Bambusa utilis pollen allergen extract, its preparation method and application

Abstract

The present application relates to the technical field of biology, and particularly relates to a broussonetia papyrifera pollen allergen extract, a preparation method and application thereof.The present application provides a broussonetia papyrifera pollen allergen extract, which comprises a peptide segment composition; and the amino acid sequence of the peptide segment composition is shown as SEQ ID NO:1 to SEQ ID NO:104.The present application provides a commercialized broussonetia papyrifera allergy diagnosis reagent, which can meet the diagnosis and treatment requirements of broussonetia papyrifera pollen allergy patients in the clinic.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to mulberry pollen allergen extract, its preparation method, and its application. Background Technology

[0002] Allergic diseases are a range of illnesses caused by an excessive immune response of the body to normally harmless substances in the environment. These include allergic rhinitis (AR), allergic conjunctivitis, allergic asthma, and food allergies. AR is one of the most common allergic diseases. Wind-borne pollen is one of the most prevalent airborne allergens. Pollen allergy (also known as hay fever) primarily manifests as seasonal AR, which may be accompanied by allergic conjunctivitis and / or allergic asthma.

[0003] Our team used active sampling to monitor airborne pollen in Chengdu throughout the year, and identified the absolute dominant pollen species of Broussonetia papyrifera, which accounted for 70.84% ​​of the total annual pollen. It is the absolute dominant species, and its pollination period is concentrated in March and April.

[0004] Due to its significant economic value (as feed, paper, and furniture raw material) and strong vitality, both artificial planting and natural growth have promoted its production and distribution. In my country, paper mulberry is mainly distributed in the southwest, south, and southeast regions.

[0005] Because of the high concentration of paper mulberry pollen in the air, the proportion of patients with paper mulberry pollen allergies is also relatively high. A few studies have reported a sensitization rate of 50.7% among patients with allergic rhinitis in Pakistan and 38.4% among student volunteers in Taiwan. However, due to the current lack of commercially available diagnostic reagents for paper mulberry allergies globally, reports on the sensitization of paper mulberry pollen are very limited.

[0006] Clinically, allergy diagnosis is primarily accomplished through in vivo testing and serological examination. In vivo testing includes skin prick tests (SPT) and nasal allergen challenges (NAC), which involve introducing trace amounts of crude extracts of the sensitizing agent into the patient via various methods and observing the occurrence of allergic reactions in the skin and nasal cavity. Serological testing involves coating the crude extract of the sensitizing agent onto different solid media and using different methods to detect the binding of specific immunoglobulin E (sIgE) in the patient's serum to the allergen in the crude extract. Therefore, there is an urgent need for standardized methods to prepare crude extracts of mulberry pollen and to perform quality control and diagnostic efficacy verification for clinical application in the diagnosis of mulberry pollen allergy. Summary of the Invention

[0007] In view of this, the present invention provides a paper mulberry pollen allergen extract, its preparation method, and its application. The present invention also provides a commercially available diagnostic reagent for paper mulberry allergy, which can meet the clinical needs of patients with paper mulberry pollen allergy.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention provides a mulberry pollen allergen extract comprising: a peptide composition; the amino acid sequence of the peptide composition is shown in SEQ ID NO:1 to SEQ ID NO:104.

[0010] The present invention also provides a method for preparing the above-mentioned mulberry pollen allergen extract, wherein the mulberry pollen is defatted, extracted, centrifuged, the supernatant protein solution is collected, dialyzed, filtered, and the mulberry pollen allergen extract is obtained.

[0011] In some embodiments of the present invention, in the above preparation method, the degreasing is performed using petroleum ether at 30-60°C.

[0012] In some embodiments of the present invention, the extraction in the above preparation method uses 10 mM PBS buffer.

[0013] In some embodiments of the present invention, the pH value of the PBS buffer in the above preparation method is 7.0.

[0014] In some embodiments of the present invention, in the above preparation method, the mass-to-volume ratio of the defatted mulberry pollen to the PBS buffer is 1:10.

