Egg yolk globulin for relieving rhinitis and application thereof

Abstract

The invention provides egg yolk globulin for relieving rhinitis as well as a preparation method and application of the egg yolk globulin. The egg yolk globulin can effectively inhibit the content of IL4, IL5 and IgE on the periphery of a mouse with allergic rhinitis, inhibit differentiation of Th2 cells and relieve rhinitis symptoms of the mouse. The preparation prepared by taking the egg yolk globulin as the raw material can effectively relieve rhinitis symptoms of people, such as nasal obstruction, running nose and rhinocnesmus. The preparation method of the specific egg yolk globulin is simple, safe and effective, and provides a new treatment means for rhinitis patients. Meanwhile, the egg yolk globulin can also be effectively used for adjuvant therapy of EBV positive nasopharynx cancer and gastric cancer, and has a relatively great application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of egg yolk globulin, and more specifically, to an egg yolk globulin for relieving rhinitis and application thereof. Background Art

[0002] Rhinitis is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, etc., which may be caused by various physical and chemical factors or certain systemic diseases. The main symptoms of rhinitis include nasal congestion, sneezing, runny nose and itchy nose, but the specific manifestations may vary according to the type of rhinitis. Acute rhinitis often manifests as dryness, burning or itching in the nose in the early stages, and eventually develops into obstructive nasal sounds, decreased sense of smell, etc. The main symptoms of chronic rhinitis are nasal congestion and increased nasal discharge, and symptoms such as headache and discomfort at the root of the nose may also occur. If the condition is severe, the inflammation may also affect other parts of the upper respiratory tract and the lower respiratory tract, causing complications such as pharyngitis, otitis media, tracheitis, bronchitis and pneumonia.

[0003] Yolk globulin refers to the specific antigen that stimulates poultry B lymphocytes, which differentiate into plasma cells and secrete specific yolk globulin into the blood circulation. When the blood flows through the ovaries, it gradually accumulates in the oocytes to form yolk globulin. Yolk globulin has the advantages of stable properties, strong specificity, low preparation cost, and oral safety. It has been widely used in the prevention and treatment of infectious diseases in poultry and livestock. The development of the product has also produced good economic and social benefits in ecological agriculture and food safety. It has good application value in the diagnosis, prevention and treatment of various diseases in humans and animals. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides an egg yolk globulin for relieving rhinitis and its application. In this study, the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα were combined as immune antigens to prepare oral egg yolk globulin. The oral egg yolk globulin can effectively inhibit the peripheral IL4, IL5 and IgE content of mice with allergic rhinitis, inhibit the differentiation of Th2 cells, and reduce the rhinitis symptoms of mice. The above experiments are sufficient to prove that the preparation prepared by the present invention using the egg yolk globulin as raw material can effectively alleviate the symptoms of rhinitis in the human population, such as nasal congestion, runny nose and itchy nose. The specific egg yolk globulin prepared by this method is simple, safe and effective, and provides a new treatment for patients with rhinitis. At the same time, the egg yolk globulin can also be effectively used in the treatment of EBV-positive nasopharyngeal carcinoma and gastric cancer, and has great application prospects.

[0005] Specifically, the present invention first provides an egg yolk globulin for relieving rhinitis, the light chain of the egg yolk globulin is shown as SEQ ID NO.1, and the heavy chain of the egg yolk globulin is shown as SEQ ID NO.2.

[0006] Furthermore, the light chain of the yolk globulin is encoded by the sequence shown in SEQ ID NO.3, and the heavy chain is encoded by the sequence shown in SEQ ID NO.4.

