WO2020186624A1 - 一种葎草花粉变应原浸提物、浸液及其制备方法 - Google Patents

一种葎草花粉变应原浸提物、浸液及其制备方法 Download PDF

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WO2020186624A1
WO2020186624A1 PCT/CN2019/088605 CN2019088605W WO2020186624A1 WO 2020186624 A1 WO2020186624 A1 WO 2020186624A1 CN 2019088605 W CN2019088605 W CN 2019088605W WO 2020186624 A1 WO2020186624 A1 WO 2020186624A1
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humulus
pollen allergen
seq
humulus pollen
ultrafiltration
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PCT/CN2019/088605
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English (en)
French (fr)
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尹佳
周俊雄
马武强
顾建青
李欣
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中国医学科学院北京协和医院
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Priority to KR1020217034016A priority Critical patent/KR20220002909A/ko
Priority to JP2021558495A priority patent/JP7394866B2/ja
Publication of WO2020186624A1 publication Critical patent/WO2020186624A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention relates to the field of biotechnology, in particular to a standardized allergen extract of Humulus humulus pollen, an extract and a preparation method thereof.
  • hay fever which is also called allergic rhinitis.
  • Noon and Freeman first used pollen extract to treat hay fever, and the history of treatment of allergic diseases began.
  • allergic diseases are one of the major global health problems. More than 25% of the population in industrialized countries are troubled by allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, among which allergic asthma is the most common. Inhalation of allergic pollen is the most important factor in allergic asthma and other respiratory allergic diseases.
  • Humulus scandens (Humulusscandens) belongs to the genus Humulus in the Rosaceae Moraceae.
  • Yin Jia "Study on the Relationship between Allergic Rhinitis and Allergic Asthma in Summer and Autumn Hay Fever Patients and Their Interrelationship” shows that Humulus hay fever accounts for 58% of Beijing’s summer and autumn hay fever patients; Zhao Jing’s "Children in Beijing The results of an epidemiological survey of respiratory allergic diseases in Beijing area for children aged 13-14 showed that 4.2% of humulus pollen sensitive persons; Ren Huali, "Adults in Beijing area” Analysis of inhaled allergen spectrum of allergic rhinitis” Selected 265 adult patients with allergic rhinitis among Beijing residents, and tested 16 standardized allergens in the two groups of perennial and seasonal inhalation, and analyzed the ranking of major allergens Among them, 18.11% of Humulus; Tian Fangjie’s Investigation of Inhaled Allergens of Allergic Rhinitis in Beijing Area investigated the main inhaled allergens of allergic rhinitis (AR) in Beijing and found that Hum
  • humulus pollen allergy is widely distributed throughout our country, and humulus pollen is the main allergen of pollen allergy in northern areas.
  • allergen-specific immunotherapy methods are mainly divided into subcutaneous injection immunotherapy and sublingual immunotherapy.
  • Subcutaneous injection of immunotherapy has a history of more than 100 years, and its safety and effectiveness have been proven.
  • the allergen sublingual drop vaccine was born.
  • the sublingual immune desensitization method has a longer treatment period than subcutaneous injection, and the curative effect is not significant in a short time.
  • the average desensitization period is 3-5 years.
  • the dose of sublingual immunodesensitization is 100 times that of subcutaneous injection. Therefore, compared with sublingual administration, subcutaneous injection of immune desensitization method has poor patient compliance, but the treatment cycle is shorter, the effect is quicker, and the treatment cost is lower. Therefore, the two modes of administration are complementary.
  • the stability of the humulus pollen allergen as a subcutaneous injection immune preparation is poor, and the protein content and biological potency will decrease over time.
  • the reagent is used to diagnose humulus pollen-induced allergic diseases after a certain period of time. At this time, the positive rate is reduced and the accuracy is low.
  • the present invention provides a humulus pollen-containing allergen extract, which has high specificity, sufficient extraction of humulus allergenic protein components and constant ratio, stable total biological potency, and expiration date. Long characteristics. It can be effectively used for skin prick test diagnosis and specific immunotherapy of allergic diseases, and can effectively diagnose allergic diseases induced by humulus pollen and perform specific immunotherapy on them.
  • a humulus pollen allergen extract which contains the humulus pollen allergen protein Humsc 1, which contains the amino acid sequence shown in SEQ ID NO.4;
  • Hum sc 2 which contains the amino acid sequence shown in SEQ ID NO.6;
  • Hum sc 3 which contains the amino acid sequence shown in SEQ ID NO.7;
  • Hum sc 4 which contains the amino acid sequence shown in SEQ ID NO.9;
  • Hum sc 5 which contains the amino acid sequence shown in SEQ ID NO.10;
  • Hum sc 6 which contains the amino acid sequence shown in SEQ ID NO.11;
  • Hum sc 7 which contains the amino acid sequence shown in SEQ ID NO.12;
  • Hum sc 8 which contains the amino acid sequence shown in SEQ ID NO.13;
  • Hum sc 9 which contains the amino acid sequence shown in SEQ ID NO.14;
  • Hum sc 10 which contains the amino acid sequence shown in SEQ ID NO.15;
  • Hum sc 11 which contains the amino acid sequence shown in SEQ ID NO.16;
  • Hum sc 12 which contains the amino acid sequence shown in SEQ ID NO.17;
  • Hum sc 13 which contains the amino acid sequence shown in SEQ ID NO.18;
  • Hum sc 14 which contains the amino acid sequence shown in SEQ ID NO.19;
  • Hum sc 15 which contains the amino acid sequence shown in SEQ ID NO.20.
  • Genes used to encode Humulus humulus pollen allergen extracts the genes encoding as shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9-20
  • the amino acid sequence corresponds to the sequence shown in SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 24, and SEQ ID NO. 26-37, respectively.
  • a humulus pollen allergen extract which contains the humulus pollen allergen extract as described above, a volume ratio of 0.2-0.4% phenol, and a volume ratio of 45- With 55% glycerin and 4.5-5.5g/L NaCl, the pH is 6.0-8.0.
  • the active concentration of the humulus pollen allergen is 50,000 to 200,000 BAU/ml
  • the total protein concentration of the humulus pollen allergen extract is 0.40 mg /ml ⁇ 1.60mg/ml
  • the allergen protein content of the humulus pollen is 0.08 ⁇ 0.32mg/ml.
