Recombinant Fel d1 allergen protein, nucleic acid molecule and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and discloses a recombinant Fel d 1 allergen protein, a nucleic acid molecule and application thereof. The recombinant Fel d 1 allergen protein is formed by sequentially and covalently connecting Fel d 1-chain1, a polypeptide linker (Linker) and Fel d 1-chain2; the amino acid sequences of the Fel < d > 1-chain1, the Fel < d > 1-chain2 and the polypeptide linker (Linker) are respectively as shown in SEQ ID NO. 1-3; and the positions of the Fel < d > 1-chain1 and the Fel < d > 1-chain2 can be exchanged. According to the recombinant Fel d 1 allergen protein obtained by adopting a specific polypeptide linker, high-level and high-solubility expression of the recombinant Fel d 1 allergen protein can be realized by utilizing an escherichia coli expression system, and it is ensured that the recombinant Fel d 1 allergen protein has a stable structure and immunological activity which are equivalent to those of natural protein; the application requirements in the field of cat allergy diagnosis, prevention and treatment can be effectively met.

Description

A recombinant Fel d1 allergen protein, nucleic acid molecule and its application Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant Fel d 1 allergen protein, nucleic acid molecule and its application. Background Technology

[0002] Allergic diseases, as a global immune system disorder, are showing an increasing incidence rate year by year. Among them, allergic reactions induced by domestic cats (Felis domesticus) are particularly common in clinical practice, leading to a series of type I hypersensitivity reactions such as allergic rhinitis, conjunctivitis, atopic dermatitis, and asthma. Epidemiological and immunological studies have confirmed that the major allergen in cats is the Fel d1 protein, and more than 90% of cat allergy sufferers have specific IgE antibodies against this protein.

[0003] Natural Fel d1 is a member of the secretoglobin superfamily, and its functional form is a structurally complex glycoprotein tetramer. This tetramer consists of two non-covalently linked heterodimers, each of which is composed of Chain 1 and Chain 2 covalently linked by three pairs of disulfide bonds. Chain 1 (UniProtKB No.: P30438) is a small polypeptide containing 92 amino acid residues, with a three-dimensional structure consisting of four α-helices. Chain 2 (UniProtKB No.: P30440) is a polypeptide containing 109 amino acid residues, also exhibiting a four-helix bundle conformation, and featuring a key N-linked complex glycan at its 33rd asparagine residue (Asn33). It is this complex quaternary structure, formed by precise covalent (disulfide) and non-covalent interactions between two different peptide chains, supplemented by post-translational glycosylation modifications, that endows Fel d 1 with a stable conformation and complete immunological activity.

[0004] However, it is precisely this complexity that leads to insurmountable obstacles when extracting and purifying Fel d1 protein from natural sources such as cat saliva and sebaceous glands using traditional biochemical methods. These obstacles include extremely low yields, high costs, poor batch-to-batch consistency, and potential risks of biocontamination, severely restricting its large-scale application in the development of in vitro diagnostic reagents and specific immunotherapy (SIT) drugs. Expressing Fel d1 protein using engineered bacteria such as E. coli requires extremely cumbersome and inefficient denaturation and renaturation processes in vitro to obtain only small quantities of protein with the correct conformation, resulting in extremely low overall yields. Furthermore, because E. coli lacks the folding environment of eukaryotic cells, the expressed peptide chains are prone to misfolding, forming inactive and difficult-to-handle inclusion bodies, making the recovery of soluble proteins extremely difficult. This production method is costly and time-consuming, completely failing to meet the needs of large-scale production.

[0005] A peptide linker is an amino acid sequence created through genetic engineering that tandemly links two or more independent proteins or functional domains at the coding level. Its fundamental function is to transform multiple subunits that would normally require translation to meet and assemble into a single polypeptide chain covalently linked during translation. This design significantly increases the effective local concentration between functional domains, thus thermodynamically promoting correct intramolecular folding and assembly, rather than intermolecular mis-aggregation. A successful linker design must ensure correct folding of functional domains without forming secondary or tertiary structures that affect function. Furthermore, its length and flexibility must be optimized to ensure appropriate spatial distance and relative orientation between functional domains, thereby mimicking the conformation of natural proteins.

