Study On Developing Vaccines Against Saliva Binding Region Of The Surface Protein Antigen From Streptococcus Mutans Aiming To Prevent Dental Caries

Dental caries is one of the most common diseases in human being. Ever since it was confirmed that dental caries is an infectious disease caused by bacteria, researchers have done a great deal of studies on the etiology and immunology concerning the disease searching for the main pathogen and main virulence factors of the pathogen which can be used as vaccine candidate, in order to develop anti-caries vaccine preventing cariogenic pathogens from initial colonization to the tooth surfaces and eventually preventing the development of dental caries.A great deal of study results indicated that Streptococcus mutans (S.mutans) is closely associated with the initiation and development of dental caries and is considered as the main causative pathogen of the disease. Reports from different research groups showed consistently that the functional domain of surface protein antigen I/II which is associated with the initial colonization of this microbe to tooth surface is located at the N-terminal one third of the molecule and play an important role in the development of dental caries and is termed saliva-binding region (SBR). Therefore, SBR become the main target in the study on prevention dental caries.In order to explore effective ways to control dental caries, we have developed the sub-unit vaccine and the genetic vaccine against SBR of S. mutans, and conducted a series of related experiments, and confirmed their immunogenicities. Firstly, through PCR and T-A cloning, we constructed a eukaryotic expression plasmid pTriEx-4-SBR, and the gene encoding SBR in the plasmid was expressed in E. coli and the protein was purified. Then we applied SBR protein to immunize mice through salivary gland to explore the its feasibility of eliciting the immune response as sub-unit vaccine and changes of related cytokines in local tissues after immune inoculation. After the above experiments were done, we began the project to construct a dual-promoter expression plasmid pCN-SSIE by using a new vector pCMVnir into which the DNA fragment encoding SBR were inserted and transform the attenuated SalmonellaSL3261 with the resultant recombinant plasmid pCN-SSIE。 Then mice were immunized through being injected with naked plasmid pCN-SSIE muscularly and through being fed with transformed Salmonella,respectively. The primary results indicated that both methods can elicit dramatic specific immune responses against SBR of S.mutans. Followings illustrate the main contents of the study.Part 1: Construction of a Prokaryotic Expression Vector pTriEx-4-SBR Containing DNA Fragment Encoding SBR of Streptococcus mutans and Preparation of SBR of Streptococcus mutans as Subunit VaccineMETHODS:1. According to the sequence of the plasmid pcDNA3.1-SBR, a pair of primers specific for amplifying the DNA fragment encoding saliva-binding region in surface protein Antigen I/II molecule were designed and synthesized. The targeted DNA fragment encoding SBR was amplified from pcDNA3.1-SBR by PCR;2. After being purified, the product of PCR was ligated into a clone vector pMD 18-T;3. The recombinant plasmid, pMD18-T-SBR was first propagated in E.coli DH5a, then extracted, purified and digested with NcoI and XhoI, was confirmed containing the DNA fragment encoding SBR by agarose gel analysis;4. The resulting NcoI-XhoI fragment which contained the SBR DNA was purifiedand ligated into prokaryotic expression vector pTriEx-4 digested with NcoI and XhoI with T4 DNA ligase. The pTriEx-4-SBR was confirmed to contain DNA sequence encoding SBR by agarose gel electrophoresis and DNA sequence analysis.5. Escherichia Coli JM109 (DE3) was transformed with pTriEx-4-SBR, and thenthe transformed E. coli was cultured to express SBR protein;6. SBR were purified with the HisTrap? protein purify kit。 RESULTS:1. The construction of the expression recombinant plasmid pTriEx-4-SBR was confirmed through restriction enzyme mapping analysis and DNA sequencing;2. SDS-PAGE showed that pTriEx-4-SBR in JM109(DE3) can express a specific protein (43.5kDa) which was conform to the expe...