  • the protein of the Humulus pollen allergen extract is mainly distributed in 10kDa (specific molecular weight 6.3kDa, 7.0kDa, 8.09kDa, 8.8kDa, 9.6kDa, 10.03kDa , 10.22kDa), 11.8kDa, 12.3kDa, 13.5kDa, 15.2kDa, 15.32kDa, 16.45kDa, 16.61kDa, 19kDa (specifically 19.04kDa, 19.18kDa, 19.99kDa), 28kDa, 37kDa, 42kDa, 45kDa, 52kDa, 66kDa, 76kDa.
  • the method for preparing the humulus pollen allergen infusion as described above includes the following steps:
  • step S4 Take the extract after step S3, centrifuge to obtain the supernatant; filter and sterilize the supernatant;
  • step S2 the pollen degreasing is degreasing treatment by pollen and acetone at a weight g volume ml ratio of 1:5 to 1:1, and repeat degreasing until the upper liquid is clear.
  • step S4 the conditions of the centrifugation are 8000 ⁇ 12000g, the centrifugation temperature is 2 ⁇ 8°C, and the time is 15 ⁇ 20min; the filtration and sterilization filtration is carried out with 4000 mesh first. After the filter cloth is filtered, it is filtered through cardboard filter, 0.45 ⁇ m and 0.22 ⁇ m filter membranes in sequence.
  • the ultrafiltration concentration is ultrafiltration with a 3KD ultrafiltration membrane.
  • the ultrafiltration is stopped; The total protein content of the permeate is >0.02mg/ml.
  • the ultrafiltration membrane After replacing the ultrafiltration membrane, repeat the ultrafiltration of the ultrafiltration permeate until the total protein content of the ultrafiltration permeate is ⁇ 0.02mg/ml.
  • the vacuum freeze-drying conditions are freezing at -50 to -35°C, drying at -25°C under a vacuum pressure of 2 to 8 mbar, and controlling the moisture content to be ⁇ 3%.
  • the step S1 further includes the steps of microscopic identification and/or DNA identification of the raw humulus pollen, wherein the DNA identification method is SEQ ID NO.1, SEQ ID NO. 2 is a primer, PCR amplifies the raw material of the identified Humulus humulus pollen, and detects the amplified product.
  • the DNA identification method is SEQ ID NO.1
  • SEQ ID NO. 2 is a primer
  • PCR amplifies the raw material of the identified Humulus humulus pollen, and detects the amplified product.
  • the phosphate-saline buffer includes 4.5-5.5g/L sodium chloride, 0.03%-0.05% potassium dihydrogen phosphate, 1.5%-2% phosphoric acid dodecahydrate Disodium hydrogen and 0.2% to 0.4% phenol.
  • the extract used includes: 0.8% sodium chloride, disodium hydrogen phosphate-citric acid buffer of pH 5.0, disodium hydrogen phosphate-citric acid buffer of pH 6.0, dibasic hydrogen phosphate of pH 7.5 Sodium-sodium dihydrogen phosphate buffer, pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.2 coca's solution, pH 8.9 Tris-hydrochloric acid buffer, pH 7.9-8.2 containing 4.5 ⁇ 5.5g/L sodium chloride, 0.03% ⁇ 0.05% potassium dihydrogen phosphate, 1.5% ⁇ 2% sodium hydrogen phosphate dodecahydrate, 0.2% ⁇ 0.4% phenol, etc.
  • the present invention also provides a freeze-dried humulus pollen allergen, which is prepared by the steps including humulus pollen collection-drying-degreasing-extraction-ultrafiltration concentration-freeze-drying:
  • Ultrafiltration concentration the filtered extract is ultrafiltered with a 3KD ultrafiltration membrane, and the ultrafiltration concentrate and ultrafiltration permeate are sampled to detect the total protein content; among them, when the ultrafiltration permeate is If the total protein content is less than 0.02mg/ml, the permeate is directly discarded; if the total protein content of the ultrafiltration permeate is >0.02mg/ml, the integrity test of the ultrafiltration membrane is performed. If the ultrafiltration membrane is not damaged, then discard Permeate: If the ultrafiltration membrane is damaged, replace the ultrafiltration membrane and repeat the ultrafiltration of the permeate;
  • the application of the humulus pollen allergen extract, the humulus pollen allergen extract, and the lyophilized product of the humulus pollen allergen as described above are used in the preparation and diagnosis of allergic diseases and their specificity Application in immunotherapy preparations, the diagnosis of allergic diseases includes allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.
  • the lyophilized product of the humulus pollen allergen extract is used to prepare tablets or orally disintegrating tablets, and the humulus pollen allergen extract is used to prepare injections, sublingual drops, allergens The original patch, allergen infusion diluent.
  • the humulus pollen allergen extract provided by the invention is used to prepare a medicine for treating humulus pollen allergic diseases.
  • the humulus pollen allergen extract or freeze-dried product provided by the present invention can be prepared for treating humulus humulus according to a therapeutically effective amount or a diagnostically effective amount of humulus pollen allergen and a pharmaceutically acceptable carrier. Medicine for allergic diseases of grass pollen.
  • the medicine for treating Humulus humulus pollen allergic disease can be made into various medically acceptable dosage forms, and the doctor can determine the beneficial dosage for the patient according to factors such as the patient's age, weight and general disease condition.
  • the dosage form of the medicine for treating Humulus pollen allergic diseases is selected from liquid dosage forms such as oral agents, (subcutaneous) injections, sublingual preparations, aerosols, nasal agents, or skin pricks; preferably (subcutaneous) injections , Sublingual oral agent, or skin prickly agent.
  • (subcutaneous) injections and sublingual oral preparations are generally used as dosage forms for specific immunotherapy, and skin pricks are commonly used dosage forms for in vivo allergen detection.
  • the humulus pollen allergen extract of the present invention is used to diagnose allergic diseases caused by humulus pollen (that is, the allergen skin prick diagnosis test), in addition to the humulus pollen allergen extract of the present invention, Generally, it should also include a negative control solution, a positive control solution, and a needle.
  • the negative control solution is generally a liquid that has no allergic reaction to the human body (for example, a mixture of glycerin and salt solution, etc.)
  • the positive control solution is generally a 1.0-5.0 mg/ml histamine phosphate/histamine hydrochloride solution.