[0006] Therefore, this invention aims to modify the two single chains of Fel d 1 protein by using a peptide linker to obtain a recombinant Fel d 1 allergen protein that can be expressed at a high level and has a stable structure and immunological activity comparable to the natural protein. Summary of the Invention

[0007] This invention aims to address at least one of the problems existing in the prior art. To this end, this invention proposes a recombinant Fel d 1 allergen protein, a nucleic acid molecule, and its applications. The recombinant Fel d 1 allergen protein obtained using a specific polypeptide linker can achieve high-level, highly soluble expression of this recombinant Fel d 1 allergen protein using an *E. coli* expression system, ensuring that it possesses a stable structure and immunological activity comparable to the natural protein, thus meeting the application needs in the diagnosis, prevention, and treatment of feline allergies.

[0008] This invention provides a recombinant Fel d 1 allergen protein, which is composed of Fel d 1-chain1, a peptide linker, and Fel d 1-chain2 covalently linked in sequence;

[0009] The amino acid sequences of Fel d 1-chain1, Fel d 1-chain2, and the polypeptide linker are shown in SEQ ID NO.1-3, respectively;

[0010] The positions of Fel d 1-chain1 and Fel d 1-chain2 can be interchanged.

[0011] The immunological activity of Fel d1 (i.e., its ability to bind to IgE antibodies) is highly dependent on its precise three-dimensional conformation. However, existing recombinant Fel d1 proteins obtained through in vitro refolding exhibit significantly reduced binding ability to IgE antibodies compared to the native protein, with some reports indicating a 25-fold decrease in activity. This is primarily attributed to the instability of non-covalent bonds between the two chains and the difficulty in precisely pairing the three key disulfide bonds in the in vitro environment.

[0012] Through design and screening, this invention discovered that by using a specific peptide linker to connect Chain 1 and Chain 2 of the Fel d 1 protein into a single-chain protein, highly soluble expression can be achieved in E. coli while maximally preserving its key IgE binding epitopes and biological activity, demonstrating promising application prospects.

[0013] The present invention also provides a nucleic acid molecule for expressing the above-mentioned recombinant Fel d 1 allergen protein.

[0014] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ.ID.NO.11.

[0015] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0016] The present invention also provides a recombinant bacterium containing the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0017] Preferably, the recombinant bacteria is Escherichia coli.

[0018] This invention also provides the application of the above-mentioned recombinant Fel d 1 allergen protein in the preparation of a feline allergy diagnostic kit.

[0019] Preferably, the feline allergy diagnostic kit is used for the detection of Fel d 1-specific IgE antibodies in the sample to be tested.

[0020] The present invention also provides the application of the above-mentioned recombinant Fel d 1 allergen protein in the preparation of anti-cat allergy products.

[0021] Preferably, the anti-cat allergy product is used for desensitization therapy.

[0022] The recombinant Fel d1 allergen protein proposed in this invention can not only be used to develop highly sensitive and specific in vitro diagnostic (IVD) reagents, but also to develop safer and more effective feline allergy-specific immunotherapy (SIT) products, which has significant clinical application value and market prospects.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention addresses the problem of the natural Fel d 1 heterodimer's difficulty in proper folding and assembly, and its extremely low expression levels. By selecting a specific peptide linker, chain 1 and chain 2 of the Fel d 1 protein are linked into a single-chain protein (recombinant Fel d 1 allergen protein). This method significantly improves the soluble expression level of recombinant Fel d 1 allergen protein in the *E. coli* recombinant expression system (expression level stabilized above 50 mg / L). Enzyme-linked immunosorbent assay (ELISA) results further demonstrate that this recombinant Fel d 1 allergen protein has good binding activity with serum IgE from cat allergic patients, and its activity is comparable to that of the natural allergen.

