A de novo-designed feline allergen Fel d1 binding protein, inhibitor and pharmaceutical uses

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a de novo designed cat allergen Fel d 1 binding protein, an inhibitor and pharmaceutical use. The amino acid sequence of the cat allergen Fel d 1 binding protein is shown in SEQ ID NO. 1 or has at least 98% identity with the sequence of SEQ ID NO. 1. The binding protein of the present application can be used as a substitute for traditional IgY antibodies and has high blocking capacity for IgE. The protein can be efficiently and solubly expressed in a conventional E. coli expression system, and the purification process is simple and easy to scale up, which can greatly reduce the production cost. The binding protein can be effectively used for the prevention and treatment of cat allergy related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a de novo-designed feline allergen Fel d1 binding protein, its inhibitor, and its pharmaceutical uses. Background Technology

[0002] The widespread adoption of companion animals poses a significant public health problem by increasing the risk of allergen exposure in the general population. Currently, cats are the most numerous pets globally, and their population expansion directly exacerbates the spread of feline allergens. Among these allergens, cat dander is a key factor in triggering allergic asthma and rhinitis. Of the eight feline allergens registered by the World Health Organization (WHO), Fel d1, exhibiting the highest IgE recognition frequency and the ability to activate basophils, has become a core target for pathogenic mechanism research and intervention strategy development.

[0003] Currently, the prevention of feline allergy relies on a comprehensive strategy that combines allergen clearance with immune regulation, such as blocking the source through IgY antibodies, reducing environmental load using air purifiers, and supplementing with nutrients such as BLG to regulate the immune response.

[0004] The key to preventing feline allergies lies in blocking the allergen at its source through IgY antibodies. However, this method faces several inherent technical bottlenecks in practical development and application: ① The large amount of antigen required during IgY antibody preparation poses a challenge to large-scale IgY production. ② Complex purification process: Egg yolk is a complex component (rich in lipids and various proteins), making the high-purity separation of IgY cumbersome and costly, severely restricting its industrialization. ③ Product consistency challenges: Traditional polyclonal IgY antibodies exhibit significant batch-to-batch heterogeneity; while the large-scale preparation technology for highly specific monoclonal IgY antibodies is still immature, making it difficult to meet uniformity requirements. ④ Insufficient antibody stability: Although IgY antibodies are more resistant to heat and pH than some mammalian antibodies, they remain unstable in complex in vivo environments and are prone to inactivation. To maintain long-term activity, they typically need to be stored under specific conditions (e.g., 2-8°C) and rely on a strict cold chain storage and transportation system, which significantly increases logistics costs and operational complexity, limiting the feasibility of its practical application. In summary, current strategies for feline allergy prevention based on IgY antibodies are still limited by core issues such as the difficulty in obtaining antibodies, poor economic efficiency in production, and insufficient formulation stability. Therefore, developing an alternative method that combines better production feasibility and physicochemical stability has become an urgent technological direction for breakthroughs in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a de novo-designed feline allergen Fel d1 binding protein, an inhibitor, and pharmaceutical applications.

[0006] In recent years, artificial intelligence (AI) technology has made groundbreaking progress in the field of protein design, especially computational simulation-based methods for the precise design of small-molecule binding proteins, which can efficiently generate binding proteins with high affinity, good expressibility, and excellent stability. Based on the aforementioned AI protein design technology, this study successfully obtained a binding protein with high affinity and high specificity for Fel d1, a major feline allergen. This protein can effectively block the binding of Fel d1 to IgE antibodies, thereby inhibiting the sensitizing activity of the allergen at its source.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution: the amino acid sequence of the feline allergen Feld 1 binding protein is as shown in SEQ ID NO.1 or has at least 98% identity with the sequence of SEQ ID NO.1.

[0008] The term "identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions during alignment, and is usually expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity.

[0009] In a preferred embodiment, the feline allergen Fel d 1 binding protein specifically targets Fel d 1 and is designed to target the IgE binding site of Fel d 1.

[0010] A second aspect of the invention is to provide the gene encoding the aforementioned de novo-designed feline allergen Fel d 1 binding protein.

[0011] A third aspect of the present invention discloses an expression vector comprising the aforementioned coding gene. The expression vector may be a pET28a(+) vector or other commonly used vectors.

