Device

Part:BBa_K2520018

Designed by: Noa Eden, Dana Kadosh   Group: iGEM17_TECHNION-ISRAEL   (2017-10-25)


CMV-Mono Display:PLA 3-hGH

This device is a complete system for displaying proteins on cells' membrane. The IgK leader (BBa_K2520029) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (BBa_K2520037) is a trans-membrane domain that anchors all the components located between the IgK leader and the PDGFR itself to the membrane. His (BBa_K2520032) 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 bee venom allergy (BBa_K2520046, phospholipases A2) and is component related to our specific project. The CMV promoter (BBa_K1119006) is a constitutive expression promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator.

Bee Venom allergen

The major Bee Venom (BV) allergen is the phospholipase A2 (PLA). Phospholipases A2 (PLA2s) are enzymes that release fatty acids from the second carbon group of glycerol. PLA2 enzymes are commonly found in mammalian tissues as well as insect venom. The venom is largely composed of melittin, which is a stimulant of PLA2. Due to the increased presence and activity of PLA2 resulting from a snake or insect bite, arachidonic acid is released from the phospholipid membrane disproportionately. As a result, inflammation and pain occur at the site (1).

Figure 1: Phospholipid with phospholipase cleavage sites (in orange)

Epitopes:

PLA 3 (our counting), is the sequence of one of the most immunogenic epitopes of PLA protein, amino acids 45-62.

Figure 2: 62 sequentially overlapping dodecapeptides (12 aa) representing the full length of the PLA molecule. The highest immunogenic response was of the 81-92 and 113-124 epitopes (2).


References

(1) Müller, Ulrich, et al. "Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom." Journal of allergy and clinical immunology 101.6 (1998): 747-754.‏

(2) Carballido, J. M., et al. "T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2." The Journal of Immunology 150.8 (1993): 3582-3591.‏


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 679
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1408


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