Temperature-sensitive Biodegradable Hydrogel Vector Anti-caries Gene Vaccine Immunization In Different Ways Experimental Study Of SD Rats

Objective:To investigate the efficiency of pVAXl-spap/A,pVAXl-gtfB/CAT, pVAXl-spap/A-gtfB/CAT to hydrogel as a carrier immunized in SD rats by different routes, and to explore the best means immune under the condition of the hydrogel.Methods:This study consists of three parts:Test one:Identification and preparation of recombinant plasmid pVAX1-spap/A,pVAXl-gtfB/CAT,pVAXl-spap/A-gtfB/CATand plasmid pVAXl. Firstly.the plasmid of pVAXl-spap/A,pVAX1-gtfB/CAT,pVAXl-spap/A-gtfB/CAT and PVAX1 were prepared in a small quantity by TIANprep Mini Plasmid Kit.Secondly, to identify those plasmids with restriction endonuclease digestion and gel electrophoresis. Thirdly, pVAXl-spap/A,pVAXl-gtfB/CAT,pVAXl-spap/A-gtfB/CAT and pVAXl were purified and measured in large quantity with E.Z.N.A. Endo-Free Plasmid Giga Kit, which were up to the requirements of the animal immunition, on the basis of correct identification.Test two:According to observe hydrogel P(CL-GA)-PEG-P(CL-GA) surface morphology of scanning electron microscopy (SEM),testing its sensibility of temperature and investigate the efficiency of pVAXl-spap/A,pVAXl-gtfB/CAT and pVAX1-spap/A-gtfB/CAT to hydrogel as the carrier in vitro release.Test three:To immunize SD rats with the recombinant plasmids to hydrogel as the carrier in order to observe the effect.Ninety 3-week-old male SD rats were randomly divided into 15 groups of 6 rat.A:vaccinated with pVAXl-spap/A via intramuscular route;B:vaccinated with pVAX1-spap/A via subcutaneous injection in the submandibalar gland region route; C:vaccinated with pVAXl-spap/A via intranasal route;D:vaccinated with pVAXl-spap/A via oral administration;E:vaccinated with pVAX1-gtfB/CATvia intramuscular route;F:vaccinated with pVAX1-gtfB/CATvia subcutaneous injection in the submandibalar gland region route;G:vaccinated with pVAXl-gtfB/CATvia intranasal route; H:vaccinated with pVAXl-gtfB/CATvia oral administration;I:vaccinated with pVAXl-spap/A-gtfB/CAT via intramuscular route;J:vaccinated with pVAXl-spap/A-gtfB/CAT via subcutaneous injection in the submandibalar gland region route; K:vaccinated with pVAXl-spap/A-gtfB/CAT via intranasal route;L:vaccinated with pVAXl-spap/A-gtfB/CAT via oral administration;M:vaccinated with pVAXl via intramuscular route(negative control group);N:vaccinated with pVAX1 and hydro gel via oral administration;O:vaccinated with saline via intramuscular route.Establish animal model of dental caries with SD rats.Every rat was immuned a dose of 100μg in a week and for three times in all(saline group, 100μl/only).Vaccinated 50μg each side of intramuscular route group,subcutaneous injection in the submandibalar gland region route group and intranasal route group,From 28 days start immune,weekly,continuous three weeks. Then some important organs (heart,liver,spleen,lung, kidney,quadriceps muscle of thigh,stomach,submandibular gland,schneiderian membrane) of the rat were separated for pathological examination. Using enzyme-linked immunosorbent assay (indirect ELISA) to test the level of specific antibodies S-IgA in saliva and IgG in serum.Results:①pVAX1-spap/A,pVAXl-gtfB/CAT,pVAX1-spap/A-gtfB/CAT and pVAXl were isolated and purifiedy by large Omega Plasmid Giga Kit in order to meet the requirements of the study;②The Lower Critical Solution Temperature(LCST) is 37℃of the P(CL-GA)-PEG-P(CL-GA);Plasmid pVAX1-spap/A,pVAX1-gtfB/CAT, pVAXl-spap/A-gtfB/CAT can slowly release from P (CL-GA)-PEG-P (CL-GA) for the sustained release system.③The antibodies of S-IgA in saliva and IgG in serum were significantly increased and continued for several weeks after immunization in SD rats.④The level of caries are significantly lower in experimental groups than the negative control groups with significant statistics (P<0.05).Conclusions:①The experiments show that temperature-sensitive biodegradable hydrogel P (CL-GA)-PEG-P (CL-GA) has a temperature sensitivity, in the solution phase when 37℃-gel phase transition;②pVAX1-spap/A, pVAXl-gtfB/CAT and pVAX1-spap/A-gtfB/CAT hydrogel as a delivery system,can effectively slow down the release of plasmid DNA, can play a slow role release;③Gene vaccine pVAXl-spap/A,pVAXl-gtfB/CAT and pVAXl-spap/A-gtfB/CAT hydrogel as a delivery system can induce to improve markedly the level of specific in SD rats,incidence of dental caries in rats.