[0007] On the other hand, the present invention also provides a method for preparing egg yolk globulin for relieving rhinitis, comprising the following steps: (1) Preparation of immune antigens: Prepare proteins containing the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα, and dissolve them in physiological saline in a mass ratio of (1-3):(1-5):(1-3):(1-5) as immune antigens; (2) Immunization procedure for laying hens: Add an equal volume of Complete Freund's adjuvant (CFA) to the immunization antigen (1-5 mL) of step (1) and mix thoroughly to form an emulsion. Disinfect the laying hens and inject 1.5-3 mL of the emulsion intramuscularly at 4-6 points under the breast and wings for the first immunization. Then, add an equal volume of Incomplete Freund's adjuvant (IFA) to the immunization antigen (1-5 mL) of step (1) and mix thoroughly to perform booster immunization twice at intervals of 1 week. Collect the eggs 1 week after the booster immunization and store at 4°C for later use. (3) Separation and purification of specific egg yolk globulin: separate and collect egg yolk liquid, add 5-10 times the volume of physiological saline, then add 10-15% (w / v) PEG-6000, let stand for 30-60 minutes, centrifuge at 3000-5000g for 20-50 minutes; filter the supernatant with absorbent cotton, and add ammonium sulfate to the supernatant to make the saturation of ammonium sulfate reach 45-55%, and let stand at 4°C for 6-12 hours; centrifuge after standing, discard the supernatant to obtain a precipitate, add 10-20mL of physiological saline per gram of precipitate to dissolve it to obtain a solution; put the solution into a dialysis bag and dialyze with distilled water to remove ammonium ions, and use physiological saline for the last dialysis; sterilize and filter the dialyzate to obtain a specific egg yolk globulin extract; after freeze-drying, store it at -80°C for later use; (4) The specific yolk globulin prepared in step (3) is sent to Beijing Anbiqi Biological Co., Ltd. for sequencing analysis.

[0008] Preferably, step (1) preparation of immune antigens: isolating the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα, and dissolving them in physiological saline in a mass ratio of 1:1:1:1 as immune antigens.

[0009] Preferably, the immunization procedure for laying hens in step (2) is as follows: the immunization antigen (1 mL) of step (1) is added with an equal volume of Complete Freund's adjuvant (CFA) and mixed thoroughly to form an emulsion, and 1.5 mL of the emulsion is intramuscularly injected at 6 points under the chest and wings of sterilized laying hens for the first immunization. Subsequently, the immunization antigen (1 mL) of step (1) is added with an equal volume of Incomplete Freund's adjuvant (IFA) and mixed thoroughly for two booster immunizations at intervals of 1 week. One week after the booster immunization, the eggs are collected and stored at 4°C for future use.

[0010] Preferably, step (3) separation and purification of specific egg yolk globulin: separate and collect egg yolk liquid, add 5 volumes of physiological saline, then add 10% (w / v) PEG-6000, let stand for 30 minutes, centrifuge at 3000g for 20 minutes; filter the supernatant with absorbent cotton, and add ammonium sulfate to the supernatant to make the saturation of ammonium sulfate reach 45%, and let stand for 6 hours at 4°C; centrifuge after standing, discard the supernatant to obtain a precipitate, add 10 mL of physiological saline per gram of precipitate to dissolve it to obtain a dissolved solution; put the dissolved solution into a dialysis bag and dialyze with distilled water to remove ammonium ions, and use physiological saline for the last dialysis; sterilize and filter the dialyzate to obtain a specific egg yolk globulin extract; after freeze-drying, store it at -80°C for use.

[0011] Furthermore, the present invention also provides the use of the above-mentioned specific egg yolk globulin in the preparation of a drug for alleviating rhinitis.

[0012] Furthermore, the present invention also provides a medicine for relieving rhinitis, characterized in that it comprises the above-mentioned specific yolk globulin.

[0013] Furthermore, the present invention also provides the use of the above-mentioned specific egg yolk globulin in the preparation of products for treating gastric cancer or nasopharyngeal cancer.

[0014] Furthermore, the present invention also provides a product for treating gastric cancer or nasopharyngeal cancer, characterized in that it comprises the above-mentioned specific yolk globulin.

[0015] Preferably, the product further comprises a pharmaceutically acceptable carrier.

[0016] Preferably, the gastric cancer or nasopharyngeal carcinoma is selected from EBV-positive gastric cancer or nasopharyngeal carcinoma.

