A feline allergen fusion protein, mRNA immunomodulator and its application

Abstract

This invention relates to the field of biomedical technology, and in particular to a feline allergen fusion protein, an mRNA immunomodulator, and their applications. The amino acid sequence of the feline allergen fusion protein of this invention is shown in SEQ ID NO. 11. The feline allergen fusion protein provided by this invention is a fusion protein obtained by fusing key sensitizing segments of Fel d2 and Fel d4 proteins with the full-length Fel d1 protein. This fusion protein can simultaneously cover the major sensitizing epitopes of Fel d1, Fel d2, and Fel d4, and can more comprehensively block the sensitizing effect of feline allergens. The feline allergen fusion protein provided by this invention enables the rapid design and synthesis of mRNA immunomodulators, allows for precise control of feline allergen fusion protein expression in poultry, results in a more durable immune response, and the prepared IgY antibody can effectively block allergen proteins in feline oral saliva.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a feline allergen fusion protein, an mRNA immunomodulator, and their applications. Background Technology

[0002] Cat allergy is a common allergic disease in humans, mainly caused by cat allergens. Fel d1, Fel d2, and Fel d4 are the main allergenic proteins in cats. They can induce the human immune system to produce specific IgE antibodies, thereby triggering allergic reactions such as sneezing, runny nose, itchy skin, asthma, etc., which seriously affect the quality of life of allergy sufferers.

[0003] Currently, treatments for cat allergies mainly include avoiding contact with cats, medication, and immunotherapy. However, avoiding contact with cats is often difficult to achieve in real life; medication can only relieve symptoms and cannot fundamentally solve the allergy problem; while immunotherapy has some effect, it has problems such as a long treatment period (often 2-3 years) and unstable efficacy. Therefore, developing an efficient, safe, and convenient method to block cat allergens is of significant clinical importance.

[0004] Existing research primarily employs protein immunization to block feline allergens. However, traditional protein immunization methods have several drawbacks, such as complex protein preparation processes, high costs, structural differences from natural antigens, and poor allergen blocking efficacy. For example, Chinese patent CN114437211A discloses a method for preparing egg yolk antibodies based on the feline allergen Fel d1, using Feld1 protein prepared through an E. coli system for immunization. However, natural Fel d1 exhibits glycosylation modification, which is absent in the E. coli expression system. Therefore, the prepared Feld1 protein differs from the natural protein, affecting the activity of the corresponding egg yolk antibody (IgY). Chinese patent CN117003848A discloses the expression, purification, and combined application of the major feline allergen proteins Feld1, Feld4, and Feld7. Immunization with a mixture of these three proteins to prepare IgY, administered at 2 mg IgY / kg body weight for 80 consecutive days, only resulted in a 50% decrease in the content of feline salivary allergen proteins. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a feline allergen fusion protein, an mRNA immunomodulator, and their applications. The feline allergen fusion protein provided by this invention enables the rapid design and synthesis of mRNA immunomodulators, allows for precise control of feline allergen fusion protein expression in poultry, resulting in a more sustained immune response. The prepared IgY antibody can effectively block allergen proteins in feline oral saliva.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a feline allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11.

[0008] The present invention provides a first nucleic acid molecule encoding the feline allergen fusion protein described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO.12.

[0009] This invention provides a second nucleic acid molecule for preparing mRNA immunomodulators, comprising a T7 promoter sequence and the first nucleic acid molecule described in the above technical solution.

[0010] Preferably, the nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO.13.

[0011] This invention provides an mRNA immunomodulator, which is prepared by in vitro transcription of the second nucleic acid molecule described in the above technical solution.

[0012] This invention provides the application of the feline allergen fusion protein described in the above-described technical solution, or the first nucleic acid molecule described in the above-described technical solution, or the second nucleic acid molecule described in the above-described technical solution, or the mRNA immunomodulator described in the above-described technical solution, in one or more of the following:

[0013] 1) As a feline allergen immunogen;

[0014] 2) Prepare products that neutralize or reduce the content of feline allergens, wherein the feline allergens include one or more of Feld1, Feld2, and Feld4;

[0015] 3) Prepare an anti-cat allergy vaccine.