  • the humulus pollen allergen extract prepared by the invention can be used in patch tests.
  • the principle is: apply a suspicious sensitizer (allergen) to the skin of the patient, enter the body through the skin or mucous membrane, and present the antigen to the T lymphocytes by the antigen presenting cells, which activates the specific T lymphocytes and induces inflammation reaction.
  • allergen a suspicious sensitizer
  • Humulus humulus is the main pollen allergen in northern regions.
  • the standardized allergen extract of humulus pollen provided by the present invention can effectively diagnose allergic diseases induced by humulus pollen and perform specific immunotherapy on it.
  • Adding stabilizer glycerin improves the stability and slow-release effect, and improves the effectiveness and safety of medication.
  • Subcutaneous injection of immune desensitization administration method for allergic diseases compared with sublingual drops administration method, has quicker effect and shorter period.
  • the dose administered throughout the treatment cycle is much less than the dose of sublingual drops (about 100 times), and the treatment cost is much less than that of sublingual drops for immunodesensitization, which reduces the medical burden of patients.
  • the present invention uses the prepared strain original infusion to do prick test with the original solution to compare with the clinical comprehensive specific diagnosis of allergy specialists and serum specific IgE (specific IgE, sIgE) diagnosis, and the intradermal test results are compared with the clinical Comprehensive specific diagnosis and consistent with the results of serum sIgE diagnosis, with good sensitivity and specificity, and good safety.
  • the humulus pollen allergen extract provided by the present invention has the characteristics of high specificity, sufficient extraction of humulus allergenic protein components, stable total biological potency, long validity period and good sterile effect; it can be effectively used for allergic diseases
  • the skin prick test diagnosis and specific immunotherapy can effectively diagnose allergic diseases induced by Humulus pollen and perform specific immunotherapy on it. Standardized control can be realized, the validity period of use can be effectively extended, and it can bring better economic benefits.
  • the present invention uses the prepared allergen extract diluted at 1:10 3 to 10 8 to do an in vitro basophil activation test, which can clinically specifically diagnose patients with humulus allergy and avoid false positive detection of sIgE in vitro At the same time, it can also avoid the risk of anaphylactic shock caused by allergen skin tests (prick or intradermal) to some Artemisia pollen allergy patients.
  • the infusion obtained by the method of the invention is more stable, the biological potency and protein content are relatively stable, and the validity period is 3 years.
  • Figure 1 shows the results of PCR electrophoresis of Humulus humulus pollen material ITS2 sequence.
  • Figure 2 shows the changing trend of fat content in different batches of Humulus pollen at different degreasing time.
  • Figure 3 shows the relationship between total protein content of Humulus pollen extract and extraction time.
  • Figure 4 shows the results of SDS-PAGE protein electrophoresis of Humulus humulus pollen allergen extract (indicating component identification).
  • Figure 5 shows the results of Western Blotting reaction between Humulus pollen allergen extract and serum of Humulus allergic patients (indicating the identification of Humulus allergen components).
  • Figure 6 shows the newly identified Humulus pollen allergenic protein 2D gel image (a) and Western Blotting image (b).
  • Figure 7 shows the test results of the pH value of Humulus pollen allergen extract in the long-term stability test.
  • Figure 8 shows the phenol content of Humulus pollen allergen extract product in the long-term stability test.
  • Figure 9 shows the test results of the sodium chloride content of the Humulus pollen allergen extract product in the long-term stability test.
  • Figure 10 shows the test results of the total protein content of Humulus pollen allergen extract in the long-term stability test.
  • Figure 11 shows the test results of the total strain of Humulus pollen allergen infusion product in the long-term stability test.
  • Figure 12 shows the test results of the main allergenic protein content Hum s 3 of Humulus pollen allergen extract in the long-term stability test.
  • Figure 13 is an example of the results of an in vitro basophil activation test of Humulus pollen allergen extract products applied to clinical Humulus pollen allergy patients.
  • this example uses DNA-specific sequence detection and microscopic examination to double identification and quality control of Humulus raw materials.
  • Tiangen rapid DNA extraction and amplification kit (Tiangen Biochemical KG203).
  • Extraction method Weigh 5 mg of Humulus pollen sample into a 1.5mL centrifuge tube, add Buffer 1 100 ⁇ l, grind the sample thoroughly with a disposable grinding pestle, add Buffer 2 100 ⁇ l, shake and mix, centrifuge at 12000r/min for 5min Then take the supernatant and place it in a new centrifuge tube as a DNA template for later use.
  • the primer sequence is as follows:
  • Hum ITS2-F (SEQ ID NO.1): 5'-CTGAGAAACGGCTACCACATC-3'
  • Hum ITS2-R (SEQ ID NO.2): 5'-GTCGGCCAAGGCTATAAACTC-3'
  • the primer sequence was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. After synthesis, aliquot and store at -20°C.
  • Humulus pollen (g) and acetone (ml) are degreased at a weight-to-volume ratio (w/v) of 1:2. Different batches (different collection times) of Humulus pollen samples with different degreasing time were tested for fat content to determine the optimal degreasing time.
  • the fat content in Humulus humulus pollen was measured by the Haineng SOX500 fat analyzer.
  • the changes in the weight of the pollen before and after extraction were compared by Sox-type extraction and dry weighing methods to obtain the corresponding fat content, and the fat content was compared after degreasing. Pollen is subject to quality control.
  • the fat content of Humulus pollen is 0.5% to 3%. Therefore, in the subsequent degreasing process, the fat content of defatted Humulus pollen should decrease by 0.5% to 3%, that is, the total pollen weight is also the same Should be reduced by 0.5% to 3%.
  • Natural shedding method collecting humulus pollen. Dry at room temperature or vacuum or fluidized bed until pollen no longer adheres. The dried pollen is passed through a 150-250 mesh sieve, which is preferably a 180-mesh sieve in this embodiment.
  • the pollen (g) and acetone (ml) obtained above are subjected to degreasing treatment at a weight-to-volume ratio (w/v) of 1:5 to 1:1. In this embodiment, it is preferably 1:2. After stirring or shaking for 30 minutes to degreasing, let it stand for layering and observe the clarification of the upper liquid. Pour out the upper liquid, add new acetone, and repeat degreasing until the upper liquid is clear.
