Cat allergen polypeptide fragment and use

Abstract

Provided are a cat allergen polypeptide fragment and use thereof. Fel d1 is divided into a plurality of polypeptides of 10-20 amino acids, which are then co-expressed with VLPs proteins; subsequently, hyperimmune serum of Fel d1 is used for screening; and 2 Fel d1 polypeptides with relatively good immunogenicity are successfully screened. The 2 polypeptides are used for co-expression with VLPs and then formulated into a vaccine to immunize a cat. The secretion amount of Fel d1 in the cat saliva is detected, and the secretion amount of Fel d1 can be reduced by 80% or more. The described polypeptides effectively reduce the secretion amount of the Fel d1 protein in the cat saliva, and reduce the spread of Fel / d1 in the air.

Description

Cat allergen polypeptide fragments and their applications Technical Field

[0001] The present invention relates to the field of immunotechnology, in particular to cat allergen polypeptide fragments and applications thereof. Background Art

[0002] Cat allergy is second only to dust mites in the incidence of allergic reactions and has become a major public health concern. Typical cat allergy symptoms include rhinitis, asthma, and conjunctivitis, which can indirectly lead to poor sleep quality, fatigue, decreased alertness, reduced work efficiency and concentration, mood changes, and potentially impaired learning in children. Current surveys indicate that approximately one-fifth of adults worldwide suffer from cat allergies, although levels of allergy appear to be higher in children. Cat allergy is not limited to cat owners; due to the ubiquitous presence of allergens, many people who do not come into direct contact with cats can also experience allergic symptoms, such as in private cars, public transportation, and buildings. Ten cat allergens have been identified, eight of which (Fel d1 to Fel d8) have been registered with the International Union of Immunology (IUIS). Fel d1 is the primary cat allergen, accounting for 96% of allergic reactions to cats in humans. Fel d1 is primarily produced in the salivary and sebaceous glands, with smaller amounts in the lacrimal and anal glands. All cats produce Fel d1, regardless of breed, age, coat length, sex, housing (indoor vs. outdoor), or weight, and there are no allergen-free or hypoallergenic cats, but Fel d1 production varies greatly between individual cats and can vary significantly in the same cat throughout the year.

[0003] Fel d1 is a secretory globin, not a lipoprotein, making cats unique among mammals. The biological function of Fel d1 remains unclear, but some suggest it may be related to skin or mucous membrane protection, while others suggest it may play a role in the transport of lipid molecules, particularly steroids, hormones, or pheromones. Fel d1 is a glycoprotein with a molecular weight of 35-39 kDa. It is composed of two non-directly linked proteins, Chain1 and Chain2, forming a heterodimer that is further bound by non-covalent bonds to form a tetramer. Fel d1 can induce IgE-mediated allergic reactions in humans, with anti-Fel d1-specific IgE detected in the serum of over 80%-95% of cat allergy sufferers. Because Fel d1 is a major cat allergen and is readily airborne and persists in indoor environments, it has become a key research target for cat allergy treatment.

[0004] Currently, the treatment methods for cat allergies include allergen-specific immunotherapy (AIT), which administers symptomatic drugs such as antihistamines or corticosteroids to patients in gradually increasing doses through subcutaneous, sublingual, or oral administration. Patients change their immune system through multiple doses of allergen treatment and improve their tolerance to allergens. The main disadvantages of AIT are that the levels of major allergens in the extract are inconsistent, so the treatment effect is uncontrollable; one is that it is dependent, so the treatment cycle is long; and one is the potential serious side effects of the treatment. In addition, there is also a method of using allergy drugs for treatment, but the treatment effect is often poor.

[0005] Currently, some researchers have reported that a Felid 1 protein vaccine, developed to immunize cats, produces antibodies against the protein, thereby eliminating Felid 1 from the body and reducing Felid 1 secretion. Furthermore, Nestlé has launched a cat food containing egg yolk antibodies against Felid 1, which neutralize Felid 1 in the cat's mouth, thereby reducing the spread of Felid 1 throughout the body.

