Temperature-sensitive Biodegradable Hydrogel Vector Anti-caries Gene Vaccine PVAX1-SpaP/P、pVAX1-GbpA/GBD And PVAX1-SpaP/P-GbpA/GBD Immunization In Different Ways Experimental Study Of SD Rats

Objective:To observe the best effective route of pVAX1-SpaP/P,pVAX1-GbpA/GBDand pVAX1-SpaP/P-GbpA/GBD with hydrogel as a carrier immunized in SD rats bydifferent routes.To explore the application of genetic vaccine used hydrogel as a sustainedrelease system and the safety index of gene vaccine by pathological examination.Method: The experiments include two parts:Part one: Identification and preparation of recombinant plasmid pVAX1-SpaP/P,pVAX1-GbpA/GBD, pVAX1-SpaP/P-GbpA/GBD and plasmid pVAX1. Firstly,the plasmid ofpVAX1-SpaP/P, pVAX1-GbpA/GBD, pVAX1-SpaP/P-GbpA/GBD and PVAX1wereprepared in a small quantity by Mini Plasmid Kit. Secondly, to identify those plasmidswith restriction endonuclease digestion and gel electrophoresis. Thirdly, pVAX1-SpaP/P,pVAX1-GbpA/GBD, pVAX1-SpaP/P-GbpA/GBD and pVAX1were purified andmeasured in large quantity with Endo-Free Plasmid Kit, which were up to the requirementsof the animal immunition, on the basis of correct identification.Part two: Immunization of SD rats by pVAX1-SpaP/P、pVAX1-GbpA/GBD and pVAX1-SpaP/P-GbpA/GBD with hydrogel as a carrier. The experimental animals are divided intoseveral groups. There are A、B、C three large group with24rats in each group, wererandomly divided into4groups, each group with6rats in each group. RespectivelypVAX1-SpaP/P, pVAX1-GbpA/GBD, pVAX1-SpaP/P-GbpA/GBD three plasmids, infour ways: the stock four myo–injection group, submandibular area subcutaneousinjection group, the nasal mucosa Infusion group, oral group. Theexperimental animals wererandomly divided into3groups,6rats ineach group. D1 group: pVAX1stock four myo-injection group;D2:empty vector pVAX1oral group; group D3: normal saline stock four myo-injection group.Establishmentof dental caries model; immune measurement: each dose is100μg,Intramuscularinjection group,submandibular gland injection group and nasal drip group on each sideof the50μg. After the3time, twenty-eighth days to start the immune, once a week,three weeks of continuous immune; sampling respectively inimmune1-10weeks afterthe collection of saliva and serumsamples;After dissection, weighing, heart, liver, spleen,lung, kidney and other organs for pathological examination; Keyess caries score;Detection of IgG antibody levels in blood and saliva S-IgA antibodies by ELISA;Immunohistochemistry.Results:①Using macro plasmid purification kit to extract the high purity and highconcentration of recombinant plasmid pVAX1-spap/P, pVAX1-gbpA/GBD,pVAX1-spap/P-gbpA/GBD and empty vector pVAX1.②Expression of recombinant protein foundin the immune animal. The stock four myo, intestinal mucosa of nasal mucosa andsubmandibular gland tissues.③Immune SD rats, can induce S-IgA specific antibodiesand IgG specific antibodies were significantly increased, and for several weeks.④Cariesassessment, with statistical significance (P <0.05).Conclusion:①The anticaries DNA vaccine pVAX1-spap/P, pVAX1-gbpA/GBD,pVAX1-spap/P-gbpA/GBD in the hydrogel as a sustained release system conditions, can effectivelyslow down the release of plasmid DNA, specific antibody could induce SD rat blood andsaliva production, reduce the incidence of dental caries in rats.②Expression ofrecombinantprotein found in the immune animal.The stock four myo, intestinal mucosa of nasalmucosa and submandibular gland tissues.