Cat allergen fusion protein, mRNA immune preparation and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to cat allergen fusion protein, an mRNA immune preparation and application of the cat allergen fusion protein and the mRNA immune preparation. The amino acid sequence of the cat allergen fusion protein disclosed by the invention is as shown in SEQ ID NO.11. The cat allergen fusion protein provided by the invention is a fusion protein obtained by fusing key segments with strong sensitization of Fel d2 and Fel d4 proteins with Fel d1 full-length protein, the fusion protein can simultaneously cover main sensitization epitopes of Fel d1, Fel d2 and Fel d4, and the sensitization effect of cat allergens can be more comprehensively blocked. According to the cat allergen fusion protein provided by the invention, an mRNA immune preparation can be rapidly designed and synthesized, the expression of the cat allergen fusion protein can be accurately controlled in poultry bodies, the immunoreaction is more durable, and the prepared IgY antibody can effectively block allergic protein in cat oral saliva.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cat allergen fusion protein, an mRNA immune preparation and applications thereof. Background Art

[0002] Cat allergy is a common allergic disease in humans, primarily triggered by cat allergens. Fel d1, Fel d2, and Fel d4 are the primary allergens in cats. They can induce the human immune system to produce specific IgE antibodies, which in turn trigger allergic reactions such as sneezing, runny nose, itchy skin, and asthma, severely impacting the quality of life of allergy sufferers.

[0003] Currently, treatments for cat allergies primarily include avoiding cats, medication, and immunotherapy. However, avoiding cats is often difficult to achieve in real life; medication can only alleviate symptoms but cannot fundamentally resolve the allergy; and while immunotherapy has some effectiveness, it suffers from long treatment cycles (often requiring 2-3 years) and inconsistent efficacy. Therefore, developing an efficient, safe, and convenient method to block cat allergens is of great clinical significance.

[0004] Existing studies have mostly used protein immunization to block cat allergens. However, traditional protein immunization methods have several drawbacks, including complex protein preparation processes, high costs, structural differences from native antigens, and poor cat allergen blocking efficacy. For example, Chinese patent CN114437211A discloses a method for preparing egg yolk antibodies based on the cat allergen Feld1. Immunization is performed using Feld1 protein produced in an Escherichia coli system. However, native Feld1 is glycosylated, which is not present in E. coli expression systems. Therefore, the prepared Feld1 protein differs from the native protein, affecting the activity of the corresponding egg yolk antibody (IgY). Chinese patent CN117003848A discloses the expression, purification, and combined application of the major cat allergen proteins Feld1, Feld4, and Feld7. These three proteins were mixed and then immunized to produce IgY. Administration of 2 mg IgY / kg body weight for 80 days reduced the allergenic protein content in cat saliva by only 50%. Summary of the Invention

[0005] To address these issues, the present invention provides a cat allergen fusion protein, an mRNA immune preparation, and their applications. The cat allergen fusion protein provided by the present invention enables the rapid design and synthesis of mRNA immune preparations, precisely controls the expression of the cat allergen fusion protein in poultry, and provides a more sustained immune response. The resulting IgY antibodies can effectively block allergenic proteins in cat oral saliva.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a cat allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11.

[0007] The present invention provides a first nucleic acid molecule encoding the cat allergen fusion protein described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO.12.

[0008] The present invention provides a second nucleic acid molecule for preparing an mRNA immune preparation, comprising a T7 promoter sequence and the first nucleic acid molecule described in the above technical solution.

[0009] Preferably, the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID NO.13.

[0010] The present invention provides an mRNA immune preparation, which is prepared by in vitro transcription of the second nucleic acid molecule described in the above technical solution through mRNA.

[0011] The present invention provides the use of the cat allergen fusion protein described in the above technical solution, or the first nucleic acid molecule described in the above technical solution, or the second nucleic acid molecule described in the above technical solution, or the mRNA immune preparation described in the above technical solution in one or more of the following: 1) As a cat allergy immunogen; 2) preparing a product that neutralizes or reduces the content of cat allergens, wherein the cat allergens include one or more of FeI d 1, FeI d 2, and FeI d 4; 3) Prepare anti-cat allergy vaccines.