[0006] Vaccines targeting Fel d1 protein currently use Fel d1 protein connected to a carrier protein, such as virus-like particles (VLPs), through a chemical coupling reagent. This method is cumbersome and requires the preparation of two proteins. After chemical coupling, the uncoupled protein must be purified to remove it. Product quality control is difficult, the production cost is high, and it is not suitable for large-scale production applications.

[0007] Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide cat allergen polypeptide fragments and applications.

[0009] The cat allergen polypeptide fragment provided by the present invention is a truncated form of Chain 1 or Chain 2 of the FeI d 1 protein.

[0010] Chain 1 of the FeI d 1 protein has an amino acid sequence as shown in SEQ ID NO: 1, and a truncated form of Chain 1 consists of 10 to 15 amino acid residues, for example, 10, 11, 12, 13, 14, or 15 residues. In some embodiments, the truncation of Chain1 consists of amino acid residues 1 to 11, or amino acid residues 6 to 16, or amino acid residues 11 to 21, or amino acid residues 15 to 25, or amino acid residues 17 to 30, or amino acid residues 21 to 31, or amino acid residues 25 to 35, or amino acid residues 31 to 41, or amino acid residues 35 to 45, or amino acid residues 41 to 52, or amino acid residues 46 to 56, or amino acid residues 51 to 61, or amino acid residues 56 to 66, or amino acid residues 61 to 71 of the amino acid sequence shown in SEQ ID NO: 1. Or a truncation formed by adding, deleting or replacing one or more amino acid residues in the aforementioned truncation, and having at least 80% sequence homology with the aforementioned truncation. Preferably, the homology is greater than 85%, more preferably, greater than 90%, more preferably, greater than 95%, and most preferably, greater than 98%, 99%, or 99.9%. In a specific embodiment, the truncated form of the FeI d1 protein Chain1 has the amino acid sequence shown in any one of SEQ ID NOs: 3-5.

[0011] Chain2 of the FeI d1 protein has an amino acid sequence as shown in SEQ ID NO: 2, and the truncated form of Chain1 consists of 10 to 15 amino acid residues, for example, 10, 11, 12, 13, 14, or 15 residues. In some embodiments, the truncation of Chain2 consists of amino acid residues 1 to 11, or amino acid residues 6 to 16, or amino acid residues 11 to 21, or amino acid residues 16 to 26, or amino acid residues 21 to 31, or amino acid residues 25 to 435, or amino acid residues 31 to 43, or amino acid residues 35 to 45, or amino acid residues 41 to 51, or amino acid residues 47 to 57, or amino acid residues 51 to 61, or amino acid residues 56 to 66, or amino acid residues 61 to 71, or amino acid residues 66 to 76, or amino acid residues 70 to 79, or amino acid residues 76 to 92 of the amino acid sequence shown in SEQ ID NO: 2. Alternatively, a truncation formed by adding, deleting, or replacing one or more amino acid residues in the aforementioned truncation has at least 80% sequence homology with the aforementioned truncation. Preferably, it has more than 85% homology, preferably more than 90% homology, more preferably more than 95% homology, and most preferably 98% homology, 99% homology, or 99.9% homology. In a specific embodiment, the truncation of the FeI d1 protein Chain2 has the amino acid sequence shown in any one of SEQ ID NOs: 6 to 8.

[0012] Furthermore, the present invention also provides a fusion protein comprising the cat allergen polypeptide fragment and VLP protein as described above.

[0013] In the present invention, the VLP is selected from any one of AP205, CPMV, CMV, PapMV, FHV, HbsAg, MS2 or M1.

[0014] In the present invention, the fusion protein is formed by fusing VLP with at least one cat allergen polypeptide fragment. As an practicable example, the number of cat allergen polypeptide fragments in the fusion protein is 1 to 5, for example, 1, 2, 3, 4 or 5. The cat allergen polypeptide fragments may all be from Chain 1 or all from Chain 2, or may be partially from Chain 1 and partially from Chain 2. The present invention is not limited to this.

[0015] The present invention does not limit the connection relationship between the VLP and the cat allergen polypeptide fragment. The VLP can be located at the N-terminus or the C-terminus of the cat allergen polypeptide fragment.