[0016] Preferably, the product includes feline allergen antibodies; the anti-feline allergy vaccine is an mRNA vaccine.

[0017] Preferably, the cat allergen antibody is an egg yolk antibody.

[0018] The present invention provides an mRNA vaccine, comprising an mRNA immunomodulator and liposomes encapsulating the mRNA immunomodulator; wherein the mRNA immunomodulator is the mRNA immunomodulator described in the above technical solution.

[0019] This invention provides a feline allergen egg yolk antibody, which is prepared by using the mRNA immunogens described in the above technical solutions or the mRNA vaccines described in the above technical solutions as immunogens.

[0020] Beneficial effects:

[0021] This invention provides a feline allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO. 11. The feline allergen fusion protein provided by this invention was developed by studying the structure and function of Fel d2 and Fel d4 proteins, screening for key segments with strong sensitizing properties, and fusing these key segments with the full-length Fel d1 protein to construct a novel fusion protein. This fusion protein can simultaneously cover the major sensitizing epitopes of Fel d1, Fel d2, and Fel d4, and can more comprehensively block the sensitizing effect of feline allergens. The feline allergen fusion protein provided by this invention enables the rapid design and synthesis of mRNA immunomodulators, allows for precise control of feline allergen fusion protein expression in poultry, results in a more durable immune response, and the prepared IgY antibody can effectively block allergen proteins in feline oral saliva. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 This is an agarose gel electrophoresis image of the purified mRNA. Detailed Implementation

[0024] This invention provides a feline allergen fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11.

[0025] The feline allergen fusion protein provided by this invention is created by studying the structure and function of Fel d2 and Fel d4 proteins, screening out key segments with strong sensitizing properties, and fusing these key segments with the full-length Fel d1 protein to construct a novel fusion protein. This fusion protein can simultaneously cover the major sensitizing epitopes of Fel d1, Fel d2, and Fel d4, and can more comprehensively block the sensitizing effect of feline allergens.

[0026] This invention provides a first nucleic acid molecule encoding the feline allergen fusion protein described in the above-described technical solution, the nucleotide sequence of which is shown in SEQ ID NO.12. The first nucleic acid molecule provided by this invention is a nucleic acid molecule encoding the feline allergen fusion protein described in the above-described technical solution, obtained by codon optimization of poultry (especially chickens), which can improve the translation efficiency and stability of mRNA in vivo.

[0027] This invention provides a second nucleic acid molecule for preparing mRNA immunomodulators, comprising a T7 promoter sequence and the first nucleic acid molecule described in the above-described technical solution. As one embodiment, the nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO. 13.

[0028] The second nucleic acid molecule provided by this invention is an optimized design 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 to the 3' end to enhance the stability of the mRNA and the translation initiation efficiency. The secondary structure of the mRNA is also optimized to reduce the possible formation of stable secondary structures, thereby improving its translation activity. It can precisely control the expression of cat allergen fusion protein in avian animals.

[0029] This invention provides an mRNA immunomodulator, which is prepared by in vitro transcription of the nucleic acid molecules described in the above technical solution.

[0030] The mRNA immunomodulator provided by this invention can be injected into the body (e.g., in birds, to obtain IgY antibodies) via appropriate immunization routes (such as intramuscular injection, subcutaneous injection, etc.). After the mRNA is taken up by cells in the body, the fusion protein is synthesized using the intracellular translation system. The fusion protein acts as an antigen to stimulate the body's immune system, inducing the production of specific antibodies (IgY) against Fel d1, Fel d2, and Fel d4.

[0031] The mRNA immunomodulator provided by this invention has the following advantages:

[0032] 1) Simple preparation: mRNA can be quickly prepared by chemical synthesis or in vitro transcription without the need for complex protein expression and purification processes, which greatly shortens the research and development cycle and reduces production costs.

[0033] 2) Uniform immunogenicity: The proteins produced by mRNA immunization are naturally synthesized in the body. Their post-translational modifications and folding patterns are more similar to those of natural proteins, resulting in more uniform immunogenicity and the ability to induce a more consistent and effective immune response.

