iGEM 2025 — Team tsinglan

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"title": 'Wet Lab',
"content": '

This page provides a detailed record of the experimental process, primarily divided into the following sections:

1. Cycle 1a-Construction of hypoallergenic derivative
2. Cycle 1b-Construction of natural allergens
3. Cycle 1-Test of hypoallergenic derivative and natural allergens
4. Cycle 2-Construction of allergen fusion protein
5. Cycle 2-Test of allergen fusion protein
6. Cell proliferation test of allergens
7. Half-life detection
8. Soluble Microneedles Preparation

',
},
{
"title": '1. Cycle 1a-Construction of hypoallergenic derivative',
"content": '
',
},
{
"title": '2. Cycle 1b-Construction of natural allergens',
"content": '
',
},
{
"title": '3. Cycle 1-Test of hypoallergenic derivative and natural allergens',
"content": '
',
},
{
"title": '4. Cycle 2-Construction of allergen fusion protein',
"content": '
',
},
{
"title": '5. Cycle 2-Test of allergen fusion protein',
"content": '
',
},
{
"title": '6. Cell proliferation test of allergens',
"content": '
',
},
{
"title": '7. Half-life detection',
"content": '
',
},
{
"title": '8. Soluble Microneedles Preparation',
"content": '
'
},
{
"title": 'Dry Lab',
"content": '
'
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] %}
{% block title %}Notebook{% endblock %}
{% block lead %}NOTEBOOK{% endblock %}
{% block leadImg %}

![](https://static.igem.wiki/teams/5747/common/notebook.webp)

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{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Overview',
"content": '

Allergen immunotherapy (AIT) is the only treatment that can change the course of allergic diseases, offering
benefits that last for years after treatment ends. However, its limitations cannot be ignored, including a limited
range of applicable cases, long treatment duration resulting in poor patient compliance, potential triggering of
more severe allergic reactions, and higher costs, etc. In order to overcome the key limitations of traditional
allergen immunotherapy (AIT), our project has developed and refined the AIT treatment strategy to achieve
systematic optimization of safety, efficacy, and patient compliance. Firstly, by rationally designing
hypoallergenic variants of six major allergens (pollen, dust mites, and seafood derivatives), we mutated the IgE
binding epitopes while retaining T-cell reactivity, thereby reducing the risk of anaphylactic shock. Secondly, by
combining the hypoallergenic units with self-assembling peptides (EFK8), we developed a broad-spectrum fusion
protein, enabling multi-allergen targeting and extending the treatment half-life.

Our project mainly consists of the following two rounds of "Design - Build - Test - Learn" (DBTL) cycles:

* Cycle 1: Construction and test of hypoallergenic derivative and natural allergens
* Cycle 2: Construction and test allergen fusion protein

![](https://static.igem.wiki/teams/5747/engineering/1.webp)

**Figure 1** Two DBTL cycles of our project.

',
"children": []
},
{
"title": 'Cycle 1 Construction and test of hypoallergenic derivative and natural allergens',
"content": '',
"children": [
{
"title": 'Design 1a',
"content": '

In this cycle, we designed and produced recombinant hypoallergenic derivatives derived from six common allergens:
Phl p1 and Bet v1 (pollen allergens), Der p1 and Der p2 (dust mite allergens), and Met e1 and Cyp c1 (seafood
allergens). Through rational protein design, we eliminated or disrupted key conformational IgE-binding epitopes
while strategically preserving T-cell epitopes, and we have named these hypoallergenic derivatives as: mPhl p1,
mBet v1, mDer p1, mDer p2, mMet e1, mCyp c1 (**Table 1**). This approach maintains the immunogenicity
required for inducing immune tolerance while significantly reducing the risk of triggering allergic reactions.

**Table 1** Design Principles for Hypoallergenic protein Sequences

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **Source** | **Hypoallergenic Mechanism** | **Ref** |
| mPhl p1 | Timothy grass pollen allergen | Phl p 1 was broken into four fragments and then reassembled as a Phl p 1 mosaic protein | (Ball et al., 2009) |
| mBet v1 | *Betula verrucosa* | 7 consecutive amino replacement | (Wallner et al., 2011) |
| mDer p1 | *Dermatophagoides pteronyssinus* | Chimeric gene of T cell epitopes | (Hong, 2018) |
| mDer p2 | Site-directed mutagenesis of IgE-binding epitopes | (Reginald & Chew, 2018) |
| mMet e1 | Shrimp tropomyosin | Site-directed mutagenesis of IgE-binding epitopes | (Wai et al., 2014) |
| mCyp c1 | Carp parvalbumin | Site-directed mutagenesis of calcium-binding domains | (Swoboda et al., 2007) |

The plasmids were designed using the pET-32a vector, which features a T7 promoter for high-level expression, a
His-tag for simplified purification, and a Trx tag to enhance soluble expression of the target allergen protein.
The gene fragments encoding the engineered ​​hypoallergenic proteins​​ were first amplified via ​​PCR​​. The
pET-32a vector was then digested with the corresponding ​​restriction enzymes​​ (BamHI and XhoI). Following
purification, the target gene fragments and vector were assembled using ​​homologous recombination​​ (**Figure 2**).

![](https://static.igem.wiki/teams/5747/engineering/2.webp)

**Figure 2** Schematic diagram of the plasmid construction.

',
},
{
"title": 'Build 1a',
"content": '

​ As shown in **Figure 3**, PCR products and linearized vector were validated via agarose gel
electrophoresis. Target gene fragments (330-720 bp) were confirmed for purity and accuracy through staining and UV
visualization. Following gel extraction and purification, six allergen genes (mPhl p1, mBet v1, mDer p1, mDer p2,
mCyp c1, mMet e1) were inserted into the linearized pET32a vector using homologous recombination technology,
completing recombinant plasmid construction.

![](https://static.igem.wiki/teams/5747/engineering/3.webp)

**Figure 3** Agarose gel electrophoresis results of (A) gene amplification and (B) vector
linearization.

The recombinant products were then transformed into *E. coli* Top 10 competent cells, and positive
clones were selected on LB agar plates, respectively. Colony PCR and DNA sequencing verified the correct insertion
and integrity of the allergen genes. Selected positive clones were sequenced, and the sequence data confirmed that
each plasmid construct contained the correct allergen gene sequence with high fidelity (**Figure 4**). The validated plasmids were subsequently transformed into *E. coli* Origami (DE3) competent cells
to enhance soluble expression of the recombinant hypoallergenic proteins.

![](https://static.igem.wiki/teams/5747/engineering/4.webp)

**Figure 4** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

',
},
{
"title": 'Design 1b',
"content": '

To validate whether our engineered hypoallergenic derivatives indeed exhibit reduced allergenicity (IgE binding
capacity), we also produced the corresponding native allergens. Notably, since native rDer p1 requires
glycosylation and cannot be produced in *E. coli*, it was commercially purchased from WOLCAVI BIOTECH.
For the other five native allergens (rPhl p1, rBet v1, rDer p2, rMet e1, rCyp c1), we introduced reversion
mutations via primers, obtained the fragments through PCR, and subsequently constructed the recombinant plasmids
using homologous recombination (**Figure 5**).

![](https://static.igem.wiki/teams/5747/engineering/5.webp)

**Figure 5** Schematic diagram of the plasmid construction of native allergens.

',
},
{
"title": 'Build 1b',
"content": '

As shown in **Figure 6**, PCR products were validated via agarose gel electrophoresis. Target gene
fragments were confirmed for purity and accuracy through staining and UV visualization. Notably, due to multiple
mutation sites in rCyp c1, its gene fragment (156 bp) was assembled from synthesized oligos and then fused with
rCyp-AMP (2847 bp) via overlap PCR, yielding a 2984 bp product. We synthesized the rPhl p1 and rMet e1 fragments
in the company. After PCR and agarose gel electrophoresis, the pET32a vector was observed to have a band near
5000 base pairs. Both rPhl p1 (777 base pairs) and rMet e1 (876 base pairs) were of the expected size.After
the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

![](https://static.igem.wiki/teams/5747/results/result-6-new.webp)

**Figure 6** Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the *E. coli* Top 10 bacteria, and then screen out the
positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we
successfully obtained the plasmid with correct sequencing (**Figure 7**). These verified plasmids
were subsequently transferred into *E. coli* Origami (DE3).

![](https://static.igem.wiki/teams/5747/engineering/7.webp)

**Figure 7** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

',
},
{
"title": 'Test 1',
"content": '

1. **Protein expression and purification**

Next, we expanded the cultivation and used IPTG to induce the expression of the hypoallergenic protein derivatives
and native allergens. Our goal was to express and purify the recombinant allergen proteins for subsequent
research. After cell lysis and centrifugation, we performed SDS-PAGE to identify the protein expression and
purification conditions. The SDS-PAGE analysis results showed that all six hypoallergenic protein derivatives and
five native allergens were successfully expressed, and the molecular weights were consistent with the expectations
(**Figure 8A**).

Among them, mPhl p1, mBet v1, and mDer p1 were mainly expressed in the precipitate part, with less background
proteins and high purity. These insoluble proteins were then denatured and refolded to obtain soluble proteins.
For the three soluble-expressed proteins: mDer p2, mMet e1, and mCyp c1, and the five natural allergen proteins
(expressed in both the supernatant and the precipitate), we used nickel columns to purify them separately. The
SDS-PAGE results indicated that we successfully obtained highly pure proteins (**Figure 8B-C**).

![](https://static.igem.wiki/teams/5747/engineering/8.webp)

**Figure 8** SDS-PAGE results of (A) hypoallergenic protein expression, (B) natural allergen
proteins, and (C-D) protein purification.

2. **Protein quantitative analysis**

Protein concentrations of both purified native allergens and their hypoallergenic derivatives were quantified
using the Bradford assay to ensure accurate loading for subsequent IgE-binding affinity assays. A standard curve
was constructed using bovine serum albumin (BSA) as a reference, exhibiting excellent linearity (R² = 0.9909) with
the regression equation Y = 0.001403x + 0.004835 (**Figure 9**). All absorbance measurements at 595
nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (**Table 2**).

