Prokaryotic expression of cat allergen protein chimeric peptide cfel d 1 based on t-cell antigen epitope, and preparation of egg yolk antibody

Abstract

Prokaryotic expression of a cat allergen protein chimeric peptide cFel d 1 based on a T-cell antigen epitope, and preparation of an egg yolk antibody. A chimeric peptide cFel d 1, comprising a 25-41th-position amino acid residue segment, a 46-59th-position amino acid residue segment, a 85-98th-position amino acid residue segment, a 132-140th-position amino acid residue segment and a 145-153th-position amino acid residue segment of a cat allergen Fel d 1 antigen amino acid residue. Experiments prove that an antibody obtained after immunizing animals by means of the chimeric peptide cFel d 1 has high titer, can specifically bind to a cat allergen complete Fel d 1 protein, and is superior to a polyclonal antibody prepared by means of a complete Fel d 1 protein (the titer is 8 times higher).

Description

Prokaryotic expression of a chimeric peptide cFel d 1 based on the T cell antigen epitope cat allergen protein and preparation of its yolk antibody Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to prokaryotic expression of a chimeric peptide cFel d 1 based on a T cell antigen epitope cat allergen protein and preparation of an egg yolk antibody thereof. Background Art

[0002] Allergies are a common problem in everyday life. Strictly speaking, allergies are an exaggerated immune response in which the body misinterprets certain foreign substances, triggering an overreaction, which in turn leads to a series of allergic reactions. Allergic symptoms induced by pet hair allergens vary widely in severity, ranging from discomfort associated with rhinitis and conjunctivitis to severe asthma, which can develop into a life-threatening condition. Cat hair allergens are considered a major risk factor for asthma and allergic rhinitis in humans. Cat hair allergens are a major source of allergens in indoor environments, causing symptoms such as nasal congestion, itchy eyes, and coughing. In severe cases, they can lead to asthma and anaphylactic shock. Exposure to cat allergens under natural conditions can lead to increased peripheral airway obstruction, which can develop into a life-threatening condition. Studies have shown that the prevalence of cat hair allergy in humans worldwide is generally around 5-20%. To date, eight cat allergens, Fel d 1 to Fel d 8, have been registered with the WHO / IUIS. Fel d 1 is the most important allergen among cat allergens, accounting for about 96% of human allergies to cat hair, with concentrations as low as 100 ng / m 3 Just 1 Fel d is enough to cause allergic symptoms.

[0003] Fel d 1 is primarily stored in cat saliva and hair and is transferred to cat hair during self-grooming. Furthermore, cat dander containing the Fel d 1 allergen is disseminated into the environment as small airborne particles. Fel d 1 is a 35 kDa tetrameric glycoprotein composed of two heterodimers linked non-covalently. Each heterodimer consists of a 70-residue peptide and an 85-, 90-, or 92-residue peptide. These chains are covalently linked within each heterodimer by disulfide bonds. The structure of Fel d 1 is similar to that of rabbit uteroglobin, with chain I sharing amino acid homology with uteroglobin, while chain II is a glycoprotein with an N-terminal oligosaccharide. An asymmetric cavity within Fel d 1 allows for binding of endogenous ligands. In vivo, the allergenicity of Fel d 1 is determined by recognition by mannose receptors on mucosal antigen-presenting cells (such as dendritic cells). As the most prominent allergen in cat allergens, Fel d 1 has become an ideal protein molecule for developing new allergen-specific immunotherapies. Studies have shown that linear epitopes of allergen proteins or short peptides that mimic epitopes can target allergen-specific T cells. Epitope vaccines or antibodies developed from these epitopes are safer and offer higher protection rates than traditional vaccines or antibodies. SUMMARY OF THE INVENTION

[0004] The technical problem to be solved by the present invention is to provide a prokaryotic expression method of a chimeric peptide cFel d 1 based on a T cell antigen epitope cat allergen protein and the preparation of an egg yolk antibody thereof. Technical Solutions

[0005] In a first aspect, the present invention provides a chimeric peptide cFel d 1, which comprises the amino acid residue segments 25-41, 46-59, 85-98, 132-140 and 145-153 of the cat allergen Fel d 1 antigen.

[0006] In the chimeric peptide cFel d 1 described above, the various segments are connected by a flexible amino acid linker.

[0007] In the chimeric peptide cFel d 1 described above, the amino acid sequence of the flexible amino acid linker is positions 18-22 of SEQ ID NO: 1.

[0008] The chimeric peptide cFel d 1 described above is any one of the following A1) to A4):

[0009] A1) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing;

[0010] A2) a protein derived from 1) with the same function as that of SEQ ID NO: 1, wherein one or more amino acid residues are substituted and / or deleted and / or added to the amino acid sequence of SEQ ID NO: 1 in the sequence listing;

[0011] A3) A protein whose sequence contains the amino acid sequence defined in A1) or A2);

[0012] A4) A fusion protein obtained by ligating a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1) to A3).

