Construction Of A Dual-Promoter Plasmid PCN-SSIE And Verification Of Its Immunogenicity As The DNA Vaccine

Dental caries is one of the most common diseases all over the world. From the latest data of WHO, the incidence of dental caries in developed courtries declined during the last 30 years, but it increased in developing courtries. It is important to find an effective and cheap vaccine against caries that is well adapted to our courtry. Streptococcus mutans is regarded as the main pathogenic bacteria of caries, because of its strong ability to adhere to tooth surface and to ferment sugar into acid. The surface protein antigen â… /â…¡ (Agâ… /â…¡) of Streptococcus mutans is one of virulence factor, and play a key role in adhering to tooth surface. A fragment of Agâ… /â…¡ rich in alanine near N-terminal called Saliva-binding region (SBR) was selected as the antigen in order to decrease the side effects of vaccine. Our purpose of this experiment is to construct a dual-promoter expression plasmid that harbors the target gene encoding SBR and can be applied as DNA vaccine especially suitable for using attenuated Salmonella as delivery vector to elicit effective mucosal immune responses because of its advantage of possessing dual-promoter.Genes encoding SBR and enhenced green fluorescence protein (EGFP) were amplified by PCR and inserted to the proper sites of vector pCMVnir. Then internal ribozyme entry site (IRES) sequence was inserted between the genes coding for SBR and EGFP. Furthermore, a DNA fragment encoding tissue-type plasminogen activator (tPA) signal peptide was fused to the 5'-end of target gene. Thereby, construction of the dual-promoter expression plasmid pCN-SSIE was completed and then the plasmid was analyzed with DNA sequencing and endonuclearase digestion mapping. The expressions of SBR protein by attenuated Salmonella SL3261 and CHO cell transformed or transfected by the plasmid were tested respectively by detecting thegreen fluorescence and Western blot. BALB/c mice were immunized through injecting intramuscularly with plasmid pCN-SSIE and anti-SBR specific IgG in serum was tested. Finally, attenuated Salmonella SL3261 carried the plasmid pCN-SSIE immuned BALB/c mice by oral administration, and anti-SBR specific IgG and IgA in serum and saliva were tested.Both DNA sequencing and endonuclearase digestion mapping showed that the construction of pCN-SSIE was succeeded. The inserted sequence and its open reading frame are correct. The expressions of SBR protein in transformed attenuated Salmonella SL3261 and transfected CHO were detected, and anti-SBR specific IgG levels in serum of immunized mice were markedly higher than the control. These results indicated that the plasmid can be expressed in vitro and in vivo both in prokaryocyte and eukaryocyte and cause effective immune response. Anti-SBR IgG and IgA levels in serum and saliva of BALB/c mice immuned by attenuated Salmonella increased respectively.The construction of the dual-promoter expression plasmid pCN-SSIE was successful which is especially suitable for using attenuated Salmonella as delivery vector. The plasmid can express in vitro and in vivo both in prokaryocyte and eukaryocyte, and elicit dramatic immune response in experimental animal. Oral administration of this DNA vaccine by attenuated Salmonella vector can induce BALB/c mice's highly immune response, especially the humoral immune response of mucosal immune system.