![](https://static.igem.wiki/teams/5747/engineering/9.webp)

**Figure 9** BSA standard curve test by Bradford assay.

(A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

**Table 2** Protein quantification results.

![](https://static.igem.wiki/teams/5747/engineering/10.webp)

3. **IgE affinity test of allergen protein**

Next, we conducted IgE binding affinity analysis, the main purpose of which is to functionally evaluate the
allergenic potential of natural and modified hypoallergenic derivatives, and fusion hypoallergenic derivatives.
This assessment is crucial because the clinical severity of allergic reactions is largely determined by the
affinity of the specific IgE antibodies against the allergen and its target epitope (Fromberg, 2006).
High-affinity IgE antibodies can trigger strong activation of mast cells and basophils even in the presence of low
concentrations of allergens, leading to significant release of inflammatory mediators and potential anaphylactic
shock. In contrast, low-affinity IgE has significantly reduced ability to bind to the FcεRI receptor, thereby
reducing the risk of severe allergic reactions (Udoye, Ehlers, & Manz, 2023). Therefore, by quantifying the
degree of reduction in IgE binding affinity, it can provide a key indicator for in vitro experiments to determine
whether our rational protein design strategy has successfully achieved the reduction of allergenicity.

The standard curve for human IgE quantification was generated using four-parameter logistic (4PL) regression in
GraphPad Prism 9.5, demonstrating excellent fit (R² > 0.998), which validated the reliability of subsequent
affinity measurements (**Figure 10A)**. The IgE-binding capacities of six purified allergens—both
native and hypoallergenic derivatives—were evaluated by ELISA. Coated proteins were incubated with 50 ng/mL human
IgE. As shown in **Figure 10B,** significant reduction in IgE binding (p < 0.0001) was observed in
four hypoallergenic derivatives compared to their native counterparts (mBet v1, mDer p2, mMet e1, mCyp c1).
Although mPhl p1 and mDer p1 also showed decreased affinity, the reduction was more modest
(p>0.05**)**. These results confirmed the effectiveness of the rational design strategy in
generating hypoallergenic proteins with markedly reduced IgE reactivity.

![](https://static.igem.wiki/teams/5747/engineering/11.webp)

**Figure 10** IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of natural
allergens and hypoallergenic derivatives.

4. **Cell proliferation experiment test**

Building upon the IgE-binding affinity results, we further evaluated the cytotoxicity of both single and fusion
hypoallergenic derivatives using a CCK-8 assay to assess their biosafety for potential
*in vivo* therapeutic applications. This evaluation is essential to ensure that engineered biologics
exhibit minimal adverse effects on cell viability before proceeding to animal studies or clinical use.

After incubating cells with each protein at concentrations of 1 nM, 10 nM, and 100 nM for 24 h and 48 h,
significant differences in cytotoxicity profiles were observed. For mBet v1 and mCyp c1, at all tested concentrations
and time points, there was almost no significant effect on
cell viability, demonstrating extremely high biocompatibility (**Figure 11B, F**). For mPhl p1 and mMet e1, the toxicity
showed a clear time and concentration dependence (**Figure 11A, E**). At low concentrations and short durations, the impact
was minimal, but as the concentrations and durations increased, the toxicity significantly intensified. For mDer p1 and
mDer p2, even at lower concentrations and shorter durations, it could cause a significant decrease in cell viability,
indicating an inherent toxicity (**Figure 11C, D**).

![](https://static.igem.wiki/teams/5747/engineering/12.webp)

**Figure 11** The results of the cell proliferation experiment.

',
},
{
"title": 'Learn 1',
"content": '

​​ The experimental results demonstrated that our engineered hypoallergenic derivatives ​​significantly reduce
IgE-binding affinity​​, confirming the effectiveness of ​​rational epitope disruption​​ in minimizing
allergenicity. This reduction in IgE reactivity aligns with the core goal of enhancing the safety profile of
allergen-specific immunotherapy (AIT). Furthermore, ​​CCK-8 cytotoxicity assays​​ revealed that these
hypoallergenic variants exhibit minimal adverse effects on cell viability, supporting their biocompatibility and
potential for *in vivo* applications.

To further broaden the therapeutic scope of AIT, we plan to develop ​​fusion constructs​​ by concatenating
multiple hypoallergenic units derived from the same allergen category and incorporating ​​self-assembling
peptides​​. This design aims to promote the formation of ​​stable oligomers or nanoparticles​​, which are expected
to ​​prolong circulatory half-life. Such a strategy could enable synchronized multi-allergen delivery while
maintaining a favorable safety profile, ultimately improving treatment efficacy and patient compliance.

',
},
]
},
{
"title": 'Cycle 2: Construction and test allergen fusion protein',
"content": '',
"children": [
{
"title": 'Design 2',
"content": '

To address the issue of multiple allergic reaction sensitivities and simplify the treatment plan, we have designed
and manufactured a new type of fusion protein. This protein integrates multiple modified hypoallergenic
derivatives that trigger similar immune responses into a single molecular structure. This strategy aims to achieve
broad therapeutic effects, potentially avoiding the cumbersome process of multiple separate injections, thereby
enhancing treatment efficiency and improving patient compliance. To achieve this, we investigated and selected the
EFK8 self-assembling peptide (FEFEFKFK), which undergoes oligomerization driven by hydrophobic stacking of
phenylalanine residues and electrostatic interactions of lysine residues. This process forms stable nanostructures
that increase molecular size and reduce renal clearance, thereby prolonging the circulation half-life (Song et
al., 2023).

![](https://static.igem.wiki/teams/5747/engineering/13.webp)

**Figure 12** The assembly process of EFK8 self-assembling peptide (Ding & Wang, 2017).

We assembled short EFK8 oligonucleotide fragments firstly. Subsequently, we separately amplified single
hypoallergenic derivative gene fragments with homologous arms through PCR. Then, we used overlapping extension PCR
to splice these fragments together to generate the complete fusion allergen gene sequence. Finally, we performed
restriction endonuclease digestion and ligation on the purified fusion gene and linearized vector backbone to
obtain the desired recombinant plasmid product. Furthermore, we also constructed a plasmid that integrates the
EFK8 peptide and the enhanced green fluorescent protein EGFP, which was used for the subsequent determination of
the half-life (**Figure 13**).

![](https://static.igem.wiki/teams/5747/engineering/14.webp)

**Figure 13** Schematic diagram of the plasmid construction.

',
},
{
"title": 'Build 2',
"content": '

As shown in **Figure 14**, PCR products were validated via agarose gel electrophoresis. Target gene
fragments were confirmed for purity and accuracy through staining and UV visualization Figure 10A-B. Then, we used
overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene
sequence (**Figure 14C**). After the fragments were recovered through gel cutting, we used homologous
recombination to recombine them.

![](https://static.igem.wiki/teams/5747/engineering/15.webp)

**Figure 14** Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the *E. coli* Top 10 bacteria, and then screen out the
positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we
successfully obtained the plasmid with correct sequencing (**Figure 15**). These verified plasmids
were subsequently transferred into *E. coli* Origami (DE3).

![](https://static.igem.wiki/teams/5747/engineering/16.webp)

**Figure 15** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

',
},
{
"title": 'Test 2',
"content": '

1. **Protein expression and purification**

We expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins.
The EFK8-mPhl p1-GS-mBet v1 (FPB) and EFK8-mDer p1-GS-mDer p2 (FDD) proteins are both expressed in the
precipitate. Subsequently, a denaturation-reconstitution procedure is required to restore their soluble state. The
EFK8-mMet e1-GS-mCyp c1 (FMC) protein was expressed in both the supernatant and the precipitate. We purified the
soluble FMC protein using a nickel column. The SDS-PAGE results indicated that we were able to successfully purify
this protein (**Figure 16)**.

![](https://static.igem.wiki/teams/5747/engineering/17.webp)

**Figure 16** SDS-PAGE results of (A) protein expression and (B) purification.

2. **Protein quantitative analysis**

Protein concentrations of purified fusion hypoallergenic derivatives were quantified using the Bradford assay to
ensure accurate loading for subsequent IgE-binding affinity assays. A standard curve was constructed using bovine
serum albumin (BSA) as a reference, exhibiting excellent linearity (R² = 0.9987) with the regression equation Y =
0.001563x + 0.002397 (**Figure 17**). All absorbance measurements at 595 nm were performed in
triplicate, and sample concentrations were interpolated based on the standard curve (**Table 3**).

![](https://static.igem.wiki/teams/5747/engineering/18.webp)

**Figure 17** BSA standard curve test by Bradford assay of

(A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

**Table 3** Protein quantification results.

![](https://static.igem.wiki/teams/5747/engineering/19.webp)

3. **IgE affinity test of allergen protein**

As illustrated in **Figure 18A**, the IgE standard curve demonstrated excellent linearity (R² =
0.9943), ensuring the reliability of the subsequent binding affinity quantification. Interestingly, comparative
analysis revealed that two fusion allergens, ​​EFK8-mPhl p1-GS-mBet v1 (FPB)​​ and ​​EFK8-mDer p1-GS-mDer p2
(FDD)​​, exhibited significantly reduced IgE binding ratios compared to their respective single hypoallergenic
constituents (mPhl p1 and mDer p1, respectively). ​​EFK8-mMet e1-GS-mCyp c1 (FMC)​​ also showed low IgE binding
affinity, which was comparable to the levels observed for its individual hypoallergenic derivatives (mMet e1 and
mCyp c1) (**Figure 18B)**. This consistent reduction across all fusion constructs suggests that the
fusion strategy effectively attenuates IgE immunoreactivity beyond the level achieved by individual engineered
allergens, likely due to structural masking of key epitopes or altered antigen presentation.