[0016] In the present invention, the VLP protein and the cat allergen polypeptide fragment can be directly linked or connected through a linker, which is not limited in the present invention. The linker is selected from at least one of GGGGS, P2A, GGGSS, GSSGG or KKK.

[0017] The preparation method of the fusion protein of the present invention can be carried out by chemical coupling or enzymatic coupling, or can be produced by genetic engineering, which is not limited by the present invention.

[0018] The present invention also provides some biomaterials, which include at least one of the following I) to VI):

[0019] 1), a composition or fusion protein formed by any two or more of the cat allergen polypeptide fragment and the fusion protein;

[0020] II), a nucleic acid encoding the cat allergen polypeptide fragment or a nucleic acid encoding the fusion protein;

[0021] III), an expression unit comprising the nucleic acid described in II);

[0022] IV), a vector containing the nucleic acid described in II) or the expression unit described in III);

[0023] V), a host transformed or transfected with the vector described in IV), or a host having the nucleic acid described in I) integrated into its genome;

[0024] VI) and V) the expression product of the host.

[0025] As mentioned above, the composition consisting of the cat allergen polypeptide fragment and the fusion protein includes at least one cat allergen polypeptide fragment and at least one fusion protein, or includes two or more fusion proteins, or includes two or more cat allergen polypeptide fragments. For example, the polypeptide fragments include those shown in SEQ ID NO: 3 and SEQ ID NO: 4, or those shown in SEQ ID NO: 3 and SEQ ID NO: 5, or those shown in SEQ ID NO: 3 and SEQ ID NO: 6, or those shown in SEQ ID NO: 3 and SEQ ID NO: 7, or those shown in SEQ ID NO: 3 and SEQ ID NO: 8, or those shown in SEQ ID NO: 4 and SEQ ID NO: 5, or those shown in SEQ ID NO: 4 and SEQ ID NO: 6, or those shown in SEQ ID NO: 4 and SEQ ID NO: 7, or those shown in SEQ ID NO: 4 and SEQ ID NO: 8, or those shown in SEQ ID NO: 5 and SEQ ID NO: 6, or those shown in SEQ ID NO: 5 and SEQ ID NO: 7, or those shown in SEQ ID NO: 5 and SEQ ID NO: 8, or those shown in SEQ ID NO: 6 and SEQ ID NO: 7, or those shown in SEQ ID NO: 6 and SEQ ID NO: 8, or those shown in SEQ ID NO: 4 and SEQ ID NO: 8. The polypeptide fragments shown in SEQ ID NO:7 and SEQ ID NO:8.

[0026] As mentioned above, the nucleic acid encoding the cat allergen polypeptide fragment may be codon-optimized or not, and the present invention does not limit this.

[0027] As mentioned above, the nucleic acid encoding the fusion protein may be codon-optimized or not, and the present invention does not limit this.

[0028] As described above, the expression unit containing the nucleic acid includes a promoter and an encoding nucleic acid. Furthermore, the expression unit may further include a terminator and / or an enhancer. The promoter may be a eukaryotic promoter or a prokaryotic promoter, but the present invention is not limited thereto. For example, the promoter may be a CMV promoter, a CAG promoter, an EF1a promoter, a PGK promoter, a U6 and H1 promoter, an EFS promoter, a CBh promoter, a SFFV promoter, an MSCV promoter, an SV40 promoter, an UBC promoter, or a TRE promoter. For example, the enhancer may be selected from the group consisting of the SV40 enhancer, the CMV enhancer, the SV-1 enhancer, the ROSA26 enhancer, the EF1α enhancer, the HARE5 enhancer, the UBC enhancer, the EF1A enhancer, the PGK enhancer, the CAGG enhancer, the COPIA enhancer, and the ACT5C enhancer. For example, the terminator is selected from the group consisting of T7 phage terminator, TO phage terminator, lambda phage terminator, SV40 terminator, CMV terminator, rrnB terminator, bGH terminator, hGH terminator or rbGlob terminator.