[0013] The chimeric peptide cFel d 1 has a size of 9 kDa. The recombinant chimeric peptide cFel d 1 has the antigenicity of a complete Fel d 1 protein and can be used for the preparation of immune antibodies.

[0014] In a second aspect, the present invention provides a nucleic acid encoding the chimeric peptide cFel d 1 according to the first aspect.

[0015] The encoding nucleic acid mentioned above is a DNA molecule described in any one of the following B1) to B4):

[0016] B1) a DNA molecule whose coding region includes the nucleotide sequence of Sequence 2 in the Sequence Listing;

[0017] B2) A DNA molecule having the nucleotide sequence of Sequence 2 in the sequence listing;

[0018] B3) a DNA molecule that hybridizes under stringent conditions with the nucleotide sequence described in B1) or B2) and encodes the chimeric peptide cFel d 1 described in the first aspect;

[0019] B4) A DNA molecule that has a nucleotide sequence homology of more than 90% with that of B1) or B2) and encodes the chimeric peptide cFel d 1 according to the first aspect.

[0020] In a third aspect, the present invention provides a recombinant vector or a transgenic cell line or a recombinant bacterium containing the encoding nucleic acid described in the second aspect.

[0021] In a fourth aspect, the present invention provides the following use, which is the use of the chimeric peptide cFel d 1 described in the first aspect in any of the following C1-C5:

[0022] C1) as an immunogen or preparation of immunomodulatory agents;

[0023] C2) Preparation of immunogen;

[0024] C3) Preparation of antibodies against FeI d 1;

[0025] C4) Preparation of anti-allergy vaccines;

[0026] C5) Preparation of products that neutralize or reduce the FeI d 1 content.

[0027] In a fifth aspect, the present invention provides the following use, which is the use of the encoding nucleic acid described in the second aspect or the recombinant vector or transgenic cell line or recombinant bacteria described in the third aspect in any of the following C1-C5:

[0028] C1) Preparation of immunomodulatory agents;

[0029] C2) Preparation of immunogen;

[0030] C3) Preparation of antibodies against FeI d 1;

[0031] C4) Preparation of anti-allergy vaccines;

[0032] C5) Preparation of products that neutralize or reduce the FeI d 1 content.

[0033] In the above applications, the antibody is a polyclonal antibody.

[0034] Furthermore, the polyclonal antibody is an animal serum antibody or an animal egg yolk antibody.

[0035] In the above-mentioned application, the antibody against FeI d 1 has the function of neutralizing the allergen FeI d 1.

[0036] In the application described above, the anti-allergic vaccine is achieved by using the chimeric peptide cFel d 1 to produce antibodies through the immune body, neutralizing the allergen Fel d 1, and achieving anti-allergy.

[0037] In the above-mentioned applications, the product for neutralizing or reducing the content of FeI d 1 is achieved by immunizing the body with the chimeric peptide cFel d 1 to produce antibodies that neutralize the allergen FeI d 1, thereby reducing the content of FeI d 1.

[0038] The above-mentioned anti-allergic vaccine is an allergy vaccine against feline hair or feline dander.

[0039] In a sixth aspect, the present invention provides an anti-allergic vaccine comprising the chimeric peptide cFel d 1 according to the first aspect.

[0040] The anti-allergic vaccine mentioned above also includes an adjuvant.

[0041] In the above-mentioned anti-allergic vaccine, the anti-allergic vaccine is an anti-feline hair allergy vaccine or an anti-feline dander allergy vaccine.

[0042] In a seventh aspect, the present invention provides an antibody against FeI d 1, which is an antibody prepared using the chimeric peptide cFel d 1 described in the first aspect as an antigen.

[0043] Among the FeI d 1 antibodies described above, the antibody prepared using the chimeric peptide cFel d 1 described in the first aspect as an antigen is an antibody obtained by immunizing an organism using the chimeric peptide cFel d 1 described in the first aspect as an immunogen.

[0044] In the above, the organism is an animal.

[0045] The animal is further specifically an avian animal, a bird animal, a reptile animal, an amphibian animal or a mammal.

[0046] The poultry animal is more specifically a laying hen.

[0047] In the above-mentioned FeI d 1 antibody, the antibody is a polyclonal antibody.

[0048] Among the antibodies mentioned above, their function is to neutralize the allergen FeI d 1.

[0049] Furthermore, the polyclonal antibody is an animal serum antibody or an animal egg yolk antibody.

[0050] Furthermore, the antibody is a mouse serum antibody or a laying hen egg yolk antibody.

[0051] The mouse serum antibody was prepared as follows: the chimeric peptide cFel d 1 was diluted with 1×PBS (8.0 g sodium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, pH 7.4), mixed with an equal volume of adjuvant and emulsified, and mice were immunized by subcutaneous injection at a dose of 25 μg / mouse. The immunizations were conducted three times in total, with the interval between the first and second immunizations being 21 days, and the interval between the second and third immunizations being 10 days. One week after the last immunization, mouse serum was collected to obtain mouse serum antibodies.