[0017] The advantages of the present invention are as follows: For the first time, the present invention uses the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα in combination as immune antigens to prepare oral yolk globulin. The oral yolk globulin can effectively inhibit the peripheral IL4, IL5 and IgE content of mice with allergic rhinitis, inhibit the differentiation of Th2 cells, and reduce the rhinitis symptoms of mice. The above experiments are sufficient to prove that the preparation prepared by the present invention using the yolk globulin as raw material can effectively alleviate the symptoms of rhinitis in the human population, such as nasal congestion, runny nose, and itchy nose. The specific yolk globulin prepared by this method is simple, safe and effective, and provides a new treatment for patients with rhinitis. At the same time, the yolk globulin can also be effectively used in the treatment of EBV-positive nasopharyngeal carcinoma and gastric cancer, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the linear detection range of the specific yolk globulin of the present invention; Figure 2 The ELISA test was used to analyze the serum IL4 and IL5 levels in the mouse model; Figure 3 The ELISA test was used to analyze the serum IgE content of the mouse model; Figure 4 The invention relates to an analysis of the proliferation ability of the specific yolk globulin on EBV-positive cancer cells. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0020] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0021] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. Wherein, w / v in the present invention represents the mass volume ratio, and the unit is g / mL.

[0022] Example 1

[0023] The present invention separates and purifies a yolk globulin for relieving rhinitis for the first time, wherein the light chain of the yolk globulin is shown in SEQ ID NO.1, and the heavy chain of the yolk globulin is shown in SEQ ID NO.2. Furthermore, the light chain of the yolk globulin is encoded by the sequence shown in SEQ ID NO.3, and the heavy chain is encoded by the sequence shown in SEQ ID NO.4.

[0024] Example 2

[0025] A method for preparing egg yolk globulin for relieving rhinitis comprises the following steps: (1) Preparation of immune antigens: Prepare proteins containing the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα, and dissolve them in physiological saline at a mass ratio of 1:1:1:1 as immune antigens; (2) Immunization procedure for laying hens: The immunization antigen (1 mL) in step (1) was added with an equal volume of Complete Freund's adjuvant (CFA) and mixed thoroughly to form an emulsion. After disinfecting the laying hens with conventional iodine and alcohol, 1.5 mL of the emulsion was intramuscularly injected at 6 points under the breast and wings for the initial immunization. Subsequently, the immunization antigen (1 mL) in step (1) was added with an equal volume of Incomplete Freund's Adjuvant (IFA) and mixed thoroughly for two booster immunizations at intervals of 1 week. One week after the booster immunization, the eggs were collected and stored at 4°C for later use.

[0026] (3) Isolation and purification of specific egg yolk globulin: separate and collect egg yolk liquid, add 5 volumes of physiological saline, then add 10% (w / v) PEG-6000, let stand for 30 minutes, centrifuge at 3000g for 20 minutes; filter the supernatant with absorbent cotton, and add ammonium sulfate to the supernatant to make the saturation of ammonium sulfate reach 45%, let stand for 6 hours at 4°C; centrifuge after standing, discard the supernatant to obtain a precipitate, add 10mL of physiological saline per gram of precipitate to dissolve it to obtain a solution; put the solution into a dialysis bag and dialyze with distilled water to remove ammonium ions, and use physiological saline for the last dialysis; sterilize and filter the dialyzate to obtain a specific egg yolk globulin extract; after freeze-drying, store it at -80°C for later use.

[0027] (4) The specific yolk globulin prepared in step (3) is sent to Beijing Anbiqi Biological Co., Ltd. for sequencing analysis.

