Difference between revisions of "Part:BBa K4645000"
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Recombinant FelD, produced by <i>E. coli</i>, 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.
 
Recombinant FelD, produced by <i>E. coli</i>, 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.
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===Sequence and Features===
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<partinfo>BBa_K4645000 SequenceAndFeatures</partinfo>
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===Usage and Biology===
 
===Usage and Biology===
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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.
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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.  
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<html>
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<head>
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<meta charset="utf-8">
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===SDS-PAGE===
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<title>无标题文档</title>
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</head>
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<body>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/feld/feld1.jpg" style="width:40%"></center>
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<center><b>Figure 1.</b> OD<sub>600</sub>-Time curve of <i>Salmonella Typhimurium</i> SL1344 in the presence or absence of HyL&alpha;. </center>
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<br>
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</body>
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</html>
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===Design===
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The plasmid we designed consists of T7 promoter, lac operator, RBS, FelD coding sequence, His tag, Flag tag and T7 terminator, which are arranged in an order on a pET28 backbone. We aim to induce the transcription of the downstream FelD by adding the IPTG to initiate the expression. The protein will then be purified and block activity to IgE was tested by Elisa. We determine the binding by coating FelD in the microtiter plates and by measuring the absorbance.
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===Materials and Method===
  
  
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===Binding activity tests of FelD to IgE===
 
===Binding activity tests of FelD to IgE===
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===Sequence and Features===
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<partinfo>BBa_K4645000 SequenceAndFeatures</partinfo>

Revision as of 13:31, 7 October 2023


FelD: Felis domesticus allergen

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. 无标题文档

Figure 1. OD600-Time curve of Salmonella Typhimurium SL1344 in the presence or absence of HyLα.

Design

The plasmid we designed consists of T7 promoter, lac operator, RBS, FelD coding sequence, His tag, Flag tag and T7 terminator, which are arranged in an order on a pET28 backbone. We aim to induce the transcription of the downstream FelD by adding the IPTG to initiate the expression. The protein will then be purified and block activity to IgE was tested by Elisa. We determine the binding by coating FelD in the microtiter plates and by measuring the absorbance.

Materials and Method

SDS-PAGE of FelD purification

Binding activity tests of FelD to IgE

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 467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]