Preparation method and application of specific IgG antibody of scylla paramamosain allergen Scy p1 hyposensitization derivative

Abstract

The invention provides a preparation method and application of a scylla paramamosain allergen Scy p1 hypoallergenic derivative specific I gG antibody, a New Zealand white rabbit is subjected to immune treatment by using a Scy p1 hypoallergenic derivative, and antiserum is purified by using a Protei n A Sepharose affinity chromatography column, so that the purity of the antibody is greatly improved, and the obtained I gG antibody is ensured to have high specificity; the purified antibody is subjected to molecular weight detection through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), and an immunoglobulin heavy chain and an immunoglobulin light chain containing 50kDa and 25kDa are defined, so that the quality of the IgG antibody is verified, and a reliable basis is provided for subsequent application; the obtained specific I gG antibody effectively blocks combination of Scy p 1 and I gE in whole blood of a patient in vitro, effectively improves allergic symptoms of mice in vivo, ensures that the prepared I gG antibody is safer and is expected to be used for immunotherapy, and side effects possibly occurring in the treatment process are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of food allergies, and in particular to a method for preparing a specific IgG antibody of a low-allergenic derivative of a mud crab allergen Scy p 1 and an application thereof. Background Art

[0002] Crustaceans are considered to be a rich source of high-quality protein, polyunsaturated fatty acids and vitamins, and play a vital role in dietary balance, but reports of food allergic reactions caused by eating crustaceans are common.

[0003] Aquatic product allergy is caused by the intake of aquatic product allergens. Avoiding contact with allergens is the most effective way to prevent allergies. However, accidental contact with allergenic foods is quite common. Therefore, it is crucial to develop effective prevention and control methods. In recent years, the induction of allergen-specific blocking IgG antibodies has been identified as one of the main mechanisms in the study of food allergy prevention and control. The concept of "blocking antibodies" is that IgG antibodies compete with serum IgE antibodies for allergens and block the formation of allergen-IgE complexes, effectively preventing cross-linking with the high-affinity receptor FcεRI on effector cells and inhibiting the release of histamine. Clinical studies of patients with inhalation allergies also emphasized that the production of "blocking antibodies" and IL-10 cytokines contributes to the long-term tolerance of patients. In addition, specific IgG antibodies induced by low-allergenic derivatives of allergens can effectively avoid potential allergic risks. Therefore, inducing the production of aquatic product allergen blocking antibodies and exploring their ability to improve allergic reactions is an important research direction in this field in the future.

[0004] In recent years, specific IgG blocking antibodies induced by hypoallergenic derivatives of allergens have been studied in fish, shrimp and other allergic proteins. Among crustacean aquatic products, Scylla paramamosain, also known as blue crab, has an annual aquaculture output that increases year by year. It is widely loved by people because of its rich nutrition, but the allergic events caused by it are also increasing. Unlike other food allergies, crustacean aquatic product allergies usually accompany lifelong, seriously affecting the quality of life of allergic people. Scy p 1 is the main allergen of blue crab, and as a pan-allergen in invertebrates, it can cause cross-reactions between different species. In view of this, this application is specially proposed. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing a specific IgG antibody of a hypoallergenic derivative of the Scy p 1 allergen of the mud crab and its application, so as to solve the above problems.

[0006] The present invention adopts the following scheme:

[0007] The present application provides a method for preparing a specific IgG antibody of a hypoallergenic derivative of a Scy p 1 allergen of a mud crab, comprising the following steps:

[0008] S1: Rabbits were immunized with a low-allergenic derivative of the Scy p 1 allergen of Scyphus pseudoburrowingii at least three times;

[0009] S2: After collecting the rabbit antiserum, it was purified using a Protein A Sepharose affinity chromatography column;

[0010] S3: The purified rabbit antiserum was analyzed by SDS-PAGE. The IgG antibody had bands at 50 kDa and 25 kDa, which corresponded to the molecular weights of the heavy chain and light chain of immunoglobulin, respectively, to obtain specific IgG antibodies.

[0011] As further improvements, it also includes:

[0012] S4: The ability of Scy p 1-IgE binding to be blocked by Scy p 1-specific IgG antibodies was analyzed by basophil activation assay.

[0013] As a further improvement, the inhibitory basophil activation assay was used to evaluate the ability of the specific IgG antibody to block the binding of Scy p 1 to IgE based on the expression levels of basophil surface markers CD63 and CD203c.

[0014] As a further improvement, Scy p 1 that had reacted with specific IgG antibodies for a preset time was added to the patient's whole blood and incubated in a cell culture incubator at a preset temperature for more than 30 minutes to detect the expression levels of CD63 and CD203c on the surface of basophils, and then analyze the blocking ability of specific IgG antibodies.