[0028] Example 3

[0029] The specific egg yolk globulin titer was determined by the ELISA method. The specific method includes the following steps: based on 1 μg / mL IL-4 concentrate, it was diluted 10 times with physiological saline (1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL) to prepare a 96-well microplate coated with the above-mentioned different concentrations, and the egg yolk globulin described in Example 1 was added, incubated at 37°C for 3.5 hours, and after washing the plate, 1:10000 diluted horseradish peroxidase-labeled rabbit anti-egg yolk globulin was added, incubated at 37°C for 3 hours, and after washing the plate, the substrate was added for color development. The absorbance value at a wavelength of 450nm was measured by an enzyme marker, and the non-immunized egg yolk solution was used as a control. Figure 1 It can be seen that the egg yolk globulin provided by the present invention can well recognize IL-4, and its recognition lower limit can reach 1 ng / mL, which has good recognition ability.

[0030] Similarly, for other IL-5, IL-4Rα, and IL-5Rα concentrates, it can reach 1 ng / mL, with good recognition ability.

[0031] Example 4

[0032] Construction of mouse model of allergic rhinitis: 4-week-old healthy female BALB / C mice were taken, with a body weight of 20-25 g. BALB / C mice were given 50 μg OVA and 4 mg gel Al(OH) 3 The suspension was injected intraperitoneally to sensitize mice, once every other day, for a total of seven times. Then, 5% OVA saline was dripped into the nose, 50 μL / mouse, once a day for 7 consecutive days, until the 15th day of nasal stimulation and sensitization, and the allergic rhinitis mouse model was established.

[0033] The above mice with allergic rhinitis were treated with specific egg yolk globulin (the control group had the same amount of normal saline) by gavage at a dose of 20 mg / mouse per day for 10 consecutive days, starting from the 20th day of modeling. The experimental endpoint was 30 days after modeling. The levels of serum IL-4, IL-5 and OVA-sIgE were detected by ELISA to evaluate the changes in the mouse nasal mucosa. The results are shown in Figure 2-3 It is shown that the egg yolk globulin can inhibit the serum IL4 and IL5 of the mouse model and the content of IgE in the serum, can effectively inhibit the differentiation of Th2 cells, and reduce the rhinitis symptoms of mice. The above experiments are sufficient to prove that the preparation prepared with the egg yolk globulin as the raw material of the present invention can effectively alleviate the symptoms of rhinitis in the human body, such as nasal congestion, runny nose and nasal itching. The specific egg yolk globulin prepared by this method is simple, safe and effective, and provides a new treatment method for patients with rhinitis.

[0034] Example 5

[0035] Effect of yolk globulin on the proliferation of EBV-positive nasopharyngeal carcinoma cell C666 TK and EBV-positive gastric carcinoma cell AGS: 1×10 7 EBV-positive nasopharyngeal carcinoma cells C666 TK or EBV-positive gastric cancer cells AGS were added to the cell culture dish, and 10 μg / mL egg yolk globulin was added after they adhered to the wall. Among them, the control group was treated with an equal amount of normal saline. After 5 days of culture, the cell killing rate was analyzed using the instructions of the LDH detection kit. The results are shown in Figure 4 As shown: the blank group had basically no killing effect on EBV-positive nasopharyngeal carcinoma C666 TK / EBV-positive gastric cancer cell AGS, while the egg yolk globulin treatment group had a significant killing effect on target cells A549 / EBV-positive gastric cancer cell AGS. The above experiments prove that the specific egg yolk globulin of the present invention can be effectively used for the treatment of EBV-positive nasopharyngeal carcinoma and gastric cancer, and has great application prospects.

[0036] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An egg yolk globulin for relieving rhinitis, characterized in that: The light chain of the egg yolk globulin is shown in SEQ ID NO.1, and the heavy chain of the egg yolk globulin is shown in SEQ ID NO.

2.

2. The egg yolk globulin according to claim 1, characterized in that The light chain is encoded by the sequence shown in SEQ ID NO.3, and the heavy chain is encoded by the sequence shown in SEQ ID NO.

4.