[0015] In some embodiments of the present invention, in the above preparation method, the centrifugation speed is 15000g and the time is 30 min.

[0016] In some embodiments of the present invention, in the above preparation method, the dialysis time is 12 hours and the temperature is 2~8°C.

[0017] The present invention also provides the application of the above-mentioned peptides, the above-mentioned mulberry pollen allergen extract and / or the mulberry pollen allergen extract obtained by the above preparation method in the preparation of products for diagnosing and / or detecting allergic diseases induced by mulberry pollen.

[0018] This invention also provides the application of the above-mentioned peptides, the above-mentioned mulberry pollen allergen extract, and / or the mulberry pollen allergen extract obtained by the above preparation method in the preparation of products for detecting serologically specific IgE.

[0019] In some embodiments of the present invention, in the above applications, the dosage form of the product includes one or more of the following: injection, aerosol, spray, drops, and skin prickling agent.

[0020] The present invention also provides a product comprising: the above-described peptide, the above-described mulberry pollen allergen extract and / or the mulberry pollen allergen extract obtained by the above-described preparation method, and acceptable adjuvants and / or excipients.

[0021] In some embodiments of the present invention, the above-mentioned products further include: detection reagents and / or kits. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0023] Figure 1 The appearance of paper mulberry pollen under an optical microscope (400×), with yellow arrows indicating paper mulberry pollen;

[0024] Figure 2 The appearance of Broussonetia papyrifera pollen under a scanning electron microscope (10000×), with red arrows indicating germination pores;

[0025] Figure 3 SDS-PAGE image of crude extract of Broussonetia papyrifera pollen; where: left lane: protein marker; right lane: Broussonetia papyrifera pollen protein;

[0026] Figure 4 Schematic diagram showing the skin reaction (++++) of SPT patients with crude extract of Broussonetia papyrifera pollen;

[0027] Figure 5 The serum sIgE of the paper mulberry pollen allergy group and the healthy control group was detected by ELISA. The positive rate was 93.3% (28 / 30), and the horizontal axis represents each patient. Detailed Implementation

[0028] This invention discloses a paper mulberry pollen allergen extract, its preparation method, and its application.

[0029] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0030] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0031] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0032] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0033] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0034] In Examples 1 to 7 of this invention, all raw materials and reagents used can be purchased from the market.

[0035] The method for preparing paper mulberry pollen provided by this invention is merely an example. For those skilled in the art, any reagents and methods that can achieve the effects of extraction and defatting can be used to prepare the paper mulberry pollen of this invention.

[0036] The present invention will be further illustrated below with reference to the embodiments:

[0037] Example 1: Collection of Broussonetia papyrifera pollen

[0038] Pollen from paper mulberry trees was collected, impurities were removed by passing it through a 180-mesh sieve, and its appearance was observed using an optical microscope and a scanning electron microscope for morphological identification.

[0039] The procedure for observation under an optical microscope is as follows: Spread pollen onto a glass slide, add one drop of basic fuchsin pollen stain as a mounting solution, and then cover with a coverslip. After the pollen has stained, observe it under a 400x optical microscope. The morphology of paper mulberry pollen under an optical microscope is as follows: Figure 1 As shown.

[0040] The scanning electron microscope (SEM) sample preparation was as follows: Pollen was mixed in 3% glutaraldehyde by blowing and stirring, fixed at 2-8℃ for 12 hours, and washed three times with ultrapure water for 10 minutes each time. It was then fixed with 1% osmium tetroxide at 2-8℃ for 1 hour, followed by washing three times with ultrapure water for 10 minutes each time. The sample was then dehydrated stepwise with 30%→50%→70%→80%→90%→95%→100% ethanol for 15 minutes each time, repeating the process three times with 100% concentration. The dried sample was then adhered to a sample holder with conductive adhesive, subjected to ion sputtering, and then observed and images were acquired using a scanning electron microscope. The morphology of Broussonetia papyrifera pollen under the scanning electron microscope is as follows: Figure 2 As shown.