[0025] This invention not only effectively solves the problems of high cost and low yield of Fel d1 allergen protein in existing solutions, but also provides key, scalable raw materials for the development of in vitro diagnostic reagents for feline allergies and the research of specific immunotherapy (desensitization therapy) products, opening up new pathways. Attached Figure Description

[0026] Figure 1 shows the SDS-PAGE results of different recombinant Fel d 1 allergen proteins after 6 hours of induction in Example 1; where lane 1 is Fel d 1-chain1+(GGGGS)1+chain2, lane 2 is Fel d 1-chain1+(GGGGS)2+chain2, lane 3 is Fel d 1-chain1+(GGGGS)3+chain2, lane 4 is Fel d 1-chain1+(EAAAK)1+chain2, lane 5 is Fel d 1-chain1+(EAAAK)2+chain2, and lane 6 is Fel d 1-chain1+(EAAAK)3+chain2.

[0027] Figure 2 shows the SDS-PAGE results of different recombinant Fel d 1 allergen proteins eluted from a nickel column in Example 1 using 20 mM Tris-HCl buffer (containing 250 mM NaCl, 250 mM imidazole, pH 8.0); where lane 1 is Fel d 1-chain1+(GGGGS)1+chain2, lane 2 is Fel d 1-chain1+(GGGGS)2+chain2, lane 3 is Fel d 1-chain1+(GGGGS)3+chain2, lane 4 is Fel d 1-chain1+(EAAAK)1+chain2, lane 5 is Fel d 1-chain1+(EAAAK)2+chain2, and lane 6 is Fel d 1-chain1+(EAAAK)3+chain2.

[0028] Figure 3 shows the enzyme-linked immunosorbent assay (ELISA) results of different Fel d 1 allergen proteins in Example 2.

[0029] Figure 4 shows the enzyme-linked immunosorbent assay (ELISA) results of the coarse cat hair extract and Fel d 1-chain1+(EAAAK)1+chain2 in Example 3.

[0030] Figure 5 is a bivariate scatter plot showing the immunogenicity of the recombinant Fel d 1-chain1+(EAAAK)1+chain2 protein and the commercial Fel d 1 protein against IgE in Example 4.

[0031] Figure 6 shows the immunogenicity data of the recombinant Fel d 1-chain1+(EAAAK)1+chain2 protein and the commercial Fel d 1 protein against IgE in Example 4. Detailed Implementation

[0032] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0033] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0034] Example 1: Preparation of recombinant Fel d1 allergen protein

[0035] The original gene sequences encoding Fel d1 allergen protein, chain 1 (Fel d1-chain1) and chain 2 (Fel d1-chain2), were synthesized by Beijing Qingke Biotechnology Co., Ltd. (sequence information for Fel d1-chain1 and Fel d1-chain2 can be obtained by searching sequence numbers P30438 and P30440 respectively on the UniPort website). The target gene fragment was amplified using polymerase chain reaction (PCR). This gene fragment, along with the linker gene, was cloned into the pET-47b plasmid expression vector to construct the recombinant expression plasmid. This plasmid was then transformed into *E. coli* BL21(DE3) host bacteria for expression, yielding recombinant Fel d1 allergen protein.

[0036] The aforementioned recombinant Fel d 1 allergen protein is composed of Fel d 1-chain1, a peptide linker, and Fel d 1-chain2 covalently linked in sequence.

[0037] The amino acid sequence of Fel d 1-chain1 is: EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLC (SEQ ID NO.1).

[0038] The amino acid sequence of Fel d 1-chain2 is: VKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR (SEQ ID NO.2);

[0039] The peptide linker is selected from one of (EAAAK)1, (EAAAK)2, (EAAAK)3, (GGGGS)1, (GGGGS)2, and (GGGGS)3, with corresponding amino acid sequences of SEQ ID NO.3-8.