[0034] 3) Immune persistence: mRNA immunity can activate the body's cellular and humoral immunity, generating more lasting immune memory, thereby providing longer-term protection.

[0035] 4) Better antibody efficacy: The proteins produced by mRNA immunization have post-translational modifications and folding patterns that are more similar to those of natural proteins, resulting in antibodies with better blocking efficiency.

[0036] Based on the above advantages, the present invention provides the application of the feline 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 immunomodulator described in the above technical solution in one or more of the following:

[0037] 1) As a feline allergen immunogen;

[0038] 2) Prepare products that neutralize or reduce the content of feline allergens, wherein the feline allergens include one or more of Feld1, Feld2, and Feld4;

[0039] 3) Prepare an anti-cat allergy vaccine.

[0040] In one embodiment, the product includes feline allergen antibodies; the anti-feline allergy vaccine is an mRNA vaccine. In another embodiment, the feline allergen antibody is an egg yolk antibody.

[0041] Based on the above advantages, the present invention provides an mRNA vaccine, comprising an mRNA immunomodulator and liposomes encapsulating the mRNA immunomodulator; wherein the mRNA immunomodulator is the mRNA immunomodulator described in the above technical solution.

[0042] Based on the above advantages, the present invention provides a feline allergen egg yolk antibody, wherein the antibody is prepared by using the mRNA immunogens described in the above technical solutions or the mRNA vaccines described in the above technical solutions as immunogens.

[0043] This invention constructs a fusion protein by fusing key sensitizing segments of the full-length Fel d1 protein, Fel d2, and Fel d4 proteins, and designs its corresponding mRNA for immunization, successfully inducing the body to produce highly effective IgY antibodies that block feline allergens. Compared with traditional single-protein immunization or combined immunization of three proteins, the fusion protein mRNA immunization method of this invention has the advantages of simple preparation, uniform immunogenicity, good immunogenicity and long-lasting immunity, and high safety, providing a new and effective strategy for the prevention and treatment of feline allergies.

[0044] Experimental verification shows that the feline allergen egg yolk antibody provided by this invention can block allergens with maximum efficiency. Compared with the existing technology of using Fel d1, Fel d2, and Fel d4 proteins alone for immunization, the IgY generated by the fusion protein mRNA immunization provided by this invention has a better sensitization blocking effect. Furthermore, its sensitization blocking effect is comparable to that of IgY generated by using all three Fel d1, Fel d2, and Fel d4 proteins simultaneously in the existing technology. The fusion protein mRNA immunization method provided by this invention can more efficiently induce the body to produce antibodies with broad neutralizing activity, thereby more effectively preventing and treating feline allergies.

[0045] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a feline allergen fusion protein, mRNA immunomodulator, and their applications provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1: Design and Construction of Fusion Proteins

[0047] 1) Screening of key sensitizing segments: By predicting allergen epitopes of Fel d2 and Fel d4 proteins, key segments with strong sensitizing properties are screened out.

[0048] The full-length amino acid sequence of Fel d2 is shown in SEQ ID NO.1, as follows:

[0049] ;

[0050] Immunogenicity analysis was performed, and fragments with epitopes 357-369aa (EYSRRHPEYSVSL, SEQ ID NO.2) and epitopes 486-498aa (VLHEKTPVSERVT, SEQ ID NO.3) were selected.

[0051] The full-length amino acid sequence of Fel d4 is shown in SEQ ID NO.4, as follows:

[0052] MKLLLLCLGLILVCAHEEENVVRSNIDISKISGEWYSILLASDVKEKIEENGSMRVFVEHIKALDNSSLSFVFHTKENGKCTEIFLVADKTKDGVYTVVYDGYNVFSIVETVYDEYILLHLLNFDKTRPFQLVEFYAREPDVSQKLKEKFVKYCQEHGIVNILDLTEVDRCLQARGSEVAQDSSVE;

[0053] Immunogenicity analysis was performed on the samples, and fragments of epitope 16-30aa (HEEENVVRSNIDISK, SEQ ID NO.5) and epitope 43-53aa (DVKEKIEENGS, SEQ ID NO.6) were selected.