[0012] Preferably, the product comprises cat allergen antibodies; and the anti-cat allergy vaccine is an mRNA vaccine.

[0013] Preferably, the cat allergen antibody is egg yolk antibody.

[0014] The present invention provides an mRNA vaccine, comprising an mRNA immune preparation and a liposome encapsulating the mRNA immune preparation; the mRNA immune preparation is the mRNA immune preparation described in the above technical solution.

[0015] The present invention provides a cat allergen egg yolk antibody, which is prepared using the mRNA immune preparation described in the above technical solution or the mRNA vaccine described in the above technical solution as an immunogen.

[0016] Beneficial effects: The present invention provides a cat allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11. The cat allergen fusion protein provided by the present invention is obtained by studying the structure and function of Fel d2 and Fel d4 proteins, screening out their key segments with strong allergenicity, and fusing these key segments with the full-length Fel d1 protein to construct a new type of fusion protein. The fusion protein can simultaneously cover the major allergenic epitopes of Fel d1, Fel d2 and Fel d4, and can more comprehensively block the sensitization effect of cat allergens. The cat allergen fusion protein provided by the present invention can quickly design and synthesize mRNA immune preparations, can accurately control the expression of cat allergen fusion proteins in poultry, and has a more sustained immune response. The prepared IgY antibody can effectively block allergenic proteins in cat oral saliva. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 Agarose gel electrophoresis of purified mRNA. DETAILED DESCRIPTION

[0019] The present invention provides a cat allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11.

[0020] The cat allergen fusion protein provided by the present invention is constructed by screening out key segments with strong allergenicity through structural and functional studies of Fel d2 and Fel d4 proteins. These key segments are then fused with the full-length Fel d1 protein to construct a new type of fusion protein. This fusion protein can simultaneously cover the major allergenic epitopes of Fel d1, Fel d2 and Fel d4, and can more comprehensively block the sensitizing effects of cat allergens.

[0021] The present invention provides a first nucleic acid molecule encoding the cat allergen fusion protein described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO. 12. The first nucleic acid molecule provided by the present invention is obtained by codon-optimizing the avian (particularly chicken) gene encoding the cat allergen fusion protein described in the above technical solution, and can improve the translation efficiency and stability of mRNA in the body.

[0022] The present invention provides a second nucleic acid molecule for preparing an mRNA immune preparation, comprising a T7 promoter sequence and the first nucleic acid molecule described in the above technical solution. As an embodiment, the nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO.13.

[0023] The second nucleic acid molecule provided by the present invention is optimized and designed based on the first nucleic acid molecule, including: adding a suitable cap structure (such as m7GpppN) to the 5' end of the mRNA and adding a poly(A) tail (poly(A) tail) to the 3' end to enhance the stability of the mRNA and the efficiency of translation initiation. The secondary structure of the mRNA is also optimized to reduce the stable secondary structure that may be formed, thereby improving its translation activity, and can accurately control the expression of cat allergen fusion protein in poultry.

[0024] The present invention provides an mRNA immune preparation, which is prepared by in vitro transcription of the nucleic acid molecule described in the above technical solution through mRNA.

[0025] The mRNA immune preparation provided by this invention can be injected into an organism (e.g., poultry, to obtain IgY antibodies) via an appropriate immunization route (e.g., intramuscular or subcutaneous injection). After the mRNA is taken up by cells in the body, it is synthesized into a fusion protein using the cellular translation system. This fusion protein acts as an antigen to stimulate the immune system, inducing the production of specific antibodies (IgY) against Fel d1, Fel d2, and Fel d4.

[0026] The mRNA immune preparation provided by the present invention has the following advantages: 1) Easy preparation: mRNA can be quickly prepared through chemical synthesis or in vitro transcription, without the need for complex protein expression and purification processes, greatly shortening the R&D cycle and reducing production costs.

[0027] 2) Uniform immunogenicity: The protein produced by mRNA immunization is naturally synthesized in the body. Its post-translational modification and folding are more similar to natural proteins, and its immunogenicity is more uniform, which can induce a more consistent and effective immune response.

[0028] 3) Immune persistence: mRNA immunity can activate the body's cellular immunity and humoral immunity, produce more lasting immune memory, and thus provide longer-term protection.