[0029] As previously described, the vector containing the nucleic acid or expression unit is a cloning vector, an expression vector, or a viral vector. In the present invention, the vector is used for storing or amplifying the nucleic acid or expression unit, or for expressing the cat allergen polypeptide fragment or fusion protein, although this is not a limitation of the present invention. In some embodiments, the vector is a plasmid vector, including pUC series plasmid vectors, pBR322 plasmid vectors, pGEM series plasmid vectors, pET series plasmid vectors, Yeast series plasmid vectors, or Gateway plasmid vectors.In some embodiments, the fusion protein is expressed using a pET series plasmid vector. As a feasible case, the pET series expression vector is selected from: pET-23c(+), pET-23(+), pET-12b(+), pET-12c(+), pET-12a(+), pET-11b(+), pET-11a(+), pET-11c(+), pET-50b(+), pET-49b(+), pET-48b(+), pET-47b(+), pET-26b(+), pET-32a(+), pET-21b(+), pET-22b(+ ), pET-14b, pET-16b, pET-15b, pET-19b, pET-20b(+), pET-21d(+), pET-21c(+), pET-21b(+), pET-21a(+), pET-24a(+), pET-24d(+) , pET-25b(+), pET-27b(+), pET-28a(+), pET-30a(+), pET-42a(+), pET-43.1c(+), pET-43.1b(+), pET-43.1a(+), pET-44a(+), pET- 44c(+), pET-46pET-37b(+), pET303 / CT-His, pET302 / NT-His, pET300 / NT-DEST, pET301 / CT-DEST, pET-5b(+), pET-17b, pET102 / D-T OPO, pET-5a(+), pET-31b(+), pET-3b(+), pET-43.1, pET-41, pET-41a(+), pET-28b(+), pET-42b(+), pET-3a(+), pET-23d(+), pET-4 1b(+), pET-44b(+), pET-42c(+), pET-41c(+), pET-45b(+), pET-33b(+), pET-39b(+), pET-32, pET-40b(+), pET-32c(+), pET-32b(+ ), pET-30, pET-32, pET-30c(+), pET-29c(+), pET-29b(+), pET-30, pET-24c(+), pET-24b(+), pET-24(+), pET-29a(+), or pET-11d(+).

[0030] As described above, the host is used for the storage and amplification of the plasmid vector, or for the expression of the cat allergen polypeptide fragment or fusion protein. In the present invention, the host is a eukaryotic host or a prokaryotic host. The eukaryotic host includes, but is not limited to, yeast, insect cells, and renal epithelial cells, while the prokaryotic host includes, but is not limited to, Escherichia coli. In the embodiments of the present invention, the host is E. coli BL21(DE3), BL21(DE3)pLysS, DH5α, JM109, JM110, TOP10, HB101, or Xl1-Blue.

[0031] As mentioned above, the expression product of the host refers to the product obtained after culturing the host, including but not limited to the culture fluid, the suspension obtained after the culture fluid is crushed, the precipitate or supernatant obtained by centrifugation of the culture fluid or suspension, or the product obtained by extracting the culture fluid, suspension, supernatant or precipitate as mentioned above.

[0032] Furthermore, the present invention also provides use of the cat allergen polypeptide fragment, the fusion protein or the biomaterial in preparing an immunoassay reagent or a vaccine.

[0033] In the present invention, the immunoassay reagent refers to a reagent for detecting FeI d 1 antibodies, including detecting the presence and / or content of FeI d 1 antibodies.

[0034] In the present invention, the immunoassay reagent comprises at least one of the cat allergen polypeptide fragment and the fusion protein and a carrier, and the carrier is a chip, glass, microspheres, magnetic beads or ELISA plate.

[0035] In the present invention, the immunoassay reagent further includes a buffer required for detection. The buffer includes but is not limited to phosphate buffered saline (PBS), Tris buffered saline (TBS), citrate buffer, borate buffer, glycine buffer, acetate buffer, disodium hydrogen phosphate-citrate buffer, glycine-sodium hydroxide buffer, sodium barbital-hydrochloric acid buffer, potassium hydrogen phthalate-sodium hydroxide buffer, etc.