  • the content of impurity particles should meet the following standards: spore content ⁇ 1%, irrelevant pollen content ⁇ 2%, and other impurities content ⁇ 10%.
  • Residual acetone content ⁇ 0.5% (volume fraction).
  • the centrifugal force is 8000 ⁇ 12000g
  • the centrifugal temperature is 2 ⁇ 8°C
  • the time is set to 15 ⁇ 20 minutes, centrifuge, and collect the supernatant
  • the filtered leachate is subjected to tangential flow ultrafiltration with a 3KD or 1KD ultrafiltration membrane.
  • a 3KD ultrafiltration membrane is used for ultrafiltration in this embodiment.
  • the dialysate is selected from 25-125 mM NH 4 HCO 3 , and in this embodiment, 50 mM NH 4 HCO 3 is preferred.
  • ultrafiltration is concentrated to an appropriate volume, and the protein content is detected to the total protein content range of the quality standard. Sampling the ultrafiltration concentrate and ultrafiltration permeate to detect the total protein content.
  • the permeate When the total protein content in the ultrafiltration permeate is less than 0.02mg/ml, the permeate is directly discarded; if the total protein content of the ultrafiltration permeate is >0.02mg/ml, the integrity test of the ultrafiltration membrane is performed. If the membrane is not damaged, discard the permeate; if the ultrafiltration membrane is damaged, replace the ultrafiltration membrane and repeat the ultrafiltration of the permeate.
  • freeze-drying process conditions freezing at -50 ⁇ -35°C, drying at -25°C under vacuum pressure of 2 ⁇ 8mbar, and controlling the moisture content ⁇ 3%; the lyophilized product of Humulus pollen allergen is obtained.
  • the lyophilized product of Humulus pollen allergen prepared in Example 4 was reconstituted with a phosphate-saline buffer (pH 6.5-7.5, see Table 5 for the formula) until the total protein content was within the range of 2 times the quality standard. Place at 2 ⁇ 8°C.
  • the semi-finished product is sterilized and filtered through a 0.22 ⁇ m filter membrane, and sterilized into finished products to obtain a light yellow to brown liquid with a pH of 6.0 ⁇ 8.0, which is the standardized allergen extract of Humulus pollen. Finished product.
  • M Genstar M223-01 Marker
  • R freeze-dried internal reference material (ie freeze-dried Humulus pollen allergen stock solution)
  • L 1 , L 2 and L 3 are different batches of Humulus pollen standardized allergen extract .
  • Its protein distribution is mainly between 6.3-76kDa, specifically distributed in 10kDa (specific molecular weight 6.3kDa, 7.0kDa, 8.09kDa, 8.8kDa, 9.6kDa, 10.03kDa, 10.22kDa), 11.8kDa, 12.3kDa, 13.5kDa, 15.2kDa, 15.32kDa, 16.45kDa, 16.61kDa, 19kDa (specifically 19.04kDa, 19.18kDa, 19.99kDa), 28kDa, 37kDa, 42kDa, 45kD
  • the product of the present invention has carried out the whole protein sequence determination of the corresponding allergenic protein (see Table 6), and carried out the nucleotide full-length sequencing of the corresponding mRNA of each allergic protein in the humulus pollen raw material, the allergenic protein code, sequence identification and The results of the full-length amino acid sequence and the corresponding full-length mRNA sequence are shown in Table 6. Among them, Hum 3 is the most important allergenic protein.
  • Humulus humulus allergenic protein contained in the present invention is as follows:
  • the full sequence of Hum sc 2 sensitizing protein is shown in SEQ ID NO. 6; the full sequence of Hum sc 3 sensitizing protein is shown in SEQ ID NO. 7;
  • Hum s 3 allergenic protein is shown in SEQ ID NO. 8 (and Hum s 3: ADB97919.1 protein sequence in the NCBI database previously discovered by the applicant, but the mRNA is synonymously substituted at position 15 );
  • Humsc 11 sensitizing protein The full sequence of Humsc 11 sensitizing protein is shown in SEQ ID NO.16;
  • the corresponding mRNA sequence is as follows
  • Humsc3 sensitizing protein gene is shown in SEQ ID NO.24;
  • Hum s3 allergenic protein gene is shown in SEQ ID NO.25; (Compared with NCBI No. FJ967117.1, the 15th nucleotide T ⁇ C is synonymously replaced)
  • Hum sC6 allergenic protein gene The full sequence of Hum sC6 allergenic protein gene is shown in SEQ ID NO.28;
  • the present invention uses high-resolution two-dimensional electrophoresis (2D) combined with Western Blotting technology (reaction with 15 cases of Humulus pollen allergy patients with positive serum pool), It is confirmed that the above-mentioned protein can bind to the sIgE in the peripheral blood of patients with Humulus pollen allergy, so it is a sensitizing protein.
  • 2D gel image is shown in Figure 6(a) and the Western Blotting result is shown in Figure 6(b).
  • Humulus humulus allergen extract and the corresponding strips of Humulus humulus allergen separated by SDS-PAGE were sampled for mass spectrometry identification, which mainly include but are not limited to the following peptides such as:
  • SEQ ID NO.56 LVKIISHHYNEK
  • SEQ ID NO.70 AYLDAHNAVR
  • the resolution of SDS-PAGE is limited.
  • the overall seropositivity rate of 10kDa protein is 66.7%.
  • Humsc3 and HumS3 are the same protein; the overall seropositivity rate of 19kDa protein is 24.1%.
  • the quality standards of the Humulus humulus allergen infusion are as shown in Table 7 after the chemical properties are tested:
  • the relative biological titer measured by competitive ELISA method is 50000BAU/ml-200000BAU/ml, and the protein concentration is 0.40 ⁇ 1.60mg/ml .
  • the double monoclonal antibody sandwich ELISA method was used to detect the main allergenic protein content in Humulus pollen allergen extract. After multiple batches of testing, it is determined that the fluctuation range is 0.08 ⁇ 0.32mg/ml.
  • the humulus pollen standardized allergen infusion product prepared in Example 5 was subjected to a long-term stability study test at 2-8°C, and three batches of samples were tested respectively at 0 o'clock, 3 months, 6 months, and 9
  • the changes in pH value between months and 12 months, phenol content, sodium chloride content, protein content, total allergen activity, and the main allergenic protein Hum 3 content are shown in Figure 7 to Figure 12.
