Egg yolk antibody for resisting canine interleukin as well as preparation method and application of egg yolk antibody

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an egg yolk antibody for resisting canine interleukin as well as a preparation method and application of the egg yolk antibody. The egg yolk antibody is prepared by taking immunocompetence antigen epitope peptide of canine interleukin-31 as an immunogen, the antigen epitope peptide is obtained by predicting key antigen epitopes of IL-31 through an Antigenic tool and performing series optimization, a coding gene of the antigen epitope peptide is optimized by an escherichia coli codon to construct a recombinant expression vector, and high-purity antigen is obtained by inducible expression and purification. A laying hen is immunized by the antigen, after basic immunization and two times of booster immunization, the egg yolk antibody is extracted from egg yolk and purified to obtain the egg yolk antibody, and the ELISA of the egg yolk antibody is obviously superior to that of an antibody prepared by commercial canine IL-31 protein immunization. The egg yolk antibody can be specifically combined with canine IL-31, the histamine level in serum of a canine with pruritus is remarkably reduced, the atopic dermatitis related pruritus symptoms are effectively relieved, an efficient and safe biological treatment preparation is provided for canine IL-31 mediated pruritus diseases, and the egg yolk antibody has wide clinical application prospects.

Description

Technical Field

[0001] This invention relates to a method belonging to the field of biomedical technology, specifically to the preparation and application of antibody drugs, and particularly to an egg yolk antibody against canine interleukin-31 and its preparation method, as well as the application of this antibody in the treatment of canine atopic dermatitis and other IL-31-mediated pruritus diseases. Background Technology

[0002] With the rapid development of the pet industry, the incidence of canine skin diseases is increasing year by year. Among them, atopic dermatitis is one of the most common chronic inflammatory skin diseases, with its core symptom being intense itching, which seriously affects the quality of life of dogs and causes great distress to pet owners. Studies have shown that canine interleukin-31 (IL-31) is a key cytokine mediating canine pruritus. It activates inflammatory signaling pathways by binding to receptors on the surface of keratinocytes and mast cells, promoting the release of pruritus mediators such as histamine, thus triggering itching perception and skin inflammation. It is the core pathogenic factor of canine atopic dermatitis and other pruritus diseases. Therefore, developing agents that can specifically block the biological activity of canine IL-31 has become a key breakthrough in the treatment of these diseases.

[0003] Currently, treatments for canine pruritus mainly include glucocorticoids, antihistamines, and immunosuppressants. However, these drugs have significant limitations: long-term use of glucocorticoids can easily lead to side effects such as obesity, osteoporosis, and immunosuppression in dogs; antihistamines have limited efficacy against IL-31-mediated pruritus; and traditional immunosuppressants have problems such as broad target range and insufficient safety. In recent years, egg yolk antibodies (IgY) have received widespread attention in the field of animal disease treatment due to their advantages such as low preparation cost, weak immunogenicity, and high specificity. Egg yolk antibodies are specific antibodies produced in egg yolks after immunization with specific antigens in hens. They are characterized by simple preparation processes, large-scale production capability, and no residual risk, and have been applied to the prevention and treatment of various animal diseases.

[0004] However, existing antibody preparation technologies targeting canine IL-31 still have shortcomings: traditional methods often use the intact IL-31 protein as an immunogen, but the intact protein suffers from problems such as masked antigenic epitopes and uneven immunogenicity, resulting in low antibody titers and poor specificity. Some technologies use prokaryotic expression systems to prepare immunogens, but they fail to accurately screen and optimize antigenic epitopes or perform codon optimization to adapt to the expression system, leading to low antigen expression levels and poor solubility, which in turn affects antibody preparation efficiency and activity. Furthermore, existing antibodies have limited therapeutic effects and short-lasting relief of itching symptoms in clinical applications, making it difficult to meet clinical treatment needs. Therefore, developing an anti-canine IL-31 egg yolk antibody with precise antigenic epitope targeting, high antibody titer, and significant therapeutic effect is of great significance for solving the clinical treatment challenges of canine pruritus. Summary of the Invention

[0005] To address the problems of significant side effects of existing drugs for treating canine pruritus, low titers of existing anti-IL-31 antibodies, and limited therapeutic effects, this invention provides an anti-canine interleukin egg yolk antibody, its preparation method, and its application. By precisely screening key antigenic epitopes of canine IL-31 and optimizing the antigen preparation process, a high-titer, high-specificity egg yolk antibody is obtained, achieving safe and effective treatment for canine IL-31-mediated pruritus.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a canine interleukin-31 (IL-31) antigenic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] This invention provides an egg yolk antibody against canine interleukin, wherein the egg yolk antibody is prepared using a canine interleukin-31 (IL-31) antigenic epitope peptide as an immunogen, and the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO:2.