![](https://static.igem.wiki/teams/5747/engineering/20.webp)

**Figure 18** IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of single and
fusion hypoallergenic derivatives.

4. **Cell proliferation experiment test**

We evaluated the cytotoxicity profiles of both individual and fused hypoallergenic derivatives. For the fusion construct
EFK8-PT-mPhl p1-GS-mBet v1, we observed that the intrinsic cytotoxicity of mPhl p1 influenced the overall profile of
the fusion protein, leading to a detectable reduction in cell viability particularly under high-concentration or
prolonged exposure conditions (**Figure 19A-C**).In contrast, the fusion protein EFK8-PT-mMet e1-GS-mCyp c1 exhibited a
different behavior: although mMet e1 alone showed time- and concentration-dependent cytotoxicity, the fusion with
mCyp c1—which itself displayed no appreciable toxicity—resulted in a composite molecule that did not induce
significant cytotoxicity, even at high doses or after extended incubation (**Figure 19D-F**). Notably, the fusion
protein EFK8-PT-mDer p1-GS-mDer p2 did not exhibit significant cytotoxicity under the tested conditions.
This is in clear contrast to its individual components, both mDer p1 and mDer p2, which caused a marked decrease
in cell viability, especially at higher concentrations (e.g., 100 nM) and with longer culture periods (48 h)
(**Figure 19G-I**). These results suggest that the fusion strategy can alter the cytotoxic properties of the constituent
hypoallergenic derivatives, in some cases mitigating the adverse effects observed with the individual components.

​ The CCK-8 results indicated that these fusion constructs (EFK8-PT-mDer p1-GS-mDer p2, and EFK8-PT-mMet e1-GS-mCyp c1)
possessed excellent biological safety characteristics
and had a negligible impact on cell viability. This highlighted that the fusion strategy not only can expand the
coverage of immunotherapy but also enhance biocompatibility. These findings supported the advancement of the
fusion of hypoallergenic derivatives towards further evaluation.

**![](https://static.igem.wiki/teams/5747/results/17.webp)**

**Figure 19** The results of the cell proliferation experiment.

5. **Half-life test**

To evaluate whether fusion with the self-assembling peptide EFK8 prolongs the half-life of hypoallergenic fusion
proteins, we employed EGFP as a model protein incubated in fetal bovine serum at 37 °C for 5 days to simulate
*in vitro* conditions. Fitting the fluorescence decay data to a one-phase exponential model revealed that
EFK8-EGFP exhibited a significantly extended half-life (1.820 days) compared to EGFP alone (0.9819 days), nearly
doubling its stability (**Figure 20**). This enhancement is likely attributable to EFK8-mediated
oligomerization, which increases molecular size and reduces degradation rate.

**![](https://static.igem.wiki/teams/5747/results/18-1.webp)**

**Figure 20** Half-life of EGFP and EFK8-EGFP.

',
},
{
"title": 'Learn 2',
"content": '

In the above experimental results, we successfully constructed and expressed the fused hypoallergenic derivative,
and tested its allergenicity (IgE affinity), biocompatibility and half-life. We found the significantly reduced
IgE binding affinity and cytotoxicity observed in our fusion allergens (EFK8-PT-mPhl p1-GS-mBet v1, EFK8-PT-mDer
p1-GS-mDer p2) compared to their monomeric counterparts. We speculated that may be attributed to several
synergistic mechanisms facilitated by the fusion strategy and the self-assembling peptide EFK8.

First, fusion-induced ​​steric hindrance​​ and ​​conformational rearrangement​​ likely occlude key IgE-binding
epitopes, thereby directly impairing antibody recognition and cross-linking capacity. Secondly, the
​​oligomerization​​ process triggered by EFK8 will increase the molecular size and alter the surface topological
structure through hydrophobic and electrostatic interactions, causing conformational changes or masking at
specific sites in these fused hypoallergenic derivatives, thereby further leading to the loss of toxic functions.
We have predicted the molecular structures of EFK8-PT-mPhl p1-GS-mBet v1 and EFK8-PT-mDer p1-GS-mDer p2, and
conducted molecular oligomerization simulations. We found that after oligomerization, some parts of the structures
of these low-allergenic derivatives would be covered. For details, please refer to the [Model](%27%2B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%2B%27) page.

Collectively, these modifications not only diminish immediate hypersensitivity risks but also mitigate direct
cellular toxicity, underscoring the potential of fusion-based designs in developing safer and more effective
allergen immunotherapies.

',
},
]
},
{
"title": 'Reference',
"content": '

Ball, T., Linhart, B., Sonneck, K., Blatt, K., Herrmann, H., Valent, P., . . . Valenta, R. (2009). Reducing
allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination. *64*(4),
569-580. doi:<https://doi.org/10.1111/j.1398-9995.2008.01910.x>

Ding, X., & Wang, Y. (2017). Weak Bond-Based Injectable and Stimuli Responsive Hydrogels for Biomedical
Applications. *Journal of Materials Chemistry B, 5*(5), 887-906. doi:10.1039/c6tb03052a

Fromberg, J. (2006). IgE as a marker in allergy and the role of IgE affinity. *61*(10), 1234-1234. doi:<https://doi.org/10.1111/j.1398-9995.2006.01222.x>

Hong, Y. (2018).
*Exploration of the mechanism of Der p1 T-cell epitope fusion peptide-specific immunotherapy in house dust
mite-sensitized asthmatic mice.*
(硕士), Available from Cnki

Reginald, K., & Chew, F. T. (2018). Conformational IgE Epitope Mapping of Der p 2 and the Evaluations of Two
Candidate Hypoallergens for Immunotherapy. *Scientific Reports, 8*(1), 3391. doi:10.1038/s41598-018-21792-1

Song, H., Su, Q., Nie, Y., Zhang, C., Huang, P., Shi, S., . . . Wang, W. (2023). Supramolecular assembly of a
trivalent peptide hydrogel vaccine for cancer immunotherapy. *Acta Biomaterialia, 158*, 535-546. doi:<https://doi.org/10.1016/j.actbio.2022.12.070>

Swoboda, I., Bugajska-Schretter, A., Linhart, B., Verdino, P., Keller, W., Schulmeister, U., . . . Spitzauer, S.
(2007). A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.
*J Immunol, 178*(10), 6290-6296. doi:10.4049/jimmunol.178.10.6290

Udoye, C. C., Ehlers, M., & Manz, R. A. (2023). The B Cell Response and Formation of Allergenic and
Anti-Allergenic Antibodies in Food Allergy. *12*(12), 1501.

Wai, C. Y. Y., Leung, N. Y. H., Ho, M. H. K., Gershwin, L. J., Shu, S. A., Leung, P. S. C., & Chu, K. H.
(2014). Immunization with Hypoallergens of Shrimp Allergen Tropomyosin Inhibits Shrimp Tropomyosin Specific IgE
Reactivity. *PLoS One, 9*(11), e111649. doi:10.1371/journal.pone.0111649

Wallner, M., Hauser, M., Himly, M., Zaborsky, N., Mutschlechner, S., Harrer, A., . . . Ferreira, F. (2011).
Reshaping the Bet v 1 fold modulates TH polarization. *Journal of Allergy and Clinical Immunology, 127*(6),
1571-1578.e1579. doi:<https://doi.org/10.1016/j.jaci.2011.01.064>

',
"children": []
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{% block title %}Engineering Success{% endblock %}
{% block lead %}ENGINEERING{% endblock %}
{% block leadImg %}

![](https://static.igem.wiki/teams/5747/common/engineering.webp)

{% endblock %}
{% block page\_content %}
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{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Preparatory work',
"content": '',
"children": [
{
"title": '1. Prepare Amp antibiotic',
"content": '

**Materials:**

Ampicillin powder,

Sterile deionized water,

Filter sterilization unit (0.22 µm pore size).

Needle-free syringe

**Equipment:**

Electronic balance

**Method (For 10 mL):**

1. Weighing 1 g of the ampicillin powder
2. Dissolve in Solvent
   1. Transfer the powder to a sterile container.
   2. Add the final volume of sterile water.
   3. Mix gently until fully dissolved.
3. Filter Sterilize
4. Pass the solution through a 0.22 µm filter into a sterile tube. This removes bacteria and ensures sterility.
5. Aliquot and Store
   1. Dispense into smaller aliquots (1 mL) to avoid repeated freeze-thaw cycles.
   2. Store at -20℃ for long-term storage.

',
},
{
"title": '2. Prepare LB medium (solid and liquid)',
"content": '

**Materials:**

Tryptone (10 g/L)

Yeast Extract (5 g/L)

NaCl (10 g/L)

Agar (for solid medium; 15 g/L)

Erlenmeyer Flask (for mixing)

Autoclave (for sterilization)

Petri dishes (for solid medium)

**Equipment:**

Electronic balance

Autoclave

Oven

**Method:**

1. Weigh Components (For 1 L): 10 g tryptone, 5 g yeast extract, 10 g NaCl
2. Add components to a flask with ~800 mL of water. Stir until fully dissolved (use a magnetic stirrer). Then add
   water to reach 1 L total volume.
3. Transfer to an autoclavable bottle. Autoclave at 121℃ for 20 minutes.
4. For solid LB:

1. After autoclaving, let the medium cool to ~50℃ (warm to touch but not boiling).
2. Add ampicillin to a final concentration of 100 μg/mL.
3. Pour 10-15 mL per 90 mm petri dish in a sterile hood.
4. Let plates solidify (30-60 minutes).
5. Invert plates and store at 4℃.