  • the humulus pollen allergen extract prepared by the present invention is effective and safe for clinical specific skin test diagnosis of humulus pollen allergy.
  • Method Choose from 2015-08-24 to 2016-09-21, in the outpatient department of Peking Union Medical College Hospital Allergy Department, suffering from allergic conjunctivitis, allergic rhinitis, allergic asthma, urticaria, atopic dermatitis 1026 outpatients with allergic diseases.
  • the subjects were subjected to the Humulus pollen allergen skin prick test (Skin prick test, SPT), using the stock solution, using the mean wheal diameter (MWD) as the interpretation standard; taking the pollen sIgE as the standard, doing ROC analysis, analyzing the accuracy of Humulus pollen allergen extract for diagnosis of Humulus pollen allergy under different diagnostic thresholds. At the same time, record adverse events and evaluate safety. Results (Results) A total of 1026 outpatients (4 to 70 years old) were enrolled in the group. There were no cases of dropout, and the rejection rate was 22.1%. There were 1026 cases in the Safety Set (SS), 990 cases in the Full Analysis Set (FAS), and 799 cases in the Per Protocol Set (PPS).
  • SPT Skin prick test
  • MWD mean wheal diameter
  • the ROC curve of the PPS set it is estimated that the best diagnostic threshold of Humulus pollen SPT is MWD 6.75mm; when the specificity reaches 95%, the diagnostic threshold is MWD 8.25mm.
  • the sensitivity of the humulus pollen allergen extract of the present invention for diagnosing humulus pollen allergies decreases successively, respectively 0.9366 (95% CI: 0.9159 ⁇ 0.9572), 0.5485 (95%CI: 0.5064 ⁇ 0.5906), 0.4216 (95%CI: 0.3798 ⁇ 0.4634); the specificity increased successively, respectively, 0.0913 (95%CI: 0.0565 ⁇ 0.1261), 0.8517 (95%) CI: 0.8088 to 0.8947), 0.9696 (95% CI: 0.9488 to 0.9903). 7 out of 1026 patients had 8 adverse events. The adverse event rate was 0.68% (7/1026).
  • the humulus pollen allergen extract of the present invention is used for diagnosing humulus pollen allergies, has high diagnostic value and good safety, and can be used as a clinical examination method for the specific in vivo diagnosis of humulus pollen allergens.
  • the humulus pollen allergen extract prepared in the present invention performs a basophil activation test on the whole blood of patients allergic to humulus pollen, and can perform clinical specific allergy in vitro diagnosis.
  • This detection method is applicable to both IgE and non-IgE-mediated allergic reactions, and can be used for the diagnosis of false-negative and false-positive patients with sIgE in vitro diagnosis, and the diagnosis of some patients with anaphylactic shock that are not suitable for skin test diagnosis.
  • Test principle The reaction of allergens with the patient’s whole blood cells can simulate the process of allergic reactions in the human body: that is, specific IgE antibodies bind to the cell surface by bridging with the corresponding allergens, and activate the intracellular signal cascade leading to basophils (CCR3 is continuously expressed in basophils and is its specific marker) activation and degranulation.
  • CCR3 is continuously expressed in basophils and is its specific marker activation and degranulation.
  • the intracellular complex affects the transmembrane protein CD63 (gp53) to make it expressed on the cell surface and exposed to the extracellular matrix. Therefore, it can rely on the principle of flow cytometry (using anti-human tendency).
  • Chemical factor receptor CCR3-phycoerythrin (anti-CCR3-PE) is used to label basophils, and anti-human CD63 monoclonal antibody-fluorescein isothiocyanate (anti-CD63-FITC) is used for the activation of Basophils are labeled, and the non-specific cell activator fMLP is used as a positive quality control), and the percentage change of basophils degranulation is used to determine whether the subject is allergic to a specific allergen.
  • Method Select healthy subjects and humulus allergic patients, take their EDTA anticoagulated whole blood samples, and use stimulation buffer (negative control), humulus allergen extract (diluted ratio of 1:10 3 ⁇ 10 10 ) Optimized, in this example, 1:10 8 ), fMLP stimulation solution (positive quality control) was used as the activator of basophils, added to the whole blood, and then added anti-CD63-FITC, anti-CCR3-PE Stain and perform detection on flow cytometer within 48 hours. Result: See Figure 13. In healthy subjects, when the negative control, Humulus allergen extract, and the positive control are basophil activation, the basophil activation rate should be ⁇ 15%, ⁇ 15%, ⁇ 15%, respectively.
  • the basophil activation rate should be ⁇ 15%, ⁇ 15%, ⁇ 15%, respectively .