[0008] The present invention provides a nucleic acid molecule encoding the canine interleukin-31 (IL-31) antigenic epitope peptide described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0009] This invention provides a recombinant expression vector containing a nucleic acid molecule of canine interleukin-31 (IL-31) antigenic epitope peptide. The expression vector is a pET-28a (+) vector that has been double-digested with BamHI and XhoI. The recombinant expression vector is named pET-28a-IL-31-P-His.

[0010] The present invention provides a recombinant engineered bacterium comprising the recombinant expression vector, wherein the engineered bacterium is Escherichia coli BL21 (DE3).

[0011] This invention provides a method for preparing the anti-canine interleukin egg yolk antibody, characterized by comprising the following steps: (1) The nucleic acid molecule was linked to the pET-28a (+) vector to construct a recombinant expression vector, which was transformed into Escherichia coli BL21 (DE3). After induction of expression, the recombinant IL-31-P antigen epitope peptide was purified by Ni²⁺-NTA affinity chromatography to obtain a purity ≥96%. (2) Using the recombinant IL-31-P antigenic epitope peptide prepared in step 1) as an immunogen, the vaccine was prepared by emulsification with the oil phase. The laying hens were given basic immunization and two booster immunizations. The dose of basic immunization and booster immunization was 1 mL / hen, and the interval between the two booster immunizations was 14 days. (3) Extraction and purification of egg yolk antibodies: Collect eggs from immunized chickens, separate and dilute the yolks, perform crude extraction by PEG-6000 gradient precipitation, and then purify by dialysis to obtain egg yolk antibodies against canine interleukin.

[0012] This invention provides the use of the anti-canine interleukin egg yolk antibody in one or more of the following: (1) Preparation of a biological agent to neutralize canine IL-31; (2) Preparation of drugs to reduce serum histamine levels in dogs; (3) Preparation of drugs for treating canine IL-31-mediated atopic dermatitis; (4) Prepare drugs for treating canine pruritus.

[0013] Preferably, the drug is administered via subcutaneous injection at a dose of 0.1 mL / kg, once a week for four consecutive weeks.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: Precise and efficient antigen design: This invention uses antigen epitope prediction technology to screen key immunologically active epitopes of canine IL-31 and constructs antigen epitope peptides by using flexible linker peptides in series, avoiding the problem of intact protein antigen epitope masking. At the same time, the expression level and solubility of the antigen are significantly improved by E. coli codon optimization. The purity of the purified antigen is ≥96%, which lays the foundation for the preparation of high-titer antibodies.

[0015] High antibody titer and strong specificity: The ELISA titer of the egg yolk antibody prepared by this invention is as high as 1:1024000, which is significantly better than the antibody prepared by commercial canine IL-31 protein immunization. It can specifically bind to canine IL-31, block its interaction with the receptor, and inhibit the activation of inflammatory signaling pathways.

[0016] Significant therapeutic effect: Clinical trials have confirmed that this egg yolk antibody can significantly reduce the level of histamine in the serum of dogs with pruritus, and quickly relieve symptoms such as skin itching, erythema, and hair loss. After 4 weeks of administration, the symptom relief rate was significantly higher than that of the control group, with no obvious side effects and high safety.

[0017] The preparation process is simple and the cost is low: This invention uses egg yolk antibody preparation technology, with laying hens as immunized animals. Antibodies can be obtained in large quantities from eggs. The preparation process does not require complex equipment, and it is highly feasible for large-scale production. The cost is significantly lower than that of traditional antibody drugs such as monoclonal antibodies.

[0018] The egg yolk antibody provided by this invention offers a new treatment option for canine IL-31-mediated atopic dermatitis and other pruritus diseases. It can be developed into a highly effective and safe animal-specific biological agent, with significant clinical application value and market potential. Attached Figure Description

[0019] Figure 1 Antigenic tools were used to predict antigenic epitopes in canine interleukin-31 that could elicit antibody responses.

[0020] Figure 2 SDS-PAGE image of canine IL-31-P protein solubility analysis. M represents protein standard, 1 represents uninduced whole bacteria, 2 represents induced whole bacteria, 3 represents induced precipitate, and 4 represents induced supernatant.