[0012] A fourth aspect of the invention provides a host cell expressing the aforementioned de novo-designed feline allergen Fel d1 binding protein. The host cell includes prokaryotic cells (e.g., *Escherichia coli*) and eukaryotic cells.

[0013] A fifth aspect of the invention discloses a de novo-designed feline allergen Fel d 1 inhibitor, comprising the aforementioned de novo-designed feline allergen Fel d 1 binding protein.

[0014] A sixth aspect of the invention provides a pharmaceutical composition comprising the aforementioned de novo-designed feline allergen Fel d1 binding protein and a pharmaceutically acceptable carrier. For example, the binding protein may be formulated in a pharmaceutically acceptable aqueous carrier medium. The pharmaceutical composition may be administered via nasal inhalation.

[0015] The seventh aspect of the invention discloses the use of a de novo-designed feline allergen Fel d1 binding protein in the preparation of a feline allergen Fel d1 inhibitor, in the preparation of a medicament for treating or preventing allergic reactions to cats, cat hair, or cat secretions, or to Fel d1 protein, wherein cat secretions include, but are not limited to, cat dander, cat saliva, etc., and the diseases include, but are not limited to, allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic dermatitis, contact urticaria, allergic pharyngitis, or other allergic reactions caused by Fel d1; or in the preparation of a feline allergen Fel d1 detection reagent, preferably an ELISA detection reagent, wherein the detection sample may be human serum.

[0016] The advantages of this invention are as follows: This invention relates to a high-affinity binding protein for Fel d1 blockade, which can serve as a substitute for traditional IgY antibodies. This binding protein was obtained through computer-aided structural and functional design combined with in vitro directed evolutionary technology screening. It exhibits high affinity for IgE and IgE blocking ability (over 80%), and can be used to prevent or treat diseases caused by allergies to cats, cat dander, or cat secretions, or to Fel d1 protein. Allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic dermatitis, contact urticaria, allergic pharyngitis, or other allergic reactions caused by Fel d1 can be alleviated. Furthermore, this protein can be efficiently and solublely expressed in a conventional E. coli expression system, and the purification process is simple and easy to scale up, significantly reducing production costs. This binding protein also possesses excellent thermal stability, maintaining its binding activity after heat treatment, which helps improve the stability of Fel d1 binding protein drugs for feline allergens. Attached Figure Description

[0017] Figure 1 Structure of the complex of TG-Fel d 1-688 and rFel d 1 (red represents TG-Fel d 1-688, green represents rFel d 1);

[0018] Figure 2 . Detection of TG-Fel d1-688 Escherichia coli expression by SDS-PAGE;

[0019] Figure 3 3-1. Effective dilution range of Fel d 1 for detection by TG-Fel d 1-688 ELISA; 3-2. Effective dilution range of recombinant Fel d 1 for detection by TG-Fel d 1-688 ELISA;

[0020] Figure 4 ELISA analysis of the blocking effect of TG-Fel d1-688 on the binding of IgE to Fel d1;

[0021] Figure 5 The effective dilution range of TG-Fel d 1-688 after heat treatment was determined by ELISA. Figure 5-1 The effective dilution range of natural Fel d 1 after TG-Fel d 1-688 heat treatment for ELISA detection. Figure 5-2 .TG-Fel d 1-688 heat-treated ELISA assay for effective dilution range of recombinant Fel d 1. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0023] Example 1: Design, screening, and expression of Fel d1 binding proteins

[0024] (1) Design and screening of Fel d1 binding proteins

[0025] This invention utilizes a self-trained protein structure prediction AI model, combined with an affinity prediction model trained from the SKEMPI2 database, to design functional proteins targeting the IgE binding sites (amino acids 23-34, 43-47, and 58-71 for P30438, and 32-41, and 102-109 for P30440) of the Fel d 1 protein (UniProt ID: P30438, P30440). In the design, a diffusion algorithm is employed, using an iterative "noise-denoising" process to simulate and learn the target structure distribution, sampling and generating protein backbones, resulting in 2500 candidate backbones. Subsequently, an MPNN-like (Message Passing Neural Network) algorithm is used for backfolding design, generating two amino acid sequences for each backbone. Based on this, a protein structure prediction AI model was used to predict the structure of all designed sequences, and the predicted structures were compared with their corresponding design backbones. The design quality was evaluated using metrics such as RMSD (Root Mean Square Deviation) and pLDDT (Predicted Local Distance Difference Test), and designed sequences meeting the structural requirements were selected. Furthermore, the affinity of candidate proteins was evaluated using a trained affinity prediction model, and high-potential sequences were selected. The top 20-30 designed proteins were expressed in *E. coli*, and their binding activity was verified by ELISA. Finally, a binding protein with both high affinity and good expressibility was obtained, named TG-Fel d1-688, and its structure is shown below. Figure 1 The amino acid sequence is shown below:

[0026] TG-Fel d1-688 amino acid sequence (SEQ ID NO.1):

[0027] MEETIKEAEKLLEELKKYAEEHP ELKKIVEREISLQKKL LESGENA ERVLNDLKVTFKLIEEREARKAA L EARLAALAARDPRARGL TGADLIRAEADAELAAL LAEVKADPEAALKRAEEAAKKAEEGD YETALEATARVAGLAA AAAQ TPEQKETLERCLELLARALKAAAEKAKE EGKKDAARMFENMAKLVEEG LKLPAEE IIAKVKKGRA

[0028] Note: The underlined area is the interaction region between the binding protein and Fel d1 (the interaction region was predicted using the protein interaction prediction software PISA).

[0029] (2) Expression of TG-Fel d1-688 protein

[0030] The coding sequence of the TG-Fel d1-688 binding protein gene was cloned into the pET28a(+) vector using NcoⅠ and Xho I restriction sites, and a double strepⅡ tag was fused to the N-terminus. This plasmid was then transformed into the *E. coli* expression strain BL21(DE3). Single colonies were picked from freshly transformed plates and inoculated into 5 mL of LB broth containing 50 µg / mL kanamycin, and cultured overnight at 37°C with shaking at 220 rpm. The overnight culture was then transferred at a 1:100 ratio to 200 mL of fresh LB broth containing antibiotics and cultured at 37°C until the OD600 reached approximately 0.6. After induction at 18°C ​​for 16 h with 0.2 mM IPTG, the cells were collected by centrifugation at 5000×g for 15 min at 4°C. The cells were resuspended in 8 mL of pre-chilled binding buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). The bacterial cells were lysed using an ultrasonic homogenizer (300 W, 3 seconds operation time, 7-second interval, total duration 3 minutes) in an ice-water bath. The lysate was centrifuged at 12,000 × g for 30 minutes at 4 °C, and the supernatant was collected as the crude extract. The crude extract was then purified by nickel column chromatography.

[0031] SDS-PAGE analysis was performed on the following fractions: before induction, after induction, sonicated supernatant (crude extract), sonicated precipitate, flow-through, sample collection 1, and sample collection 2. Figure 2 The results showed that the crude extract of TG-Fel d1-688 exhibited a distinct band between 25-30 kDa, and only a small amount of the target protein was found in the ultrasonic precipitate, indicating that the protein was efficiently and solublely expressed in E. coli. The high-purity elution fractions were combined, and the protein was replaced with storage buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) using a PD-10 desalting column to a final concentration of 0.8 mg / ml for subsequent experiments.

[0032] TG-Fel d 1-688 encoded sequence (SEQ ID NO.2):

[0033] 5'-ATGGAGGAAACCATTAAGGAAGCCGAAAAACTGCTGGAAGAACTGAAAAAATACGCAGAAGAACACCCGGAACTGAAAAAGATTGTGGAACGTGAAATCAGCCTGCAGAAAAAACTGCTGGAGAGCGGCGAAAATGCAGAACGCGTGCTGAATGATCTGAAAGTTACCTTTAAGCTGATCGAAGAACGTGAAGCCCGCAAAGCAGCACTGGAAGCCCGTTTAGCCGCCTTAGCCGCACGTGATCCGCGTGCAAGAGGTTTAACCGGTGCCGATCTGATTCGTGCCGAAGCAGATGCAGAACTGGCAGCACTGCTGGCAGAAGTTAAAGCAGATCCGGAAGCAGCCCTGAAACGTGCAGAAGAAGCAGCCAAAAAAGCCGAAGAAGGCGATTATGAAACCGCCCTGGAAGCCACCGCACGTGTTGCAGGTCTGGCAGCAGCTGCTGCTCAGACACCTGAACAGAAAGAAACCCTGGAACGCTGTCTGGAACTGCTGGCACGTGCCTTAAAAGCAGCCGCAGAAAAAGCAAAAGAAGAAGGCAAAAAGGACGCAGCCCGTATGTTTGAAAATATGGCCAAACTGGTGGAAGAAGGCCTGAAACTGCCGGCAGAAGAAATTATTGCCAAAGTTAAAAAGGGTCGCGCC-3'

