Difference between revisions of "Part:BBa K2520004"

Latest revision as of 11:13, 31 October 2017

TRE-Mono display:MOG1-hGH

This device is a complete system for displaying proteins on cells' membrane. The IgK leader (BBa_K2520024) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (BBa_K2520026) is a trans-membrane domain that anchors all the components located between the IgK leader and the PDGFR itself to the membrane. HA (BBa_K2520030) is a tag that binds to specific antibodies and provide an indirect method for verifying the display of proteins on the membrane. The protein that we chose to express on the membrane is an epitope that is known target of multiple sclerosis (BBa_K2520038) and is component related to our specific project.

TRE promoter

The TRE promoter (BBa_K2520025) is an inducible expression promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator. The addition of TRE promoter allows inducible expression in specific conditions. We used the tet-off system. In the Tet-Off system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from Escherichia coli fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).

The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the target gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.

MS disease

In MS, the immune system attacks the protective sheath (myelin) that covers nerve fibers and causes communication problems between your brain and the rest of your body. Eventually, the disease can cause the nerves themselves to deteriorate or become permanently damaged.

In MS disease, T cells attack 3 types of glycoprotein:

1) Myelin basic protein (MBP)

2) Proteolipid protein (PLP)

3) myelin oligodendrocyte glycoprotein (MOG)

Experimental autoimmune encephalomyelitis (EAE):

EAE is a CD4' T cell-mediated inflammatory disease of the central nervous system (CNS) induced in experimental animals with CNS homogenate, myelin or myelin components. In its chronic form, EAE is a well accepted experimental model for multiple sclerosis (MS).

Epitopes:

MOG glycoprotein is the only CNS autoantigen known to induce both a T-cell response and a demyelinating autoantibody response in EAE

Therefore, we chose the three epitopes that induced the most significant immune response. MOG1 is the first epitope that appears in the MOG glycoprotein sequence (1-21)

References

Mendel, Itzhack, Nicole Kerlero de Rosbo, and Avraham Ben‐Nun. "A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H‐2b mice: Fine specificity and T cell receptor Vβ expression of encephalitogenic T cells." European journal of immunology 25.7 (1995): 1951-1959.‏ APA

Sequence and Features