[0015] As a further improvement, the preset time duration is not less than 30 minutes, and the preset temperature is within the normal temperature range of the human body.

[0016] As further improvements, it also includes:

[0017] S5: Further analysis of the ability of Scylla sylvaticum allergen-specific IgG blocking antibodies to improve allergic symptoms in sensitized mice.

[0018] As a further improvement, a Balb / c mouse treatment model was established, and specific IgG antibodies were injected before high-dose stimulation. The effect of specific IgG antibodies in improving allergic symptoms in mice was evaluated based on indicators such as pathological detection of the cecum and jejunum tissues of sensitized mice.

[0019] The present application further provides an IgG antibody specific for a hypoallergenic derivative of the Scy p 1 allergen of the mud crab, which is prepared according to the above-mentioned preparation method.

[0020] The present application also provides an application of a specific IgG antibody drug of a hypoallergenic derivative of the mud crab allergen Scy p 1, which can be used for immunotherapy or prevention of allergic diseases of crustacean aquatic products.

[0021] By adopting the above technical solution, the present invention can achieve the following technical effects:

[0022] 1. The method for preparing IgG antibodies specific to the hypoallergenic derivative of the mud crab allergen Scy p 1 of the present application is to immunize rabbits with the hypoallergenic derivative of Scy p 1 and purify the antiserum using a Protein A Sepharose affinity chromatography column, thereby greatly improving the purity of the antibody and ensuring that the obtained IgG antibody has high specificity.

[0023] 2. The molecular weight of the purified antibody was tested by SDS-PAGE, which clearly showed that the heavy and light chains of immunoglobulins were 50 kDa and 25 kDa, respectively. This verified the quality of the IgG antibody and provided a reliable basis for subsequent applications.

[0024] 3. Furthermore, the blocking effect of specific IgG antibodies was analyzed using an in vitro basophil activation test, and the ability of specific IgG blocking antibodies to improve allergic symptoms was analyzed using an in vivo mouse treatment model, which is expected to be used for the immunotherapy and prevention of allergic diseases caused by crustacean aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow chart of the method for preparing specific IgG antibodies of the hypoallergenic derivative of the mud crab allergen Scy p 1 according to an embodiment of the present invention;

[0026] Figure 2 The invention relates to a low-sensitivity derivative of the allergen Scy p 1 of the mud crab purified by SDS-PAGE analysis and a specific IgG antibody thereof; wherein:

[0027] M: molecular weight standard protein; Lane 1: purified low-sensitivity derivative of the mud crab allergen Scy p 1; Lane 2: purified specific IgG antibody.

[0028] Figure 3 The inhibitory basophil activation test of the embodiment of the present invention analyzes the blocking effect of specific IgG antibodies; wherein,

[0029] Figure 3A: The effect of specific IgG antibody on the ratio of CD63 positive cells on the surface of basophils;

[0030] Figure 3 B: Effect of specific IgG antibody on the ratio of CD203c positive cells on the surface of basophils;

[0031] NC group: negative control; wtScy p 1 group: positive control; Rabbit anti-mtScy p 1-High and Rabbit anti-mtScy p 1-Low groups: sample groups.

[0032] Figure 4 The mouse treatment model of the embodiment of the present invention is used to analyze the ability of specific IgG blocking antibodies to improve the allergic symptoms of sensitized mice; wherein,

[0033] Figure 4 A: Design of mouse therapeutic model;

[0034] Figure 4 B: Effects of specific IgG blocking antibodies on mouse feces and cecum;

[0035] Figure 4 C: Effect of specific IgG blocking antibody on inflammation of mouse jejunum tissue (200 times). DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0037] Example

[0038] Combination Figures 1 to 4 This embodiment provides a method for preparing a specific IgG antibody of a hypoallergenic derivative of the allergen Scy p 1 of the mud crab, comprising the following steps:

[0039] S1: Rabbits were immunized with a low-allergenic derivative of the Scy p 1 allergen of Scyphus pseudoburrowingii at least three times;

[0040] S2: After collecting the rabbit antiserum, it was purified using a Protein A Sepharose affinity chromatography column;

[0041] S3: The purified rabbit antiserum was analyzed by SDS-PAGE. The IgG antibody had bands at 50 kDa and 25 kDa, which corresponded to the molecular weights of the heavy chain and light chain of immunoglobulin, respectively, to obtain specific IgG antibodies.

[0042] In the above, the rabbits were immunized with low-sensitization derivatives of Scyp 1, and the antiserum was purified with Protein A Sepharose affinity chromatography column, which greatly improved the purity of the antibody and ensured that the obtained IgG antibody had high specificity and efficient blocking ability. The molecular weight of the purified antibody was detected by SDS-PAGE, and the 50kDa and 25kDa immunoglobulin heavy and light chains were clearly distinguished, so that the quality and specificity of the IgG antibody were verified, providing a reliable basis for subsequent applications.