',
},
]
},
{
"title": 'Molecular cloning',
"content": '',
"children": [
{
"title": '1. PCR amplification',
"content": '

**Materials:**

PrimeStar Max Premix (2×)

Primers (10 μM)

Plasmid

ddH2O

**Equipment:**

Thermal cycler

PCR tubes

**Method:**

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| PrimeStar Max Premix (2×) | 25 | Denaturation: 98 ℃ for 10 sec |
| Primer F (10 μM) | 1 | Annealing: Tm for 15 sec |
| Primer R (10 μM) | 1 | Extension: 72 ℃, 30 sec/kb |
| DNA template | 1 (10 ng) | Back to step 2 (30 times) |
| ddH2O | 22 | Final elongation: 72 ℃ for 2 min |
| Total | up to 50 | 10 ℃ hold |

',
},
{
"title": '2. Agarose gel electrophoresis',
"content": '

**Materials:**

Agarose powder

1×TAE buffer (Tris-acetate -EDTA)

DNA loading dye (6×)

DNA ladder

YeaGreen DNA stain

**Equipment:**

Electronic balance

Microwave

Electrophoresis equipment

UV transilluminator

**Method:**

1. Prepare Gel (1%):
   1. Mix agarose 1 g powder with 100 mL TAE buffe.
   2. Heat to dissolve agarose, cool to ~50°C.
   3. Add 10 μL nucleic acid dye.
   4. Pour into gel tray with comb; let solidify.
2. Load Samples:
   1. Mix DNA samples with loading dye.
   2. Load into gel wells, and load DNA size marker in a separate well.
3. Run Gel:
   1. Submerge gel in TAE buffer.
   2. Apply 180 V until bands separate (About 15 min).
4. Visualize Bands
   1. Place gel on UV transilluminator.
   2. Image bands.

',
},
{
"title": '3. DNA Fragment extraction',
"content": '

**Materials:**

Gel extraction kit

Ethanol

**Equipment:**

Centrifuge

Water bath

**Procedure:**

1. Add 96 mL ethanol into wash solution. Adjust water bath to 50 ℃.
2. Cut the agarose gel containing the DNA fragment and put into micro-centrifuge tube.
3. Weight the gel and add 3 times of Buffer B2. Heat in 50 ℃ water bath for 10 min.
4. Add DNA gel solution into the spin column, 8,000 *×g* for 1 min. Discard the filtrate in collection
   tube, then return column back to the collection tube.
5. Add 500 μL of Wash Solution into the column, then into the spin column, 8,000 *×g* for 1 min. Discard
   the filtrate in collection tube, then return column back to the collection tube. Repeat this step once.
6. Centrifuge column at 8,000 *×g* for 2 min.
7. Place column into a 1.5 mL tube, then add 30 μL of ddH₂O into the column.
8. Put in 50 ℃ dryer for 5 min.
9. Centrifuge column at 8,000 ×*g* for 4 min. Store centrifuge solution in -20 °C refrigerator.

',
},
{
"title": '4. Homologous recombination',
"content": '

**Materials:**

ClonExpress Ultra One Step Cloning Kit

**Equipment:**

Thermal cycler

PCR tubes

**Procedure:**

|  |  |
| --- | --- |
| **Reagent** | **Volume (µL)** |
| 2×Clonexpress mix | 5 |
| Gene | 3 |
| pET32a Linearized vector | 1 |
| ddH2O | 1 |
| Total | 10 |
| 50 ℃，10 min | |

',
},
{
"title": '5. Restriction Enzyme Digestion',
"content": '

**Materials:**

BamHI restriction enzymes

XhoI restriction enzymes

10×CutSmart buffer

Plasmid DNA

Target fragment

Nuclease-free water

**Equipment:**

Thermal cycler

PCR tubes

**Procedure:**

|  |  |
| --- | --- |
| **Reagent** | **Volume (μL)** |
| 10×Cutsmart buffer | 2 |
| Plasmid DNA | 3 |
| BamHI | 2 |
| XhoI | 2 |
| ddH2O | up to 20 μL |
| 37℃, 15 min; 65℃, 10 min | |

',
},
{
"title": '6. Digestion Product Purification',
"content": '

**Materials:**

PCR product purification kit

Ethanol

**Equipment:**

Centrifuge

Water bath

**Procedure:**

1. Add 5 volumes of Buffer PB directly to the digestion product
2. Transfer the entire mixture to a spin column seated in a 2 mL collection tube.
3. Centrifuge at 10,000 ×g for 60 seconds. Discard flow-through and reassemble column
4. Add 700 μL Buffer PW (wash buffer containing ethanol) to the column. Centrifuge at 10,000 ×g for 60 sec. Discard
   flow-through.
5. Repeat with 500 μL Buffer PW.
6. Centrifuge the empty column at 10,000 ×g for 2 minutes to remove residual ethanol.
7. Transfer column to a clean 1.5 mL microcentrifuge tube.
8. Add 30 μL ddH2O directly to the center of the membrane.
9. Incubate at room temperature for 2 minutes
10. Centrifuge at 10,000 ×g for 2 minutes to collect purified DNA.
11. Using a Microspectrophotometer to Detect DNA Concentration

',
},
{
"title": '7. DNA Ligation',
"content": '

**Materials:**

T4 DNA Ligase

10×T4 DNA Ligase buffer

Vector and insert DNA

Nuclease-free water

**Equipment:**

Thermal cycler

PCR tubes

**Procedure:**

|  |  |
| --- | --- |
| **COMPONENT** | **20 μL REACTION** |
| T4 DNA Ligase Buffer (10X) | 2 μL |
| T4 DNA Ligase | 1 μL |
| pET32a-V (5329 bp) | 5 μL |
| Insert DNA (3: 1) | / |
| Nuclease-free water | to 20 μL |

',
},
{
"title": '8. Transformation',
"content": '

**Materials:**

*E. coli* Top10 and Origami (DE3) competent cells,

Recombination product after Gibson assembly,

Antibiotic-free LB liquid, Solid medium with antibiotics

**Equipment:**

Ice bath

42°C water bath

Incubator

Shaker

**Procedure:**

1. Added recombination mix (10 μL) to pre-chilled competent Top10 cells, incubated on ice 30 min.
2. Heat-shocked at 42 °C for 90 sec.
3. Immediately placed on ice 2 min.
4. Added 900 μL LB medium and incubated at 37 °C for 1 h.
5. Plating: Spread 100 μL of recovered cells on LB agar + ampicillin.
6. Incubated plates overnight at 37 °C to select for transformants.

',
},
{
"title": '9. Colony PCR',
"content": '

**Materials:**

PCR buffer

dNTPs

T7-pro and T7-ter primers

2×Hieff® Ultra-Rapid II HotStart PCR Master Mix

**Equipment:**

Thermal cycler

PCR tubes

**Procedure:**

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| PCR Rapid mix (2×) | 5 | Denaturation: 95 ℃ for 10 sec |
| T7-pro (10 μM) | 0.4 | Annealing: Tm for 15 sec |
| T7-ter (10 μM) | 0.4 | Extension: 72 ℃, 3 sec/kb |
| Single colony | / | Back to step 1 (20 times) |
| ddH2O | 4.2 | Final elongation: 72 ℃ for 10 sec |
| Total | 10 | 10 ℃ hold |

* After amplification, analyze 3.5 μL of the PCR reaction by agarose gel electrophoresis. The procedure is as previously
  described.
* Inoculate the positive transformants in an LB culture medium containing antibiotics at 220 rpm, 37 ℃ overnight.

',
},
{
"title": '10. Plasmid Extraction',
"content": '

**Materials:**

DiaSpin Plasmid Mini-Preps Kit

Ethanol

**Equipment:**

Centrifuge

Oven

**Procedure:**

(1) Culture bacterial fluid: Inoculate strains into 10 mL of liquid LB medium supplemented with 100 μg/mL Ampicillin.

(2) Place a column into a collection tube. Add 500 μL of Buffer S to column, centrifuge at 12,000 ×g for 1 min. Discard the
filtrate in the collection tube, then place column back to the collection tube.

(3) Collect 10 mL of overnight bacteria culture. Centrifuge at 4,000 ×g for 10 min to pellet the bacteria. Discard the
supernatant.

(4) Resuspend the bacteria pellet in 500 μL of Buffer SP1 by vortexing. Make the pellet completely resuspended.

(5) Add 500 μL of Buffer SP2 and mix by gently inverting the tube for 10 times.

(6) Add 700 μL of Buffer SP3 and mix by gently inverting the tube for 10 times.

(7) Centrifuge at 12,000 ×g for 10 min. Transfer the supernatant to the column, then centrifuge at 8,000 ×g for 1 min.
Discard the filtrate in the collection tube.

(8) Add 700 μL of Wash Solution into the column, then centrifuge at 9,000 ×g for 1 min. Discard the filtrate in collection
tube, then return column back to the collection tube. Repeat this step once.

(9) Place column into a 1.5 mL tube, then add 50 μL of ddH₂O into the column.

(10) Put in 50 ℃ oven for 5 min.

(11) Centrifuge column for 2 min at 9,000 ×g. Store centrifuge solution in -20 °C refrigerator.

',
},
]
},
{
"title": 'Protein expression and purification',
"content": '',
"children": [
{
"title": '1. Protein expression',
"content": '

**Materials:**

Seed culture (Origami DE3 strains with recombinant plasmids)

LB liquid medium (with 100 μg/mL Amp)

1 M IPTG solution

**Equipment:**

Shaker

Clean bench

Centrifuge

**Procedure:**

1. Inoculated at 1: 50 ratios: 200 μL seed culture + 10 mL LB medium (with 10 μL Amp antibiotic).
2. Incubated at 37°C with shaking (200 rpm) for 2–3 h until OD600 reached 0.4–0.6 (log phase).
3. IPTG Induction:

1. Reduced culture temperature to 18°C (ice bath for 10 min).
2. Added IPTG to final concentration of 0.2 mM (2 μL 1 M IPTG + 10 mL LB medium).
3. Induced overnight (12–16 h) at 18°C with shaking (200 rpm).