[0029] 4) Good antibody effect: The post-translational modification and folding of the protein produced by mRNA immunization are more similar to those of natural proteins, and the prepared antibodies have better blocking efficiency.

[0030] Based on the above advantages, the present invention provides the use of the cat allergen fusion protein described in the above technical solution, or the first nucleic acid molecule described in the above technical solution, or the second nucleic acid molecule described in the above technical solution, or the mRNA immune preparation described in the above technical solution in one or more of the following: 1) As a cat allergy immunogen; 2) preparing a product that neutralizes or reduces the content of cat allergens, wherein the cat allergens include one or more of FeI d 1, FeI d 2, and FeI d 4; 3) Prepare anti-cat allergy vaccines.

[0031] In one embodiment, the product comprises cat allergen antibodies; the anti-cat allergy vaccine is an mRNA vaccine; and in one embodiment, the cat allergen antibodies are egg yolk antibodies.

[0032] Based on the above advantages, the present invention provides an mRNA vaccine, including an mRNA immune preparation and a liposome encapsulating the mRNA immune preparation; the mRNA immune preparation is the mRNA immune preparation described in the above technical solution.

[0033] Based on the above advantages, the present invention provides a cat allergen egg yolk antibody, which is prepared using the mRNA immune preparation described in the above technical solution or the mRNA vaccine described in the above technical solution as an immunogen.

[0034] This study constructs a fusion protein by fusing key allergenic regions of the full-length FeI d1 protein, FeI d2, and FeI d4 proteins, and designs corresponding mRNA for immunization, successfully inducing the production of IgY antibodies that effectively block cat allergens. Compared with traditional single-protein immunization or combined immunization with three proteins, the fusion protein mRNA immunization method of this invention offers advantages such as simple preparation, uniform immunogenicity, good immune persistence, and high safety, providing a new and effective strategy for the prevention and treatment of cat allergies.

[0035] Experimental verification has shown that the cat allergen egg yolk antibodies provided by the present invention can block allergens with the highest efficiency. Compared to the prior art immunization using individual Fel d1, Fel d2, and Fel d4 proteins, the IgY produced by immunization with the fusion protein mRNA provided by the present invention has a superior allergy-blocking effect. Furthermore, its sensitization-blocking effect is comparable to that of IgY produced by immunization using the three proteins Fel d1, Fel d2, and Fel d4 simultaneously, as is the case with the prior art. The fusion protein mRNA immunization method provided by the present invention can more efficiently induce the production of antibodies with broad neutralizing activity, thereby more effectively preventing and treating cat allergies.

[0036] To further illustrate the present invention, the cat allergen fusion protein, mRNA immune preparation and applications thereof provided by the present invention are described in detail below with reference to the examples and drawings. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 Design and construction of fusion protein 1) Screening of key allergenic segments: By predicting the allergen epitopes of Fel d2 and Fel d4 proteins, the key segments with strong allergenicity are screened out.

[0038] The full-length amino acid sequence of FeI d2 is shown in SEQ ID NO. 1, and is as follows: ; Immunogenicity analysis was performed, and a fragment with epitope 357-369aa (EYSRRHPEYSVSL, SEQ ID NO. 2) and a fragment with epitope 486-498aa (VLHEKTPVSERVT, SEQ ID NO. 3) were selected.

[0039] The full-length amino acid sequence of FeI d4 is shown in SEQ ID NO. 4, and is as follows: MKLLLLCLGLILVCAHEEENVVRSNIDISKISGEWYSILLASDVKEKIEENGSMRVFVEHIKALDNSSLSFVFHTKENGKCTEIFLVADKTKDGVYTVVYDGYNVFSIVETVYDEYILLHLLNFDKTRPFQLVEFYAREPDVSQKLKEKFVKYCQEHGIVNILDLTEVDRCLQARGSEVAQDSSVE; The immunogenicity of the fragments was analyzed, and the fragments of epitope 16-30aa (HEEENVVRSNIDISK, SEQ ID NO. 5) and epitope 43-53aa (DVKEKIEENGS, SEQ ID NO. 6) were selected.