[0034] Example 2 Affinity test of TG-Fel d 1-688

[0035] a. Preparation of coated plate

[0036] Take a polystyrene 96-well microplate and use phosphate buffer (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to dilute the natural nFel d1 protein (Indoor Biotech, Catalog #LTN-FD-1) and recombinant rFel d1 (Fel d1 chain 2 (NP_001041619.1) amino acid sequence 18-109 is located at the N-terminus, directly fused with Fel d1 chain 1 (NP_001041618.1) amino acid sequence 23-92 (containing D27G mutation) and attached to the N-terminus with a double strepII-hexahistidine tag, expressed in CHO cells) to a final concentration of 0.5 μg / mL. Add Fel d1 (nFel d1 / rFel d1) to each well of the microplate. 1) 100 μL protein dilution buffer. Seal the microplate with sealing film and incubate overnight at 4°C. Aspirate the Fel d 1 protein solution from each well and add 300 μL of PBS buffer containing 0.05% (v / v) Tween-20 (0.05% (v / v) Tween-20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to each well, washing 5 times. Add 200 μL of PBS blocking buffer containing 1% (w / v) bovine serum albumin (1% (w / v) bovine serum albumin, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to each well and incubate at room temperature for 2 hours. Discard the blocking buffer, wash 3 times with PBST, and then wash once with PBS. Invert the plate onto sterile filter paper and pat it dry. Place it in a vacuum desiccator to dry. Immediately after drying, vacuum seal the plate in an aluminum foil bag, add desiccant, and store at 2-8℃.

[0037] b. Affinity range of TG-Fel d1-688 detected by ELISA

[0038] TG-Fel d 1-688 was serially diluted with sample diluent (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to obtain the following concentrations: 2 μg / ml, 1 μg / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, 15.625 ng / ml, 7.813 ng / ml, and 3.906 ng / ml. Each concentration of protein solution was added to 100 μL of Fel d 1 (n Fel d 1 / r Fel d 1) coated wells, with three replicates per concentration. The solutions were incubated at 37°C for 1 hour. After washing three times, 100 μL of SA-HRP working solution (1 μg / ml SA-HRP, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) was added, and the solution was incubated at 37°C in the dark for 30 minutes. After washing three times, 100 μL of luminol working solution (purchased from Tiandi Renhe Biotechnology Co., Ltd., catalog number H31020, solution A and solution B were mixed at a volume ratio of 1:1) was added, and the solution was incubated at room temperature in the dark for 5 minutes. Immediately afterwards, the luminescence value at approximately 425 nm in each well was measured using a chemiluminescence detector (results are shown in Tables 1 and 2). Figure 3 , Figure 3 (Log value was taken for medium concentration). The results showed that the effective dilution range of TG-Fel d 1-688 was 1 ng-1 ug / ml, and there was no significant difference between natural Fel d 1 and recombinant Fel d 1.

[0039]

[0040] Table 1. Effective dilution range for TG-Fel d1-688 ELISA detection of natural Fel d1 (nFel d1)

[0041] Table 2. Effective dilution range for TG-Fel d1-688 ELISA detection of recombinant Fel d1 (r Fel d1).

[0042] The luminescence value of 20513963 is the upper limit of the instrument's detection range.

[0043] When performing ELISA testing, a negative control is set up simultaneously (a control in which TG-Fel d 1-688 is not added to the sample dilution).

[0044] Example 3: Assay of TG-Fel d1-688 Blockade against IgE Antibody

[0045] a. Preparation of the covering board

[0046] Take a polystyrene 96-well microplate and dilute the anti-Fel d 1 human IgE antibody (abinScience, FP447083, referred to as Fel d 1 IgE antibody, IgE antibody, or IgE in this paper) to a final concentration of 5 μg / mL with carbonate buffer at pH 9.6. Add 100 μL of Fel d 1 IgE antibody protein dilution to each well of the microplate. Seal the microplate with sealing film and incubate overnight at 4°C. Aspirate the Fel d 1 IgE antibody solution from the wells and add 300 μL of PBS buffer containing 0.05% (v / v) Tween-20 to each well, repeating the washing 5 times. Add 200 μL of PBS blocking buffer containing 1% (w / v) bovine serum albumin to each well and incubate at room temperature for 2 hours. Discard the blocking buffer, wash 3 times with PBST, and then wash once with PBS. Invert the plate onto sterile filter paper and pat it dry. Place it in a vacuum desiccator to dry. Immediately after drying, vacuum seal the plate in an aluminum foil bag, add desiccant, and store at 2-8℃.