4. Cell Pellet Collection: Centrifuged at 4,000 rpm for 10 min. Discarded supernatant; stored pellets at -80°C.

',
},
{
"title": '2. Protein purification',
"content": '

**Materials:**

Lysis buffer (with protease inhibitors)

Ni-NTA resin

Binding buffer (20 mM phosphate buffer, pH 7.4, 500 mM NaCl),

Wash buffer (20 mM imidazole), elution buffer (250 mM imidazole)

Native buffer (20 mM Tris-HCl, pH 8.0)

Urea

**Equipment:**

Sonicator

Ice bath

Centrifuge

**Procedure:**

1. **Cell Lysis**

* ![](https://static.igem.wiki/teams/5747/common/xing.webp) Resuspend pellets in lysis buffer (5–10× pellet volume).
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Sonicated on ice**: 30% amplitude, 5 sec ON/OFF pulses, 10 min total.
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Centrifuged**: 12,000 rpm, 20 min, 4°C; collected supernatant.

2. **Ni-NTA Affinity Chromatography:**

* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Equilibration:** Washed resin with 5 column volumes of binding buffer.
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Binding**: Incubated lysate with resin on ice for 1 h with gentle agitation.
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Wash:** Centrifuged (1,000 g, 10 sec); washed twice with wash buffer.
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Elution:** Collected target protein with elution buffer.
* ![](https://static.igem.wiki/teams/5747/common/xing.webp) **Principle:** His-tag binds Ni2+; imidazole competes for elution.

3. **Protein Denaturation & Renaturation:**

* Denaturation

1. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Prepare protein sample in native buffer (20 mM Tris-HCl, pH 8.0).
2. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Add ​​denaturant​​ (8 M urea) to unfold and reduce disulfide bonds.
3. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Incubate for ​​2–4 hours at room temperature​​ or ​​37°C​​ with gentle agitation.
4. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Centrifuge at ​​12,000 × g for 20 min​​ to remove insoluble aggregates.

* ​​Renaturation by Dialysis​​

1. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Transfer denatured protein to dialysis tubing (MWCO 10 kDa).
2. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Dialyze sequentially against buffers with ​​stepwise decreasing denaturant​​ (4 M → 2 M → 0 M urea) to promote disulfide
   reformation.
3. ![](https://static.igem.wiki/teams/5747/common/xing.webp) Each step requires ​​4–6 hours at 4°C​​.

',
},
{
"title": '3. SDS-PAGE and Staining',
"content": '

**Materials:**

12% SDS-PAGE Gel Rapid Preparation Kit

Fast Stain Solution

**Equipment:**

SDS-PAGE adhesive dispensing device

**Procedure:**

1. **Gel Casting Setup​​**

1. Clean glass plates, spacers, and comb thoroughly. Assemble the gel cassette securely to prevent leaks

2. **​​Prepare Separating Gel (Lower Gel)​​**

1. Mix ​**​**2.5 mL​​ of pre-mixed Separating Gel Solution
2. Add ​​50 μL​​ of Modified Coagulant (provided instead of TEMED)
3. Mix gently by swirling to avoid bubble formation
4. Immediately pour into gel cassette, leaving appropriate space for stacking gel
5. Overlay with ​​isopropanol or water​​ to flatten the surface
6. Polymerization occurs within ​​5-10 minutes​​

3. **​​Prepare Stacking Gel (Upper Gel)​​**

1. After separating gel polymerization, remove the overlay liquid and dry the residual moisture
2. Mix ​​1.0 mL​​ of pre-mixed Stacking Gel Solution
3. Add ​​20 μL​​ of Modified Coagulant
4. Mix gently without introducing bubbles
5. Pour onto polymerized separating gel
6. Insert the comb without trapping air bubbles
7. Polymerization completes within ​​5-10 minutes​​

4. **Run the sample on the gel​​**

1. Sample preparation: Mixed 5 μL 5× SDS-PAGE loading buffer + 20 μL sample, then denatured at 98°C for 5 min
2. Carefully remove comb after complete polymerization
3. Rinse wells with electrophoresis buffer to remove residual acrylamide
4. Mount gel in electrophoresis chamber and fill with running buffer
5. Set the electrophoresis program: 150V, 1 h

5. **Staining & Destaining:**

1. Transfer gel to a tray, rinse with distilled water.
2. Stain with Fast Stain Solution (5–30 min with gentle agitation).
3. Destain with water until bands are clear (change water as needed).

',
},
{
"title": '4. Protein Quantification',
"content": '

**Materials:**

Bradford solution

BSA

PBS

**Equipment:**

Microplate reader

**Procedure:**

* **Preparation:**

1. Equilibrate all reagents to room temperature.
2. Prepare BSA standard dilutions using the same buffer as samples (0, 10, 20, 40, 60, 80, 100, 200 μg/mL).

* Standard Curve Setup

1. Add 5 μL of each BSA standard or sample to a 96-well plate .
2. Add 250 μL of Bradford solution to each well.

* **Incubation**

Incubate at room temperature for 5 minutes (avoid exceeding 1 hour).

* **Measurement**

1. Read absorbance at 595 nm using a microplate reader.
2. Generate a standard curve by plotting absorbance vs. BSA concentration.
3. Calculate sample protein concentration from the standard curve equation

',
},
]
},
{
"title": 'IgE affinity test',
"content": '

**Material:**

IgE ELISA Kit (Sangon Biotech)

PBS (pH 7.2)

Blocking buffer (1% BSA in PBS)

Wash buffer (PBS containing 0.05% Tween-20)

**Equipment:**

Microplate reader

**Procedure:**

1. **Protein-Coated 96-Well Plate**
   1. Dilute the allergen protein to a concentration of 10 μg/mL using PBS (pH 7.2).
   2. Add 0.1 mL of the diluted allergen protein solution to each well of an ELISA plate, and incubate overnight
      at 4°C.
2. **Blocking**

1. Add 100 µL of blocking buffer (1% BSA in PBS) to each well.
2. Incubate at 37°C for 1 hour.
3. Wash the wells to remove unbound blocking reagent.

3. **Preparation of IgE Standards**

1. Centrifuge the lyophilized IgE standard at 10,000 × g for 1 minute.
2. Reconstitute by adding 1.0 mL of sample dilution buffer, invert gently, and allow to dissolve for 10 minutes.
   Mix thoroughly.
3. Prepare a 200 ng/mL stock solution, then perform serial two-fold dilutions to obtain the following
   concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.13, 0 ng/mL.
4. Use 7 tubes, each preloaded with 500 µL dilution buffer. Add 500 µL from the previous tube, mix well, and
   continue stepwise. The final tube (0 ng/mL) serves as the blank. Add the diluted sample to the 96-well plate.

4. **IgE Binding to Allergen-Coated Wells**

1. Add 100 µL of IgE working solution (50 ng/mL) to each well. For the standard curve, use the full dilution
   series.
2. Incubate at 37°C for 90 minutes.

   *Note: Add samples gently to the center of the well, avoiding the walls; mix gently without bubbles. Complete
   sample loading within 10 minutes.*
3. Prepare biotin-labeled anti-IgE antibody by diluting the 100× stock 1:100 in SD2 buffer (calculate required
   volume based on 100 µL per well). Use within 30 minutes.
4. Discard the well contents and tap dry. Add 100 µL of the biotin-labeled antibody to each well.
5. Incubate at 37°C for 60 minutes, then wash 4 times with 350 µL wash buffer (soak 1–2 minutes per wash).

5. **Streptavidin-HRP Conjugate Binding**

1. Dilute the 100× HRP-labeled streptavidin stock 1:100 in SD3 buffer (post-centrifugation), based on 100 µL/well.
   Use within 30 minutes.
2. Add 100 µL of the HRP conjugate to each well.
3. Incubate at 37°C for 30 minutes, then wash 4 times using 300 µL per wash (30 s intervals).

6. **Color Development**

1. Add 90 µL of TMB substrate to each well (protect from light).
2. Incubate at 37°C in the dark for ~15 minutes.

7. **Reaction Termination**

Add 50 µL stop solution to each well and immediately read absorbance at 450 nm using a microplate reader (within 5
minutes).

8. **Data Analysis**

1. Subtract the OD value of the blank (0 ng/mL) from all standards and sample readings.
2. Plot the standard curve using IgE concentrations on the X-axis and corresponding OD values on the Y-axis.
3. Fit the curve using a four-parameter logistic regression model to quantify IgE binding in unknown samples.

',
},
{
"title": 'Cell Proliferation test',
"content": '

**Materials:**

NIH/3T3 cells

DMEM complete medium

1× DPBS

0.25% trypsin solution

T75 cell culture flasks

15 mL centrifuge tubes

hypoallergenic derivative, natural allergens, allergen fusion protein

CCK-8

**Equipment:**

Biosafety cabinet

Inverted microscope

CO₂ incubator (37 °C, 5% CO₂)

Centrifuge (200 ×g)

Pipettes and sterile tips

Microplate reader

**Procedure:**

**(1) Cell Revival:**

1. Rapidly take out frozen NIH-3T3 cells from liquid nitrogen.
2. Thaw the vial in a 37 °C water bath with gentle shaking. Do not exceed 1.5 min.
3. In a biosafety cabinet, slowly add the 1 mL thawed cell suspension to 9 mL pre-warmed complete DMEM medium
   in a centrifuge tube.
4. Mix gently by pipetting up and down.
5. Centrifuge at 200 ×g for 5 min.
6. Discard the supernatant carefully.
7. Resuspend the cell pellet in 1 mL of fresh complete DMEM medium.
8. Transfer the suspension into a T75 flask containing 9 mL complete DMEM medium.
9. Mix gently by crosswise shaking and place the flask into a 37 °C, 5% CO₂ incubator.
10. Replace medium with fresh complete medium on the second day, then every other day.

**(2) Cell Passaging:**

1. Observe cells under an inverted microscope; proceed when ~80% confluent.
2. In biosafety cabinet, discard old medium.
3. Add 3 mL DPBS, gently shake, discard; repeat wash once.
4. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
5. Incubate at 37 °C, 5% CO₂ for ~2 min.
6. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
7. Pipette up and down to disperse cell clumps.
8. Transfer to centrifuge tube, spin at 200 ×g for 5 min, discard supernatant.
9. Resuspend pellet in 2 mL complete medium.
10. Add 9 mL fresh medium to a new T75 flask; seed appropriate volume of cell suspension.
11. Mix crosswise and place in incubator (37 °C, 5% CO₂).

