Study On The Construction And The Immunological Activity Of The DNA Vaccine Coexpression Vector About Influenza A Virus

Nucleic acid vaccines are eukaryotic expression plasmid containing gene sequence encoding certain kind of antigen, which can be introduced into zooblast. It synthesizes antigen protein by transcription system in host cell and activate immunologic system, which can introduce host produce immunologic response against certain antigen to achieve immunity. Nucleic acid vaccine makes the body produce both humoral immunity and cellular immunity, moreover, the plasmid can be injected into the muscle directly and applied easily. Then muscle cells transfer antigenic message by MHC-Ⅰand MHC-Ⅱmolecule without considering MHC restriction, and it will induce CTL effect that many subunit vaccine can't induce. After expression plasmas containing DNA encoding certain antigen being injected locally, they are absorbed by local histiocytes, APC (antigen presenting cells) and other cells, and the plasmas are transcribed and translated into protein in cytoplasm. These proteins are degraded into short peptide fragments by protein enzyme complex. Part of short peptides are transported into reticulum, combined with MHC-Ⅰmolecule and expressed on the surface of cell memebrane. They can be recognized by precursor cell CD8+T cell, assisted with interaction between CD28 on T cell and B7 on APCs, stimulated by lymphokine secreting by lymphocyte, precursor CD8+T cell can be activated to CTL to kill target tumor cells. The other part of short peptides may enter lysosome / endosome region, combined with MHC-Ⅱmolecule, which are transported on the surface of cell memebrane and recognized and activated by CD4+T cells, and they can activated CD8+T cell or CD4+T cells, at the same time the complex presents on B cells, assisted with CD4+T cells, they can stimulate B cell to produce neutralizing antibody, inducing humoral immunity.Nowadays, the keypoint of nucleic acid vaccines study is how to elevate the immunogenicity and anti-infective immunoprotection of DNA vaccines. It will relate how to transform Nucleic acid vaccines into the products. In the past, people constructed dual promoter expression vector, recombinating plasmas expressing fusion protein or carrying different antigen gene and adjunvant gene in order to construct multivalency nucleic acid vaccine and enhance its immunogenicity. Though dual promoters can make two genes coexpression, mutual interference between promotors may result in transcription effect discrepancy, attenuate or lost certain gene expression. Multi-vector's co-immunitive efficiency is low, and can't satisfy us.Internal ribosome entry site (IRES) is the first DNA sequence in 5′noncoding region found in animal virus's genome. It has translation initiation function without depending on 5′cap sequence, and can make two open reading frame came from the same mRNA express correctly, which is the most important method of multi-gene expression. Green fluorescent protein is convenient labeled molecule in detecting foreign protein.Taking pVAXI vector as framework, we have constructed nucleic acid vaccines'double expression vector pVI containing IRES of parvovirus. In order to test the vectors expressive effect, we choose Green fluorescent protein gene (EGFP) and neomycin phosphotransferase gene (neor) as report gene and ligate them on upstream and downstream multiple clone site of pVI vector to form eukaryotic expression vector pEIN. Transfecting it into COS-7 cell and adding G418 to cultivate, we get anti- G418 cell clone, which suggests it express neomycin phosphotransferase, at the same time, we observe green fluorescence under microscope, which suggests EGFP gene in the upstream of IRES expresses at the same time. It proves that the vector expresses two exogenous gene successfully.Variation of Influenza virus is the main cause of influenza prevalence, especially when new subtype of influenza virus were founded, which causes virus strain of influenza vaccine should be changed each year. It much inconvenient for influenza vaccine preparation, and if vaccine doesn't match with prevalent strain, it can't produce enough immunoprotection.M2 of Influenza virus A is a kind of transmembrane protein, containing 97 amino acids, with a highly conservative structure, which plays an important role in virus replication. Conservation in structure and its special immunologic effect make M2 gene become one of important target gene in studying DNA vaccine and general vaccine. However, M2 protein is a transmemberane protein, and the molecular weight of its protein outside membrane is small, so it may induce much lower immunologic reaction. GM-CSF is a multifunctional cytokine, which may enhance the maturation of APCs and activate them, so it can increase the number and function of APC cell, and enhance secretion of IL-2 and proliferation of CD4+,CD8+T cell in order to increase antigen. At the same time, GM-CSF induce local inflammatory reaction, which may play an important role in antigen location, macrophage activation and inducing local cytokine enrichment to absorb APC migrating to immunoreactive location from lymph tissue, so it increases DNA vaccine increasing humoral and cellular immunological reaction.On the basis of having constructed double expression vector pVI, we inoculate Influenza virus Caledonia/20/99 (H1N1) strain into allantoic fuild of embryonated egg, and extract total RNA of influenza virus by Trizol, then separate M2 gene of influenza virus by RT-PCR method, and construct its DNA vaccines'double expression vector pMIG, then select pMIG with right sequence and transfect it into COS7 cell, finally the result of Western blot shows that M2 protein of influenza virus A were expressed.We extract plasmas by alkaline lysis, and purify plasmas by Sepharose-CL 4B column chromatography. Then we measure A260 and A280 ratio by ultraviolet spectrophotometer to determine purification and concentration, and test DNA integrity by agarose electrophoresis.We divide BALB/c mice into 5 group randomly, and 10 in one group, the groups are as follow: influenza vaccine control group, keno-vector pVAXI group, pVAX/GM-CSF control group, pVAX/M2 experimental group, pMIG experimental group.The plasmas are injected into the tibial muscle of mice, the dose is 10ug/100ul, the antigen's dose of the influenza split vaccine control group is 2ug in each mouse. Mice should be immunized for three time, respectively in week 0,2,4, the dose and injection site are still the same. We should fetch blood in vena orbitalis of mice every week since one week after the first immunization to test its immune effcet.According to analysis of variance, influenza split vaccine control group had produced specific hemagglutinin antibody one week after immunization, and in the fourth week, it became to the peak. 13 weeks after immunization, the antibody hadn't disappeared. We hadn't found antibody in other group.The level of CD4+, CD8+ and CD4+/CD8+ in T cell were tested with two-color flow cytometry, and it suggests that the level of CD4+/CD8+ and cytokine (IL-4,IFNγ)produced in influenza nucleic acid vaccines is much higher than that in influenza split vaccine control group.So, influenza nucleic acid vaccines have an advantage over influenza split vaccines in immunoprotection: protective efficiency of influenza split vaccines are 4/10, however, protective efficiency of influenza nucleic acid vaccines are 7/10, which is higher nearly for one time.