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ABSTRACT: To construct a dual-promoter expression plasmid that harbors the target gene encoding SBR of Streptococcus mutans 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 green fluorescence protein gene (EGFP) were amplified by PCR and inserted to the proper sites of vector pCMVnir. Then 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. Finally, BALB/c mice were immunized through injecting intramuscularly with plasmid pCN-SSIE and anti-SBR specific IgG in serum was tested.
Both DNA sequencing and endonuclearase digestion mapping showed that the construction of pCN-SSIE was successful with its open reading frame being 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.
The construction of the dual-promoter expression plasmid pCN-SSIE was successful and the plasmid can express in prokaryocyte and eukaryocyte and elicit dramatic immune response when applied as DNA vaccine in experimental animal.
Shanghai kou qiang yi xue = Shanghai journal of stomatology 11/2005; 14(5):479-84.