[0052] The egg yolk antibody was prepared as follows: the chimeric peptide cFel d 1 was diluted with 1×PBS (same as above) and then mixed with an equal volume of adjuvant for emulsification. Layers were immunized by intramuscular injection at a dose of 300 mg / hen for a total of four immunizations, with an interval of two weeks between each immunization. Eggs from weeks 3 to 16 were collected and extracted by polyethylene glycol precipitation to obtain the egg yolk antibody.

[0053] In an eighth aspect, the present invention provides the use of the antibody according to the seventh aspect in any of the following:

[0054] D1) Preparation of products for treating allergies;

[0055] D2) Preparation of products with reduced FeI d 1 content.

[0056] In the above-mentioned applications, the antibody is used as an active ingredient in a product for treating allergies or a product for reducing the content of FeI d 1.

[0057] In a ninth aspect, the present invention provides a product for treating allergies, comprising the antibody described in the seventh aspect.

[0058] In the above, the products are medicines, compositions, health products, functional foods, foods for special medical purposes or other biological products.

[0059] Furthermore, the product for treating allergies or the product containing the antibody described in the seventh aspect can be cat food, cat strips, cat canned food, etc. for special medical purposes. The product contains the antibody described in the seventh aspect as an anti-allergic drug.

[0060] In a tenth aspect, the present invention provides a method for preventing allergy, comprising administering to the subject a product comprising a preventively effective amount of the chimeric peptide cFel d 1 according to the first aspect.

[0061] In the eleventh aspect, the present invention provides a method for treating allergy, which comprises administering to the subject a therapeutically effective amount of a product comprising the antibody described in the seventh aspect, wherein the administered dose comprises an antibody added in an amount of 1%-3% of the feed intake / day / 3-4 kg.

[0062] In the above method, the administered dosage comprises an antibody added at 1.5% of the feed intake / day / 3-4 kg.

[0063] In the above, subjects include humans or animals who are patients being treated for a disease or preventing a disease. The methods described herein can be used to treat animal subjects belonging to any category. Examples of such animals include mammals.

[0064] The mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorphs (order Logomorpha) mammals, such as rabbits. The mammals can be of the order Carnivora, including felines (cats) and canines (dogs). The mammals can be of the order Artiodactyla, including bovines (cows) and suids (pigs), or of the order Perssodactyla, including equines (horses). The mammals can be of the order Primate, Ceboid, or Simoid (monkeys) or Anthropoid (humans and apes). In some embodiments, the mammal is a cat or a human.

[0065] In an embodiment of the present invention, the method for treating allergies is specifically to administer to cats cat food, cat strips, cat cans and other products containing a therapeutically effective amount of the antibody described in the seventh aspect, thereby reducing the allergen FeI d 1 content in the cat's body and achieving the purpose of anti-allergic drug treatment.

[0066] In a twelfth aspect, the present invention provides a protein for use as a medicine, wherein the protein is the chimeric peptide cFel d 1 according to the first aspect.

[0067] In the above-mentioned protein for use as a medicament, the medicament is used for preventing allergies.

[0068] Further specifically, it is to prevent allergies such as feline hair or dander.

[0069] In a thirteenth aspect, the present invention provides an immunogen, the active ingredient of which is the chimeric peptide cFel d 1 described in the first aspect.

[0070] In a fourteenth aspect, the present invention provides an immunomodulator, the active ingredient of which is the chimeric peptide cFel d 1 described in the first aspect.

[0071] In a fifteenth aspect, the present invention provides a product for neutralizing or reducing the content of FeI d 1, wherein the active ingredient is the chimeric peptide cFel d 1 described in the first aspect.

[0072] In a sixteenth aspect, the present invention provides an antibody for use as a medicine, wherein the antibody is prepared by immunizing an organism with the chimeric peptide cFel d 1 described in the first aspect as an antigen.

[0073] In the above-mentioned antibody for use as a medicament, the medicament is used for treating allergies.

[0074] The above allergies are anti-feline hair allergies or anti-feline dander allergies, etc.

[0075] The dosage form of the above-mentioned medicine or composition is injection, lyophilized powder for injection, aerosol, large infusion, dripping pill, pill, powder, granule, tablet, capsule, oral solution or emulsion.

[0076] The pharmaceutically acceptable carriers mentioned above may be excipients, stabilizers, suspending agents or diluents, etc., which are well known to those skilled in the art.

[0077] Furthermore, the carrier material includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make the unit dosage form into a tablet, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations. Beneficial effects

[0078] The antibody titer obtained after immunizing mice with 25 μg / mouse chimeric peptide cFel d 1 was 1:34500-1:301500; the antibody titer obtained after immunizing laying hens with 300 mg / mouse chimeric peptide cFel d 1 was 1:1048576 (i.e. 1:2 20 ), the highest antibody titer was obtained after nine weeks, which was 1:4194304 (i.e. 1:2 22 ), the antibody titer obtained after sixteen weeks was 1:131072 (i.e. 1:2 17 After feeding cats with chimeric peptide cFel d 1-specific egg yolk antibodies at 1.5% and 2% supplementation for four weeks, the Fel d 1 content in their saliva decreased by 73.1% and 66.7%, respectively, which is better than similar products on the market (42.0%). BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 shows the prediction analysis of the cat allergen protein Fel d 1. A. Flexibility prediction; B. β-turn prediction; C. Surface accessibility prediction; D. Antigenicity prediction; E. Hydrophilicity prediction.