3. A method for preparing egg yolk globulin for relieving rhinitis, characterized in that: The steps include: (1) Preparation of immune antigens: Prepare proteins containing the extracellular region sequences of IL-4, IL-5, IL-4Rα and IL-5Rα, and dissolve them in physiological saline in a mass ratio of (1-3):(1-5):(1-3):(1-5) as immune antigens; (2) Immunization procedure for laying hens: 1-5 mL of the immunization antigen in step (1) is added to an equal volume of Freund's complete adjuvant and mixed thoroughly to form an emulsion. After disinfection, 1.5-3 mL of the emulsion is distributed and injected intramuscularly at 4-6 points under the breast and wings of the laying hens for the first immunization; then 1-5 mL of the immunization antigen in step (1) is added to an equal volume of Freund's incomplete adjuvant and mixed thoroughly for two booster immunizations at intervals of one week; one week after the booster immunization, the eggs are collected and stored at 4°C for later use; (3) Separation and purification of specific egg yolk globulin: separate and collect egg yolk liquid, add 5-10 times the volume of physiological saline, then add 10-15% (w / v) PEG-6000, let stand for 30-60 minutes, centrifuge at 3000-5000g for 20-50 minutes; filter the supernatant with absorbent cotton, and add ammonium sulfate to the supernatant to make the saturation of ammonium sulfate reach 45-55%, and let stand at 4°C for 6-12 hours; centrifuge after standing, discard the supernatant to obtain a precipitate, add 10-20mL of physiological saline per gram of precipitate to dissolve it to obtain a solution; put the solution into a dialysis bag and dialyze with distilled water to remove ammonium ions, and use physiological saline for the last dialysis; sterilize and filter the dialyzate to obtain a specific egg yolk globulin extract; after freeze-drying, store it at -80°C for later use; (4) The specific yolk globulin prepared in step (3) is sent to Beijing Anbiqi Biological Co., Ltd. for sequencing analysis.

4. The method according to claim 3, characterized in that Step (1) Preparation of immune antigens: Isolate the extracellular domain sequences of IL-4, IL-5, IL-4Rα and IL-5Rα, and dissolve them in physiological saline in a mass ratio of 1:1:1:1 as immune antigens.

5. The method according to claim 3, characterized in that Step (2) immunization procedure for laying hens: 1 mL of the immunization antigen of step (1) is added to an equal volume of Freund's complete adjuvant and mixed thoroughly to form an emulsion. After disinfection, 1.5 mL of the emulsion is distributed and injected intramuscularly at 6 points under the breast and wings of the laying hens for the first immunization; then 1 mL of the immunization antigen of step (1) is added to an equal volume of Freund's incomplete adjuvant and mixed thoroughly and boosted twice at intervals of 1 week; 1 week after the booster immunization, the eggs are collected and stored at 4°C for later use.

6. The method according to claim 3, characterized in that Step (3) separation and purification of specific egg yolk globulin: separate and collect egg yolk liquid, add 5 volumes of physiological saline, then add 10% (w / v) PEG-6000, let stand for 30 minutes, centrifuge at 3000g for 20 minutes; filter the supernatant with absorbent cotton, and add ammonium sulfate to the supernatant to make the saturation of ammonium sulfate reach 45%, let stand for 6 hours at 4°C; centrifuge after standing, discard the supernatant to obtain a precipitate, add 10mL of physiological saline per gram of precipitate to dissolve it to obtain a solution; put the solution into a dialysis bag and dialyze with distilled water to remove ammonium ions, and use physiological saline for the last dialysis; sterilize and filter the dialyzate to obtain a specific egg yolk globulin extract; after freeze-drying, store it at -80°C for use.

7. Use of the egg yolk globulin according to claim 1 or 2 in the preparation of a drug for relieving rhinitis.

8. Use of the egg yolk globulin according to claim 1 or 2 in the preparation of a product for treating gastric cancer or nasopharyngeal cancer.

9. A product for relieving rhinitis or treating gastric cancer or nasopharyngeal cancer, characterized in that: It comprises the egg yolk globulin according to claim 1 or 2.

10. The product according to claim 9, characterized in that The gastric cancer or nasopharyngeal cancer is selected from EBV-positive gastric cancer or nasopharyngeal cancer.

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