[0036] Accordingly, the present invention provides a method for detecting FeI d 1 antibodies, wherein the method comprises detecting a sample using the aforementioned immunoassay reagent. The sample includes, but is not limited to, serum, blood, plasma, saliva, tears, sputum, nasal secretions, etc. The detection method includes, but is not limited to, antigen-antibody reaction, enzyme-linked immunosorbent assay (ELISA), immunofluorescence technique, immunoblotting technique, or flow cytometry.

[0037] The present invention also provides a vaccine comprising: at least one of the cat allergen polypeptide tablets or the fusion protein and an adjuvant. The vaccine of the present invention may be in the form of an oral dosage form, an injectable dosage form, or an aerosol dosage form.

[0038] The oral dosage forms include but are not limited to oral sugar pills, oral liquids or granules. The injectable dosage forms include but are not limited to injectable liquids or injectable powders.

[0039] Adjuvants for the vaccine include, but are not limited to, aluminum adjuvants, oily adjuvants, immunostimulatory complex adjuvants, peptide adjuvants, cytokine adjuvants, small molecule compound adjuvants, and nanoadjuvants. Aluminum adjuvants include, but are not limited to, aluminum hydroxide and / or aluminum phosphate; oily adjuvants include, but are not limited to, mineral oil and / or vegetable oil; small molecule compound adjuvants include, but are not limited to, imidazolinone and / or imidazolate; and adjuvants may also include viral vectors, polymers, dendritic cells, and the like used in gene therapy.

[0040] Accordingly, the present invention also provides a method for inhibiting FeI d 1 secretion, comprising immunizing an animal with the vaccine. Immunization methods include, but are not limited to, inhalation, oral administration, injection, mucosal administration, or topical administration. Mucosal administration includes, but is not limited to, nasal and / or rectal administration. The animal includes, but is not limited to, mammals. The animal includes bovines, equines, ovines, porcines, canines, felines, rodents, and primates, for example, cats, dogs, mice, or rabbits.

[0041] The present invention separates Feld1 into multiple polypeptides of 10 to 20 amino acids, which are then co-expressed with VLPs. Screening with Fel d1 hyperimmune serum successfully identified two Fel d1 polypeptides with excellent immunogenicity. These two polypeptides were co-expressed with VLPs and then prepared into vaccines to immunize cats. Testing of Fel d1 secretion in cat saliva revealed a reduction of over 80%, effectively reducing the amount of Fel d1 protein in cat saliva and the spread of Fel d1 in the air. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the electrophoresis diagram after co-expression of AP205 and each fragment;

[0043] Figure 2 shows the titers of each antibody. DETAILED DESCRIPTION

[0044] The present invention provides cat allergen polypeptide fragments and applications. Those skilled in the art can draw upon the present disclosure and appropriately modify process parameters to achieve these results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described using preferred embodiments. It is readily apparent that those skilled in the art will be able to modify, adapt, and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the scope, spirit, and spirit of the present invention.

[0045] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0046] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0047] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0048] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0049] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0050] The numerical ranges and parameters used in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0051] The invention divides FeI d 1 into multiple polypeptides of 10 to 20 amino acids, co-expresses the polypeptides with VLPs proteins, and then uses FeI d 1 high immune serum for screening.

[0052] The polypeptide fragment constructed by the present invention is Chain1 15～25:It is connected with different VLPs, and the obtained fusion proteins are Chain1 15～25 -AP205, Chain1 15～25 -CPMV, Chain1 15 ～25 -PapMV, Chain1 15～25 -FHV, Chain1 15～25 -HbsAg, Chain1 15～25 -CMV, Chain1 15～25 -M1, Chain1 15～25 -MS2;

[0053] Or the polypeptide fragment constructed by the present invention is Chain1 17～30 :It is connected with different VLPs, and the obtained fusion proteins are Chain1 17～30 -AP205, Chain1 17～30 -CPMV、Cha in1 17～30 -PapMV, Chain1 17～30 -FHV, Chain1 17～30 -HbsAg, Chain1 17～30 -CMV, Chain1 17～30 -M1, Chain1 17～30 -MS2;

