Part:BBa_K782060
Interferon alpha-2a
Introduction
Interferons are cytokines that play important role in the early immune response, exibit antiproliferative effects on cells and have immunomodulatory and antiviral function (Thomas et al., 2007). Type I interferons (IFN-alpha and beta) mediate signaling through the IFNAR receptor, the STAT1 and STAT2 components of the JAK/STAT-signal transduction pathways and finally, the interferon stimulated response element (ISRE) promotor element. IFN-alpha has been used since 1980s in the treatment of chronic hepatitis and still represents an important part of the management of chronic hepatitis C infection. Initial studies used IFN-alpha monotherapy, but current treatments are a combination therapy consisting of ribavirin and IFN-alpha (Feld and Hoofnagle, 2005). Side effects of treatment are still very common, with half of the patients suffering from flu like symptoms and a third experiencing emotional problems. Anxiety, sleep disorders and irritability are frequently observered and can in some cases lead to severe behavioral or phsycological disorders. This can in turn leed to the discontinuation of therapy. With our biological delivery system we aim to overcome some of the adverse effects of the IFN therapy and make the therapy more efficient and affordable. We constructed INF-alpha-2a under the CMV promoter (Figure 1).
Read more on our Wiki page (iGEM team Slovenia[http://2012.igem.org/Team:Slovenia/ImplementationHepatitisC]).
Figure 1. Shematic representation of INF-alpha-2a construct under CMV promoter.
- IFN-alpha-2a was obtained from Sino Biological Inc.
Characterization
We tested the biological activity of IFN-alpha-2a produced by HEK293T cells. We used a STAT1/STAT2-responsive luciferase construct that encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the interferon stimulated response element (ISRE). We designed the experiment as a co-culture of HEK293T cells transfected with either the IFN-alpha-2a encoding plasmid or an empty vector, and HEK293T cells transfected with the reporter vector. Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-alpha-2a encoding plasmids. As a positive control, we used the response of the same reporter to recombinant IFN-beta or TLR3 stimulation (TLR3 is an innate immune receptor that also signals through the ISRE promoter element).
Figure 2. IFN-alpha-2a expressed in HEK293T cells under a constitutive promoter induces expression of an ISRE-dependant reporter. A co-culture of HEK293T cells transfected with either the IFN-alpha-2a encoding plasmid or an empty vector and HEK293T cells transfected with firefly luciferase reporter with ISRE was prepared. Additionally, HEK293T cells were cotransfected with both the reporter and the IFN-alpha-2a encoding or mock plasmids. After 24 hours of incubation, a dual luciferase reporter assay was preformed. Transfection of HEK293T with TLR3 or stimulation with IFN-beta served as experiment controls.
We also performed an enzyme-linked immunosorbent assay (ELISA) of the IFN-alpha-2a production from HEK293T cells. We measured that on average a single cell produced 4,6 *10^-9 μg of IFN-alpha in 24 hours.
Refrences
Feld, J.J. and Hoofnagle, H. (2005) Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 436, 967-72.
Thomas, C., Moraga, I., Levin, D., Krutzik, P.O., Podoplelova, Y., Trejo, A., Lee, C., Yarden, G., Vleck, S.E., Glenn, J.S., Nolan, G.P., Piehler, J., Schreiber, G., Garcia, K.C. (2011) Structural linkage between ligand discrimination and receptor activation by type I interferons. Cell. 146(4), 621–632.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 220
Illegal BsaI.rc site found at 301
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