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Induction of antigen-specific cytotoxic T lymphocytes (CTLs) by plasmid DNA immunization: What are the requirements for promotor driving the antigen expression?
The immune system has a powerful ability to react against foreign protein antigens in a very specific manner. This allows mounting of antigen-specific immune responses by means of immunization.
We study induction of antigen-specific antibodies and cytotoxic T lymphocytes (CTLs) after immunizations with plasmid DNA encoding an antigen of interest. Plasmid DNA used for immunizations should contain an antigen under the control of a strong viral promotor, which will drive sufficient expression of the antigen in vivo .
Aim of the project:
We have previously constructed a plasmid DNA encoding the Prostate Specific Antigen (PSA) under the control of human cytomegalovirus (CMV) immediate early promotor. Immunizing with this plasmid induce PSA-specific CTLs that can be quantified ex vivo by intracellular cytokine staining assay (ICCS).
The aim of this study is to construct several plasmids, containing PSA under the control of other viral promoters and quantify amounts of PSA-specific CTLs induced after immunizations with these plasmids. This will allow to identify a viral promotor that is optimal for induction of antigen-specific CTL responses after plasmid DNA immunization .
1)Construct several plasmid constructs containing PSA under control of different viral promoters.
2)Compare levels of PSA-specific CTLs induced after immunizations with each of the plasmids.
·Recombinant DNA clonings
·Detection of protein expression in vitro (transfection of mammalian cells with plasmid DNA and Western blotting)
·Immunization with plasmid DNA and assessment of antigen-specific CTL responses (Stimulation of CD8+ T cells with antigen-derived peptides; Intracellular cytokine staining; flow-cytometry analysis)
1. Addison, C.L., Hitt, M., Kunsken, D. & Graham, F.L. Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression by adenoviral vectors. J Gen Virol 1997, 78 ( Pt 7), 1653-1661.
2. Garmory, H.S., Brown, K.A. & Titball, R.W. DNA vaccines: improving expression of antigens. Genet Vaccines Ther 2003, 1(1), 2.
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