[0040] 2) Fusion protein construction: The coding sequence for the full-length Fel d1 protein was linked to the coding sequences for the key allergenic regions of the Fel d2 and Fel d4 proteins via a linker peptide (GGGGS, SEQ ID NO. 7) to construct the gene encoding the fusion protein. The constructed gene was cloned into an expression vector, and the protein was expressed and purified to verify the structure and function of the fusion protein.

[0041] The amino acid sequence of the α chain of the FeI d1 protein is shown in SEQ ID NO. 8, and is as follows: MKGACVLVLLWAALLLISGGNCEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLC; The amino acid sequence of the b chain of the FeI d1 protein is shown in SEQ ID NO.9, and is as follows: MRGALLVLALLVTQALGVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR; where MRGALLVLALLVTQALG (SEQ ID NO.10) is the signal peptide; The double-chain Fel d1 protein and the epitope polypeptides of Fel d2 and Fel d4 are combined to form a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11, and is as follows: MKGACVLVLLWAALLLISGGNCEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPER TAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRGGGGSEYSRRHPEYSVSLGGGGSVLHEKTPVSERVTGGGGSHEEENVVRSNIDISKGGGGSDVKEKIEENGS; where GGGGS (SEQ ID NO.7) is Linker.

[0042] The fusion protein codon was optimized to chicken (Gallus gallus), and the optimized coding sequence is shown in SEQ ID NO. 12, as follows: 5'-ATGAAGGGCGCCTGCGTGCTGGTGCTGCTGTGGGCCGCCCTGTTGCTGATTAGCGGCGGAAATTGCGAGATCTGCCCAGCCGTGAAGCGGGACGTGGATCTGTTCCTGACTGGGACACCTGATGAGTACGTGGAGCAGGTGGCCCAGTACAAGGCACTGCCTGTGGTGCTGGAGAACGCTAGGATCCTGAAAAACTGCGTTGACGCAAAAATGACAGAGGAGGATAAGGAAAATGCCCTGAGCGTGCTGGATAAAATCTACACCTCCCCACTGTGCGGAGGGGGCGGGTCCGTGAAGATGGCAGAGACGTGCCCCATCTTTTACGACGTGTTCTTCGCCGTGGCTAACGGCAACGAGCTGCTGCTCGATCTGAGCCTGACAAAAGTGAACGCTACTGAACCCGAGCGGACAGCTATGAAAAAGATTCAGGATTGCTACGTGGAGAACGGACTGATCTCTAGGGTGCTGGACGGACTGGTGATGACAACTATCAGCTCTAGCAAGGACTGCATGGGAGAGGCTGTGCAGAACACCGTGGAAGATCTGAAGCTGAATACACTGGGAAGAGGAGGGGGGGGAAGCGAATACTCTAGGCGGCACCCTGAATACTCTGTGTCTCTGGGCGGAGGCGGGAGCGTGCTGCACGAGAAAACCCCCGTGTCTGAGAGAGTGACTGGCGGAGGCGGCAGCCACGAGGAAGAGAATGTGGTGAGGTCCAATATCGATATTAGCAAAGGCGGAGGAGGGAGCGACGTGAAGGAAAAGATCGAGGAGAACGGGAGC-3'.

[0043] Example 2 Design and synthesis of mRNA corresponding to the fusion protein 1. mRNA sequence design: Based on the fusion protein designed in Example 1, the corresponding mRNA sequence was designed using the codon-optimized coding sequence (SEQ ID NO. 12). The human α-globin UTR sequence and the T7 promoter sequence were added to the 5' end of the mRNA. The m7GpppN cap structure was added to the 5' end of the mRNA, and the 3' UTR sequence was added after the 3' stop codon. A polyadenylation tail with 100 adenine nucleotides was added. The secondary structure of the mRNA was also predicted and optimized to reduce the potential formation of stable secondary structures. The designed sequence is shown in SEQ ID NO. 13, which is as follows:

[0044] 2. Template full gene synthesis: Send the designed sequence to a gene company for full gene synthesis into the EcoRI-HindIII restriction site of the puc57 vector to obtain the target plasmid.