[0054] Or the polypeptide fragment constructed by the present invention is Chain1 46～56 :It is connected with different VLPs, and the obtained fusion proteins are Chain1 46～56 -AP205, Chain1 46～56 -CPMV、Cha in1 46～56 -PapMV, Chain1 46～56 -FHV, Chain1 46～56 -HbsAg, Chain1 46～56 -CMV, Chain1 46～56 -M1, Chain1 46～56 -MS2;

[0055] Or the polypeptide fragment constructed by the present invention is Chain2 31～43 :It is connected with different VLPs to obtain fusion proteins Chain2 31～43 -AP205, Chain2 31～43 -CPMV, Chain2 31～43 -PapMV, Chain2 31～43 -FHV, Chain2 31～43-HbsAg, Chain2 31～43 -CMV, Chain2 31～43 -M1, Chain2 31～43 -MS2;

[0056] Or the polypeptide fragment constructed by the present invention is Chain2 47-57 :It is connected with different VLPs to obtain fusion proteins Chain2 47-57 -AP205, Chain2 47-57 -CPMV, Chai n2 47-57 -PapMV, Chain2 47-57 -FHV, Chain2 47-57 -HbsAg, Chain2 47-57 -CMV, Chain2 47-57 -M1, Chain2 47-57 -MS2;

[0057] Or the polypeptide fragment constructed by the present invention is Chain2 70-79 :It is connected with different VLPs to obtain fusion proteins Chain2 70-79 -AP205, Chain2 70-79 -CPMV, Chai n2 70-79 -PapMV, Chain2 70-79 -FHV, Chain2 70-79 -HbsAg, Chain2 70-79 -CMV, Chain2 70-79 -M1, Chain2 70-79 -MS2;

[0058] In some cases, the vaccine may include only one of the fusion proteins described above, or may include two or more of the fusion proteins, which is not limited in the present invention. 17～30 -AP205 and Chain2 31～43 -AP205, or including Chain1 17～30 -AP205 and Chain2 47～57 -AP205, or including Chain1 17～30 -AP205 and Chain2 70～79 -AP205,

[0059] or including Chain1 46～56 -AP205 and Chain2 31～43 -AP205, or including Chain1 46～56 -AP205 and Chain247～57 -AP205, or including Chain1 46～56 -AP205 and Chain2 70～79 -AP205,

[0060] or including Chain1 15～25 -AP205 and Chain2 31～43 -AP205, or including Chain1 15～25 -AP205 and Chain2 47～57 -AP205, or including Chain1 15～25 -AP205 and Chain2 70～79 -AP205.

[0061] The above polypeptide fragments, or fusion proteins, or combinations of fusion proteins can reduce the secretion of animal Fel d1 protein to a certain extent, and can achieve better inhibitory effects compared to Chain 1 of Fel d1 or Chain 2 of Fel d1, for example, by increasing the inhibitory effect by 1 to 100 times, specifically by 1 to 10 times, preferably by 1 to 5 times, and more preferably by 1 to 2 times.

[0062] In addition, the fragments obtained by screening were combined with different adjuvants for screening, and the final results showed that multiple adjuvants could promote the results.

[0063] The Examples and Comparative Examples of the present invention describe some examples. The Examples illustrate certain implementations of the present invention. However, this does not mean that the effects of the present invention are achieved only in these examples. In addition, many attempts were made during the research and development process, such as using different components, different polypeptide fragments, different VLP proteins, or different encoding nucleic acids, vectors, hosts, and / or different adjuvants. However, the effects of these attempts were not as good as those of the Examples and will not be repeated here.

[0064] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0065] The present invention will be further described below in conjunction with the embodiments:

[0066] Example 1 Design of FeI d1 polypeptide

[0067] The amino acid sequence of Chain 1 of Fel d1 is:

[0068] The amino acid sequence of Chain 2 of Fel d1 is:

[0069] Chain 1 and Chain 2 of Fel d1 were split into 15 to 17 amino acids, as shown in Table 1.

