Immunogenicity Of Genetic Recombinant Vaccines Against Foot-and-Mouth Disease Virus (FMDV)

Along with the great development of molecular biology and genetics and immunology,many kinds of new generation vaccine had been prepared with the character of higher purity and specificity,but they were hard to induce the orgnism to produce the effective immune response for their small molecule and weak immunogenicity.So it need to some kinds of material to be in accordence with this effect,under the cirsumstance, immunoadjuvant was to be braught about in order to inhance the immunogenicity and the conservitive response of host to the antigen.to utilize the technology of molecular biology immunology molecular virology biochemistry vacuum freeze-drying and so on ,we prepared the chitosan nanoparticle with the function of adjuvant and delivering carrier.in other wise we preliminary screened two sets of recombined Variola avium virus vivi-carrier vaccine freeze-drying protective agent,and evaluated their function in vivo of mouse and pig,and also detected immunogenicity of FMD recombined Variola avium virus vivi-carrier vaccine and nucleinic acid vaccine.As fallowof outline content:Chitosan is a kind of natural positive ion glycans,it can be degraded and has favourable biocompatibility and histocompatibility,can effectively bond with DNA and protect it from degrading by nuclease.As it is electropositivity and easily approched to cellular membrane,it can be conducted as a new kind of non-viral vector,make the plasmid sended to intra-cellular,prior to this, chitosan can be absorbed by APC to induce much stronger immunoreaction.We used the physico-chemical property of Chitosan to prepared the nanoparticle gene-carrier with ambo- coacervation,it indicated that nanoparticle had narrow distribution range and the unifom size.zeta electric potential is an important parameter to measure colloid disperse system, the result of nanoparticle particle size analysis displayed,gene-carrier chitosan nanoparticle had positive charge,and 21mV's zeta electric potential,illustrated that the chitosan nanoparticle prepared in the experiment was stable in the given time. For chitosan nanoparticle had positive charge ,but negative charge on the surface of cellular membrane, they were easily to combine with each other,so chitosan formed endosome insuring the normal process of particleis being swallowed. Gel-blocking analytic result indicated chitosan could wrap up plasmid DNA avoiding binding with EB dye,so foreign matter was impossible contacted with DNA to protect plasmid DNA from degrading.Results of indirec immunofluorescence and RT-PCR showed that nanoparticle could deliver plasmid gene to intra-cellular ,expressed under the mRNA level,and certificated the possiblity as gene delivering carrier.As borreliola avium virus was sensitive to heating and freeze thawing again and again,it needed hash store condition to guarantee its immunization effect.We optimized freeze-dry curve and freeze-dry protective agent prescription making use of vacuum freeze-drying technology, preliminary screened team V and VI protective agent prescription and a set of eligible freeze-dry curve,as using that protective agent prescription, freeze dried vaccine had better physical character, virus titer decreased lower than 0.1 LgPFU/ml before and after freeze-drying,three weeks under 37℃, virus titer decreased lower than 1 LgPFU/ml, accord ed with nation pharmacopoeiai's standardization.Based on the O-type FMDV multi-epitope recombinant mVP1 nucleinic acid vaccine plasmid constructed by our discussion orgnization, and assisted by gene-carrier chitosan nanoparticle, adjuvant A and superantigen SEA immunized Balb/c mouse, to compare the immune enhancement effect with different adjuvant.According to indirect ELISA,specific CTL kill activity and T lymphocyte subtype detection,the level of cytoimmunity exp group induced significantly higher than control group, chitosan group also higher than other adjuvant group. the number of T lymphocyte subtype existed in mouse SPC in djuvant A group slightly higher than other groups,but the difference was not significantly.Based on DNA vaccine and recombinant fowl-pox virus vaccine constructed by our discussion orgnization,chose better enhancement effect adjuvant to immunize pig, and through lymphproliferation response, neutralizing antibody experiment and liquid phase interruption ELISA to detect the immunization effect.Results showed, specific humoral immunity and cellullar immunologic response induced by recombinant virus higher than DNA vaccine.in adjuvant groups,the effect of chitosan was the best,and all the results matched with the experiment lay the foundation to the effective immune presentation and industrialization of genetically engineering vaccine.