| Influenza virus (IV) can cause widespread acute respiratory infections in the crowd. Based onthe viral protein, World Health Organization first divided the virus into three types: A, B and C.Influenza virus are categorized into different subtypes according to the difference of the structuralproteins hemagglutinin (Hemagglutinin HA) and neuraminidase (Neuraminidase NA) on the virussurface. It has been found that there are17HAsubtypes and10NAsubtypes so far. H1and H3subtypes of influenza can cause influenza in humans, pigs, poultry, and the disease epidemicbrought huge economic losses to humans every year. Fast variability of the epitopes of influenzavirus and too many subtypes brought great difficulties to the research on the new vaccine againstinfluenza. The surface of the influenza virus subtype epitopes were screened and combined withgenetic engineering techniques to construct the corresponding DNA vaccine, the multi-epitopevaccine, which can prevent variety of subtypes of influenza. Therefore, advantages ofmultiple-epitope DNA vaccine against the flu which spread quickly, fast variation, serotypes ofinfectious diseases are more obvious.The constructed plasmid (pVAX1-H3-EHA-H1HA1new, pVAX1-H3-H1HA new,pVAX1-EHA), optionally are fermented with fermentation tank, based on the exploration of thesmall-scale fermentation conditions and optimize the fermentation process to get the best of thefermentation process. According to the experimental data, the optimum fermentation conditionsare as follows: the culture conditions of the system20L and inoculate liquid2L,37°C for10h,best dissolved oxygen is30%, the optimum pH is7.25. The additional glucose culture processflow can effectively prolong the logarithmic phase of growth of E. coli, thereby to obtain a higherbacterial cell wet weight. The broth obtained after the extraction of a large number of plasmid, theresulting plasmid was purified by anion exchange chromatography (Q Sepharose XL). The resultsshow that the two-step filtration method can effectively remove the cell debris and other impuritiesand purified by column chromatography can effectively remove RNA, proteins and host genomicDNA, to obtain a high purity plasmid DNA. This experiment proved to establish the feasibility ofthe preparation process for large-scale production of DNA vaccines and also offer the technical data for it.Finally, the immune experimental study of DNA vaccine was proceeded in accordance withthe principles of animal model experiment. Grind spleen after immunizing mice with QuilA asadjuvant to classify lymphatic for the detection of various test indexes, the results indicated thatthe multi-epitope vaccine group could stimulate H1subtype-specific IgG antibody levels in micecorresponding with immunity group of co-expression of H1, H3subtype of influenza HA protein.Moreover, mice of multi-epitope vaccine immunity group showed better effectiveness of TLymphocyte Transformation than immunity group of co-expression of H1, H3subtype ofinfluenza HA protein which explained the adjunction of multi-epitope box could improve bodyimmune response, effectively improved the cellular immune function in mice. In addition, thecytokine levels of IL-4in multi-epitope vaccine group showed that the B-cell epitope contained inepitope box could stimulate Th2cytokines in mice which inhibited Th1cell activation andtriggered significant improvement of humoral immune response, the detection result wasconsistent with the IgG antibody test. The results indicate that the immunity group DNA vaccinecan effectively develop cellular and humoral immune. |
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