[0080] Figure 2 shows the prokaryotic expression and purification of the cat allergen protein chimeric peptide cFel d 1. A. Colony PCR identification of transformants. M: DNA marker; 1: negative control; 2: Colony PCR of positive transformants. B. SDS-PAGE electrophoresis of IPTG-induced expression of the chimeric peptide cFel d 1 in BL21 cells. M: protein marker; 1: protein expression before IPTG induction; 2: protein expression after IPTG induction. C. SDS-PAGE electrophoresis of cells before and after sonication. M: protein marker; 1: cell pellet after sonication; 2: supernatant after sonication. D. Purification of the chimeric peptide cFel d 1. M: protein marker; 1: flow-through; 2, 3: 50 mM imidazole eluents; 4: 100 mM imidazole eluents; 5, 6: 500 mM imidazole eluents.

[0081] FIG3 shows the determination of serum IgG titer in mice immunized with the recombinant chimeric peptide cFel d 1.

[0082] FIG4 shows the determination of the yolk antibody IgY titer in laying hens immunized with the recombinant chimeric peptide cFel d 1.

[0083] Figure 5 shows the determination of allergen levels in saliva of cats fed with specific egg yolk antibody IgY for four weeks.

[0084] Figure 6 shows the changes in allergen levels in saliva of cats fed with specific egg yolk antibody IgY for four weeks. Modes for Carrying Out the Invention

[0085] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0086] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0087] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0088] In the following examples, the formula of 1×PBS is as follows: 8.0 g sodium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, and the balance is water, with a pH of 7.4.

[0089] Example 1: Obtaining cat allergen protein chimeric peptide cFel d 1 based on T cell antigen epitope recombination

[0090] The T cell antigen epitopes of cat allergen protein Fel d 1 were predicted by online tools. The appropriate epitopes were screened according to surface accessibility, antigenicity, hydrophilicity, β-turn flexibility, etc. to determine the recombinant protein sequence. The recombinant plasmid was further constructed, expression was induced, and expression conditions were optimized before ultrasonic treatment. 2+ Column purification, dialysis and freeze drying were performed to complete the preparation of cat allergen protein chimeric peptide cFel d 1 based on T cell antigen epitope.

[0091] The plasmid vectors and strains used in the examples of the present invention are all commercially available products, and the reagents used are all commercially available products; the primers and DNA sequences used are all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0092] The details are as follows:

[0093] 1. Obtaining the chimeric peptide cFel d 1 protein gene

[0094] First, the genes encoding chain I and chain II of the cat allergen protein Fel d 1 were linked together to obtain the full-length gene sequence. Then, the ProPred program was used to predict the Th epitope of Fel d 1 protein, and the CTL epitope of Fel d 1 protein was predicted using the CTLpred software. The antigenic epitope, surface accessibility, antigenicity, hydrophilicity, β-turn flexibility, etc. of the cat allergen protein Fel d 1 were predicted as shown in Figure 1. The antigenic determinant prediction software provided by Nanjing Detai Bioinformatics Tools (http: / / www.detaibio.com / peptide-antigen-prediction) was used to perform multiple prediction algorithms to predict the antigenicity, hydrophilicity, β-turn, surface accessibility, and flexibility of the Fel d 1 protein sequence. After comprehensive analysis of the prediction results, the amino acid sequences at positions 25-41, 46-59, 85-98, 132-140, and 145-153 of the natural cat allergen Fel d 1 antigen (chain I + chain II) were selected and connected using a flexible linker (GGGGS) to obtain the chimeric peptide cFel d 1 protein, approximately 9 kDa in size.

[0095] The amino acid sequence of the chimeric peptide cFel d 1 protein is sequence 1, VAQYKALPVVLENARILGGGGSDAKMTEEDKENALSGGGGSFAVANGNELLLDLSGGGGSLVMTTISSSGGGGSGEAVQNTVE, and the underlined element is the flexible amino acid linker.

[0096] To increase expression, the gene encoding the chimeric peptide cFel d 1 protein was codon-optimized for Escherichia coli and sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis. The nucleotide sequence of the gene encoding the chimeric peptide cFel d 1 protein is shown in SEQ ID NO: 2. The chimeric peptide cFel d 1 gene is 243 bp in size: GTGGCACAATACAAAGCACTACCTGTAGTATTGGAAAATGCCAGAATACTGGGAGGAGGAGGATCGGATGCAAAAATGACAGAAGAGGATAAGGAGAATGCTCTCAGCGGAGGAGGAGGATCGGCGGTGGCCAATGGAAATGAATTACTGTTGGACTTGTCCGGAGGAGGAGGATCGCTAGTCATGACAACCATCAGCTCCAGCGGAGGAGGAGGATCGGAAGCAGTTCAGAACACCGTAGAA. The underlined element is the gene encoding the flexible amino acid linker.