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Abstract

一种葎草花粉变应原浸提物、浸液及其制备方法。该葎草花粉变应原浸提物，其中含有葎草花粉变应原蛋白如SEQ ID NO.4-SEQ ID NO.20所示的氨基酸序列。一种葎草花粉变应原浸液经葎草花粉收集、干燥、脱脂、提取、超滤浓缩、冷冻干燥、复溶等工艺方法制得。该葎草花粉变应原浸液具有特异性高的特点，葎草致敏蛋白组分提取充分，总生物效价稳定，有效期长，无菌效果好；其原液可有效用于变态反应疾病的皮肤点刺试验诊断、经适当稀释后(10 -2～10 -20)可用于体外嗜碱性粒细胞活化试验诊断、皮内试验诊断及特异性免疫治疗，可有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。

Description

一种葎草花粉变应原浸提物、浸液及其制备方法 技术领域
本发明涉及生物技术领域，具体涉及一种葎草花粉标准化变应原浸提物、浸液及其制备方法。
背景技术
人们对变态反应疾病的认识始于“花粉热”，花粉热又称过敏性鼻炎。1911年Noon和Freeman首次利用花粉提取液来治疗花粉热，变态原疾病治疗的历史从此开始。目前，变态反应性疾病是全球性重大卫生问题之一。工业化国家超过25％的人口被变应性哮喘、变应性鼻结膜炎及变应性皮炎困扰，其中以变应性哮喘最为常见。吸入致敏花粉是引发变应性哮喘及其他呼吸道变态反应性疾病的最重要因素。VRTA—L.A等调查发现近50％的变态反应性疾病患者对草类花粉过敏。葎草(Humulusscandens)属蔷蔽目桑科葎草属，为一年或多年生茎蔓草本植物，生长条件要求低，好生于沟边和路旁荒地，花期为7-9月。除新疆和青海外，在全国各省区均有分布，是常见杂草之一。律草在国外也有分布，如日本、韩国、越南。葎草花粉症在中国北方地区非常常见。夏秋季节，中国北方大部分城市葎草花粉在空气中含里仅次于蒿属花粉，个别地区甚至高于蒿属花粉。
尹佳的《夏秋季花粉症患者合并过敏性鼻炎和过敏性哮喘关系的情况及其相互关系研究》表明葎草花粉症占北京地区夏秋季花粉症患者的58％；赵京的《北京地区儿童呼吸道过敏性疾病与皮肤过敏原试验的调查》针对13-14岁儿童进行的北京地区呼吸道变态反应性疾病流行病学调查结果显示，葎草花粉敏感者为4.2％；任华丽的《北京地区成人过敏性鼻炎吸入过敏原谱分析》择北京居民中明确诊断为过敏性鼻炎的成人患者265例，对其吸人常年性、季节性两组共16种标准化过敏原进行检测，分析主要过敏原排序，其中葎草18.11％；田芳洁的《北京地区变应性鼻炎的吸入性变应原调查》对北京地区变应性鼻炎(allergic rhinitis，AR)的主要吸入性变应原进行调查发现，其中葎草占17.29％；李勇的《台州地区1013例变应性鼻炎患者变应原谱分析》调查台州地区变应性鼻炎患者的变应原分布情况，其中，葎草花粉过敏者248例，占30.58％；刘杰的《青岛地区气传花粉及其致敏性调查》发现青岛市气传花粉的种类、数量和飘散规律，主要是松属花粉居多，其次就是葎草和蒿属；兰海燕的《河北迁安地区气传花粉调查》调查迁安地区气传花粉种类,飘散规律及致敏情况，气传花粉飘散高峰为春季、秋季；花粉种类为松属、杉木属、悬 铃木属、梨属、柏属、柳属、蒿属、葎草、豚草等；朱香顺的《潍坊市花粉症患者主要致敏花粉种属分析》调查分析潍坊市花粉症患者主要致敏花粉种类致敏花粉为葎草,占68％,其次为葎草、夏秋季花粉Ⅰ、豚草、法桐；孙立英的《上海中心城区气传花粉调查》明确上海市全年气传花粉的种类和数量及其分布特征，秋季以莠草类及禾本科花粉为主,主要为葎草属(59.01％)、禾本科(18.45％)和蒿属(5.32％)。
以上葎草花粉流行病学调查研究显示，葎草花粉过敏广泛分布在我国各地，在北方地区葎草花粉是花粉过敏的主要致敏原。
现在变应原特异免疫治疗的方法主要分为皮下注射给药免疫治疗和舌下给药免疫治疗。皮下注射给药免疫治疗已有100多年历史，其安全性和有效性已经被得到证明。上世纪90年代初，变应原舌下滴剂疫苗诞生，1998年，WHO宣布了变应原舌下滴剂疫苗安全和有效。
但是舌下给药免疫脱敏的方式相比皮下注射免疫给药，治疗周期长，短时间内疗效不显著，平均脱敏周期3-5年。在一个治疗周期中，舌下给药方式免疫脱敏给药量是皮下注射给药量的100倍。所以皮下注射给药免疫脱敏的方式相比舌下给药，虽然病人依从性差，但是治疗周期短，见效快，治疗费用低。所以两种给药方式是相辅相成。目前，用于葎草花粉变应原的作为皮下注射免疫制剂的稳定性差，蛋白含量和生物效价都会随时间下降，成分不确定试剂存放一定时间后用于诊断葎草花粉诱发的变态反应疾病时，阳性率降低，准确性低。
发明内容
为了解决上述技术问题，本发明提供一种含葎草花粉变应原浸液，该浸液具有于特异性高，葎草致敏蛋白组分提取充分且比例恒定，总生物效价稳定，有效期长的特点。其可有效用于变态反应疾病的皮肤点刺试验诊断及特异性免疫治疗，可有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。
为实现上述目的，本发明采用以下的技术方案为：
一种葎草花粉变应原浸提物，其含有葎草花粉变应原蛋白Hum sc 1，其含有如SEQ ID NO.4所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 2，其含有如SEQ ID NO.6所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 3，其含有如SEQ ID NO.7所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 4，其含有如SEQ ID NO.9所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 5，其含有如SEQ ID NO.10所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 6，其含有如SEQ ID NO.11所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 7，其含有如SEQ ID NO.12所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 8，其含有如SEQ ID NO.13所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 9，其含有如SEQ ID NO.14所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 10，其含有如SEQ ID NO.15所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 11，其含有如SEQ ID NO.16所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 12，其含有如SEQ ID NO.17所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 13，其含有如SEQ ID NO.18所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 14，其含有如SEQ ID NO.19所示的氨基酸序列；