[0041] Example 2 Preparation of crude extract of Broussonetia papyrifera pollen

[0042] Add 10 times the volume of petroleum ether (boiling range 30-60°C) to the paper mulberry pollen obtained in Example 1 for defatting. After placing it on a shaker at room temperature for 2-5 hours, let it stand and discard the upper layer of petroleum ether. Repeat this process 3-5 times until the petroleum ether is no longer turbid. Discard the petroleum ether and place the pollen in a fume hood to allow the remaining petroleum ether to completely evaporate.

[0043] Weigh 1 gram of defatted pollen and add it to 10 mL of pH 7.0, 10 mM PBS buffer. Incubate at 2–8 °C for 12 h on a shaker. Then, centrifuge the suspension at 15000 × g for 30 min at 4 °C and collect the supernatant protein solution. Place the solution in a dialysis bag with a molecular weight cutoff of 1 kDa, and then in 5 L of pH 7.0, 10 mM PBS buffer. Dialyze at 2–8 °C for 12 h, followed by filtration through a 0.45 μm filter membrane. Measure the protein concentration in the crude pollen extract using the BCA method.

[0044] Example 3: Polyacrylamide gel electrophoresis for the detection of proteins in crude extracts

[0045] Pollen proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 5× denaturing-reducing protein loading buffer was added to 4 volumes of crude pollen extract and mixed thoroughly. The mixture was boiled at 95°C for 10 min, centrifuged, and then a sample containing 100 μg of protein was added to each well of the PAGE gel. The gel was then placed in Tris-MOPS-SDS electrophoresis buffer and electrophoresed at 140 V for 60 min. After electrophoresis, the gel was washed with ultrapure water, stained with Coomassie brilliant blue solution, and stained on a shaker for 30 min. The gel was then destained with tap water before imaging.

[0046] Depend on Figure 3 It can be seen that the protein distribution in the crude extract of Broussonetia papyrifera pollen is mainly in the range of 10-100 kDa.

[0047] Example 4: Mass Spectrometry Detection

[0048] Mass spectrometry was used to detect the total protein content of the crude extract of Broussonetia papyrifera pollen, and characteristic peptides with high abundance were obtained.

[0049] Table 1

[0050]

[0051]

[0052]

[0053] Example 5: Skin prick test

[0054] Before performing SPT, patients should discontinue the use of H1 receptor antagonists, omalizumab, phenothiazines, etc. The SPT procedure is as follows: Dilute the crude extract of Broussonetia papyrifera pollen obtained in Example 2 to 2 mg / mL with sterile PBS solution. Disinfect the palmar side of the subject's forearm with 75% ethanol. After the ethanol evaporates, apply approximately 10 μL of prick test kit to the skin surface. Use a metal prick test needle to gently prick the skin beneath the droplet, allowing a small amount of prick test kit to enter the epidermis. Avoid excessive force during prick testing to prevent significant bleeding. Simultaneously, use the prick test kit solvent and 10 mg / mL histamine solution as negative and positive controls, respectively. Blot away any remaining solution with absorbent paper. After 15 minutes, interpret the SPT results and calculate the skin index. Skin index = (longest diameter of wheal + corresponding vertical diameter) / 2, graded according to the following criteria: 0.3 ≤ + < 0.5; 0.5 ≤++ < 1.0; 1.0 ≤+++ < 2.0; 2.0 ≤ ++++. The SPT results are shown in Table 2. Examples of patients with strongly positive skin reactions (++++) are shown below. Figure 4 .

