Preparation And Characterisation Of Chitosan-based Anti-caries Gene Vaccine Carrier

Objective: Using chitosan as the main component of the carrier,prepare a chitosan/sodium alginate composite carrier carrying the anti-caries gene vaccine,and estimate its physical and chemical properties and biological properties through in vitro experiments and animal experiments.Methods:1.Preparation of chitosan/sodium alginate(CS/SA)composite carrier: Mix chitosan solution with a small amount of sodium alginate and keep the whole solution at positive charge state,and then sodium tripolyphosphate is added to make it cross-linked,and than coat a layer of sodium alginate on the outside.Use calcium ions cross-link with sodium alginate to form a calcium shell layer.Finally a composite with chitosan and sodium alginate was prepared.2.Preparation of chitosan/sodium alginate composite carrier loaded with p VAX1-Spap/A plasmid: Based on the preliminary grasp of the appropriate conditions(stirring speed,dropping rate,reagent ratio,temperature,etc.)of the carrier preparation in the previous experiment.Add the extracted genetic vaccine-p VAX1-Spap/A plasmid into the chitosan solution,and add a small amount of protamine DNA into the solution to reduce the degradation of the genetic vaccine by the nuclease in the body environment when it is released.Then add sodium tripolyphosphate to make it cross-linked,and make it coat with a layer of sodium alginate.Control the ratio of the inner chitosan and the outer sodium alginate,and test the physical and chemical properties of the composite carrier in an in vitro simulated environment3.Animal experiment: 21 clean-grade female SD rats were randomly divided into 3 groups,which are the blank control group,the CS/SA vaccine carrier group,and the empty carrier group.Immunization was started on the 28 th day.The experimental group was immunized with a dose of 1 m L(100 μg/m L)each for 3 weeks.Blood and saliva were collected 1 day before the first immunization and 7 days after each immunization until the 7th week.The rats are sacrificed after the 7th week sample collection.Using the indirect ELISA method to detect the change levels of Ig G in serum and Ig A in saliva during 1-7 weeks.Results:1.The CS/SA gene vaccine composite carrier loaded with p VAX1-Spap/A plasmid was prepared on the basis of the preparation of the empty carrier.The composite carrier has good mechanical properties,and its freeze-dried powder presents a porous agglomerated structure,and has a good encapsulation rate.The encapsulation rate under various CS/SA ratios is about 90% or more.The release experiment of the carrier loaded with the gene vaccine in an in vitro simulated gastric juice environment(p H=2.1)shows that the release rate of the vaccine within 24 hours is 0.3%-3.2%.The release experiment in an in vitro simulated intestinal fluid environment(p H=7.4)shows that the release rate of the vaccine within 24 hours is 31.2%-68.7%.2.The CS/SA composite carrier group loaded with recombinant plasmid p VAX1-Spap/A is immunogenic and can induce the body to produce specific antibodies.The difference in antibody levels compare to the control group(blank control group and empty carrier group,the same below)is statistically significant.Conclusion:1.The CS/SA composite carrier loaded with the p VAX1-Spap/A plasmid exhibits a p H-sensitive,controls and slow-releases effect in vitro,and shows a certain degree of protection for genetic vaccines in a low p H environment.2.The antibody levels between the CS/SA composite carrier loaded with plasmid p VAX1-Spap/A and the control group is statistically different,and it can carry the gene vaccine to cause an immune response.