[0045] 3. Endotoxin-free plasmid extraction: The target plasmid was transformed into E. coli for amplification, and the amplified target plasmid was extracted using the Endotoxin-free Plasmid Extraction Kit (Cat. No. DP117) from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0046] 4. Plasmid linearization: The extracted plasmid was linearized using BsaI-HFV2 from New England Biolabs (NEB) as follows: Prepare the enzyme digestion system by adding 2 μg of plasmid template, 5 μl of 10× rcutsmart buffer, and the rest of double-distilled water to every 50 μl system. Incubate the enzyme digestion at 37°C for 4 h and at 65°C for 20 min.

[0047] Purification was performed by agarose gel electrophoresis, and the purity was determined by spectrophotometry and gel electrophoresis.

[0048] 5. mRNA in vitro transcription: Using the linearized plasmid obtained in step 4 as a template, perform capping transcription using the capping transcription kit from Nanjing Novozymes Biotech Co., Ltd. The steps are as follows: Mix all the components and centrifuge them briefly, then add the components listed in Table 1 in sequence.

[0049] Table 1 mRNA in vitro transcription reaction system

[0050] The components were gently mixed with a pipette, collected by brief centrifugation, and incubated at 37°C for 2 h.

[0051] 6. mRNA purification: 1) Dilute the product to 180 μl by adding 160 μl RNase-free ddH2O. 2) Add 20 μl of 3 M sodium acetate (pH 5.2) to the diluted product and mix thoroughly with a pipette; 3) Add 200 μl of phenol / chloroform mixture (v / v, 1:1), centrifuge at 10,000 rpm for 5 min at room temperature, and transfer the upper aqueous phase to a new centrifuge tube; 4) Repeat the extraction twice; 5) Add 2 volumes of anhydrous ethanol, mix well, incubate at -20°C for 30 min, centrifuge at 15,000 rpm at 4°C for 15 min, and discard the supernatant; 6) Add 500 μl of pre-chilled 80% ethanol to wash the RNA pellet, centrifuge at 15,000 rpm for 15 min at 4°C, and discard the supernatant. 7) Open the lid and let it dry for 2 minutes. Add 20-50 μl of RNase-free ddH2O to dissolve the RNA precipitate.

[0052] 7. mRNA identification: by agarose gel electrophoresis ( Figure 1 ) and UV spectrophotometer to identify the purified mRNA.

[0053] Example 3 Immunity Experiment 1. Selection of immune animals: Select healthy chickens in the laying period as immune animals.

[0054] 2. Immunization Procedure: The prepared mRNA immune preparation corresponding to the fusion protein is encapsulated in liposomes using an LNP (Lipid Nanoparticle) encapsulation kit and injected intramuscularly into chickens. The initial dose is 10 μg per chicken, based on the weight of the mRNA immune preparation. Booster immunizations are administered every two weeks at a dose of 5 μg per chicken, for a total of two booster immunizations.

[0055] 3. Antibody collection: After the final booster immunization, eggs were collected and the egg yolks were diluted with ultrapure water at a volume ratio of 1:9. The pH was adjusted to 5.0-5.2 with HCl and the eggs were allowed to stand overnight at 4°C for stratification. The supernatant was added with pre-cooled glacial acetic acid (final concentration 60%) and the precipitate was collected by centrifugation. The precipitate was dissolved with 0.9% NaCl solution, filtered to remove impurities, and IgY was separated. The lyophilized egg yolk powder (containing anti-Fel d1, Fel d2, and Fel d4 specific egg yolk antibodies) was further verified for its neutralization activity against cat allergens.

[0056] 4. Antibody titer test 1) Perform ELISA on the isolated lyophilized egg yolk powder.

[0057] 2) Plate coating: Dilute Fel d1, Fel d2, and Fel d4 proteins to 2 μg / mL in 0.1 M NaHCO3 buffer and coat the plate overnight at 4°C.

[0058] 3) Washing: Wash five times with PBST (PBS containing 0.1% Tween 20).

[0059] 4) Blocking: Add 1% casein and block at 37°C for 1 hour. Wash five times with PBST.

[0060] 5) Sample addition: Dilute the IgY antibody with PBS at 5000, 10000, 20000, 40000, 80000, 160000, or 320000 times. Add 100 μl to the well plate and incubate at 37°C for 1 h.