[0043] Preferably, the effect is particularly significant by immunizing New Zealand white rabbits with a hypoallergenic derivative of Scy p 1.

[0044] In this embodiment, it also includes:

[0045] S4: The ability of Scy p 1-IgE binding to be blocked by Scy p 1-specific IgG antibodies was analyzed by basophil activation assay.

[0046] Specifically, the ability of the specific IgG antibody to block the binding of Scy p 1 to IgE was evaluated based on the expression levels of basophil surface markers CD63 and CD203c using an inhibitory basophil activation assay.

[0047] Furthermore, Scy p 1 that had reacted with specific IgG antibodies for a preset time was added to the patient's whole blood and incubated in a cell culture incubator at a preset temperature for more than 30 minutes to detect the expression levels of CD63 and CD203c on the surface of basophils, and then analyze the blocking ability of specific IgG antibodies.

[0048] Preferably, the preset time duration is not less than 30 minutes, and the preset temperature is within the normal temperature range of the human body.

[0049] In this embodiment, it also includes:

[0050] S5: Further analysis of the ability of Scylla sylvaticum allergen-specific IgG blocking antibodies to improve allergic symptoms in sensitized mice.

[0051] Specifically, a Balb / c mouse treatment model was established, and specific IgG antibodies were injected before high-dose stimulation. The effect of specific IgG antibodies in improving allergic symptoms in mice was evaluated based on indicators such as pathological detection of the cecum and jejunum tissues of sensitized mice.

[0052] Among them, the specific IgG antibody obtained effectively blocked the binding of Scy p 1 with IgE in the patient's whole blood in vitro, and effectively improved the allergic symptoms of mice in vivo, ensuring that the prepared IgG antibody is safer and is expected to be used in immunotherapy and reduce possible side effects during treatment.

[0053] In addition, this embodiment provides a specific IgG antibody of a hypoallergenic derivative of the Scy p 1 allergen of Scyphus pseudoburrowing, which is prepared according to the above-mentioned preparation method.

[0054] Furthermore, this embodiment also provides an application of a specific IgG antibody drug of a hypoallergenic derivative of the mud crab allergen Scy p 1, which can be used for immunotherapy or prevention of allergic diseases of crustacean aquatic products.

[0055] In this example, the purification of IgG antibodies specific to the low-sensitivity derivative of the mud crab allergen Scy p 1 is specifically as follows:

[0056] Purified low-sensitivity derivatives of Scy p 1 were immunized in New Zealand white rabbits three times, 1.5 mg / time. After immunization, rabbit serum was collected and purified using Protein A Sepharose affinity chromatography column: rabbit antiserum diluted with washing buffer (0.15 mol / L NaCl, 20 mmol / L Na2HPO4, pH 7.4) was loaded on pre-equilibrated Protein A Sepharose, and after washing the impurities with washing buffer, the target protein was eluted with elution buffer (0.1 mol / L citric acid, pH 2.5-3.0) and neutralized with 1 mol / L Tris-HCl solution (pH 8.5), and the protein concentration was determined using A280 nm.

[0057] SDS-PAGE analysis of the purified low-sensitivity derivatives of the mud crab allergen Scy p 1 and its specific IgG antibody. Figure 2 As shown in the figure, the molecular weight of the purified Scy p 1 low-sensitivity derivative is 35 kDa. The purified specific IgG antibody has obvious bands at 50 kDa and 25 kDa, corresponding to the immunoglobulin heavy chain and light chain, respectively, indicating that the specific IgG antibody has been successfully purified.

[0058] In this example, the inhibitory basophil activation test analyzes the blocking effect of specific IgG antibodies, specifically:

[0059] A basophil activation test was established. Different concentrations of specific IgG antibodies were reacted with Scy p 1 for 30 minutes. The reaction solution was then added to the whole blood of patients with crab allergy and incubated in a cell culture incubator at 37°C for 30 minutes. After the stimulation, the cells were collected by centrifugation and stained with fluorescent labeled antibodies (anti-human CD63, anti-human CD203c, anti-humanHLA-DR) in the dark. After staining, the cells were placed on a flow cytometer and the expression levels of CD63 and CD203c on the surface of basophils were analyzed.

[0060] like Figure 3 As shown in the results, wtScy p 1 can significantly upregulate the expression of CD63 (50.5%) and CD203c (46.3%) on the surface of basophils, while after wtScy p 1 reacted with different concentrations of specific IgG antibodies, the expression levels of CD63 (30.7%-36.2%) and CD203c (27.8%-33.2%) on the surface of basophils were significantly reduced, indicating that the specific IgG antibody effectively blocked the binding of Scy p 1 to IgE in the whole blood of patients.

