Difference between revisions of "Part:BBa K782060"

Revision as of 18:33, 24 September 2012

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).

Figure 1. Shematic representation of INF-alpha-2a construct under CMV promoter.

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 promotor 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