Coding

Part:BBa_K4645000

Designed by: Ruixuan Zhu   Group: iGEM23_HZAU-China   (2023-10-01)
Revision as of 12:53, 7 October 2023 by Ruixuan Zhu (Talk | contribs)


FelD: Felis domesticus allergen


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Recombinant FelD, produced by E. coli, exhibits comparable immunogenicity to Fel d 1 extracted from cats and is capable of inducing allergies in the human body. Its structure comprises the 93 amino acids of chain 2 of natural FelD that is directly fused to the 70 amino acids of chain 1. This is followed by the addition of 6*His tag and Flag tag to the end of the C-terminal. By binding with IgE in individuals allergic to cats, FelD mediates allergy symptoms in the affected individuals. In our study, FelD was utilized to investigate the blocking effect of our scFvs (single-chain fragment variables) on cat allergies.

Usage and Biology

Of all known cat allergens, feline domestic allergen 1 (Fel d 1) is the most well-studied. IgE antibodies to Fel d 1 are present in sera in more than 90%±95% of feline allergy patients. Fel d 1 is a 38 kDa acidic glycoprotein with N-linked carbohydrate content, found in feline pelt, saliva and lacrimal glands. The allergen consists of two 18 kDa non-covalently linked heterodimers,, each consisting of two chains, chain 1 (8 kDa) and chain 2 (10 kDa), encoded by different genes. Using the direct fusion of 93 amino acids of chain 2 and 70 amino acids of chain 1, we succeeded in creating in vitro conditions for proper folding of both chains. Stable recombinant Fel d1 (FelD) acts in a very similar way to natural allergens. It displays the same disulfide bond pattern as the natural protein and forms homodimers with a similar secondary structure. Most importantly, it also acts as a native allergen in terms of in vitro immune reactivity. In addition, we combined 6*His tag and Flag tag at the C-terminal to better obtain and verify FelD.


SDS-PAGE

SDS-PAGE of FelD purification

Binding activity tests of FelD to IgE

[edit]
Categories
Parameters
None