含有葎草花粉变应原蛋白Hum sc 15，其含有如SEQ ID NO.20所示的氨基酸序列。
用于编码葎草花粉花粉变应原浸提物的基因，所述基因为编码如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.9-20所示的氨基酸序列，分别对应如SEQ ID NO.21、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.26-37所示的序列。
一种的葎草花粉变应原浸液，所述变应原浸液中含如上所述的葎草花粉变应原浸提物、体积比为0.2～0.4％的苯酚、体积比为45～55％的甘油和4.5～5.5g/L的NaCl，其pH值为6.0～8.0。
如上所述的葎草花粉变应原浸液，优选地，所述葎草花粉变应原的活性浓度为50000～200000BAU/ml，所述葎草花粉变应原浸液的总蛋白浓度0.40mg/ml～1.60mg/ml，所述葎草花粉的变应原蛋白含量0.08～0.32mg/ml。
进一步地，通过SDS-PAGE和Western Blotting检测，所述所述葎草花粉变应原提取物的蛋白主要分布在10kDa(具体分子量6.3kDa、7.0kDa、8.09kDa、8.8kDa、9.6kDa、10.03kDa、10.22kDa)、11.8kDa、12.3kDa、13.5kDa、15.2kDa、15.32kDa、16.45kDa、16.61kDa、19kDa(具体为19.04kDa、19.18kDa、19.99kDa)、28kDa、37kDa、42kDa、45kDa、52kDa、66kDa、76kDa。
如上所述的葎草花粉变应原浸液的制备方法，其包括如下步骤：
S1、采集葎草花粉，常温干燥或真空干燥或流化床干燥；
S2、对干燥后的花粉脱脂、干燥；
S3、将脱脂干燥后的葎草花粉按重量g体积ml比1:50～1:10加入pH为7.9～8.2磷酸盐—盐水缓冲液，2～8℃搅拌22～26h进行浸提；
S4、取步骤S3后的浸提液，离心取上清液；将上清液过滤及除菌过滤；
S5、将过滤后的上清液进行超滤浓缩，获得超滤浓缩液；
S6、将所述超滤浓缩液经过二次过滤和除菌过滤后，真空冻干获得葎草花粉变应原 冻干品；
S7、将所述葎草花粉变应原冻干品用pH 6.5～7.5磷酸盐—盐水缓冲液复溶配置为葎草花粉变应原原液，2～8℃放置，与等体积灭菌的甘油混匀，调节溶液pH值至6.0～8.0。
如上所述的制备方法，优选地，在步骤S2中，所述花粉脱脂采用花粉与丙酮以1：5～1:1的重量g体积ml比进行脱脂处理，重复脱脂至上层液体澄清。
如上所述的制备方法，优选地，在步骤S4中，所述离心的条件为8000～12000g，离心温度2～8℃，时间为15～20min；所述过滤及除菌过滤为先用4000目滤布过滤后，再通过纸板过滤、0.45μm和0.22μm滤膜依次过滤。
如上所述的制备方法，优选地，在步骤S5中，所述超滤浓缩用3KD超滤膜超滤，当超滤透过液总蛋白含量≤0.02mg/ml，停止超滤；当超滤透过液总蛋白含量>0.02mg/ml，更换超滤膜之后对超滤透过液重复超滤，直至超滤透过液总蛋白含量≤0.02mg/ml为止。
如上所述的制备方法，优选地，在步骤S6中，所述真空冻干的条件为-50～-35℃冻结，2～8mbar真空压力下，-25℃干燥，控制水分含量≤3％。
如上所述的制备方法，优选地，所述步骤S1还包括对原料葎草花粉进行镜检鉴别和/或DNA鉴定的步骤，其中DNA鉴定方法为，以SEQ ID NO.1、SEQ ID NO.2为引物，对鉴别葎草花粉原料进行PCR扩增，并检测扩增产物。
如上所述的制备方法，优选地，所述磷酸盐—盐水缓冲液包括4.5～5.5g/L的氯化钠、0.03％～0.05％的磷酸二氢钾、1.5％～2％十二水合磷酸氢二钠和0.2％～0.4％的苯酚。
在制备葎草花粉变应原进行浸提时，发明人尝试了多种浸提液，并且比较了它们的浸提效率。所使用的浸提液包括：0.8％的氯化钠、pH5.0的磷酸氢二钠-柠檬酸缓冲液、pH6.0的磷酸氢二钠-柠檬酸缓冲液、pH7.5的磷酸氢二钠-磷酸二氢钠缓冲液、pH8.0的磷酸氢二钠-磷酸二氢钠缓冲液、pH8.2的coca’s液、pH8.9的Tris-盐酸缓冲液、pH为7.9～8.2的含有4.5～5.5g/L的氯化钠及重量百分比含量为0.03％～0.05％的磷酸二氢钾，1.5％～2％十二水合磷酸氢二钠，0.2％～0.4％的苯酚等。实验结果证实，使用pH为7.9～8.2的含有4.5～5.5g/L的氯化钠及0.03％～0.05％的磷酸二氢钾，1.5％～2％十二水合磷酸氢二钠，0.2％～0.4％的苯酚的浸提效率较高，且制备的葎草花粉变应原浸液可长期有效的保存，稳定性高。
本发明还提供一种葎草花粉变应原冻干品，其由包括葎草花粉收集-干燥-脱脂-提取-超滤浓缩-冻干的步骤制得：
(1)收集：用自然脱落法收集葎草花粉；
(2)干燥：常温干燥或真空干燥或流化床干燥，直至花粉不再粘附，将干燥后的花 粉过150～250目分样筛；
(3)脱脂：将(2)所得到的花粉与丙酮分别按g与ml计，以1：5～1:1的重量体积比进行脱脂处理，振荡脱脂30分钟后，静置分层，倒出上层液，加入新的丙酮，重复脱脂至上层液体澄清，将脱脂后的花粉均匀摊开，室温下干燥48小时至72小时；
(4)提取：将脱脂干燥后的葎草葎草花粉按重量体积比1:50～1:10加入10L，pH7.9～8.2磷酸盐—盐水缓冲液，2～8℃搅拌22～26h进行浸提；取提取后的浸提液，在离心力8000～12000g，离心温度2～8℃，时间设定为15～20分钟，进行离心，收取上清；先用4000目滤布过滤后，将滤液依次通过纸板过滤、0.45μm和0.22μm滤膜依次过滤；