[0047] b. ELISA analysis of TG-Fel d1-688's inhibition of IgE binding to Fel d1

[0048] A constant concentration (15 ng / ml) of recombinant Fel d 1 (N-terminal fused with a double strep II tag) was premixed with a series of dilutions of TG-Fel d 1-688 (N-terminal without a strep II tag, concentration gradients of 15 µg / ml, 1.5 µg / ml, 150 ng / ml, 15 ng / ml, 1.5 ng / ml, 150 pg / ml) at room temperature and incubated in a rotary mixer for 1 hour to allow the interaction between Fel d 1 and TG-Fel d 1-688 to reach equilibrium. The constant concentration of 15 ng / ml refers to the concentration of recombinant Fel d 1 in the mixture of recombinant Fel d 1 and TG-Fel d 1-688, and the aforementioned concentration gradients refer to the concentration of TG-Fel d 1-688 in the mixture of recombinant Fel d 1 and TG-Fel d 1-688. 100 μL of the above-mentioned TG-Fel d 1-688-rFel d 1 mixture was added to IgE-coated plates, with three replicates per concentration, and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μL of SA-HRP working solution was added, and the plates were incubated at 37°C in the dark for 30 minutes. After washing three times, 100 μL of luminol working solution was added, and the plates were incubated at room temperature in the dark for 5 minutes. Immediately afterwards, the luminescence value at approximately 425 nm in each well was measured using a chemiluminescence detector. The results are shown in Table 3 and... Figure 4TG-Fel d 1-688 showed a blocking rate of over 80% against the binding of recombinant Fel d 1 to IgE.

[0049]

[0050] Table 3. ELISA analysis of TG-Fel d1-688's inhibition of IgE binding to Fel d1.

[0051] Example 4: Thermal stability determination of TG-Fel d 1-688

[0052] The diluted TG-Fel d1-688 protein from Example 2 was incubated at 37°C for 14 days. After removal, ELISA detection was performed according to the steps in Example 2 (results are shown in Tables 4 and 5). Figure 5 The results showed that there was almost no difference between the values ​​detected before being placed at 37°C, indicating that the TG-Fel d1-688 protein has good thermal stability, which is significantly better than that of traditional antibodies.

[0053]

[0054] Table 4. Effective dilution range for ELISA detection of natural Fel d1 after TG-Fel d1-688 heat treatment.

[0055] Table 5. Effective dilution range of recombinant Fel d1 after TG-Fel d1-688 heat treatment for ELISA detection.

[0056] Example 5: Comparison of TG-Fel d1-688 and reported antibody blocking effects on IgE antibodies.

[0057] Prepare the Feld 1 IgE antibody-coated plate according to Example 3a. A constant concentration (15 ng / ml) of recombinant Feld 1 (N-terminal fused with a double strep II tag) was premixed with a series of dilutions of REGN1908-1909 antibody (purchased from Abin Science FP447036 and FP447046, mixed 1:1, with total concentration gradients of 15 µg / ml, 1.5 µg / ml, 150 ng / ml, 15 ng / ml, 1.5 ng / ml, and 150 pg / ml) at room temperature and incubated in a rotary mixer for 1 hour to allow the interaction between Feld 1 and REGN1908-1909 antibody to reach equilibrium. Add 100 μL of the above REGN1908-1909-rFeld 1 mixture to the IgE-coated plate, with three replicates per concentration, and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL of SA-HRP working solution, and incubate at 37°C in the dark for 30 minutes. After washing three times, add 100 μL of luminol working solution and incubate at room temperature in the dark for 5 minutes. Immediately afterwards, use a chemiluminescence detector to measure the luminescence value of each well at approximately 425 nm. The results (Table 6) show that the blocking rate of TG-Fel d 1-688 against the binding of recombinant Fel d 1 to IgE is comparable to the blocking rate reported by the REGN1908-1909 antibody in the literature.