**(3) Cell culture plate and CCK-8 treatment**

1. Observe cells under an inverted microscope; proceed when ~80% confluent.
2. In biosafety cabinet, discard old medium. Add 3 mL DPBS, gently shake, discard; repeat wash once.
3. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
4. Incubate at 37 °C, 5% CO₂ for ~2 min.
5. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
6. Prepare a cell suspension with a density of 1×10⁵ cells/mL using complete medium. Transfer 90 μL of the cell
   suspension into a 96-well plate.
7. Preparation of drugs: Dilute each sample to 1000, 100, and 10 nM (the concentration after adding to the
   cells will be 100, 10, and 1 nM respectively). Add 10 μL of drug solutions at different concentrations to a
   96-well cell culture plate.
8. Add an equal volume of DPBS solution to the outer perimeter of the 96-well plate to prevent excessive
   evaporation.
9. After 48h of incubation, add 10μL CCK-8 reagent to each well. Incubate at 37°C with 5% CO₂ for 30 min, then
   measure the absorbance at 450 nm using a microplate reader.
10. Calculate cell viability using the following formula:

Cell viability (%) = Pc/Pb×100%

*Pe represents the absorbance of the experimental group and Pb represents the absorbance of the control
group.*

*Control group: Cells + PBS + complete medium + CCK-8*

*Blank group: Complete medium + CCK-8*

*Experimental group: Cells + drug + complete medium + CCK-8*

'
},
{
"title": 'Half-life detection',
"content": '

**Materials​​**

Purified ​​FEFKFEFK-EGFP​​ fusion protein (experimental group)

Purified ​​non-fused EGFP​​ (control group)

​​ Fetal bovine serum (FBS), sterile-filtered

PBS (pH 7.4) for dilution

**​​Equipment​​:**

Thermostatic incubator (37°C)

Microplate reader

Black 96-well plates (to minimize background fluorescence).

Pipettes and sterile tips.

**Procedure**

**1. Sample Preparation:**

1. Dilute both FEFKFEFK-EGFP and non-fused EGFP to 500 µg/mL in FBS.
2. Aliquot 200 µL of each protein-serum mixture into wells of a 96-well plate (triplicates per time point).
3. Include blank controls-FBS

**2.Incubation:**

Seal the plate to prevent evaporation, and incubate at 37°C for 5 days.

**3. Fluorescence Measurement:**

1. At each time point, the culture plate is briefly shaken first to prevent the precipitates from aggregating
   together.
2. Measure fluorescence (excitation 488 nm, emission 507 nm).

**4.Data processing**

The fluorescence intensity was processed using GraphPad 8.0.2, and the half-life of the sample was predicted using
the Exponential decay - one phase model.

',
},
{
"title": 'Soluble Microneedles Preparation',
"content": '

**Materials​​**

**​** PMSF microneedle molds

Sodium hyaluronate (50,000 MW)

PVA

Pigment

**​​Equipment​​:**

Centrifuge

Water bath

**Procedure**

**1. Clean PMSF molds:**

Place molds in a beaker containing water and briefly sonicate; remove excess moisture (using absorbent paper),
then place in a 60°C oven.

**2. Prepare soluble gel**

1. Weigh 0.3g sodium hyaluronate (50,000 MW) and add sequentially (3 additions total) to 1mL drug solution. After
   each addition, vigorously stir using a 1mL pipette tip until completely dissolved (needle layer gel).
2. Weigh PVA and dissolve in water to prepare a 250mg/mL backing layer gel solution.
3. Centrifuge both needle layer and backing layer gels at 3000 rpm for 3 min to degas;
4. Transfer gel into molds using pipette tip. Cover mold cavities with non-absorbent sealing film, cap with another
   mold, and centrifuge in a centrifuge tube.
5. Repeat gel addition and centrifugation as needed.
6. Scrape off surface gel.

**3. Prepare the base layer - PVA**

Add an appropriate amount of backing gel solution to fill the mold completely. Dry at room temperature for 3-4
days.

**4. Water Solubility Testing**

(1) Immerse microneedles in pre-warmed PBS (37℃​​) in a Petri dish

(2) Maintain temperature using a water bath (37°C​​ to simulate skin conditions)

(3) Observe the dissolution of the needle tip on the light-transmitting plate.

',
},
] %}
{% block title %}Experiments{% endblock %}
{% block lead %}EXPERIMENTS{% endblock %}
{% block leadImg %}

![](https://static.igem.wiki/teams/5747/common/experiments.webp)

{% endblock %}
{% block page\_content %}
{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Overview',
"content": '

To overcome the key limitations of traditional allergen immunotherapy (AIT), we integrated three innovative
strategies aimed at enhancing safety, efficacy, and patient compliance.

* Firstly, by rationally designing hypoallergenic variants of six major allergens (pollen, dust mites, and seafood
  derivatives), we mutated the IgE binding epitopes while retaining T-cell reactivity, thereby reducing the risk
  of anaphylactic shock.
* Secondly, by combining the hypoallergenic units with self-assembling peptides (EFK8), we developed a
  broad-spectrum fusion protein, enabling multi-allergen targeting and extending the treatment half-life.
* Thirdly, we implemented a micro-needle-based delivery platform for painless and continuous antigen release,
  improving accessibility and long-term compliance.

These methods collectively addressed the challenges of artificial immunotherapy (AIT) in terms of side effects,
complexity, and invasiveness, while inducing a strong immune tolerance.

',
},
{
"title": 'Part 1: Hypoallergenic derivatives preparation',
"content": '

In this part, we designed and produced recombinant hypoallergenic derivatives derived from six common allergens:
Phl p1 and Bet v1 (pollen allergens), Der p1 and Der p2 (dust mite allergens), and Met e1 and Cyp c1 (seafood
allergens). Through rational protein design, we eliminated or disrupted key conformational IgE-binding epitopes
while strategically preserving T-cell epitopes, and we have named these hypoallergenic derivatives as: mPhl p1,
mBet v1, mDer p1, mDer p2, mMet e1, mCyp c1 (**Table 1**). This approach maintains the immunogenicity
required for inducing immune tolerance while significantly reducing the risk of triggering allergic reactions.

**Table 1** Design Principles for Hypoallergenic protein Sequences

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **Source** | **Hypoallergenic Mechanism** | **Ref** |
| mPhl p1 | Timothy grass pollen allergen | Phl p 1 was broken into four fragments and then reassembled as a Phl p 1 mosaic protein | (Ball et al., 2009) |
| mBet v1 | *Betula verrucosa* | 7 consecutive amino replacement | (Wallner et al., 2011) |
| mDer p1 | *Dermatophagoides pteronyssinus* | Chimeric gene of T cell epitopes | (Hong, 2018) |
| mDer p2 | Site-directed mutagenesis of IgE-binding epitopes | (Reginald & Chew, 2018) |
| mMet e1 | Shrimp tropomyosin | Site-directed mutagenesis of IgE-binding epitopes | (Wai et al., 2014) |
| mCyp c1 | Carp parvalbumin | Site-directed mutagenesis of calcium-binding domains | (Swoboda et al., 2007) |

The plasmids were designed using the pET-32a vector, which features a T7 promoter for high-level expression, a
His-tag for simplified purification, and a Trx tag to enhance soluble expression of the target allergen protein.
The gene fragments encoding the engineered ​​hypoallergenic proteins​​ were first amplified via ​​PCR​​. The
pET-32a vector was then digested with the corresponding ​​restriction enzymes​​ (BamHI and XhoI). Following
purification, the target gene fragments and vector were assembled using ​​homologous recombination​​ (**Figure 1**).

![](https://static.igem.wiki/teams/5747/results/1.webp)

**Figure 1** Schematic diagram of the plasmid construction.

​ As shown in **Figure 2**, PCR products and linearized vector were validated via agarose gel
electrophoresis. Target gene fragments (330-720 bp) were confirmed for purity and accuracy through staining and UV
visualization. Following gel extraction and purification, six allergen genes (mPhl p1, mBet v1, mDer p1, mDer p2,
mCyp c1, mMet e1) were inserted into the linearized pET32a vector using homologous recombination technology,
completing recombinant plasmid construction.

![](https://static.igem.wiki/teams/5747/results/2.webp)

**Figure 2** Agarose gel electrophoresis results of (A) gene amplification and (B) vector
linearization.

The recombinant products were then transformed into *E. coli* Top 10 competent cells, and positive
clones were selected on LB agar plates, respectively. Colony PCR and DNA sequencing verified the correct insertion
and integrity of the allergen genes. Selected positive clones were sequenced, and the sequence data confirmed that
each plasmid construct contained the correct allergen gene sequence with high fidelity (**Figure 3**). The validated plasmids were subsequently transformed into *E. coli* Origami (DE3) competent cells
to enhance soluble expression of the recombinant hypoallergenic proteins.

![](https://static.igem.wiki/teams/5747/results/3.webp)

**Figure 3** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

Next, we expanded the cultivation and used IPTG to induce the expression of the hypoallergenic protein
derivatives. Our goal was to express and purify the recombinant allergen proteins for subsequent research. After
cell lysis and centrifugation, we performed SDS-PAGE to identify the protein expression and purification
conditions. The SDS-PAGE analysis results showed that all six hypoallergenic protein derivatives were successfully
expressed, and the molecular weights were consistent with the expectations (**Figure 4**
**A**). Among them, mPhl p1, mBet v1, and mDer p1 were mainly expressed in the precipitate part, with
less background proteins and high purity. These insoluble proteins were then denatured and refolded to obtain
soluble proteins. For the three soluble-expressed proteins: mDer p2, mMet e1, and mCyp c1, we used nickel columns
to purify them separately. The SDS-PAGE results indicated that we successfully obtained highly pure proteins
(**Figure 4** **B**).