[0021] Figure 3 SDS-PAGE purification and validation of the elution peak of canine IL-31-P protein.

[0022] Figure 4 ELISA titer detection of anti-canine interleukin egg yolk antibody. Detailed Implementation

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] Example 1: Design and Construction of Canine Interleukin Antigen Peptide interleukin-31 precursor [Canis lupus familiaris]: MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDVRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLTSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLESVFKSLNSGPQ (SEQ ID NO: 1); Antigenic tools are used to predict antigenic epitopes in protein sequences that can elicit antibody responses. This is a practical tool for screening fragments with B-cell immune activity (http: / / imed.med.ucm.es / Tools / antigenic.pl). Prediction results are shown below. Figure 1 By sequentially tandemly connecting the antigenic epitope peptides, the following immune-enhancing canine IL-31-P antigenic epitope peptides were obtained: SRFALFLLCSMETLLSSGGGGGSGGGGSMAPTHQLPPSDVRKIILELQPLSRGGGGGSGGGGSRTLLLCLTSGGGGGSGGGGSQPPRLNSSAILPYFRAIRPGGGGGSGGGGSIDKIIEQLDKLKFGGGGGSGGGGSTEISVPADTFECKSFILTILQQFSACLESVFK (SEQ ID NO: 2); For the immune-enhancing canine IL-31-P antigenic epitope peptide (SEQ ID NO:2), Nanjing Genscript Biotech Co., Ltd. was commissioned to optimize the E. coli codons, obtaining the optimized nucleotide sequence SEQ ID NO:3. The company then prepared the target gene fragment using a chemical synthesis method and performed sequence verification.

[0025] The codons of the canine IL-31-P antigenic epitope peptide were optimized using E. coli, and the optimized sequence is as follows: TCTCGTTTTGCTTTATTTTTATTATGTTCTATGGAAACTTTATTATCTTCTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTATGGCTCCTACTCATCAATTACCTCCTTCTGATGTTCGTAAAA TTATTTTAGAATTACAACCTTTATCTCGTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCGTACTTTATTATTATGTTTAACTTCTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAACCT CCTCGTTTAAATTCTTCTGCTATTTTACCTTATTTTCGTGCTATTCGTCCTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTATTGATAAAATTATTGAACAATTAGATAAATTAAAATTTGGTGG TGGTGGTGGTTCTGGTGGTGGTGGTTCTACTGAAATTTCTGTTCCTGCTGATACTTTTGAATGTAAATCTTTTATTTTAACTATTTTACAACAATTTTCTGCTTGTTTAGAATCTGTTTTTAAA (SEQ ID NO:3); Primers containing BamHI and XhoI restriction sites were designed, and the target gene was amplified by PCR using SEQ ID NO:3 as a template. Simultaneously, the pET-28a (+) expression vector (with a C-terminal 6×His tag) was double-digested with BamHI and XhoI, and the target gene fragment and vector backbone were recovered by agarose gel electrophoresis. The recombinant expression vector pET-28a-IL-31-P-His was constructed by ligation overnight at 16°C using T4 DNA ligase. This vector was transformed into *E. coli* DH5α competent cells, and plasmids were extracted after single colony culture. Double enzyme digestion and sequencing confirmed the correct vector construction. The verified recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, plated on LB agar plates containing kanamycin, and incubated at 37°C for 12 h. Positive single colonies were inoculated into LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking until OD. 600 =0.6-0.8, add IPTG to a final concentration of 0.5 mmol / L, and induce expression at 30℃ for 4 h. Collect the induced bacterial culture, centrifuge to collect the bacterial cells, resuspend in PBS, sonicate to disrupt, centrifuge to collect the supernatant, and analyze by SDS-PAGE. See [link to SDS-PAGE analysis]. Figure 2 .

[0026] Figure 2 The results showed that the IL-31-P protein was present in the supernatant in a soluble state, and its molecular weight was as expected.