[0040] (GGGGS) nLinker sequences are the most widely used and representative flexible linkers. Their building blocks contain only glycine (G) and serine (S). Glycine's side chain has only one hydrogen atom, making it the least sterically hindered of all amino acids. This grants the polypeptide backbone a great deal of conformational freedom, allowing it to rotate with minimal energy barriers, thus exhibiting high flexibility. Serine, on the other hand, is a small, polar, hydrophilic amino acid whose hydroxyl side chain can form hydrogen bonds with water molecules. This helps improve the solubility of the linker region and even the entire fusion protein, reducing the risk of aggregation in the cytoplasm due to hydrophobic effects. Therefore, (GGGGS) n Connectors are generally considered to be biologically inert, unstructured, flexible spacers that effectively connect two functional domains while minimizing interference with their independent folding.

[0041] (EAAAK) n This sequence is a typical example of a rigid linker. It is rich in alanine (A), glutamic acid (E), and lysine (K). Alanine has an extremely high tendency to form α-helices and is the most common amino acid residue constituting α-helical structures. By tandemly connecting multiple EAAAK units, a predictable and conformationally stable α-helix can be constructed. This helical structure acts as a rigid intramolecular "scaffold," fixing the two connected functional domains at a relatively defined spatial distance and rotation angle. The charged glutamic acid (negatively charged) and lysine (positively charged) are typically designed on the hydrophilic side of the helix to enhance the solubility of the entire fusion protein.

[0042] The specific preparation steps are as follows:

[0043] (1) Using pET-47b plasmid as a template, primers were used for amplification to obtain the linear plasmid pET-47b-6his, which contains a 6his fusion tag sequence. Using a ready-to-use seamless cloning kit from Sangon Biotech, the obtained linear plasmid was seamlessly ligated with the Fel d 1 chain1 gene, Linker gene, and Fel d 1 chain2 gene obtained by two PCR amplification techniques. The ligation product was transformed into T10 competent cells and plated on LB agar plates containing 50 ng / μL kanamycin, and incubated overnight at 37°C. After single colonies grew, colony PCR was performed for verification. Plasmids containing the target band were cultured and extracted, and sent to a sequencing company to check the sequence accuracy. The correctly sequenced plasmid pET-47b-6his-Fel d 1-chain1+Linker+chain2 was retained for later use, with the His tag located at the N-terminus of the Fel d 1 sequence.

[0044] Using pET-47b plasmid as a template, the following PCR amplification primers were designed to obtain the linear plasmid pET-47b-6his.

[0045] Forward: 5'-TAAGCGGCCGCAGAGCTCGCTCTGGT-3' (SEQ.ID.NO.9);

[0046] Reverse: 5'-GTGATGGTGGTGGTGATGTGCCATATGTA-3' (SEQ.ID.NO.10).

[0047] The nucleotide sequences of Fel d 1-chain1+(EAAAK)1+chain2, Fel d 1-chain1+(EAAAK)2+chain2, Fel d 1-chain1+(EAAAK)3+chain2, Fel d 1-chain1+(GGGGS)1+chain2, Fel d 1-chain1+(GGGGS)2+chain2, and Fel d 1-chain1+(GGGGS)3+chain2 are shown in SEQ.ID.NO.11-SEQ.ID.NO.16, respectively.

[0048] (2) Transform the correctly sequenced plasmid pET-47b-6his-Fel d 1-chain1+Linker+chain2 into *E. coli* (BL21(DE3)). Spread the plasmid on LB agar plates containing 50 ng / μL kanamycin and incubate overnight at 37°C to screen for positive colonies. Verify positive transformants by PCR. In LB agar, the target strain is cultured at 37°C and shaken at 220 rpm until the OD600 reaches 0.6-0.8. Subsequently, isopropyl-β-D-thiogalactopyranoside (IPTG) is added at a final concentration of 0.3-0.5 mM to induce protein expression. The culture is usually carried out at 21-37°C with shaking for 4-8 hours.

[0049] (3) After expression, the bacterial cells were collected by centrifugation and resuspended in a buffer solution with the following formula: 20 mM Tris-HCl, 250-500 mM NaCl, 10-20 mM imidazole, pH 8.0. The cells were then broken up and the proteins were released by high-pressure cell disruption. The soluble protein supernatant was obtained by centrifugation and purified.