[0097] 2. Expression and purification of chimeric peptide cFel d 1 protein

[0098] 1) Preparation of recombinant vector

[0099] The gene encoding the chimeric peptide cFel d 1 protein of sequence 2 was used to replace the sequence between the XhoI and NcoI restriction sites of the pET-28a (+) vector to obtain the recombinant vector pET-28a-cFel d 1.

[0100] 2) Protein expression

[0101] The recombinant vector pET-28a-cFel d 1 was transformed into the Escherichia coli expression strain BL21(DE3) (hereafter referred to as the BL21 strain) and cultured on LB plates (50 μg / mL kanamycin) for 16-18 hours at 37°C. Positive transformants were screened by colony PCR to obtain the recombinant strain BL21(DE3) / pET-28a-cFel d 1.

[0102] The primer sequences for colony PCR are as follows:

[0103] F: 5'-GGAATTCCATATGATGGTGGCACAATAC-3' (sequence 3)

[0104] R: 5'-CCGCTCGAGTTCAACGGTGTTCT-3' (sequence 4)

[0105] The screening results of positive transformants are shown in Figure 2A. The positive transformants contain about 300 bp of the target gene sequence, indicating that the chimeric peptide cFel d 1 gene sequence has been successfully transformed into the BL21 strain.

[0106] A single colony of BL21(DE3) / pET-28a-cFel d 1 was inoculated into LB liquid medium and cultured at 37°C (220 rpm) for 16-18 hours to obtain a bacterial suspension. The bacterial suspension was then transferred to LB medium supplemented with 50 μg / mL kanamycin and cultured at 37°C (220 rpm) until the optical density at 600 nm (OD) reached 0.6. 0.2 mM IPTG was added, and the culture was induced at 37°C and 220 rpm for 2 hours. The culture was then centrifuged at 10,000 rpm for 20 minutes at 4°C to collect the bacterial pellet.

[0107] The expression of chimeric peptide cFel d1 in bacterial precipitates before and after IPTG induction was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0108] The results are shown in Figure 2B . After IPTG induction, a chimeric peptide cFel d 1 with a size of approximately 9 kDa was obtained.

[0109] To isolate the expressed target protein, add an appropriate amount of lysis buffer (approximately 20 mL of lysis buffer (25 mM Tris, 150 mM NaCl, pH 7.5) to 100 mL of bacterial culture), fully dissolve it on an ultrasonicator (15% power, ultrasonication for 15-20 min), and centrifuge it (4°C, 10,000 rpm, 15 min), and collect the supernatant and precipitate.

[0110] The supernatant and precipitate after sonication were subjected to 12% SDS-PAGE electrophoresis, and the recombinant chimeric peptide was analyzed using imaging software.

[0111] The results are shown in Figure 2C. After the bacteria were disrupted by ultrasound, a target band of approximately 9 kDa appeared in the supernatant, which is consistent with the theoretical molecular size of the chimeric peptide cFel d 1.

[0112] 3) Protein purification

[0113] Using Ni 2+ The expressed chimeric peptide cFel d 1 was separated and purified using an affinity chromatography column. The details are as follows:

[0114] Filter the supernatant from step 2) through a 0.22 μm pore size filter membrane and purify the target protein using a purifier. Elute with different concentrations of imidazole solution (50-500 mM) and collect the eluates after elution with different concentrations of imidazole.

[0115] SDS-PAGE electrophoresis detection results are shown in Figure 2D. After the crude protein solution was eluted with 100 mM imidazole, the chimeric peptide cFel d 1 with a molecular weight of approximately 9 kDa was obtained. The target band was not obtained with other concentrations of imidazole elution.

[0116] The eluate with 100 mM imidazole was collected, dialyzed, and then freeze-dried in a vacuum freeze dryer to obtain the chimeric peptide cFel d 1 (recombinant chimeric peptide cFel d 1). The chimeric peptide cFel d 1 was then dissolved in 1×PBS to obtain a chimeric peptide cFel d 1 solution.

[0117] The protein concentration of the chimeric peptide cFel d 1 solution was detected by the CBB method.

[0118] Example 2: Preparation of polyclonal antibodies against the cat allergen protein chimeric peptide cFel d 1 based on T cell antigen epitopes

[0119] 1. Antibody Preparation

[0120] 1) Mouse polyclonal IgG

[0121] The chimeric peptide cFel d 1 solution obtained in Example 1 was diluted with 1× PBS to a concentration of 500 μg / mL. 50 μL of the solution was taken from each mouse and emulsified with an equal volume of Freund's adjuvant. The mice were immunized by subcutaneous injection. A total of three immunizations were performed, with an antigen dose (cFel d 1) of 25 μg per mouse. The specific vaccination cycle was:

[0122] First immunization: On day 0, mice were immunized with a recombinant chimeric peptide cFel d 1 solution mixed with Freund's complete adjuvant at a ratio of 1:1 and fully emulsified. Simultaneously, an adjuvant control group was immunized with a 1:1 mixture of 1× PBS and Freund's incomplete adjuvant. A blank control group received no treatment. The day of immunization was designated as day 0.