[0027] Engineered bacteria expressing IL-31-P were collected, resuspended in lysis buffer (containing 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 1 mmol / L PMSF, pH 8.0), and then sonicated to disrupt the bacterial cells. The cells were centrifuged at 12000 rpm for 30 min at 4 °C, and the supernatant (containing soluble His-tagged IL-31-P) was collected and filtered through a 0.22 μm filter to remove impurities. A Ni²⁺-NTA affinity chromatography column was taken, and the column bed was first washed with 5 column volumes of deionized water to remove the preservative solution. Then, the column was equilibrated with 10 column volumes of PBS buffer containing 20 mmol / L imidazole (137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na₂HPO₄, 2 mmol / L KH₂PO₄, pH 7.4). Column stability (baseline stability) was ensured by UV monitoring. The pretreated supernatant was loaded at a flow rate of 0.5 mL / min to ensure sufficient binding of the His tag to Ni²⁺. After loading, elution was continued with 20 mmol / L imidazole-PBS for 5 column volumes until the UV absorbance returned to baseline to remove non-specifically bound proteins. Elution was then performed with PBS buffer containing 500 mmol / L imidazole at a flow rate reduced to 0.3 mL / min. Elution peaks were collected in fractions, and absorbance at 280 nm was measured using a UV spectrophotometer. The protein-containing eluent peaks were combined. The combined eluent was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in PBS buffer (pH 7.4) at 4°C. The dialysate was changed every 4 hours for a total of 3 times to completely remove imidazole. The dialysis samples were then verified by SDS-PAGE electrophoresis. Figure 3 .

[0028] Figure 3 The results showed that the purified IL-31-P band with the HIS tag was single, the molecular weight was close to the expected value of 16.42 kDa, and there were no obvious contaminating proteins, with a purification rate of more than 96%.

[0029] Example 2: Preparation and detection of egg yolk antibodies against canine interleukin Experimental group: The immunogen was the recombinant IL-31-P antigenic epitope peptide purified in Example 1, with a purity of ≥96% verified by SDS-PAGE and a theoretical molecular weight of 16.42 kDa; Control group: The immunogen used was commercial canine IL-31 protein from Starter Biotech, product name IL-31 Protein, Canine, catalog number UA040130.

[0030] Immunized animals: Thirty healthy Hy-Line Brown laying hens were selected and randomly divided into an experimental group and a control group, with 15 birds in each group. All chickens were acclimatized for one week before the experiment, during which standard feed and clean drinking water were provided, the ambient temperature was controlled at 22-25℃, and the light cycle was 16 hours of light / 8 hours of darkness to ensure that the animals were in a consistent health state.

[0031] Aqueous phase preparation: The experimental group IL-31-P antigenic epitope peptide and the control group IL-31 protein were diluted with PBS buffer to a concentration of 1 mg / mL. Tween-80 was added to each dilution to a final concentration of 4%, and the mixture was magnetically shaken for 30 min until the Tween-80 was completely dissolved. The mixture was then stored at 4°C for later use.

[0032] Oil phase preparation: Mix white oil and Span-80 at a volume ratio of 94:6, add 2% aluminum stearate (mass-volume ratio), shake in a 50°C water bath until the solution is pale yellow, clear and transparent, autoclave at 121°C for 20 min, cool to room temperature and store at 4°C.

[0033] Emulsion preparation: Two portions of the oil phase were placed in a tissue homogenizer and stirred slowly at 2000 rpm for 2 minutes. During this time, one portion of the corresponding aqueous phase was slowly added (controlling the drop rate to 1-2 mL / min) to ensure that the aqueous phase was uniformly dispersed in the oil phase. The speed was then adjusted to 10000 rpm, and emulsification was carried out for 3 minutes, followed by a 5-minute pause. This operation was repeated 4 times to prepare a water-in-oil emulsion vaccine. A small amount of vaccine was dropped into cold water. If the vaccine drop formed a spherical shape and did not spread, the emulsification was deemed successful. The vaccine was then sealed and stored at 4°C.

[0034] Animal immunization and sample collection: Both groups were immunized by subcutaneous injection in the neck. The immunization schedule was as follows: the basic immunization dose was 1 mL / bird; the first booster immunization was given on day 14 after the first immunization, with a dose of 1 mL / bird; the second booster immunization was given on day 14 after the first booster immunization, with a dose of 1 mL / bird. All chicken eggs were collected before the first immunization as a negative control; egg collection began on day 7 after each booster immunization and continued for 21 days. After collection, the eggs were refrigerated at 4°C, and the yolks were separated within 24 hours for later use.

[0035] Egg yolk antibody extraction: Gently crack the egg along the blunt end, separate the yolk and remove the attached egg white, dilute the yolk with PBS buffer at a volume ratio of 1:2, and vortex for 10 minutes until well mixed.

