Studies On The Preparation Of New DNA-loaded Nanoparticle Vaccine For Anti-caries And Immunological Effect

In recent years, with the improvement of life quality and development of agingsociety, more and more people begin to pay attention to oral hygiene. Incidence of dentalcaries is high, just second to the flu. Mild dental caries affect chewing function. Severecaries can cause periapical and dental pulp disease, complications such as oralmaxillofacial inflammation, seriously affect human health and quality of life. Due to thelarge population, limited medical resources in our country, anti-caries vaccine which canprevent tooth decay, as well as reduce the incidence of caries disease is particularlyimportant to guarantee the national health.Streptococcus mutants (s. mutants) are generally considered to be one of the mainfactors to cause caries. One of surface molecules response for the pathogenicity of s.mutants is wall-associated protein A(wapA), which participates in s. mutants adhesionand plaque formation.In the first part, We amplified the wapA fragment from strain UA159and cloned itinto the plasmid pVAX1to construct a new recombinant plasmid pVAX-wapA as ananti-caries DNA vaccine and established its prokaryotic expression system escherichiacoli (e. coli). The results of restriction digestion and DNA sequencing confirmed thesuccess of construction correct recombinant plasmid, without frameshift mutations andpieces of DNA missing. The positive clones were propagated and a large amount ofplasmid pVAX-wapA was extracted by alkaline lysis method. Purified plasmid was usedfor the following prescription design and evaluation of in vivo and in vitro experiments.The second part, we expressed wapA protein in Rosetta-gami B prokaryoticexpression system. The time, temperature and IPTG concentration of inductionexpression condition were optimized, results showed that the best induction conditionswas0.1mM IPTG,37oC,5h. After purification by affinity gel filtration chromatography,we got5.2mg,90%purity of recombinant protein wapA.In the third part, methyl acryloyl oxygen ethyl trimethyl ammonium chloride(TMAEMC) modified chitosan was synthesized by free radical polymerization.Quaternary ammonium chitosan (CSTM) has good solubility in water. We used H1-NMRand FT-IR to confirm its structure. The quaternary ammonium degree of CSTM is52%.We established a plasmid quantitative fluorescence spectrophotometry, optimal condition was emission wavelength of464nm, excitation wavelength of350nm, slit width5nm.Plasmid concentration within the scope of0.07-1.75ug/ml linear good, recovery rate was97.6-99.9%. Chitosan and its derivatives CSTM were used as carrier materials for blanknanoparticles preparation. We observed related effects of the crucial factors for theformation of nanoparticles: chitosan solution concentration, PH value, N/P ratio and TPPconcentration, with nanoparticles size and zeta potential as index. Based on the previouswork, the preparation methods of chitosan nanoparticles encapsulated plasmids wereoptimized. Ultimately determine the conditions for CS solution concentration is0.25mg/ml, pH5.5buffer system,0.02mM TPP, pVAX-wapA150ng/μl, and CSTM solutionconcentration of5mg/ml, pH5.0buffer system,0.02mM TPP, pVAX-wapA300ng/μl.CSTM nanoparticles size is222.5nm, Zeta potential of19.6mV, the encapsulationefficiency was87.66%, drug loading rate of4.96%. CS nanoparticles size is219.2nm,Zeta potential of24.7mV, encapsulation efficiency was91.24%, drug loading rate of34.22%.In the fourth part, Cell transfection test was carried out to indicate that improvementof cell transfection efficiency of nanocarrier. Results showed naked plasmidpVAX1-wapA transfection cells almost undetectable wapA mRNA transcription of genes.With lipo2000pVAX1-wapA transcription mRNA level was1, CSTM NPs loadedpVAX1-wapA transcription mRNA level was0.87,3.18fold of chitosan nanoparticlesloaded pVAX1-wapA. In vitro cell transfection results showed that CS nanoparticles andCSTM nanoparticles could help pVAX1-wapA enter cells, and transcribed successfully.The naked plasmid almost cannot be translated.In the fifth part, early builded DNA vaccine and drug delivery system were used toimmune normal mice, saliva IgA and serum IgG levels were determined by ELISA. Itwas found that DNA vaccine can successfully stimulate the immune system in micethrough muscle injection, raise IgG antibody levels, proved that pVAX1-wapA hadimmunogenicity. CS nanoparticles delivery systems can improve the immunogenicity ofthe vaccine via intramuscular injection, but the intramuscular immunization does notinduce mucosal immunity, the experimental results observed no IgA antibody levels. Tomeasure specific anti-wapA IgG and IgA antibodies, indirect ELISA method wasestablished. Variation coefficient of repetitive experiments between batch and batch of isless than10%. In order to further confirm our DNA vaccine anti-caries effect, cariesmodel rats were established. Nasal immunization DNA vaccine nanoparticles group showed minimum caries score and this group’s saliva IgA and serum IgG antibody levelswere also significantly increased. Intramuscular injection of DNA vaccines and DNAvaccine nanoparticles group had the loss of scores, but the score higher thannanoparticles nasal immunization group, although intramuscular injection of DNAvaccine and DNA nanoparticles induced the high levels of serum IgG, but saliva IgAlevels were lower than the nasal immunization group. Results showed that CSTMnanoparticles have good immune effect, good for the carrier of nasal immunization.To sum up, we construct a new recombinant plasmid pVAX1-wapA.DNA vaccinepVAX1-wapA elicited mucosal and systemic immune responses with or withoutnanoparticles delivery vector and showed caries protection activity, suggesting that wapAprotein can be a candidate for anti-caries vaccine. CSTM is a chitosan derivatives whichexerts advantages as a vaccine carrier due to its immune stimulating activity andbioadhesive properties that enhance cellular uptake and antigen protection. We usedCSTM nanoparticles as the DNA vaccine delivery system against dental caries for thefirst time. The superiority of delivery system over naked plasmid was attractive in boththe in vivo and in vitro outcomes evaluated. CSTM nanoparticles were proved to be anefficient DNA vaccine delivery vector on the basis of the cell and animal experimentalresults. Of particular concern is the mucosal adjuvant effect of CSTM nanoparticle. It canbe applied to caries prevention.