[0050] (4) Thoroughly mix the soluble protein supernatant with the nickel column packing material. After loading the column, remove the flow-through solution and wash away non-specifically bound proteins with a buffer solution containing 20 mM Tris-HCl, 250-500 mM NaCl, 10-20 mM imidazole, and pH 8.0. Then, replace the buffer solution with another buffer solution containing 20 mM Tris-HCl, 250-500 mM NaCl, 200-250 mM imidazole, and pH 8.0 to elute the bound proteins. Finally, perform SDS-PAGE analysis on the samples generated in each step of the purification process to verify protein expression and purity.

[0051] The SDS-PAGE results are shown in Figures 1 and 2. As shown in Figure 1, the target band is clearly present in the bacterial cell lysate at a protein size of 18 kDa, accounting for 35% of the total protein in that lane, with a target protein yield of up to 300 mg / L. As shown in Figure 2, after elution with a high-concentration imidazole buffer, the target protein in the lane was fully eluted, and the proportion of target protein in the purified eluent reached over 95% of the total protein.

[0052] Example 2: Enzyme-linked immunosorbent assay (ELISA) of different Fel d1 allergen proteins

[0053] Using patient serum containing cat hair-specific IgE (89.4 kU / L), the IgE binding activity of Fel d 1-chain1+chain2 protein expressed in the Pichia pastoris system and various recombinant Fel d 1 allergen proteins expressed in the Escherichia coli system in Example 1 was compared by enzyme-linked immunosorbent assay.

[0054] The experimental procedure included: coating different Fel d 1 proteins (10 mg / L) onto 96-well plates and incubating overnight at 4°C; blocking with 5% BSA for 2 hours, adding 1:4 diluted patient serum (cat fur IgE = 89.4 IU / L), incubating at 37°C for 2 hours, incubating with HRP-labeled anti-human IgE secondary antibody (1:5000) for 2 hours; incubating with TMB at 37°C for 0.5 hours for color development, and measuring the OD value at 450 nm.

[0055] As shown in Figure 3, all recombinant proteins with a linker introduced between the two peptide chains exhibited significantly higher binding activity to feline hair-specific IgE serum than proteins without a linker (i.e., Fel d 1-chain1+chain2 proteins). Among them, the Fel d 1-chain1+(EAAAK)1+chain2 protein, employing a rigid linker peptide (EAAAK)1, showed the best binding ability, with a corrected absorbance of 1.4312 at OD 450 nm, representing a 1.5-fold increase in immunomodulatory activity compared to Fel d 1 without a linker. Furthermore, for (EAAAK)... n and (GGGGS) n The activity of the series of linker peptides decreased with increasing linker length. In summary, introducing linkers between peptide chains is an effective strategy to enhance the immunomodulatory activity of recombinant Fel d 1 protein, and (EAAAK)1 is the optimal sequence in this study.

[0056] Example 3: Enzyme-linked immunosorbent assay (ELISA) of crude cat hair extract and Fel d 1-chain1+(EAAAK)1+chain2

[0057] To further verify the immunological activity advantages of the recombinant Fel d 1 allergen protein in this invention, the optimal conformation protein Fel d 1-chain1+(EAAAK)1+chain2, modified from a specific rigid linker peptide (EAAAK)1, was directly compared with a crude extract of natural allergens from cat hair using an enzyme-linked immunosorbent assay (ELISA). It must be noted that the crude cat hair extract is a complex mixture containing Fel d 1 and various other minor cat hair allergen components; its detection signal is the cumulative effect of all these allergens binding to IgE.

[0058] As shown in Figure 4, the recombinant Fel d 1-chain1+(EAAAK)1+chain2 protein and crude cat hair extract were used as experimental subjects. Serum samples from patients with different allergy levels (IgE values ​​ranging from 8.63 kU / L to >100 kU / L) were used for testing. Data showed that in almost all serum grades, the absorbance (OD 450nm) of the Fel d 1-chain1+(EAAAK)1+chain2 recombinant protein was comparable to that of the complex crude cat hair extract, and even showed higher IgE binding capacity in several samples. This comparative result is highly convincing, clearly highlighting the superior effect of modifying the two peptide chains of Fel d 1 using the (EAAAK)1 peptide linker. It clearly demonstrates that although the natural crude extract contains multiple sensitizing components, the binding capacity of the single-chain recombinant Fel d 1 protein prepared by this invention through the ingenious design of the (EAAAK)1 peptide linker to specific IgE antibodies has reached or even exceeded the overall cumulative level of natural mixed allergens.