[0061] 6) Wash plate: Wash 5 times with PBST.

[0062] 7) Secondary antibody: Add goat anti-chicken IgY-HRP purchased from Luoyang Bioton, dilute 10,000-fold with PBS, take 100 μl and add to the well plate, incubate at 37°C for 1 hour.

[0063] 8) Wash the plate: Wash 5 times with PBST.

[0064] 9) Substrate: Add 100 μl of HRP substrate and incubate at room temperature in the dark for 15 minutes.

[0065] 10) Termination: Add 2M sulfuric acid to terminate the reaction.

[0066] 11) Reading: Read the sample on a microplate reader at 450 nm. See Table 2 for results.

[0067] Table 2 Antibody titer test results (OD 450 value)

[0068] Note: The x in the table is the dilution factor in step 5). 5000x means the IgY antibody is diluted 5000 times with PBS.

[0069] The results showed that the titers of the prepared anti-Fel d1, Fel d2 and Fel d4 IgY were all greater than 320,000 (based on 2.1 times the background OD as the titer determination point), and the titers were good.

[0070] Example 4 Verification of sensitization blocking effect The egg yolk freeze-dried powder obtained in Example 3 (containing egg yolk-specific antibodies against Fel d1, Fel d2, and Fel d4) was added to regular cat food at a mass ratio of 0.5% and fed to 10 experimental cats. The egg yolk freeze-dried powder was fed for a baseline period of one week before feeding and for six consecutive weeks during the experimental period. Saliva clinical samples from the ten cats, collected over a total of seven weeks during the baseline and experimental periods, were analyzed for Fel d1, Fel d2, and Fel d4 levels using Inbio's Fel d1 and Fel d4 ELISA kits and a conventional Fel d2 ELISA, respectively, and the results were statistically analyzed. The conventional Fel d2 ELISA method included the following steps: Fel d2 protein was used to prepare Fel d2 rabbit polyclonal antibody; ELISA plates were coated with Fel d2 rabbit polyclonal antibody, and saliva clinical samples were added for 1 hour. After washing, HRP-labeled Fel d2 rabbit polyclonal antibody was added and reacted for 1 hour. After washing, TMB substrate solution was added for 15 minutes. The reaction was terminated by adding 2M sulfuric acid, and the reading was read at 450 nm to calculate the Fel d2 protein content.

[0071] The results are shown in Table 3.

[0072] Table 3 Fel d1, Fel d2 and Fel d4 content test results (µg / mL)

[0073] The results showed that the IgY antibody prepared by the present invention can effectively block the allergic protein in the cat's oral saliva.

[0074] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A cat allergen fusion protein, characterized in that: The amino acid sequence is shown in SEQ ID NO.

11.

2. The first nucleic acid molecule encoding the cat allergen fusion protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

12.

3. A second nucleic acid molecule for preparing an mRNA immune preparation, characterized in that: The method comprises a T7 promoter sequence and the first nucleic acid molecule according to claim 2.

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

13.

5. An mRNA immune preparation, characterized in that The mRNA immune preparation is prepared by in vitro transcription of the second nucleic acid molecule according to claim 3 or 4.

6. Use of the cat allergen fusion protein according to claim 1, the first nucleic acid molecule according to claim 2, the second nucleic acid molecule according to claim 3 or 4, or the mRNA immune preparation according to claim 5 in one or more of the following: 1) As a cat allergy immunogen; 2) preparing a product that neutralizes or reduces the content of cat allergens, wherein the cat allergens include one or more of FeI d 1, FeI d 2, and FeI d 4; 3) Prepare anti-cat allergy vaccines.

7. The use according to claim 6, characterized in that The product includes cat allergen antibodies; the anti-cat allergy vaccine is an mRNA vaccine.

8. The use according to claim 7, characterized in that The cat allergen antibody is egg yolk antibody.

9. An mRNA vaccine, characterized in that It comprises an mRNA immune preparation and a liposome encapsulating the mRNA immune preparation; the mRNA immune preparation is the mRNA immune preparation according to claim 5.

10. A cat allergen egg yolk antibody, characterized in that The antibody is prepared using the mRNA immune preparation according to claim 5 or the mRNA vaccine according to claim 9 as an immunogen.

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