[0054] 2) Construction of fusion protein: The coding sequence of the full-length Fel d1 protein was linked with the coding sequences of the key sensitization regions of Fel d2 and Fel d4 proteins using a linker peptide (GGGGS, SEQ ID NO.7) to construct the coding gene of the fusion protein. The constructed gene was cloned into an expression vector for protein expression and purification to verify the structure and function of the fusion protein.

[0055] The amino acid sequence of the α chain of the Fel d1 protein is shown in SEQ ID NO.8, as follows:

[0056] MKGACVLVLLWAALLLISGGNCEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLC;

[0057] The amino acid sequence of the b chain of the Fel d1 protein is shown in SEQ ID NO.9, as follows:

[0058] MRGALLVLALLVTQALGVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR; where MRGALLVLALLVTQALG (SEQ ID NO.10) is the signal peptide;

[0059] The double strand of Fel d1 protein and the epitope peptides of Fel d2 and Fel d4 were combined to form a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.11, as follows:

[0060] MKGACVLVLLWAALLLISGGNCEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPER TAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRGGGGSEYSRRHPEYSVSLGGGGSVLHEKTPVSERVTGGGGSHEEENVVRSNIDISKGGGGSDVKEKIEENGS; where GGGGS (SEQ ID NO.7) is Linker.

[0061] The codons of the fusion protein were optimized for chicken (Gallus gallus), and the optimized coding sequence is shown in SEQ ID NO. 12, as follows:

[0062] 5'-ATGAAGGGCGCCTGCGTGCTGGTGCTGCTGTGGGCCGCCCTGTTGCTGATTAGCGGCGGAAATTGCGAGATCTGCCCAGCCGTGAAGCGGGACGTGGATCTGTTCCTGACTGGGACACCTGATGAGTACGTGGAGCAGGTGGCCCAGTACAAGGCACTGCCTGTGGTGCTGGAGAACGCTAGGATCCTGAAAAACTGCGTTGACGCAAAAATGACAGAGGAGGATAAGGAAAATGCCCTGAGCGTGCTGGATAAAATCTACACCTCCCCACTGTGCGGAGGGGGCGGGTCCGTGAAGATGGCAGAGACGTGCCCCATCTTTTACGACGTGTTCTTCGCCGTGGCTAACGGCAACGAGCTGCTGCTCGATCTGAGCCTGACAAAAGTGAACGCTACTGAACCCGAGCGGACAGCTATGAAAAAGATTCAGGATTGCTACGTGGAGAACGGACTGATCTCTAGGGTGCTGGACGGACTGGTGATGACAACTATCAGCTCTAGCAAGGACTGCATGGGAGAGGCTGTGCAGAACACCGTGGAAGATCTGAAGCTGAATACACTGGGAAGAGGAGGGGGGGGAAGCGAATACTCTAGGCGGCACCCTGAATACTCTGTGTCTCTGGGCGGAGGCGGGAGCGTGCTGCACGAGAAAACCCCCGTGTCTGAGAGAGTGACTGGCGGAGGCGGCAGCCACGAGGAAGAGAATGTGGTGAGGTCCAATATCGATATTAGCAAAGGCGGAGGAGGGAGCGACGTGAAGGAAAAGATCGAGGAGAACGGGAGC-3'.

[0063] Example 2 Design and Synthesis of mRNA Corresponding to the Fusion Protein

[0064] 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). A human α-globin UTR sequence and a T7 promoter sequence were added to the 5' end of the mRNA. An m7GpppN cap structure was added to the 5' end of the mRNA, and a 3' UTR sequence was added after the 3' stop codon. A polyadenylated tail with 100 adenosine nucleotides spaced was added. Simultaneously, the secondary structure of the mRNA was predicted and optimized to reduce the potential formation of stable secondary structures. The designed sequence is shown in SEQ ID NO. 13, as follows:

[0065]

[0066] 2. Template whole gene synthesis: The designed sequence is sent to a gene company for whole gene synthesis into the EcoRI-HindIII restriction site of the puc57 vector to obtain the target plasmid.