[0123] Second immunization: On day 21, the recombinant chimeric peptide cFel d 1 solution was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and fully emulsified for immunization. Simultaneously, the adjuvant control group was immunized with 1× PBS mixed with Freund's incomplete adjuvant at a ratio of 1:1. The blank control group received no treatment.

[0124] Third immunization: On day 31, the recombinant chimeric peptide cFel d 1 solution was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and fully emulsified for immunization. Simultaneously, the adjuvant control group was immunized with 1× PBS mixed with Freund's incomplete adjuvant at a ratio of 1:1. The blank control group received no treatment.

[0125] One week after the three immunizations, blood was collected from the mouse eyeballs, allowed to stand at room temperature for 1 hour, and then centrifuged for 10 minutes (3000 rpm). The supernatant was the polyclonal antibody (mouse antiserum).

[0126] 2) Egg yolk antibody IgY

[0127] (1) Immunity

[0128] The chimeric peptide cFel d 1 solution obtained in Example 1 was diluted to a concentration of 600 μg / mL using 1× PBS. 500 μL of the solution was emulsified with an equal volume of Freund's adjuvant and inoculated into 140-day-old Hy-Line Brown laying hens via multiple intramuscular injections. A total of four immunizations were performed, with an antigen dose of 300 mg per chicken. The specific inoculation steps were as follows:

[0129] First immunization: On day 0, mice were immunized with a recombinant chimeric peptide cFel d 1 solution mixed with Freund's complete adjuvant at a ratio of 1:1 and fully emulsified. Simultaneously, an adjuvant control group was immunized with a 1:1 mixture of 1× PBS and Freund's incomplete adjuvant. A blank control group received no treatment. The day of immunization was designated as day 0.

[0130] Second immunization: On day 14, mice were immunized with a recombinant chimeric peptide cFel d 1 solution mixed with Freund's incomplete adjuvant at a ratio of 1:1 and fully emulsified. Simultaneously, the adjuvant control group was immunized with 1× PBS mixed with Freund's incomplete adjuvant at a ratio of 1:1. The blank control group received no treatment.

[0131] Third immunization: On day 28, mice were immunized with a recombinant chimeric peptide cFel d 1 solution mixed with Freund's incomplete adjuvant at a ratio of 1:1 and fully emulsified. Simultaneously, the adjuvant control group was immunized with 1× PBS mixed with Freund's incomplete adjuvant at a ratio of 1:1. The blank control group received no treatment.

[0132] Fourth immunization: On day 42, mice were immunized with a recombinant chimeric peptide cFel d 1 solution mixed with Freund's incomplete adjuvant at a ratio of 1:1 and fully emulsified. Simultaneously, an adjuvant control group was immunized with 1× PBS mixed with Freund's incomplete adjuvant at a ratio of 1:1. A blank control group received no treatment.

[0133] After the first immunization, eggs were collected every day.

[0134] (2) Extraction of yolk antibodies

[0135] The yolk antibody was extracted using polyethylene glycol precipitation method. The specific steps are as follows:

[0136] ① Wash the collected eggs, disinfect them with 75% alcohol, and carefully peel off the eggshells to separate the egg white and yolk, removing as much of the egg white as possible;

[0137] ② Transfer the egg yolk to filter paper, roll it to remove the remaining egg white, then cut the yolk membrane with a lancet or pipette tip and pour the egg yolk into a graduated cylinder;

[0138] ③ Add 1× PBS (4 times the volume of egg yolk) and then add PEG6000 powder to a final concentration of 3.5% (mass to volume ratio, g:mL). Mix thoroughly, let stand, and centrifuge at 4°C for 20 min (10,000 rpm). Collect the supernatant and filter through folded filter paper.

[0139] ④ Add PEG6000 to the collected supernatant to a final volume of 8.5% (volume percentage), mix thoroughly for 10 min, centrifuge at 4°C for 20 min (10,000 rpm), and discard the supernatant;

[0140] ⑤ Dissolve the precipitate in 1× PBS using a glass rod and vortexer, add the same volume of PBS as the original volume, and add PEG6000 to a final volume of 12%. Mix thoroughly for 10 minutes, centrifuge at 4°C for 20 minutes (10,000 rpm), and discard the supernatant.

[0141] The precipitate was dissolved in 1× PBS to obtain crude IgY (egg yolk antibody) and stored at -20°C.