![](https://static.igem.wiki/teams/5747/results/4.webp)

**Figure 4** SDS-PAGE results of (A) protein expression and (B) purification

',
},
{
"title": 'Part 2: Natural allergens preparation',
"content": '

To validate whether our engineered hypoallergenic derivatives indeed exhibit reduced allergenicity (IgE binding
capacity), we also produced the corresponding native allergens. Notably, since native rDer p1 requires
glycosylation and cannot be produced in *E. coli*, it was commercially purchased from WOLCAVI BIOTECH.
For the other five native allergens (rPhl p1, rBet v1, rDer p2, rMet e1, rCyp c1), we introduced reversion
mutations via primers, obtained the fragments through PCR, and subsequently constructed the recombinant plasmids
using homologous recombination (**Figure 5**).

![](https://static.igem.wiki/teams/5747/results/5.webp)

**Figure 5** Schematic diagram of the plasmid construction of native allergens.

As shown in **Figure 6**, PCR products were validated via agarose gel electrophoresis. Target gene
fragments were confirmed for purity and accuracy through staining and UV visualization. Notably, due to multiple
mutation sites in rCyp c1, its gene fragment (156 bp) was assembled from synthesized oligos and then fused with
rCyp-AMP (2847 bp) via overlap PCR, yielding a 2984 bp product. We synthesized the rPhl p1 and rMet e1 fragments
in the company. After PCR and agarose gel electrophoresis, the pET32a vector was observed to have a band near
5000 base pairs. Both rPhl p1 (777 base pairs) and rMet e1 (876 base pairs) were of the expected size.After
the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

![](https://static.igem.wiki/teams/5747/results/result-6-new.webp)

**Figure 6** Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the *E. coli* Top 10 bacteria, and then screen out the
positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we
successfully obtained the plasmid with correct sequencing (**Figure 7**). These verified plasmids
were subsequently transferred into *E. coli* Origami (DE3).

![](https://static.igem.wiki/teams/5747/results/7.webp)

**Figure 7** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

Next, we expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins.
The SDS-PAGE analysis results showed that all five natural allergen proteins were detectably expressed in both the
supernatant and the precipitate, and their molecular weights were consistent with the expected values (**Figure 8A**). Subsequently, we purified them using a nickel column. The SDS-PAGE results indicated that we successfully
obtained highly purified proteins (**Figure 8B**).

![](https://static.igem.wiki/teams/5747/results/8.webp)

**Figure 8** SDS-PAGE results of (A) protein expression and (B-C) purification.

',
},
{
"title": 'Part 3: Fused allergens preparation',
"content": '

To address the issue of multiple allergic reaction sensitivities and simplify the treatment plan, we have designed
and manufactured a new type of fusion protein. This protein integrates multiple modified hypoallergenic
derivatives that trigger similar immune responses into a single molecular structure. This strategy aims to achieve
broad therapeutic effects, potentially avoiding the cumbersome process of multiple separate injections, thereby
enhancing treatment efficiency and improving patient compliance. To achieve this, we investigated and selected the
EFK8 self-assembling peptide (FEFEFKFK), which undergoes oligomerization driven by hydrophobic stacking of
phenylalanine residues and electrostatic interactions of lysine residues. This process forms stable nanostructures
that increase molecular size and reduce renal clearance, thereby prolonging the circulation half-life (Song et
al., 2023).

We assembled short EFK8 oligonucleotide fragments firstly. Subsequently, we separately amplified single
hypoallergenic derivative gene fragments with homologous arms through PCR. Then, we used overlapping extension PCR
to splice these fragments together to generate the complete fusion allergen gene sequence. Finally, we performed
restriction endonuclease digestion and ligation on the purified fusion gene and linearized vector backbone to
obtain the desired recombinant plasmid product. Furthermore, we also constructed a plasmid that integrates the
EFK8 peptide and the enhanced green fluorescent protein EGFP, which was used for the subsequent determination of
the half-life (**Figure 9**).

![](https://static.igem.wiki/teams/5747/results/9.webp)

**Figure 9** Schematic diagram of the plasmid construction.

As shown in **Figure 10**, PCR products were validated via agarose gel electrophoresis. Target gene
fragments were confirmed for purity and accuracy through staining and UV visualization (**Figure 10A-B**). Then, we used
overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene
sequence (**Figure 10C**). After the fragments were recovered through gel cutting, we used homologous recombination to
recombine them.

![](https://static.igem.wiki/teams/5747/results/10.webp)

**Figure 10** Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the *E. coli* Top 10 bacteria, and then screen out the
positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we
successfully obtained the plasmid with correct sequencing (**Figure 11**). These verified plasmids
were subsequently transferred into *E. coli* Origami (DE3).

![](https://static.igem.wiki/teams/5747/results/11.webp)

**Figure 11** (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

We expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins.
The EFK8-mPhl p1-GS-mBet v1 (FPB) and EFK8-mDer p1-GS-mDer p2 (FDD) proteins are both expressed in the
precipitate. Subsequently, a denaturation-reconstitution procedure is required to restore their soluble state. The
EFK8-mMet e1-GS-mCyp c1 (FMC) protein was expressed in both the supernatant and the precipitate. We purified the
soluble FMC protein using a nickel column. The SDS-PAGE results indicated that we were able to successfully purify
this protein (**Figure 12)**.

![](https://static.igem.wiki/teams/5747/results/12.webp)

**Figure 12** SDS-PAGE results of (A) protein expression and (B) purification.

',
},
{
"title": 'Part 4: IgE affinity test of allergen protein',
"content": '

Protein concentrations of both purified native allergens and their hypoallergenic derivatives were quantified
using the Bradford assay to ensure accurate loading for subsequent IgE-binding affinity assays. A standard curve
was constructed using bovine serum albumin (BSA) as a reference, exhibiting excellent linearity (R² = 0.9987) with
the regression equation Y = 0.001563x + 0.002397 (**Figure 13**). All absorbance measurements at 595
nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (**Table 2**).

![](https://static.igem.wiki/teams/5747/results/13.webp)

**Figure 13** BSA standard curve test by Bradford assay. (A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

**Table 2** Protein quantification results.

![](https://static.igem.wiki/teams/5747/results/14.webp)

Next, we conducted IgE binding affinity analysis, the main purpose of which is to functionally evaluate the
allergenic potential of natural and modified hypoallergenic derivatives, and fusion hypoallergenic derivatives.
This assessment is crucial because the clinical severity of allergic reactions is largely determined by the
affinity of the specific IgE antibodies against the allergen and its target epitope (Fromberg, 2006).
High-affinity IgE antibodies can trigger strong activation of mast cells and basophils even in the presence of low
concentrations of allergens, leading to significant release of inflammatory mediators and potential anaphylactic
shock. In contrast, low-affinity IgE has significantly reduced ability to bind to the FcεRI receptor, thereby
reducing the risk of severe allergic reactions (Udoye, Ehlers, & Manz, 2023). Therefore, by quantifying the
degree of reduction in IgE binding affinity, it can provide a key indicator for in vitro experiments to determine
whether our rational protein design strategy has successfully achieved the reduction of allergenicity.

The standard curve for human IgE quantification was generated using four-parameter logistic (4PL) regression in
GraphPad Prism 9.5, demonstrating excellent fit (R² > 0.998), which validated the reliability of subsequent
affinity measurements (**Figure 14A)**. The IgE-binding capacities of six purified allergens—both
native and hypoallergenic derivatives—were evaluated by ELISA. Coated proteins were incubated with 50 ng/mL human
IgE. As shown in **Figure 14B,** significant reduction in IgE binding (p < 0.0001) was observed in
four hypoallergenic derivatives compared to their native counterparts (mBet v1, mDer p2, mMet e1, mCyp c1).
Although mPhl p1 and mDer p1 also showed decreased affinity, the reduction was more modest
(p>0.05**)**. These results confirmed the effectiveness of the rational design strategy in
generating hypoallergenic proteins with markedly reduced IgE reactivity.

![](https://static.igem.wiki/teams/5747/results/15.webp)

**Figure 14** IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of natural
allergens and hypoallergenic derivatives.

As illustrated in **Figure 15A**, the IgE standard curve demonstrated excellent linearity (R² =
0.9943), ensuring the reliability of the subsequent binding affinity quantification. Interestingly, comparative
analysis revealed that two fusion allergens, ​​EFK8-mPhl p1-GS-mBet v1 (FPB)​​ and ​​EFK8-mDer p1-GS-mDer p2
(FDD)​​, exhibited significantly reduced IgE binding ratios compared to their respective single hypoallergenic
constituents (mPhl p1 and mDer p1, respectively). ​​EFK8-mMet e1-GS-mCyp c1 (FMC)​​ also showed low IgE binding
affinity, which was comparable to the levels observed for its individual hypoallergenic derivatives (mMet e1 and
mCyp c1) (**Figure 15B)**. This consistent reduction across all fusion constructs suggests that the
fusion strategy effectively attenuates IgE immunoreactivity beyond the level achieved by individual engineered
allergens, likely due to structural masking of key epitopes or altered antigen presentation.

![](https://static.igem.wiki/teams/5747/results/16.webp)

**Figure 15** IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of single and
fusion hypoallergenic derivatives.

',
},
{
"title": 'Part 5: Cell proliferation experiment test',
"content": '

Building upon the IgE-binding affinity results, we further evaluated the cytotoxicity of both single and fusion
hypoallergenic derivatives using a CCK-8 assay to assess their biosafety for potential in vivo therapeutic
applications. This evaluation is essential to ensure that engineered biologics exhibit minimal adverse effects on
cell viability before proceeding to animal studies or clinical use.