[0070] Table 1 Fel d1 polypeptide segmentation sequence

[0071] Example 2 Co-expression of AP205 with the above polypeptides

[0072] The amino acid sequence of AP205 (SEQ ID NO: 9):

[0073] (1) Construction of recombinant plasmid: After codon optimization, the nucleic acid sequence expressing AP205-Peptide was commissioned to a gene synthesis company to be synthesized between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the recombinant plasmid AP205-Peptide-pET28a;

[0074] (2) Transfection of the recombinant plasmid into the expression strain: The AP205-Peptide-pET28a recombinant plasmid was transformed into the Escherichia coli BL21 (DE3) expression strain to obtain the recombinant expression strain AP205-Peptide-pET28a-BL21;

[0075] (3) Cultivation of recombinant bacteria and induction of target protein expression: The recombinant expression strain AP205-Peptide-pET28a-BL21 was spread on a plate containing 100 μg / mL kanamycin resistance and cultured at 37°C overnight. A single colony was picked and inoculated into 10 mL of LB medium containing 100 μg / mL kanamycin and shaken at 37°C 200 rpm until the OD600 of the bacterial solution was 0.8-2.0. The colony was transferred into 500 mL of LB medium containing 100 μg / mL kanamycin and shaken at 37°C 200 rpm until the OD600 of the bacterial solution was about 0.8-1.5. β-D-galactoside (IPTG) inducer was added at a final concentration of 0.5 mmol / L and expression was induced at 20°C overnight.

[0076] (4) Bacterial cell collection and disruption: The bacterial cell suspension was centrifuged at 8000 rpm and the cells were collected. The cells were resuspended in 0.01 mol / L PBS solution at a weight to volume ratio of 1:10. The cells were then homogenized twice at 2-8°C using a high-pressure homogenizer at 900 bar.

[0077] (5) Ammonium sulfate precipitation: After the cells are disrupted, centrifuge at 10,000 rpm to collect the supernatant. Add ammonium sulfate solution with a final concentration of 1 mol / L and mix at 2-8°C for 1 hour. After centrifugation at 10,000 rpm, collect the precipitate, add 0.01 mol / L PBS solution to reconstitute it, and centrifuge at 15,000 rpm to remove the precipitate. Collect the supernatant.

[0078] (6) Endotoxin Removal: Add Triton X-114 to the supernatant collected in step (5) to a final concentration of 1.5% and stir at 2-8°C for 1 hour. After treatment, return the sample temperature to 30°C and maintain for 40 minutes. Centrifuge to remove the precipitate and collect the supernatant. Repeat the above process twice.

[0079] (7) Protein purification: The protein solution after endotoxin removal was concentrated and replaced with a 100KD membrane, and then chromatographically purified using an S300 molecular sieve chromatography filler. The target protein peak was collected, which was the purified AP205 virus-like particle protein.

[0080] Protein expression is shown in Figure 1 , and AP205 can be co-expressed with each fragment smoothly.

[0081] Example 3 Identification of the Binding Activity of the Expressed AP205-Peptide to Fel d1 Positive Serum

[0082] The purified AP205-Peptide was diluted to 2 mg / ml and mixed with 1313 adjuvant at a ratio of 1:1 for vaccine preparation. 35 C57BL / 6J mice aged 6 to 8 weeks were randomly divided into 7 groups.

[0083] Each group consisted of 5 mice. Six mice received the vaccine subcutaneously behind the ears, while another 5 mice received the same dose of PBS solution as a control. The immunization dose was 0.1 ml per mouse, and the mice were immunized three times, 14 days apart. Twenty-one days after the three immunizations, blood was collected from each mouse in the control and immunized groups, and serum was isolated and tested for Fel d1 antibodies. The specific method is as follows:

[0084] (1) Coating: Dilute the expressed AP205-peptide protein to 1 μg / mL, add 100 μL per well to an empty ELISA plate, place it at 4°C overnight for coating, wash it five times with a plate washer, and pat dry;

[0085] (2) Add 200 μL of 3% sodium caseinate blocking solution to each well and incubate at 37°C for 1 h. After blocking, wash the plate five times with a plate washer and pat dry.