[0036] Crude antibody extraction: Add PEG-6000 to the diluted egg yolk solution to a final concentration of 3.5%, vortex at 200 rpm for 10 min, centrifuge at 4℃ and 10000 rpm for 20 min, and collect the supernatant; add PEG 6000 to the supernatant to a final concentration of 8.5%, repeat the above vortexing and centrifugation steps, discard the supernatant, and resuspend the precipitate in 10 mL of PBS buffer.

[0037] Purification and dialysis: Add PEG 6000 to the resuspension to a final concentration of 12%, centrifuge at 10000 r / min for 20 min at 4 °C, and collect the precipitate; reconstitute the precipitate with 800 μL PBS buffer, transfer it to a dialysis bag with a molecular weight cutoff of 20 kDa, and dialyze overnight at 4 °C in 500 mL PBS buffer, changing the dialysis buffer every 4 h to obtain purified IgY antibodies for the experimental and control groups, which were then aliquoted and stored at -20 °C.

[0038] ELISA antibody titer assay: Coating: Dilute canine IL-31 protein to 2 μg / mL with 0.1M NaHCO3 buffer (pH 9.6), add 100 μL to each well, and coat overnight at 4°C.

[0039] Blocking and incubation: Discard the coating solution, wash the plate 5 times with PBST (PBS containing 0.1% Tween-80), 3 min each time; add 150 μL of 1% casein blocking solution to each well and incubate at 37℃ for 1 h; after washing, dilute the two groups of IgY antibodies with PBS at serial dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, and 1:2048000, add 100 μL of dilution to each well and incubate at 37℃ for 1 h; after washing, add 100 μL of goat anti-chicken IgY-HRP secondary antibody diluted 1:10000 to each well and incubate at 37℃ for 1 h.

[0040] Color development and reading: After washing the plate, add 100 μL of HRP substrate solution (TMB chromogenic solution) to each well and incubate at room temperature in the dark for 15 min; stop the reaction by adding 50 μL of 2M sulfuric acid solution, and measure the OD value at 450 nm using a microplate reader. Simultaneously, measure the OD values ​​of the experimental group, control group, and blank group (pre-immunization yolk antibody). See [link to relevant documentation]. Figure 4 .

[0041] Figure 4 The results showed that the IgY antibody titer of the recombinant IL-31-P antigenic epitope peptide in the experimental group reached 1:1024000, which was significantly better than the IgY antibody of canine IL-31 protein in the control group.

[0042] Example 3: Application of anti-canine interleukin egg yolk antibody in the treatment of canine pruritus. Sixty healthy clinical dogs from Aikon Pet Hospital (China) were selected, including Golden Retrievers, Labrador Retrievers, and Bichon Frises, aged 1-5 years and weighing 8-25 kg, with half being male and half female. All dogs underwent clinical examination to rule out parasitic infections, fungal / bacterial infections, and other systemic diseases. They were not given antihistamines, corticosteroids, or immunosuppressants for 14 days prior to the experiment, and underwent a 7-day acclimatization period to eliminate environmental stress and ensure a consistent experimental baseline.

[0043] The core of the experiment was to establish a standardized canine pruritus-treatment model. The specific procedure was as follows: each dog was given recombinant canine interleukin-31 (cIL-31) protein via intravenous injection at a dose strictly controlled at 3 μg / kg to induce a stable pruritus response. Behavioral observation was conducted via video recording 30 minutes after the injection of cIL-31 protein, and the recording time lasted for 2 hours. The frequency of pruritus-related behaviors was the main focus.

[0044] The experimental group IgY antibody (purified product of the experimental group in Example 2, concentration 2 mg / mL), the control group IgY antibody (purified product of the control group in Example 2, concentration 2 mg / mL), and physiological saline (negative control) were all stored at 4°C for later use. The canine histamine ELISA kit (catalog number PD30837, Periman) was prepared according to the instructions.

[0045] Group design: Sixty dogs with pruritus were randomly divided into an experimental group, a control group, and a negative control group, with 20 dogs in each group. There were no statistically significant differences in breed, age, weight, and initial pruritus scores among the three groups (P>0.05), ensuring balance among the groups.

[0046] Dosage regimen: All three groups received subcutaneous injection at a dose of 2 mg / kg, once weekly for 4 weeks. The experimental group received the experimental group's IgY antibody, the control group received the control group's IgY antibody, and the negative control group received an equal volume of physiological saline. During the treatment period, all dogs were fed allergen-free prescription food, provided with clean drinking water, and kept in a consistent living environment.