[0142] 2. Determination of antibody titer

[0143] Steps:

[0144] 1) Coating: The purified chimeric peptide cFel d 1 expressed in prokaryotes prepared in Example 1 was used as an antigen, diluted to 1.5 μg / mL with coating solution, and 100 μL / well was added to the ELISA plate and incubated at 4°C overnight.

[0145] 2) Washing: The next day, discard the liquid in the 96-well plate and wash four times with washing solution.

[0146] 3) Blocking: Add blocking solution (200 μL / well) and incubate at 37°C for 2 h.

[0147] 4) Washing: Wash 4 times with detergent.

[0148] 5) Add the sample to be tested (primary antibody): Serially dilute the mouse antiserum (polyclonal antibody) or egg yolk antibody (crude IgY) obtained in step 1 above with sample diluent and add (100 μL / well) to the coated ELISA plate. Use the sample diluent as a negative control. Incubate at 37°C for 1 h.

[0149] 6) Washing: Wash 4 times with washing solution.

[0150] 7) Add enzyme-labeled secondary antibody: Dilute the secondary antibody (mouse anti-chicken IgY-HRP, bersee, BIR798) proportionally with sample diluent, add to the plate (100 μL / well), and incubate at 37°C for 1 h.

[0151] 8) Washing: Wash 4 times with detergent.

[0152] 9) Color development: Add TMB color development solution (100 μL / well) to a 96-well plate and incubate at 37°C in the dark for 10-15 min.

[0153] 10) Stop the reaction and determine the titer: Add stop solution (100 μL / well) to a 96-well plate. After 5 minutes, measure the absorbance of each well at 450 nm using a microplate reader. The maximum dilution with a positive reaction is the titer of the antibody to be tested.

[0154] Specifically, the reagents used for the titer detection of specific IgG antibodies in antiserum and laying hen egg yolk antibodies are as follows:

[0155] ①Coating buffer (pH 9.6 carbonate buffer)

[0156] NaHCO3 2.93 g

[0157] Na2CO3 1.59 g

[0158] Add ddH2O to 1 L.

[0159] ② Washing buffer (PBST, pH 7.4)

[0160] NaCl 8.0 g

[0161] Na2HPO4 1.2 g

[0162] KH2PO 4 0.2 g

[0163] KCl 0.2 g

[0164] Tween-20 0.5 mL

[0165] Add ddH2O to 1 L.

[0166] ③Blocking solution:

[0167] 5.0 g of skim milk powder was added with PBST to make up to 100 mL.

[0168] ④Blocking solution:

[0169] 5 g of skim milk powder was added with PBST to make up to 100 mL.

[0170] ⑤Sample diluent:

[0171] 0.5 g of skim milk powder was added with PBST to make up to 100 mL.

[0172] ⑥ Stop solution (1 M H2SO4):

[0173] Add 21.7 mL of concentrated sulfuric acid (98%) to 178.3 mL of distilled water and adjust the volume to 400 mL.

[0174] TMB colorimetric solution was purchased from Solebol.

[0175] The titer test results are shown in Figures 3 and 4. After three immunizations, the titer of specific antibody IgG in mouse serum was 1:34500-1:301500 (Figure 3). The titer of egg yolk antibody IgY reached the highest level at the ninth week of immunization in laying hens, which was 1:4194304 (i.e. 1:2 22 ) (Figure 4).

[0176] Example 3: Experiment on the feeding effect of specific egg yolk antibody IgY

[0177] The egg yolk (containing egg yolk antibody IgY) obtained from the immunized laying hens in 1-2) of Example 2 was freeze-dried and pulverized, and then mixed into the cats' diet at a feeding amount of 1.5% (denoted as E-1.5% in the figure, 9 cats) and 2% (denoted as E-2% in the figure, 9 cats) by mass. The cats were fed for 4 weeks. The control group was a similar product on the market (LOVIDOVI Lolipoli (Nanjing Maxiwake Biotechnology Co., Ltd.), denoted as CK in the figure, 5 cats). Saliva samples were collected from the cats before feeding (denoted as 0 week in the figure), 1 week after feeding (denoted as 1 week in the figure), 2 weeks after feeding (denoted as 2 weeks in the figure), 3 weeks (denoted as 3 weeks in the figure), and 4 weeks after feeding (denoted as 4 weeks in the figure). The Fel d 1 protein content was detected using a Fel d 1 ELISA 2.0 kit (Indoor Biotech).

[0178] The test results are shown in Figures 5 and 6. After cats consumed specific egg yolk antibodies IgY for one to four weeks, the Fel d 1 content in the saliva of cats in the 1.5% addition treatment group decreased by an average of 41.1-73.1% (except for one cat with an abnormal value), the Fel d 1 content in the saliva of cats in the 2% addition treatment group decreased by an average of 46.5-79.3%, and the Fel d 1 content in the saliva of cats in the control group decreased by an average of 32.3-60.5%. This shows that egg yolk antibodies can reduce the content of cat allergen Fel d 1 and can be used as anti-allergic drugs.

