Study On The Immune Effect Of DNA Vaccine Expressing Different Proteins Of Avian Influenza Virus

Avian influenza(AI)is an infectious disease caused by avian influenza virus(Avian influenza virus,AIV).AIV hemagglutinin(HA)and neuraminidase(NA)are the main protective antigens that play a key role in immunoprotection and the packaging and release of AIV.Vaccination remains the most effective means of preventing avian flu.DNA vaccine is one of the most promising genetic engineering vaccines,but DNA immunization commonly used immunization route for intramuscular injection,with a large dose,the target gene immunogenicity is weak,low level of protein expression defects,while intramuscular injection drugs cause most of the plasmid DNA to be degraded before delivery to antigen-presenting cells(APCs),resulting in a significant reduction in bioavailability.At present,nano-materials as a non-viral carrier,with good biocompatibility,mucosal adsorption,unique physical and chemical properties,such as easy processing modification,surface properties control,promote functional molecules into the cell,protect DNA and protein from degradation,in the vaccine adjuvant and drug delivery carrier research has become a hot topic of concern.In this study,six eukaryotic expression plasmids(pβH5,pβH5SH9,pSH9,pβH9,pβH9N1,pβH9N1SH5)which contain single genes or multiple genes were constructed using the optimized HA of H9N2,HA and NA genes of H5N1.Through animal experiments screened the better immune effect plasmid(pβH9N1SH5),according to the electrostatic adsorption with polylysine dendrimer(DGL)plasmid package,prepared the nano DNA vaccine(pβH9N1SH5/DGL-NPs)at the same time expressing the H9N2 subtype AIV HA protein,H5N1 subtype of AIV HA and NA.And the physical and chemical properties,stability,transfection efficiency and immune effect were studied in detail.The results showed as follow:1.Six eukaryotic expression plasmids(pβH5,pβH5SH9,pSH9,pβH9,pβH9N1,pβH9N1SH5)which contain single genes or multiple genes were constructed;2.Animal experiments showed that the plasmid pβH9N1SH5,which was inserted into the three gene fragments,was significantly better than the plasmid DNA into which one or two gene fragments were inserted.At the same time,the mouse immunization test results also show that pβH9N1SH5 eukaryotic plasmid immunization effect is best;3.DGL cytotoxicity test results showed that DGL toxicity was small,can be used as pβH9N1SH5 delivery vector;4.Preparation of pβH9N1SH5/DGL-NPs morphological rules,an average particle size of(44±2.26)nm,Zeta potential(119.33±0.93)mV,Encapsulation efficiency(93.1±1.5)%,Drug loading(23.3±0.38)%;5.In vitro DNase I digestion test results showed that,DGL can protect pβH9N1SH5 from DNase I degradation;6.In vitro release test results showed that,pβH9N1SH5/DGL-NPs was the first continuous release after the release process,continued until 216h;7.In vitro expression test results showed that the use of DGL wrapped eukaryotic expression plasmid pβH9N1SH5,verify that the plasmid DNA biological activity remains intact;8.Mice immunization test results showed that pβH9N1SH5/DGL-NPs intramuscular immunization,humoral immune antibody levels higher than other immunized groups.Cellular immune level results showed that IFN-γ,IL-2 secretion and splenic lymphocyte transformation rate were significantly changed,T lymphocyte proliferation,the number of CD3+CD8+ and CD8+/CD4+ T lymphocytes rapidly rised,induced the body to produce a certain cellular immune response.In this study,we constructed a single gene or a multi-gene eukaryotic expressionplasmid,and the plasmid DNA was screened by animal experiment.The DGL DNA vaccine was prepared by connecting it with the nanostructured DGL.To develop new avian influenza vaccine or Improve the existing vaccine and vaccine release provides a reference to the research ideas and methods.