[0047] Sample collection and testing: Venous blood was collected from dogs before administration (T0), 2 weeks after administration (T2), and 4 weeks after administration (T4). Serum was separated by centrifugation at 3000 rpm for 15 min and stored at -20℃ for testing. Histamine levels in serum were detected using an ELISA kit, strictly following the kit instructions. Each sample was tested in triplicate, and the average value was used as the result.

[0048] Clinical symptom scoring: The canine atopic dermatitis severity scoring system (CADESI-4) was used to assess pruritus symptoms, scoring on three dimensions: frequency of pruritus, skin lesions (erythema, hair loss, crusting), and number of lesion sites. The total score ranged from 0 to 100, with higher scores indicating more severe pruritus symptoms. Two veterinarians scored the pruritus in a blinded manner at time points T0, T2, and T4, and the average score was used as the final score.

[0049] Clinical symptom scoring results: At T0, there was no significant difference in CADESI-4 scores among the three groups (P>0.05); at T2, the score of the experimental group decreased to (32.5±4.2) points, the control group decreased to (48.3±5.1) points, and the negative control group decreased to (65.7±6.3) points. The score of the experimental group was significantly lower than that of the control group and the negative control group (P<0.05); at T4, the score of the experimental group decreased to (18.2±3.5) points, the control group decreased to (35.6±4.8) points, and the negative control group decreased to (58.9±5.7) points. The score of the experimental group was still significantly better than that of the control group (P<0.05), and the itching symptoms of 8 dogs in the experimental group basically disappeared (score <20 points).

[0050] Changes in serum histamine levels: At T0, there was no significant difference in serum histamine levels among the three groups (P>0.05); at T2, the histamine level in the experimental group decreased to (125.4±16.8) ng / mL, the control group decreased to (186.7±19.5) ng / mL, and the negative control group was (268.9±22.6) ng / mL; at T4, the histamine level in the experimental group decreased to (98.5±14.2) ng / mL, the control group decreased to (152.3±17.9) ng / mL, and the negative control group was (256.4±20.8) ng / mL. The histamine regulation effect in the experimental group was significantly better than that in the control group (P<0.05).

[0051] The recombinant IL-31-P antigenic epitope peptide IgY antibody prepared in Example 2 significantly reduced serum histamine levels in dogs with pruritus, alleviating skin itching and lesion symptoms. Its therapeutic effect was significantly better than that of the commercial canine IL-31 protein immunization-prepared IgY antibody in the control group, providing a highly efficient candidate biological agent for the clinical treatment of canine pruritus.

[0052] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An egg yolk antibody against canine interleukin, characterized in that, The egg yolk antibody was prepared using canine interleukin-31 antigenic epitope peptide as an immunogen, and the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the antigenic epitope peptide of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

3.

3. A recombinant expression vector, characterized in that, The expression vector is a pET-28a (+) vector that has been double-digested with BamHI and XhoI, and the recombinant expression vector is named pET-28a-IL-31-P-His.

4. A recombinant engineered bacterium, characterized in that, The recombinant expression vector of claim 3 is included, wherein the engineered bacterium is Escherichia coli BL21 (DE3).

5. The method for preparing the anti-canine interleukin egg yolk antibody according to claim 1, characterized in that, Includes the following steps: (1) The nucleic acid molecule described in claim 2 is linked to the pET-28a (+) vector to construct a recombinant expression vector, which is then transformed into Escherichia coli BL21 (DE3). After induction of expression, the recombinant IL-31-P antigen epitope peptide is purified by Ni²⁺-NTA affinity chromatography to obtain a purity ≥96%. (2) Using the recombinant IL-31-P antigenic epitope peptide prepared in step (1) as an immunogen, the vaccine was prepared by emulsification with the oil phase. The laying hens were given basic immunization and two booster immunizations. The dose of basic immunization and booster immunization was 1 mL / hen, and the interval between the two booster immunizations was 14 days. (3) Extraction and purification of egg yolk antibodies: Collect eggs from immunized chickens, separate and dilute the yolks, perform crude extraction by PEG-6000 gradient precipitation, and then purify by dialysis to obtain egg yolk antibodies against canine interleukin.

6. The use of the anti-canine interleukin egg yolk antibody of claim 1 in one or more of the following: (1) Preparation of a biological agent to neutralize canine interleukin-31; (2) Preparation of drugs to reduce serum histamine levels in dogs; (3) Preparation of drugs for treating canine interleukin-31 mediated atopic dermatitis; (4) Prepare drugs for treating canine pruritus.

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