[0061] In this example, the mouse treatment model was used to analyze the ability of specific IgG blocking antibodies to improve allergic symptoms in sensitized mice. Specifically:

[0062] Establishment of Balb / c mouse treatment model (such as Figure 4 As shown in A, female Balb / c mice were randomly divided into 4 groups, 6 in each group. Except for the PBS group, the other 3 groups of mice were intraperitoneally injected with wtScy p 1 + aluminum adjuvant on days 0, 7, 14, and 21, and 2 groups of mice were intraperitoneally injected with different concentrations of specific antibodies on days 28 to 30 for treatment; on days 35, 37, and 39, they were gavaged with wtScy p 1 for high-dose stimulation; on day 40, the mice were killed and the intestinal tissues were removed.

[0063] The results showed that the feces of mice in the PBS group were full and granular, while the feces of mice in the wtScy p 1 group were loose. The feces of mice treated with different concentrations of specific antibodies were normal in shape (e.g. Figure 4 (B) HE staining was used to observe the inflammation of the jejunum tissues of mice in each group.

[0064] The results are as follows Figure 4 As shown in C, the jejunal tissue of mice in the PBS group was normal, and the villi were arranged in an orderly manner; the jejunal tissue of mice in the wtScy p 1 group was severely inflamed, with pathological phenomena of villus atrophy and a large number of lymphocyte infiltration; after treatment with different concentrations of specific antibodies, the jejunal tissue of mice had relatively mild pathological changes. This result shows that specific antibodies have the ability to improve the allergic symptoms of mice.

[0065] Thus, by immunizing New Zealand white rabbits, we obtained specific IgG antibodies against the hypoallergenic derivative of the mud crab allergen Scy p 1, which effectively blocked the binding of Scy p 1 to IgE in the patient's whole blood. Moreover, in the mouse model, compared with the wild-type Scy p 1, the specific IgG antibodies against the Scy p 1 derivatives can effectively improve the allergic symptoms, and can be used to develop desensitization preparations for the prevention or treatment of crustacean allergic diseases.

[0066] The above are only preferred implementations of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention.

Claims

1. A method for preparing IgG antibodies specific to the hypoallergenic derivative of the Scy p 1 allergen of Scyphus pseudoburrowing, characterized in that: The steps include: S1: Rabbits were immunized with a low-allergenic derivative of the Scy p 1 allergen of Scyphus pseudoburrowingii at least three times; S2: After collecting the rabbit antiserum, it was purified using a Protein A Sepharose affinity chromatography column; S3: The purified rabbit antiserum was analyzed by SDS-PAGE. The IgG antibody had bands at 50 kDa and 25 kDa, which corresponded to the molecular weights of the heavy chain and light chain of immunoglobulin, respectively, to obtain specific IgG antibodies.

2. The method for preparing the IgG antibody specific to the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 1, characterized in that: Also includes: S4: The ability of Scy p 1-IgE binding to the allergen-specific IgG antibody of Scy p 1 was analyzed by basophil activation assay.

3. The method for preparing the IgG antibody specific to the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 2, characterized in that: The ability of the specific IgG antibody to block the binding of Scy p 1 to IgE was evaluated using an inhibitory basophil activation assay based on the expression levels of basophil surface markers CD63 and CD203c.

4. The method for preparing the specific IgG antibody of the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 3, characterized in that: Scy p 1 that had reacted with specific IgG antibodies for a preset time was added to the patient's whole blood and incubated in a cell culture incubator at a preset temperature for more than 30 minutes to detect the expression levels of CD63 and CD203c on the surface of basophils, and then analyze the blocking ability of specific IgG antibodies.

5. The method for preparing IgG antibody specific to the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 4, characterized in that: The preset time is not less than 30 minutes, and the preset temperature is within the normal temperature range of the human body.

6. The method for preparing the specific IgG antibody of the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 1, characterized in that: Also includes: S5: Further analysis of the ability of Scylla sylvaticum allergen-specific IgG blocking antibodies to improve allergic symptoms in sensitized mice.

7. The method for preparing IgG antibody specific to the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 6, characterized in that: A Balb / c mouse treatment model was established. Specific IgG antibodies were injected before high-dose stimulation. The effect of specific IgG antibodies in improving allergic symptoms in mice was evaluated based on indicators such as pathological detection of the cecum and jejunum tissues of sensitized mice.

8. An IgG antibody specific for the hypoallergenic derivative of the mud crab allergen Scy p 1, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 7.

9. The use of the specific IgG antibody drug of the hypoallergenic derivative of the mud crab allergen Scy p 1 according to claim 8 can be used for immunotherapy or prevention of allergic diseases of crustacean aquatic products.

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