H7 subtypes of pVAX1-H5-H7 DNA vaccine ,against influenza virus H5, H3 and H7 subtypes of The pVAX1-H5-H7-H3 nucleic acid vaccine.In this study, influenza virus, mainly in the major protective antigen gene HA as the core, using the successful model of multi-gene tandem construct multivalent influenza DNA vaccine. To make fusion gene can be expressed effectively, we have added two genes between the flexible linker (G4S) 3 to promote the expressed protein conformation correctly folded conformation to reduce the mutual influence that may arise against the pathogenic effects. We also joined self-cleavage function of foot and mouth disease 2A protein linker, its accession to various antigens can be effectively cracking region, and can improve the level of downstream gene expression, its function is more powerful than traditional IRES, and show a good role in many vectors or during the pairs of genes expression . In order to promote more efficient fusion protein expression, Our translation initiation of the "ATG" have been added Kozak sequence.DNA recombinant pVAX1-H5-H3, pVAX1-H5-H7, pVAX1-H5-H7-H3 transfected BHK cells. Confirmed by RT-PCR, the H5HA-H3HA1, H5HA-H7HA1, H5HA-H7HA1-H3HA1 gene can be transcription correctly in BHK cells ; by indirect immunofluorescence method of identification that can occur with the corresponding antibodies specific binding. The results showed that exogenous gene to be the correct expression, and the antigen well, and further proved the effectiveness and feasibility of the recombinant nucleic acid of the candidate vaccines .In the previous study, based on the mouse as a mammalian model, we evaluated the multivalent DNA vaccine immunogenicity. During the experiment, we set up a variety of antigen expression by immunohistochemistry as a separate component of control, the multivalent vaccine group data containing immune-associated antigen by immunohistochemistry data, the system comparison.Antibodies are important immune effector molecule during prevention viral infection, which reflects the strength of the body ability to rid of the virus can block viral infection of tissues damage. ELISA results showed that all DNA vaccine groups were effective in stimulating the immune mice produced specific antibodies. With the number of immune antibody levels increased with the increase, especially after the third immunization, antibody levels increased most rapidly,that are according with the classic theory of immune. 45d, the anti-H5, H3 subtypes of influenza virus-specific ELISA antibody test results are basically consistent and the pVAX1-H5-H7-H3 have the highest level, but the immunity was no significant difference between the groups, and the polyvalent vaccine group can be induced with the Individual expression of HA, pVAX1-H5 or equivalent pVAX1-EH3 group subtype H5 or H3 antibody levels in ELISA. 45d, the anti-H7 subtypes of influenza virus-specific ELISA antibody detection showed that the group of immune pVAX1-H5-H7-H3 have advantage in inducing H7 subtypes antibodies on ELISA test and pVAX1-H5-H7 are inducible expression alone H7HA1 of pVAX1-EH7 group equivalent H7 subtype ELISA antibody levels.Activated T lymphocytes are important immune-mediated protection mechanisms in DNA vaccine. To evaluate the DNA vaccine induced cellular immune response levels, we conducted a T lymphocyte proliferation testing, flow cytometry, and IFN-γELISPOT test. Lymphocyte transformation test results showed that all DNA vaccine groups were effective to stimulate the cellular immune responses in mice. When stimulated with ConA, the SI index are the highest in the group of immune pVAX1-H5-H7-H3, and significantly better than other immune groups, the model shows three HA molecules play a functional, causing a comprehensive immune response, effectively improving cellular immune function . The overall level of cell-mediated immunity induced by the best. Where the H5 subtype of antigen stimulation,the pVAX1-H5-H7-H3 showed the highest level, but each immunization was no significant difference between groups. which illustrated the SI index of H5 subtype may be a major factor in the decision H5HA gene and may be related to a stronger immunogenicity H5HA own decision. When stimulate with the H7, H3 subtypes of antigen, pVAX1-H5-H7-H3 are the highest, and significantly higher than the other immunization groups, indicating the model number HA molecules contribute to a single subtype-specific cellular immunity ,different HA molecules can provide a certain degree of cross-protection capability, and have better protection in implementation of multi-subtype of influenza A virus.The number of lymphocyte sub-category test results showed that pVAX1-H5-H7-H3 have the leaching Puerto highest about CD3 + CD4 + and CD3 + CD8 + T subtype, analysis the CD4/CD8 ratio, each group is normal range and proves that not unusual immunity . Comprehensive Evaluation of pVAX1-H5-H7-H3 immune mice, the optimal level of cellular immunity is desirable to establish a balanced response pattern of Th1/Th2 .IFN-γELISPOT analysis showed, when with the H5, H7 or H3 subtypes of antigen stimulate, the pVAX1-H5-H7-H3 have the largest number of spots, which indicated that Sanya COMBINED immunity group improve level of immunity for single-subtype-specific cell-mediated. When stimulate with the H5 and H3 subtypes, the immunity was no significant difference in the groups, indicating specificity of HA-specific genes in the induction of IFN-γsecretion have more advantage. When stimulate with the H3 subtype of antigen,the pVAX1-H5-H7-H3 were significantly higher than the number of spots the pVAX1-EH3. A number of HA molecules that help to improve the body pattern of H3 subtype-specific the capacity of IFN-γsecretion and the different HA molecules demonstrated cross-protection capabilities to deduce that it can better achieve the multi-protective effect of influenza virus subtype .This study was designed gene containing tandem HA multivalent DNA vaccine, and systematic evaluated the multivalent DNA vaccine immunogenicity from multiple subtypes of influenza humoral and cellular immunity level. we found that in terms of multivalent DNA vaccine induced humoral and cellular immune aspects are significant advantages in search, the present study cross-species protection against influenza research laid the foundation.