[0067] 3. Endotoxin-free plasmid large-scale extraction: The target plasmid was transformed into E. coli for amplification, and the amplified target plasmid was extracted using the endotoxin-free plasmid large-scale extraction kit (catalog number DP117) from Tiangen Biotech (Beijing) Co., Ltd.

[0068] 4. Plasmid linearization: The extracted plasmid was linearized using BsaI-HFV2 from New England Biolabs (NEB). The steps are as follows: Prepare the enzyme digestion system, add 2 μg of plasmid template, 5 μl of 10×rcutsmart buffer, and the remainder double-distilled water to each 50 μl system, digest at 37℃ for 4 h, and then at 65℃ for 20 min.

[0069] Agarose gel electrophoresis was used for gel purification, and the purity was identified by spectrophotometry and gel electrophoresis.

[0070] 5. In vitro mRNA transcription: Using the linearized plasmid obtained in step 4 as a template, capped transcription was performed using the capped transcription kit from Nanjing Novizan Biotechnology Co., Ltd., as follows:

[0071] Mix all components thoroughly and centrifuge briefly, then add the components listed in Table 1 in sequence.

[0072] Table 1. mRNA in vitro transcription reaction system

[0073]

[0074] Gently mix the components with a pipette, collect by brief centrifugation, and incubate at 37°C for 2 hours.

[0075] 6. mRNA purification:

[0076] 1) Add 160 μl of RNase-free ddH2O to dilute the product to 180 μl;

[0077] 2) Add 20 μl of 3 M sodium acetate (pH 5.2) to the diluted product and mix thoroughly with a pipette;

[0078] 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;

[0079] 4) Repeat the extraction twice;

[0080] 5) Add 2 volumes of anhydrous ethanol, mix well, incubate at -20℃ for 30 min, centrifuge at 15000 rpm for 15 min at 4℃, and discard the supernatant;

[0081] 6) Add 500 μl of pre-cooled 80% ethanol to wash the RNA precipitate, centrifuge at 15000 rpm for 15 min at 4℃, and discard the supernatant;

[0082] 7) Open the lid and dry for 2 min, then add 20~50 μl of RNase-free ddH2O to dissolve the RNA precipitate.

[0083] 7. mRNA identification: by agarose gel electrophoresis ( Figure 1 The purified mRNA was identified using a UV spectrophotometer.

[0084] Example 3 Immunoassay

[0085] 1. Selection of immunized animals: Select healthy chickens during their egg-laying period as immunized animals.

[0086] 2. Immunization Program: The prepared fusion protein-corresponding mRNA immunomodulator was encapsulated in liposomes using an LNP (Lipid Nanoparticle) encapsulation kit and injected intramuscularly into the chickens. The initial immunization dose was 10 μg / bird based on the mass of the mRNA immunomodulator, with booster immunizations every two weeks at a dose of 5 μg / bird, for a total of two booster immunizations.

[0087] 3. Antibody collection: After the last booster immunization, eggs were collected. Egg yolks were diluted with ultrapure water at a volume ratio of 1:9, and the pH was adjusted to 5.0-5.2 with HCl. The mixture was then allowed to stand overnight at 4°C for separation. The supernatant was collected and pre-cooled glacial acetic acid (final concentration 60%) was added. The precipitate was collected by centrifugation. The precipitate was dissolved in 0.9% NaCl solution, filtered to remove impurities, and IgY was obtained. The IgY was then lyophilized to obtain lyophilized egg yolk powder (containing specific egg yolk antibodies against Fel d1, Fel d2, and Fel d4) to further verify its neutralizing activity against feline allergens.

[0088] 4. Antibody titer test

[0089] 1) The isolated freeze-dried egg yolk powder was analyzed by ELISA.

[0090] 2) Plate coating: Dilute Fel d1, Fel d2 and Fel d4 proteins to 2 μg / mL in 0.1M NaHCO3 buffer and coat overnight at 4°C.