[0058]

[0059] Table 6. ELISA analysis of REGN1908-1909 inhibition of IgE binding to Fel d1

[0060] Example 6: Comparison of binding protein affinity between TG-Fel d1-688 and other de novo designs from the same batch of this invention.

[0061] Prepare the recombinant rFel d1 coated plate as described in Example 2a. Two other de novo designed binding proteins, TG-Fel d1-1152 and TG-Fel d1-462, were serially diluted with sample diluent to the following concentrations: 2 μg / ml, 1 μg / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, 15.625 ng / ml, 7.813 ng / ml, and 3.906 ng / ml. Add 100 μL of each protein solution to each rFel d1 coated well, with three replicates per concentration. Incubate at 37°C for 1 hour. After washing three times, add 100 μL of SA-HRP working solution and incubate at 37°C in the dark for 30 minutes. After washing three times, add 100 μL of luminol working solution and incubate at room temperature in the dark for 5 minutes. Subsequently, the luminescence value of each well at approximately 425 nm was immediately measured using a chemiluminescence detector (results are shown in Tables 7 and 8). The results showed that the detection range and luminescence value of TG-Fel d 1-1152 and TG-Fel d 1-462 were significantly weaker than those of the selected TG-Fel d 1-688.

[0062]

[0063] Table 7. Effective dilution range for TG-Fel d1-1152 ELISA detection of recombinant Fel d1 (r Fel d1)

[0064] Table 8. Effective dilution range for TG-Fel d1-462 ELISA detection of recombinant Fel d1 (r Fel d1)

[0065] TG-Fel d1-1152 amino acid sequence (SEQ ID NO.3):

[0066] MEETIKEAEKLLEELKKYAEEHP ELKKIVEREISLQKKLLESGENAERVLNDLKVTFKLIEEREARDAE L KARYGTDSV EEARALRAAAARAELAAL LAAVRADPEAALKRAEEAAKKAEEGD YETALEATARVAGLAAAAA QTPEQKETLERCLELLARALKAAAEKAKEEG KKDAARMFENMAKLVEEGLKDVDAVQATAQANAARYAEWRAAEEAAR AAALAG

[0067] TG-Fel d 1-462 amino acid sequence (SEQ ID NO.4):

[0068] AEAEARLKKLAEEAEKYKKEAKKLLEEAKKATPEEREKL LEKRKEYREKAYALFDERQALLRAQQSPEE RAARLAAADAEA AEILKLVREDPEAALKRAEEAAKKAEEGD YETALEATARVAGLAAAAAQ TPEQKETLERCLELLARALKAAAEKAKEEG KKDAARMFENMAKLVEEGLKKPFEEELAARAKARARLAAA

[0069] Note: The underlined area is the interaction region between the binding protein and Fel d1 (the interaction region was predicted using the protein interaction prediction software PISA).

[0070] This invention utilizes a protein artificial intelligence design method to screen and obtain a Fel d1 blocking protein with high affinity, easy expression, and high stability. This binding protein can effectively replace traditional IgY antibodies, significantly reducing production costs through an E. coli expression system while maintaining high blocking efficacy (over 80%). Its excellent thermostability helps reduce the cost burden of storage and transportation, improves the stability of Fel d1 binding protein drugs for feline allergens, and is more suitable for widespread and convenient applications such as home use in detection.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A de novo-designed feline allergen Fel d1 binding protein, characterized in that, The amino acid sequence of the feline allergen Fel d 1 binding protein is shown in SEQ ID NO.

1.

2. The de novo-designed feline allergen Fel d1 binding protein according to claim 1, characterized in that, The aforementioned feline allergen Fel d 1 binding protein specifically targets Fel d 1 and is designed to target the IgE binding site of Fel d 1.

3. The gene encoding the de novo-designed feline allergen Fel d 1 binding protein as described in claim 1 or 2.

4. An expression vector comprising the encoding gene of claim 3.

5. Host cells expressing the de novo-designed feline allergen Fel d 1 binding protein as described in claim 1 or 2.

6. A de novo-designed feline allergen Fel d1 inhibitor, characterized in that, Includes a de novo-designed feline allergen Fel d 1 binding protein as described in claim 1 or 2.

7. A pharmaceutical composition, characterized in that, Includes a de novo-designed feline allergen Fel d 1 binding protein as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

8. The use of the de novo-designed feline allergen Fel d1 binding protein according to claim 1 or 2 in the preparation of a feline allergen Fel d1 detection reagent.

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