| With the deepening of immunology research and the rapid development of thegeneticengineering techniques, to avoid large doses of inactivated vaccine inoculation and disadvantageof attenuated virulence back strong and immunity is slow. For different diseases have beendevelop a variety of new genetically engineered vaccine. But these vaccines ubiquitous smallmolecule, low immunogenicity, it is difficult to induce the body to produce an effective immuneresponse, thus require some carrier or adjuvant to make up the shortfall. But there are manydefects in conventional carrier or adjuvant, such as cause local inflammation, cancer, and difficultto degrade in the body, so it is necessary to develop new carrier or adjuvant to overcome the abovedrawbacks.Chitosan is a natural polysaccharide polymer from chitin, which can be hydrolysis bylysozyme and chitosan enzymatic, in vivo biodegradable. It is a mucosal absorption enhancer,having mucoadhesive characteristics. It has been used as mucosal administration wherein thecarrier. Polylactic acid-glycolic acid copolymer (PLGA) is a lactic acid and glycolic acid byrandom polymerization. It is a biodegradable organic polymer, having a good biocompatibility,encysted and film and non-toxic. In the United States, PLGA has been certified by the FDA, aspharmaceutical excipients were officially included into the United States Pharmacopeia. Mannose,a kind of glyconutrients only used in the clinical, currently. which can be utilized directlysynthesized glycoproteins. Immunomodulatory effects on the immune cells involved in endocyticand phagocytosis by mannose receptor. Further activation of T lymphocytes, increase MHC-I andMHC-II molecules in antigen presentation. Thus the use of the biodegradable material chitosan,PLGA and mannose as a new carrier or adjuvant vaccine has great potential.In this study, used the theory techniques of molecular biology, from Pseudomonasaeruginosa standard strain ATCC27853and Pseudomonas aeruginosa isolates strain DL9genomeof mink obtained OprF190-342-OprI21-83through fusion PCR. Bioinformatics analysis ofOprF190-342-OprI21-83by DNAStar, DNAMAN and multiple biological sites. The nucleotidesequence of OprF190-342-OprI21-83in P. aeruginosa is highly conserved among different strains;protein is hydrophilic, secondary structure mainly α helix. Subsequently integrated multiconditions conducive to direct expression of active recombinant protein, in E. coli expressionsystem successfully soluble expression outer membrane protein OprF190-342-OprI21-83(FI) ofPseudomonas aeruginosa standard strains ATCC27853. The use of thrombin to remove the GSTtag on its carrier, achieve soluble expression of label-free outer membrane protein OprF190-342-OprI21-83(FI) of Pseudomonas aeruginosa standard strains ATCC27853. Furtherpreparation for Pseudomonas aeruginosa vaccine and to further explore the role of outermembrane fusion protein FI in the immunology provide materials.Mannose receptor (MR) is expressed in the immune cell surface, such as macrophages anddendritic cells. If the modified vector with mannose, is through receptor-mediated phagocytosis toenhance drug uptake to the cells. Thus, under catalytic cyano sodium borohydride, condensationreaction of carboxyl groups of mannose and amino groups of chitosan, the mannose-modifiedchitosan to obtain a mannose-modified chitosan derivatives. Pseudomonas aeruginosa outermembrane fusion protein FI has been obtained as a model antigen, prepared the chitosanmicrospheres vaccine (FI-CS-MPs), the mannose-modified chitosan microspheres vaccine(FI-MCS-MPs), PLGA microspheres of chitosan-coated vaccine (FI-CP-MPs) and PLGAmicrospheres of mannose-modified chitosan-coated vaccine (FI-MCP-MPs) containing the antigenFI by the ion condensation and the double emulsion method. And to characterize these fourbiological polysaccharide microspheres vaccine were tested by scanning electron microscopy,particle size analyzer, BCA protein concentration kit and other methods. The results showed thatfour kinds of microsphere vaccine are sphere, The results showed that four kinds of microspherevaccine are sphere, regular shape, good dispersion, the surface is not smooth, a rough state,wrinkles; average particle size in2~3μm; Degradable polysaccharide microspheres vaccineswithin the first12hours there is a significant burst release, release of about30%to50%theantigen FI. And the release rate through the mannose-modified microspheres vaccine is alwayshigher than without the mannose-modified microspheres vaccine, but the difference was notsignificant. Successful obtained biodegradable polysaccharide microspheres vaccine, detected thecharacterize of them for the next immunization microspheres vaccine research has laid a goodfoundation.FI antigen markers on FITC fluorescence, compare murine macrophage RAW264.7uptake offour biodegradable polysaccharide microsphere vaccine by flow cytometry and confocal laser. Theresults