[0091] 3) Washing: Wash 5 times with PBST (PBS containing 0.1% Tween 20).

[0092] 4) Blocking: Add 1% casein and block at 37°C for 1 hour, then wash 5 times with PBST.

[0093] 5) Sample addition: Dilute the IgY antibody with PBS at 5000, 10000, 20000, 40000, 80000, 160000, and 320000 times, take 100 μl and add it to the well plate, and react at 37℃ for 1 h.

[0094] 6) Wash plate: Wash 5 times with PBST.

[0095] 7) Secondary antibody: Add sheep anti-chicken IgY-HRP purchased from Luoyang Baitong, dilute with PBS 10,000 times, take 100 μl and add it to the well plate, react at 37℃ for 1 h.

[0096] 8) Wash plate: Wash 5 times with PBST.

[0097] 9) Substrate: Add 100 μl of HRP substrate and incubate at room temperature in the dark for 15 min.

[0098] 10) Termination: Add 2M sulfuric acid to terminate the reaction.

[0099] 11) Reading: The microplate reader reading was 450 nm. The results are shown in Table 2.

[0100] Table 2 Antibody titer test results (OD) 450 value)

[0101]

[0102] Note: x in the table is the dilution factor in step 5). 5000x means that the IgY antibody is diluted 5000 times with PBS.

[0103] The results showed that the prepared anti-Fel d1, Fel d2 and Fel d4 IgY titers were all >320,000 (based on the baseline OD 2.1 times as the titer criterion), indicating good titers.

[0104] Example 4: Verification of sensitization blocking effect

[0105] The freeze-dried egg yolk powder obtained in Example 3 (containing specific egg yolk 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 freeze-dried egg yolk powder was fed for one week before baseline and continuously for six weeks during the experimental period. Saliva clinical samples from the 10 cats during the baseline and experimental periods (a total of seven weeks) were collected. The levels of Fel d1, Fel d2, and Fel d4 were detected using the Inbio Fel d1, Fel d4 ELISA kit and the conventional Fel d2 ELISA, respectively, and the results were statistically analyzed. The conventional Fel d2 ELISA method includes the following steps:

[0106] Fel d2 rabbit polyclonal antibody was prepared using Fel d2 protein; Fel d2 rabbit polyclonal antibody was coated onto ELISA plates, and saliva clinical samples were added and reacted for 1 h. After washing, HRP-labeled Fel d2 rabbit polyclonal antibody was added and reacted for 1 h. After washing, TMB substrate solution was added for 15 min, and the reaction was terminated by adding 2M sulfuric acid. The reading was taken at 450 nm, and the Fel d2 protein content was calculated.

[0107] The results are shown in Table 3.

[0108] Table 3. Results of Fel d1, Fel d2 and Fel d4 content detection (µg / mL)

[0109]

[0110] The results showed that the IgY antibody prepared by this invention can effectively block allergenic proteins in cat oral saliva.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

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

11.

2. A first nucleic acid molecule encoding the feline allergen fusion protein of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

12.

3. A second nucleic acid molecule for preparing mRNA immunomodulators, characterized in that, It includes the T7 promoter sequence and the first nucleic acid molecule as described in claim 2.

4. The second 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 immunomodulator, characterized in that, The mRNA immunomodulator is prepared by in vitro transcription of the second nucleic acid molecule as described in claim 3 or 4.

6. The use of the feline allergen fusion protein of claim 1, the first nucleic acid molecule of claim 2, the second nucleic acid molecule of claim 3 or 4, or the mRNA immunomodulator of claim 5 in one or more of the following: 1) Prepare egg yolk antibodies that neutralize or reduce the content of feline allergens, wherein the feline allergens include one or more of Fel d1, Fel d2 and Feld4; 2) Prepare an anti-cat allergy vaccine.

7. The application according to claim 6, characterized in that, The anti-cat allergy vaccine is an mRNA vaccine.

8. An mRNA vaccine, characterized in that, It includes an mRNA immunoassay agent and liposomes encapsulating the mRNA immunoassay agent; the mRNA immunoassay agent is the mRNA immunoassay agent according to claim 5.

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