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Mentions: A prokaryotic expression cassette was developed in which the gfp gene was fused in-frame with an E coli β-galactosidase α-fragment sequence (N-terminus) (Figure 1). The gfp gene was amplified and cloned into pGEM-Teasy plasmid vector. The β-galactosidase α-fragment with DNA sequence (5'-ATG ACC ATG ATT ACG CCA AGC TAT TTA GGT GAC ACT ATA GAA TAC TCA AGC TAT GCA TCC AAC GCG TTG GGA GCT CTC CCA TAT GGT CGA CCT GCA GGC GGC CGC GAA TTC ACT AGT GAT-3') had 24 amino acids (MTMITPSYLG DTIEYSSYAS NALGALPYGR PAGGREFTSD) and the peptide was 4.2 kDa in size. A small linker (L) sequence with 15 codons (5-TAT GGC GCC AAA GAC TCC GGC TCC GCC GGT TCC GCC GGC TCA GCT-3) was incorporated between the β-galactosidase α-fragment and gfp. The linker peptide had 15 amino acids (YGAKDSGSAG SAGSA) and a molecular weight of 1.266 kDa. The gfp gene had 237 amino acids (SKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV TTFSYGVQCF SRYPDHMKRH DFFKSAMPEG YVQERTISFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY ITADKQKNGI KANFKIRHNI EDGSVQLADH YQQNTPIGDG PVLLPDNHYL STQSALSKDP NEKRDHMVLL EFVTAAGITH GMDELYK) and a molecular weight of 26.6 kDa. The GFP contains a Balb/C mouse CD8+ T cell epitope, HYLSTQSAL. The whole β-galactosidase-GFP fusion protein was 32.1 kDa. A preferred translation stop codon (TAAG) which was incorporated in the PCR primer, GR, was found at the end of the gfp gene. There was also an extra stop codon, TAAT, one codon downstream the end of the gfp gene.
An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice
Bottom Line: It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay.The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05).The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05).
Affiliation: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Anzio Rd, Observatory 7925, Cape Town, South Africa. Nyasha.Chinombe@uct.ac.za
Background: The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated.
Results: High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-gamma and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05).
Conclusion: These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.