showed that murine macrophage RAW264.7uptake of microspheres vaccine containingFITC-FI were highter than the free FITC-FI; And mannose-modified microspheres vector vaccineuptake rate was the highest. May be due to mannose-modified microspheres vector vaccinestargeting the mannose receptor, endocytosis occurs in mediating cell, to increase their uptake. InVivo and vitro, the mannose-modified microspheres vaccine can target macrophage mannosereceptor of murine macrophages RAW264.7and mice NALT tissue by immunohistochemistry andconfocal laser.Biodegradable polysaccharide microspheres vaccine intranasally immunized BALB/c mice,PBS, and AL adjuvant group (FI-AL) as the control group, Various time points after immunizationof mice to detect mucosal and systemic humoral antibodies and T lymphocyte subtypes levelsituation, by indirect ELISA and flow cytometry. The results showed that the antibody firstdetected is FI specific IgM in the lungs slurry. As a continuation of its immune time the antibody titers were decreased, mannose-modified microspheres vaccine group were significantly higherantibody titers than control group; In nasal lavage and bronchial lavage, mannose-modifiedchitosan microspheres group-specific IgA antibody titers were significantly higher in the earlyimmunization, and continued until the end of the immune observed (tenth week after a free); Andin the distal small mucosal sites intestine lavage, mannose-modified chitosan microspheres groupspecific IgA antibody levels were also significantly increased compared to the control group; While activating mucosal sites antibody levels, specific IgA and IgG levels were also significantlyincreased in serum, mannose-modified chitosan microspheres group was significantly higher. Inthe lungs, small intestine and spleen, mannose-modified chitosan microspheres group of Tlymphocyte subtypes of CD3+, CD3+CD4+, CD3+CD8+were significantly higher, and isaccompanied by cell culture supernatant IL-4and IFN-γ expression, Biodegradable polysaccharidemicrospheres vaccine can significantly increase mice survival, FI-CS-MPs, FI-CP-MPs,FI-MCS-MPs and FI-MCP-MPs the survival rate of the mice were60%,55%,75%and80%.In this study, used mouse macrophage RAW264.7and NF-κB and MAPK signaling pathwayskit by Western-blot method, revealing biodegradable polysaccharide microspheres vaccine cansignificantly activate mouse macrophage RAW264.7NF-κB and MAPK signaling pathways. Mannan inhibited mannose receptors, mannose-modified chitosan microspheres vaccine activatesthese two signaling pathways phenomenon apparently disappeared. Lay a good foundation tofurther elucidate the mechanism of chitosan microspheres vaccine antigen presentation andactivation of the body’s immune response.Studies have shown that the immune effects of experimental animal models can not replaceanimal body immune effects, so this study chooseas mink a research object. Biodegradablepolysaccharide microsphere vaccine intranasal immunization American Shorthair black mink,research changes in antibody levels and immune effects by HE staining, indirect ELISA andhistological. Reveal the protective effect of the microspheres vaccines through absolutely lethaldose of Pseudomonas aeruginosa isolates mink challenge protection experiments. The resultsshowed that mannose-modified microspheres vaccine group FI-MCS-MPs and FI-MCP-MPs weresignificantly higher serum IgG antibody levels in the initial immunization, and continued until theend of the immune observed. Chitosan microspheres vaccine can significantly improve theabsolutely lethal poison attack mink aeruginosa survival, FI-CS-MPs, FI-CP-MPs, FI-MCS-MPsand FI-MCP-MPs group of mink survival were62.5%,62.5%,75%and62.5%. However, due topoison attack during the experiment process for acute infection, so there was no significantdifference between mannose-modified chitosan microsphere vaccine group and withoutmannose-modified chitosan microsphere vaccine group. Histological observation, chitosanmicrospheres vector vaccine can significantly reduce the mink of Pseudomonas aeruginosa attacklung and spleen inflammation.In conclusion, this study successfully prepared mannose-modified chitosan biodegradablemicrospheres vaccine (FI-MCS-MPs and FI-MCS-MPs) containing Pseudomonas aeruginosa outer membrane fusion protein FI. It can be targeted to macrophage mannose receptor in vivo andvitro. After intranasal immunization BALB/c and mink, mannose-modified chitosan biodegradablemicrospheres vaccine can significantly enhance early mucosal and systemic humoral and cellularimmune responses, showing a mixed Th1/Th2response. Pseudomonas aeruginosa in mice andmink attacks have good protection rate. Thus, mannose-modified chitosan biodegradablemicrosphere carrier is a promising biodegradable carrier intranasally vaccines. Biodegradablepolysaccharide microspheres vaccines provide reliable theoretical and experimental basis in minkand other economic fur animals. |
