Coding

Part:BBa_K4645001

Designed by: Ruixuan Zhu   Group: iGEM23_HZAU-China   (2023-10-02)
Revision as of 14:33, 10 October 2023 by Ruixuan Zhu (Talk | contribs)


NeuA: scFv ( single-chain fragment variable)that inhibits the immune response to the feline

Velocimmune mice were immunized with recombinant dimeric Fel D1 and produced polyclonal antibodies. Among the resulting polyclonal antibodies, a monoclonal antibody named REGN1908 was found to be the most effective in blocking the immune response to the recombinant dimeric Fel D1. Through a literature review, we discovered both light chain and heavy chain genes of REGN1908. By fusing the C-terminal of the light chain with the N-terminal of the heavy chain using a rigid linker (GGGGS)3, an scFv was obtained. An OmpA signal peptide was fused to the N terminal of the scFv to secrete it into the periplasmic space, where it can more effectively neutralize the antigen.

Protein Molecular Structures

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Figure 1. Protein molecular structures of NeuA when it bind to FelD. The blue part is NeuA.

Design

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

Figure 2. Expression construct used for NeuA production.

Materials and Method

1.Expression and Purification

    1) Plasmid pET-28a(+)-NeuA (with His-tag) is transformed to Escherichia coli BL21(DE3). The E. coli strain is cultured in LB medium containing 50 μg/mL kanamycin.

    2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 2 mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China).


2.Blocking Elisa

    1) Microtiter plates were coated overnight at 4 ℃ with FelD.

    2) Plates were blocked with 1% BSA for 1 h at RT.

    3) Adding 200 ul ELISA Washing Buffer to wash.

    4) Different combinations of proteins(NeuA, NeuB, NeuA-NeuB, ClyR) were mixed on the microtiter plates for 90 min at 37℃.

    5) Adding 200 ul ELISA Washing Buffer to wash 2 time.

    6) Incubating 100 ul Standard IgE sample (25 ng/ul) or 100 ul sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.

    7) Incubating 100 ul biotin-labeled IgE antibody at 37℃ for 1 h.

    8) Adding 350 ul ELISA Washing Buffer to wash 4 time.

    9) Adding 100 ul Streptavidin was labeled by HRP at 37℃ for 30 min.

    10) Adding 300 ul ELISA Washing Buffer to wash 4 time.

    11) Adding 90 ul color developer (dark) at 37℃ for 15 min.

    12) Adding 50 ul termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength.

Result

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Figure 3. SDS-PAGE analysis of FelD with His-tag expression.

The molecular weight of FelD is around 18.93 kDa. Thus, the result of SDS-PAGE above indicating that there do have the expression of FelD in our chassis E. coli BL21(DE3) (Figure 3). Compared with the control groups, absorbance at 450 nm of IgE from allergic human donor sera has an obvious increase. We successfully express and purify the FelD in the Escherichia coli. And we confirmed that FelD can bind to IgE from allergic human donor. The results of our experiment are shown in the figure below (Figure 4). 无标题文档

Figure 4. OD450-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.

Reference

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 315
    Illegal SpeI site found at 75
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 315
    Illegal SpeI site found at 75
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 315
    Illegal XhoI site found at 693
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 315
    Illegal SpeI site found at 75
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 315
    Illegal SpeI site found at 75
  • 1000
    COMPATIBLE WITH RFC[1000]


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