[0059] Example 4: Comparison of Fel d 1-chain1+(EAAAK)1+chain2 with commercially available Fel d 1 protein measurements

[0060] To accurately verify the performance breakthrough of the optimal Fel d 1-chain1+(EAAAK)1+chain2 recombinant protein constructed using the rigid linker (EAAAK)1 of this invention, this study conducted rigorous parallel validation on 35 samples, comparing the measured values ​​of the protein of this invention with those of commercial Fel d 1 protein to assess its immunomodulatory activity against IgE. The experimental results strongly support the superiority of the technical solution of this invention.

[0061] As shown in Figures 5 and 6, in the comparative test of 35 samples on the flow cytometry platform, the bivariate scatter plot visually revealed the systematic upward shift of the measured data, that is, the vast majority of data points were distributed above the Y=X isoline, indicating that the Fel d 1-chain1+(EAAAK)1+chain2 recombinant protein proposed in this invention has higher IgE immunogenicity. Through specific analysis of the 35 samples, as many as 80% of the samples (28 samples) showed that the measured value of the protein of this invention was higher than that of the corresponding commercial Fel d 1, which fully demonstrates the broad effectiveness of this invention in improving immunogenicity. This significant trend irrefutably confirms that the design strategy of this invention has successfully optimized the antigen conformation, greatly improving the binding efficiency and diagnostic sensitivity to specific IgE antibodies. In addition, the coefficient of variation (CV=90.4%) of the protein of this invention is lower than that of commercial Fel d 1 (CV=118.6%), indicating that it maintains high activity while having better batch-to-batch quality uniformity. In summary, this invention surpasses existing technologies in both the two core technical indicators of systemic sensitivity and batch stability.

[0062] The above experiments strongly demonstrate that the introduction of the (EAAAK)1 peptide linker in this invention successfully optimizes the spatial conformation of the Fel d1 protein, completely avoiding the inactivation caused by subunit dissociation of the natural heterodimer. This results in a more fully exposed and structurally more stable Fel d1 protein with its key IgE binding epitope. The novel modified construct not only outperforms the original natural structure in IgE immunomodulation but also surpasses the currently commercially available control Fel d1. The results ultimately prove that the (EAAAK)1 peptide linker approach of this invention is the key and optimal strategy for enhancing the immunomodulation of Fel d1, and has unparalleled application value in the development of high-precision diagnostic reagents and high-titer desensitization therapeutics.

[0063] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

Claims

1. A recombinant Fel d1 allergen protein, characterized in that, The recombinant Fel d 1 allergen protein is composed of Fel d1-chain1, a peptide linker, and Fel d 1-chain2 covalently linked in sequence; the amino acid sequences of Fel d 1-chain1, Fel d 1-chain2, and the peptide linker are shown in SEQ ID NO.1-3, respectively; the positions of Fel d 1-chain1 and Fel d 1-chain2 are interchangeable.

2. A nucleic acid molecule, characterized in that, Used to express the recombinant Fel d 1 allergen protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ.ID.NO.

11.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant bacterium, characterized in that, It comprises the nucleic acid molecule of claim 2 or 3 or the recombinant expression vector of claim 4.

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria is Escherichia coli.

7. The use of the recombinant Fel d 1 allergen protein according to claim 1 in the preparation of a feline allergy diagnostic kit.

8. The application according to claim 7, characterized in that, The feline allergy diagnostic kit is used to detect Fel d 1-specific IgE antibodies in the test sample.

9. The use of the recombinant Fel d 1 allergen protein according to claim 1 in the preparation of anti-cat allergy products.

10. The application according to claim 9, characterized in that, The anti-cat allergy product is used for desensitization therapy.

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