[0055] Example 6 Nasal mucosal provocation test

[0056] Outside of the mulberry pollen season, the "gold standard" nasal allergen challenge (NAC) test was performed on patients. Patients were instructed to discontinue any medications that might affect the results before testing. The procedure was as follows: The crude mulberry pollen extract obtained in Example 2 was diluted to 0.5 mg / mL with sterile PBS solution. Approximately 100 μL of the crude mulberry pollen extract was sprayed onto the inferior turbinate mucosa of both nostrils of the subject sequentially using the solvent (Shanxi Taiyuan Pharmaceutical Co., Ltd., allergen (antigen) solvent), diluted 1:10000, 1:1000, 1:100, and 1:10, until a positive result was obtained or the challenge reagent reached its highest concentration. Subjective symptoms were assessed using the Lebel score before the test and 15-20 minutes after each administration. Objective nasal ventilation was measured bilaterally using a four-phase nasal resistance meter. A Lebel score increase of ≥5 points compared to baseline was considered a subjective positive result; an increase in nasal resistance of ≥100% or a decrease in nasal ventilation of ≥40% was considered an objective positive result. A positive result for NAC is determined when both subjective and objective results are positive. The results are shown in Table 2.

[0057] Table 2 Summary of NAC results for crude extracts of Broussonetia papyrifera pollen

[0058]

[0059] Example 7 Enzyme-linked immunosorbent assay

[0060] The binding capacity of crude mulberry pollen protein to sIgE was quantitatively determined using an indirect enzyme-linked immunosorbent assay (ELISA). The crude pollen extract solution was diluted to 40 μg / mL with 50 mM carbonate buffer (pH 9.4). 100 μL / well of coating buffer (i.e., 4 μg pollen protein per well) was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed 5 times before detection, with 300 μL / well each time. 100 μL of 5% bovine serum albumin (BSA) solution was added to each well and the plate was blocked at 37°C for 1 h. After 5 washes, 100 μL of 1:10 diluted subject serum was added to each well and the plate was incubated at 37°C for 1.5 h. After washing the plate five times, add 100 μL of HRP-conjugated anti-human IgE antibody diluted 1:5000 to each well and incubate at 37°C for 1 h. Add 3,3',5,5'-tetramethylbenzidine (TMB) substrate for color development for 5-30 min, then add TMB stop solution to stop the reaction and perform analysis as soon as possible. A positive serum sIgE result is defined as: the optical density (OD) of the test sample is greater than twice the average OD value of healthy control serum. Figure 5The serum sIgE of the paper mulberry pollen allergy group and the healthy control group was detected by ELISA, and the positive rate was 93.3% (28 / 30).

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Broussonetia papyrifera pollen allergen extract, characterized in that, Comprise: peptide segment composition; amino acid sequences of the peptide segment composition are shown as SEQ ID NO: 1 to SEQ ID NO: 104; the preparation method of the broussonetia papyrifera pollen allergen extract comprises the following steps: after defatting the broussonetia papyrifera pollen, extracting, centrifuging, collecting the upper protein solution, dialysis, filtering, obtaining the broussonetia papyrifera pollen allergen extract; the defatting adopts petroleum ether with a boiling range of 30~60℃; the extraction adopts PBS buffer solution with a concentration of 10 mM and a pH value of 7.0; the centrifugation speed is 15000g, and the time is 30 min; the dialysis time is 12h, the temperature is 2~8℃, the dialysis adopts PBS buffer solution with a pH value of 7.0 and a concentration of 10 mM, and the dialysis molecular weight cut-off is 1kDa.

2. The use of the broussonetia papyrifera pollen allergen extract according to claim 1 in the preparation of products for diagnosing and / or detecting broussonetia papyrifera pollen-induced allergic diseases.

3. Use according to claim 2, wherein the compound is ###0002### The use of the broussonetia papyrifera pollen allergen extract in the preparation of products for detecting serological specific IgE.

4. The use according to claim 2, wherein the compound is ###0002### The dosage form of the product is one or more of the following: spray, drop and skin prick.

5. A product characterised in that, Comprise: the broussonetia papyrifera pollen allergen extract according to claim 1 and acceptable adjuvants and / or excipients.

6. The product of claim 5, wherein, The product is a detection reagent and / or a detection kit.

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