[0179] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0180] CROSS-REFERENCE TO RELATED APPLICATIONS

[0181] This application claims priority to the Chinese patent application (application number 202311319699.3) filed on October 12, 2023, the entire contents of which are incorporated herein by reference. Industrial Applicability

[0182] Experiments of the present invention have shown that the antibodies obtained after immunizing animals with the chimeric peptide cFel d 1 have a high titer and can specifically bind to the complete FeI d 1 protein of cat allergens. They are superior to the existing polyclonal antibodies prepared with the complete FeI d 1 protein (with a titer 8 times higher).

Claims

1. A chimeric peptide cFel d 1, comprising the amino acid residue segments 25-41, 46-59, 85-98, 132-140 and 145-153 of cat allergen Fel d 1 antigen amino acid residues.

2. The chimeric peptide cFel d 1 according to claim 1, characterized in that: The various segments are connected by a flexible amino acid linker.

3. The chimeric peptide cFel d 1 according to claim 1, characterized in that: The amino acid sequence of the flexible amino acid linker is positions 18-22 of sequence 1.

4. The chimeric peptide cFel d 1 according to any one of claims 1 to 3, characterized in that: The chimeric peptide cFel d 1 is any one of the following A1)-A4): A1) a protein consisting of the amino acid sequence of sequence 1 in the sequence listing; A2) a protein derived from A1) in which the amino acid sequence of sequence 1 in the sequence list is replaced and / or deleted and / or added with one or more amino acid residues and has the same function; A3) A protein whose sequence contains the amino acid sequence defined in A1) or A2); A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1) to A3).

5. A nucleic acid encoding the chimeric peptide cFel d 1 according to any one of claims 1 to 4.

6. The coding nucleic acid according to claim 5, characterized in that: The encoding nucleic acid is a DNA molecule described in any one of the following B1)-B4): B1) A DNA molecule whose coding region includes the nucleotide sequence of sequence 2 in the sequence list; B2) A DNA molecule whose nucleotide sequence is the nucleotide sequence of Sequence 2 in the sequence list; B3) a DNA molecule that hybridizes with the nucleotide sequence described in B1) or B2) under stringent conditions and encodes the chimeric peptide cFel d 1 described in any one of claims 1 to 4; B4) A DNA molecule having a nucleotide sequence homology of more than 90% with that of B1) or B2) and encoding the chimeric peptide cFel d 1 according to any one of claims 1 to 4.

7. A recombinant vector, transgenic cell line or recombinant bacterium containing the encoding nucleic acid according to claim 5 or 6.

8. Use, characterized in that: The use is the use of the chimeric peptide cFel d 1 according to any one of claims 1 to 4 in any one of the following C1 to C5: C1) as an immunogen or preparation of immunomodulators; C2) Preparation of immunogen; C3) Preparation of antibodies against Fel d 1; C4) Preparation of anti-allergy vaccines; C5) Preparation of products with neutralized or reduced FeI d 1 content.

9. Use, characterized in that: The use is the use of the encoding nucleic acid according to claim 5 or 6 or the recombinant vector or transgenic cell line or recombinant bacteria according to claim 7 in any of the following C1-C5: C1) Preparation of immunomodulators; C2) Preparation of immunogen; C3) Preparation of antibodies against Fel d 1; C4) Preparation of anti-allergy vaccines; C5) Preparation of products with neutralized or reduced FeI d 1 content.

10. The use according to claim 8 or 9, characterized in that: The antibody is a polyclonal antibody.

11. An anti-allergic vaccine comprising the chimeric peptide cFel d 1 according to any one of claims 1 to 4.

12. The anti-allergic vaccine according to claim 11, characterized in that: The anti-allergic vaccine is an anti-feline hair allergy vaccine or an anti-feline dander allergy vaccine.

13. An antibody against FeI d 1, which is an antibody prepared using the chimeric peptide cFel d 1 according to any one of claims 1 to 4 as an antigen.

14. The antibody according to claim 13, characterized in that: The antibody prepared using the chimeric peptide cFel d 1 described in any one of claims 1 to 4 as an antigen is an antibody obtained by immunizing an organism using the chimeric peptide cFel d 1 described in any one of claims 1 to 4 as an immunogen; The organism is a poultry animal, a bird animal, a reptile animal, an amphibian animal or a mammal.

15. The antibody according to claim 13 or 14, characterized in that: The antibody is a polyclonal antibody.

16. Use of the antibody according to any one of claims 13 to 15 in any of the following: D1) Preparation of products for treating allergies; D2) Preparation of products with reduced Fel d 1 content.

17. A product for treating allergy, comprising the antibody according to any one of claims 13 to 15.

18. A method for preventing allergy, comprising administering to a subject a product comprising a preventively effective amount of the chimeric peptide cFel d 1 according to any one of claims 1 to 4.

19. A method for treating allergy, comprising administering to a subject a product comprising a therapeutically effective amount of the antibody according to any one of claims 13 to 15, wherein the administered dose comprises an antibody added in an amount of 1% to 3% of the feed intake / day / 3 to 4 kg.

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