Construction Of A Dual-Promoter Plamid PCN-SSISG And Verification Of Its Immunogenicity As The DNA Vaccine

Background and Objective:Dental caries is a common disease with high prevalence in humans and is considered by modern etiology as an infectious disease caused by bacteria and related to many factors among which the formation of dental plaque on the surface of teeth is essential and prerequisite.Many researches have indicated that Streptococcus mutans (S.mutans)is of great importance in the formation of dental plaque and the process of dental caries,and is regarded as the main causative organism of human dental caries. Attachment to,and accumulation on tooth surface is vital for the cariogenicity of S.mutans.So the study of immune means against dental caries,by way of active or passive immunization,has been focusing on preventing S.mutans from adhering to tooth surface by blocking adhensins and inactivating virulence factors on S.mutans' surface that play an important role in S.mutans' adherence.In recent years with the rapid development of gene therapy much more attention has been paid on DNA vaccine for dental caries.In previous studies we have successfully constructed the dual-promoter expression vector-pCN-SSIE harboring Saliva-binding region(SBR) gene and prokaryotic expression vector-pTriEx-4-SBR containing the Glucan-binding region(GBR)gene of S.mutans GTF-I,and also proved their immunogenicity.In the following study we are going to construct a dual-promoter bivalent recombinant plasmid pCN-SSISG containing SBR and GBR to test its expression in attenuated Salmonella as delivery vector and its immune effects elicited in BALB/c mice when administered orally.Methods:According to the sequence of the glucosyltransferase(gtfB and gtfC)genes of S.mutans published by GenBank,a pair of primers was designed and synthesized which was specific for amplifying the DNA fragment encoding Glucan-binding region (GBR),with the 5' end of the upstream primer containing the site of restriction enzyme Hindâ…¢and that of the downstream primer including XhoI site.The targeted DNA fragment encoding GBR was amplified from the plasmid harboring the gtfB gene of S.mutans by PCR.Then the GBR gene with the upstream linked DNA fragment which encodes tissue-type plasminogen signal peptide(tPA-SP)was directedly cloned into the plasmid pCN-SSIE which contains sSBR gene encoding tPA-SP and saliva binding region(SBR)of antigen proteinâ… /â…¡(Agâ… /â…¡)from S. mutans to form plasmid pCN-SSISG.After being proved to be successfully constructed by DNA sequencing and endonuclearase digestion mapping,the plasmid pCN-SSISG was employed to transform attenuated Salmonella SL3261 and then the recombinants were used to immunise BALB/c mice by oral administration.The expressions of SBR and GBR proteins in attenuated Salmonella SL3261 and the humoral and mucosal immune responses induced by attenuated Salmonella SL3261 carrying the plasmid pCN-SSISG were tested respectively by ELISA and Westernblotting.Results:The results of agarose gel electrophoresis and DNA sequence analysis proved the successful construction and correct open reading frame of recombinant plasmid pCN-SSISG.SDS-PAGE showed that pCN-SSISG in JM109(DE3)can express two specific fusion proteins which conformed to the expectancy.The expression was also confirmed by Western blot.These results indicated that the plasmid can work properly in prokaryocyte.The murine antibody IgG which was elicited by oral administration in BALB/c mice was specifically combined with recombinant SBR and GBR proteins when tested by ELISA and Western blot.The result of ELISA showed that there were considerably higher levels of specific antibody IgG and IgA in serum and saliva of BALB/c mice orally immunized by attenuated Salmonella SL3261 carrying the plasmid pCN-SSISG than those of control group.Conclusion:We have successfully used molecularbiological techniques such as PCR,T-A cloning,directed cloning and so on,to insert SBR and GBR gene into expression vector pCMVnir to construct a new dual-promoter plasmid pCN-SSISG as bivalent anti-caries DNA vaccine especially suitable for using attenuated Salmonella as delivery vector.Both SBR and GBR protein can be expressed well in attenuated prokaryotic Salmonella.And this DNA vaccine pCN-SSISG which is contained in attenuated Salmonella can elicit dramatic immune response in experimental animals. All these efforts have laid a solid basis for future studies.