[0086] (3) Serially dilute Fel d1-positive serum 3-fold with sodium caseinate solution, add 100 μL per well, incubate at 37°C for 1 h, wash 5 times with a plate washer, and pat dry;

[0087] (4) Add rabbit anti-cat HRP (used at a 1:500 dilution with casein), 100 μL per well, incubate at 37°C for 1 h, wash five times with a plate washer, and pat dry;

[0088] (5) Add TMB colorimetric solution. The color development time should not be too long (should not exceed 6 minutes at room temperature). Before the blank well changes color, add 50 μL of 1 mol / L dilute sulfuric acid / hydrochloric acid to each well to stop the reaction. Read the OD450 value.

[0089] The results showed that the AP205-Peptide immunization groups of SEQ ID NO: 4 and SEQ ID NO: 6 had the highest antibody titers after three immunizations, which were 1:89100 and 1:105300, respectively, as shown in FIG2 .

[0090] Example 4: Experimental study on immunization of cats with peptide vaccines of SEQ ID NO: 4 and SEQ ID NO: 6

[0091] The AP205-peptide vaccines of SEQ ID NO: 4 and SEQ ID NO: 6 were administered subcutaneously to healthy cats aged 8 to 10 weeks, five per group. Five cats were also vaccinated with the same dose of adjuvant as controls. The immunization dose was 1 ml per cat, and three immunizations were administered 21 days apart. Saliva was collected from each cat in the control and immunized groups before immunization and 21 days after the three immunizations. Saliva concentrations of Fel d1 protein were measured using a commercially available ELISA kit, according to the kit's instructions. Blood was also collected 21 days after the three immunizations for Fel d1 antibody testing.

[0092] Table 2 Results of FeI d1 concentration detection in cat saliva after vaccination

[0093] The results showed that in cats immunized with the AP205-Peptide vaccine expressing SEQ ID NO: 4 and SEQ ID NO: 5, the concentration of Fel d1 in saliva was significantly reduced 21 days after the three vaccinations, both decreasing by more than 80%. There was no significant difference between the two groups, and both were significantly higher than the control group. The results are shown in Table 2.

[0094] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cat allergen polypeptide fragment which is a truncated form of Chain 1 or Chain 2 of the FeI d 1 protein.

2. The fragment according to claim 1, characterized in that It has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 8, or a sequence having at least 80% sequence homology thereto.

3. A fusion protein comprising at least one of the cat allergen polypeptide fragments according to claim 1 or 2 and a VLP protein.

4. The fusion protein according to claim 3, characterized in that The VLP is selected from any one of AP205, CPMV, CMV, PapMV, FHV, HbsAg, M2S or M1.

5. A biomaterial comprising at least one of the following I) to V): 1), the cat allergen polypeptide fragment according to claim 1 or 2, and the fusion protein according to claim 3 or 4, wherein a composition or fusion protein formed by any two or more of them; II), a nucleic acid encoding the cat allergen polypeptide fragment of claim 1 or 2 or a nucleic acid encoding the fusion protein of claim 3 or 4; III), an expression unit comprising the nucleic acid described in II); IV), a vector containing the nucleic acid described in II) or the expression unit described in III); V), a host transformed or transfected with the vector described in IV), or a host having the nucleic acid described in I) integrated into its genome; VI) and V) the expression product of the host.

6. Use of the cat allergen polypeptide fragment according to claim 1 or 2, the fusion protein according to claim 3 or 4, or the biomaterial according to claim 5 in the preparation of an immunoassay reagent or a vaccine.

7. An immunoassay reagent, characterized in that The invention comprises at least one of the cat allergen polypeptide fragment according to claim 1 or 2, the fusion protein according to claim 3 or 4, and a carrier, wherein the carrier is a chip, glass, microspheres, magnetic beads or ELISA plate.

8. A vaccine, characterized in that include: At least one of the cat allergen polypeptide fragment according to claim 1 or 2, the fusion protein according to claim 3 or 4, and an adjuvant.

9. A method for detecting feline Feld1 antibodies, comprising detecting a sample with the immunodetection reagent according to claim 7.

10. A method for inhibiting Feld1 secretion, comprising immunizing an animal with the vaccine according to claim 8.

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