After incubating cells with each protein at concentrations of 1 nM, 10 nM, and 100 nM for 24 h and 48 h,
significant differences in cytotoxicity profiles were observed. For mBet v1 and mCyp c1, at all tested concentrations
and time points, there was almost no significant effect on
cell viability, demonstrating extremely high biocompatibility (**Figure 16B, H**). For mPhl p1 and mMet e1, the toxicity
showed a clear time and concentration dependence (**Figure 16A, G**). At low concentrations and short durations, the impact
was minimal, but as the concentrations and durations increased, the toxicity significantly intensified. For mDer p1 and
mDer p2, even at lower concentrations and shorter durations, it could cause a significant decrease in cell viability,
indicating an inherent toxicity (**Figure 16D, E**).

Next, we compared the cytotoxicity profiles of both individual and fused hypoallergenic derivatives. For the fusion construct
EFK8-PT-mPhl p1-GS-mBet v1, we observed that the intrinsic cytotoxicity of mPhl p1 influenced the overall profile of
the fusion protein, leading to a detectable reduction in cell viability particularly under high-concentration or
prolonged exposure conditions (**Figure 16A-C**).In contrast, the fusion protein EFK8-PT-mMet e1-GS-mCyp c1 exhibited a
different behavior: although mMet e1 alone showed time- and concentration-dependent cytotoxicity, the fusion with
mCyp c1—which itself displayed no appreciable toxicity—resulted in a composite molecule that did not induce
significant cytotoxicity, even at high doses or after extended incubation (**Figure 16D-F**). Notably, the fusion
protein EFK8-PT-mDer p1-GS-mDer p2 did not exhibit significant cytotoxicity under the tested conditions.
This is in clear contrast to its individual components, both mDer p1 and mDer p2, which caused a marked decrease
in cell viability, especially at higher concentrations (e.g., 100 nM) and with longer culture periods (48 h)
(**Figure 16G-I**). These results suggest that the fusion strategy can alter the cytotoxic properties of the constituent
hypoallergenic derivatives, in some cases mitigating the adverse effects observed with the individual components.

​ The CCK-8 results indicated that most hypoallergenic variants, especially fusion constructs, possess favorable
biosafety profiles with minimal impact on cell viability. The marked reduction in cytotoxicity observed for the
fusion form of mDer p1, compared to its single allergen counterpart, highlights the potential of fusion strategies
not only to broaden immunotherapeutic coverage but also to improve biocompatibility. These findings support the
advancement of fusion hypoallergenic derivatives, particularly EFK8-based constructs, toward further evaluation.

**![](https://static.igem.wiki/teams/5747/results/17.webp)**

**Figure 16** The results of the cell proliferation experiment.

',
},
{
"title": 'Part 6: Half-life test',
"content": '

To evaluate whether fusion with the self-assembling peptide EFK8 prolongs the half-life of hypoallergenic fusion
proteins, we employed EGFP as a model protein incubated in fetal bovine serum at 37 °C for 5 days to simulate
*in vitro* conditions. Fitting the fluorescence decay data to a one-phase exponential model revealed that
EFK8-EGFP exhibited a significantly extended half-life (1.820 days) compared to EGFP alone (0.9819 days), nearly
doubling its stability (**Figure 17**). This enhancement is likely attributable to EFK8-mediated
oligomerization, which increases molecular size and reduces degradation rate.

**![](https://static.igem.wiki/teams/5747/results/18-1.webp)**

**Figure 17** Half-life of EGFP and EFK8-EGFP.

',
},
{
"title": 'Part 7: Microneedle preparation',
"content": '

To fabricate the dissolvable microneedles (MNs), sodium hyaluronate (50,000 MW) was utilized as the tip-layer
material and polyvinyl alcohol (PVA) as the backing layer using a 10 mm × 10 mm mold. The resulting MNs, as shown
in the **Figure 18A,** exhibited integrated structure. Subsequent solubility testing demonstrated
that the needle tips dissolved completely within 5 minutes of incubation at 37 °C, indicating their characteristic
suitable for efficient drug delivery applications (**Figure 18B)**.

**![](https://static.igem.wiki/teams/5747/results/19.webp)**

**Figure 18** (A) Preparation and (B) Water Solubility Test Soluble Microneedles.

',
},
{
"title": 'Discussion',
"content": '

In the above experimental results, we found the significantly reduced IgE binding affinity and cytotoxicity
observed in our fusion allergens (EFK8-PT-mPhl p1-GS-mBet v1, EFK8-PT-mDer p1-GS-mDer p2) compared to their
monomeric counterparts. We speculated that may be attributed to several synergistic mechanisms facilitated by the
fusion strategy and the self-assembling peptide EFK8.

First, fusion-induced ​​steric hindrance​​ and ​​conformational rearrangement​​ likely occlude key IgE-binding
epitopes, thereby directly impairing antibody recognition and cross-linking capacity. Secondly, the
​​oligomerization​​ process triggered by EFK8 will increase the molecular size and alter the surface topological
structure through hydrophobic and electrostatic interactions, causing conformational changes or masking at
specific sites in these fused hypoallergenic derivatives, thereby further leading to the loss of toxic functions.
We have predicted the molecular structures of EFK8-PT-mPhl p1-GS-mBet v1 and EFK8-PT-mDer p1-GS-mDer p2, and
conducted molecular oligomerization simulations. We found that after oligomerization, some parts of the structures
of these hypoallergenic derivatives would be covered. For details, please refer to the [Model](https://2025.igem.wiki/tsinglan/model) page.

Collectively, these modifications not only diminish immediate hypersensitivity risks but also mitigate direct
cellular toxicity, underscoring the potential of fusion-based designs in developing safer and more effective
allergen immunotherapies.

',
},
{
"title": 'Future plan',
"content": '

To comprehensively evaluate our engineered EFK8-fusion hypoallergenic derivative, we will conduct a multi-tiered
assessment in the future. This includes analyzing their ​​aggregation propensity​​ via biophysical techniques to
ensure structural stability, followed by ​​T-cell stimulatory activity assays​​ to confirm preserved
immunogenicity for tolerance induction. Furthermore, ​​*in vivo* immunogenicity studies​​ in murine models
will be performed to measure IgG-blocking antibody production and long-term desensitization efficacy. Finally, we
aim to develop a ​​microneedle-based biosensor​​ for real-time, minimally invasive monitoring of histamine release
in the dermal interstitial fluid, enabling direct pharmacodynamic correlation between treatment and reduction in
allergic response.

1. **Aggregation Propensity Assessment of EFK8-Fusion Proteins​**

To ensure the structural integrity and stability of the novel EFK8 fusion with a hypoallergenic derivative, we
will systematically evaluate its aggregation tendency. Specific methods include: quantitative analysis of soluble
oligomers via size exclusion chromatography (SEC), determination of hydrodynamic particle size distribution using
dynamic light scattering (DLS), and monitoring via exogenous dye binding assays (*e.g.*, bis-fluorescein
succinimide). These studies will determine how self-assembled EFK8 peptides influence aggregation kinetics or
promote oligomerization, thereby ensuring the stability of subsequent preparations (Grillo et al., 2001).

2. **T-cell stimulatory activity assessment**

To further validate the T-cell stimulatory activity of our engineered hypoallergenic derivatives, future work will
include functional T-cell proliferation and cytokine profiling assays using human peripheral blood mononuclear
cells (PBMCs) from sensitized donors (Raaphorst et al., 2025). We plan to utilize standardized methods such as EdU
incorporation and dye dilution assays to track proliferation dynamics and measure IL-2, IFN-γ, and IL-10 secretion
to evaluate Th1/Tr1 responses. Additionally, we will assess the ability of our constructs to induce
antigen-specific T-cell activation in comparison with native allergens, using ELISpot and intracellular
staining(van Lier, Brouwer, Rebel, van Noesel, & Aarden, 1989).

3. ***In Vivo* Immunogenicity Assessment​​**

*In vivo* immunogenicity assessment evaluates the ability of engineered hypoallergenic derivatives to
stimulate a measurable immune response in a living organism, typically mouse models. This goes beyond simple
binding assays to assess complex biological outcomes, such as ​​T-cell activation, cytokine production
(*e.g*., IFN-γ, IL-10), and the generation of allergen-specific IgG blocking antibodies​​. In the future,
if permission is granted, this would involve immunizing mice (*e.g.*, BALB/c) with our fusion
hypoallergenic derivatives and then analyzing splenocytes or serum. Key readouts include ​​ELISPOT or
intracellular cytokine staining for Th1/Tr1 responses​​ and ​​serum IgG titers​​ via ELISA (Thalhamer et al.,
2010).

4. **Microneedle-Based Histamine Monitoring for Therapeutic Evaluation**

To objectively evaluate the *in vivo* efficacy and pharmacodynamic profile of our novel hypoallergenic
therapeutics, we propose developing a dedicated microneedle (MN)-based wearable biosensor for continuous,
minimally invasive monitoring of histamine levels in dermal interstitial fluid (ISF). ​​The primary purpose of
detecting histamine is to directly quantify mast cell degranulation and allergic response intensity in
real-time​​, providing a dynamic and objective measure of therapeutic intervention effectiveness. This approach
leverages the core advantages of MN technology, including painless penetration, direct ISF biomarker access, and
compatibility with real-time monitoring. We will explore two sensing strategies tailored for histamine detection:

Immunosensor: Utilizing a sandwich ELISA format on MNs tips, where captured histamine is quantified via
enzyme-labeled secondary antibodies generating colorimetric/fluorescent signals (Ying et al., 2024).

Electrochemical Sensor: Immobilizing histamine oxidase on MN-integrated electrodes to catalyze histamine,
producing hydrogen peroxide, which is then electrochemically oxidized to generate a current signal proportional to
histamine concentration (García-Guzmán, Pérez-Ràfols, Cuartero, & Crespo, 2021).

This platform aims to provide direct pharmacological evidence of reduced histamine release following treatment,
enabling precise correlation between drug administration and therapeutic response *in vivo*.

![descript](https://static.igem.wiki/teams/5747/results/20.webp)

**Figure 19** Microneedle based electrochemical sensing (García-Guzmán et al., 2021).

',
},
{
"title": 'Reference',
"content": '

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