Construction And Expression of an Anti-caries Plasmid pCDGLU/GFP

Objectives: The cellular localization of an antigen expressed from a DNA vaccine can greatly influence the strength and Th-bias of the resulting immune responses. Here we constructed a Green fluorescent protein (GFP)-labeled anti-caries plasmid pCDGLU/GFP encoding secreted form of Streptococcus mutans glucan-binding domain and observed the protein expression both in vitro and in vivo, in order to get some understanding of the mechanism following anti-caries DNA vaccine immunization. Methods: The CD5 leader sequence (CD5L) for secretion was cloned from the human T cells. pCDGLU/GFP was then constructed by replacing the CTLA4 fragment of pGJGLU/GFP with the CD5L. The COS7 cells were transfected with the pCDGLU/GFP plasmid using the Sofast^TM transfection reagent and the GFP fusion protein was observed under a fluorescence microscopy 36 hours later . The secreted protein in the supernatant was analyzed by ELISA. BALB/c mice were either injected with the naked pCDGLU/GFP plasmid via the intramuscular route or delivered with the pCDGLU/GFP-Sofast^TM complex via the intranasal route. 36 hours later, the muscle tissue and the nasal-associated lymphoid tissue (NALT) were colleted for the frozen sections. And the protein expression in vivo was observed under a fluorescence microscopy. Results: Sequencing and electrophoresis results showed the correctness of the cloned CD5L fragment. The GFP fusion protein expression was evident after transfection in vitro and represented a granular form. The concentration of the secreted protein in the supernatant was 340pg/ml. The frozen sections of the injected muscle tissue showed that the granular GFP fluorescence was present both in the plasma and under the membrane of the myocytes. Speckled GFP fluorescence was also observed in the NALT, although it was much weaker as compared with that in the myocytes. Conclusions: The GFP-labeled anti-caries plasmid pCDGLU/GFP was constructed and could express proteins both in vitro and in vivo. Supported by NSFC grant 30330660.