(5)超滤浓缩：将过滤后的浸提液，用3KD超滤膜超滤，取样超滤浓缩液、超滤透过液，检测其总蛋白含量；其中，当超滤透过液中总蛋白含量≤0.02mg/ml，则直接排弃透过液；超滤透过液总蛋白含量>0.02mg/ml，则对超滤膜进行完整性测试，超滤膜未破损，则排弃透过液；若超滤膜破损，则更换超滤膜之后对透过液重复超滤；
(6)冻干：将超滤浓缩液经过二次过滤和除菌过滤后，按照冻干工艺条件，-50～-35℃冻结，2～8mbar真空压力下，-25℃干燥，控制水分含量≤3％，获得葎草花粉变应原冻干品。
如上所述葎草花粉变应原浸提物、所述葎草花粉变应原浸液及葎草花粉变应原冻干品的应用，用于在制备诊断变态反应疾病并对其进行特异性免疫治疗制剂中的应用，所述诊断变态反应疾病包括过敏性哮喘、变应性鼻炎、特应性皮炎和慢性荨麻疹。
进一步，所述葎草花粉变应原浸提物冻干品用于制备成片剂、口崩片，所述葎草花粉变应原浸液用于制备注射液、舌下滴剂、变应原斑贴剂、变应原浸液稀释液。本发明提供的葎草花粉变应原浸液用于制备治疗葎草花粉过敏性疾病的药物使用。
具体而言，采用本发明提供的葎草花粉变应原浸液或冻干品按治疗有效量或诊断有效量的葎草花粉变应原、以及药学上可接受的载体可制备用于治疗葎草花粉过敏性疾病的药物。
用于治疗葎草花粉过敏性疾病的药物可以制成各种医学上可接受的剂型，并可由医师根据患者年龄、体重和大致疾病状况等因素确定对病人有益的剂量进行施用。用于治疗葎草花粉过敏性疾病的药物的剂型选自口服剂、(皮下)注射剂、舌下含服剂、气雾剂、鼻腔剂、或皮肤点刺剂等液体剂型；优选(皮下)注射剂、舌下含服剂、或皮肤点刺剂。其中，(皮下)注射剂、舌下含服剂一般是作为特异性免疫治疗时常用的剂型，而皮肤点刺剂是作为体内变应原检测时常用的剂型。
当采用本发明葎草花粉变应原浸液应用于诊断由葎草花粉引起的过敏性疾病时(即 变应原皮肤点刺诊断试验)，除了本发明葎草花粉变应原浸液外，一般还应包括阴性对照液、阳性对照液、以及点刺针。阴性对照液一般为对人体无过敏性反应的液体(例如甘油与盐溶液的混合物等)，阳性对照液一般为1.0～5.0mg/ml的磷酸组胺/盐酸组胺溶液。
本发明制备的葎草花粉变应原浸液可应用于斑贴试验中。其原理为：将可疑致敏物质(变应原)敷贴于患者皮肤上，通过皮肤或粘膜进入机体后由抗原呈递细胞将抗原呈递给T淋巴细胞，使特异性T淋巴细胞活化，诱发炎症反应。
本发明的有益效果在于：
1、葎草作为北方地区主要花粉过敏原，本发明提供的葎草花粉标准化变应原浸液可以有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。
2、加入稳定剂甘油，提高了稳定性和缓释作用，提高了用药的有效性和安全性。
3、变态反应疾病皮下注射免疫脱敏给药方式，相比舌下含服滴剂给药方式，见效快，周期短。整个治疗周期给药剂量远小于舌下滴剂给药量(约100倍)，其治疗费用远小于舌下滴剂给药免疫脱敏，减轻患者的医疗负担。
4、本发明通过使用制备的应变原浸液以原液做点刺试验分别与变态反应专科医生临床综合特异性诊断和血清特异性IgE(specific IgE，sIgE)诊断进行对比，皮内试验结果与临床综合特异性诊断及与血清sIgE诊断的结果一致、具有较好的灵敏度和特异度，安全性好。
本发明提供的葎草花粉变应原浸液具有特异性高的特点，葎草致敏蛋白组分提取充分，总生物效价稳定，有效期长，无菌效果好；可有效用于变态反应疾病的皮肤点刺试验诊断及特异性免疫治疗，可有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。可实现标准化控制，使用有效期有效延长，能带来较好的经济效益。
5、本发明通过使用制备的变应原浸液以1：10 3～10 8稀释后做体外嗜碱性粒细胞活化试验，能临床特异性诊断葎草过敏患者，避免部分体外sIgE检测假阳性，同时也能避免变应原皮肤试验(点刺或皮内)给部分蒿属花粉过敏患者带来过敏性休克的风险。
本发明方法通过冻干复溶后得到的浸液，相比北京协和医院研制的葎草花粉变应原注射原液更稳定，生物效价和蛋白含量都比较稳定，且效期为3年。
附图说明
图1为葎草花粉原料ITS2序列PCR电泳结果。
图2为不同批次葎草花粉不同脱脂时间脂肪含量变化趋势。
图3为葎草花粉浸液总蛋白含量与浸提时间的关系。
图4为葎草花粉变应原浸液SDS-PAGE蛋白电泳结果(示意成分鉴别)。
图5为葎草花粉变应原浸液与葎草过敏患者血清Western Blotting反应检测结果(示意葎草变应原成分鉴别)。
图6为新鉴定的葎草花粉致敏蛋白2D胶图(a)及Western Blotting图(b)。
图7为葎草花粉变应原浸液产品pH值在长期稳定性试验中的测试结果。
图8为葎草花粉变应原浸液产品苯酚含量在长期稳定性试验中的测试结果。
图9为葎草花粉变应原浸液产品氯化钠含量在长期稳定性试验中的测试结果。
图10为葎草花粉变应原浸液产品总蛋白含量在长期稳定性试验中的测试结果。
图11为葎草花粉变应原浸液产品总应变原活性在长期稳定性试验中的测试结果。
图12为葎草花粉变应原浸液产品主要致敏蛋白含量Hum s 3在长期稳定性试验中的测试结果。
图13为葎草花粉变应原浸液产品应用于临床葎草花粉过敏患者体外嗜碱性粒细胞活化试验结果示例。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下，对本发明所作的修饰或者替换，均属于本发明的范畴。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 葎草花粉原料的鉴别
由于原料的鉴别和纯度对于后续变应原诊断及治疗制剂极为重要，本实施例采用DNA特异性序列检测加显微镜检对葎草原料进行双重鉴别及质控。
1、葎草花粉DNA提取
采用天根快速DNA提取扩增试剂盒(天根生化KG203)。
提取方法：称取5mg葎草花粉样品置于1.5mL离心管中，加入Buffer 1 100μl后用一次性研磨杵将样品彻底研碎后加入Buffer 2 100μl后震荡混匀，离心机12000r/min离心5min后取上清液于新的离心管中作为DNA模版备用。
2、引物设计及合成
引物序列如下：
Hum ITS2-F(SEQ ID NO.1)：5'-CTGAGAAACGGCTACCACATC-3'
Hum ITS2-R(SEQ ID NO.2)：5'-GTCGGCCAAGGCTATAAACTC-3'
引物序列由上海生工生物工程有限公司合成。合成后分装-20℃保存。
3、PCR反应体系
PCR扩增试剂盒(天根生化)。对提取的DNA采用如下50μl体系的配制PCR反应体系见表1：
表1.葎草花粉ITS2序列PCR反应体系
Figure PCTCN2019088605-appb-000001
4、PCR反应条件见表2.
表2.葎草花粉ITS2序列PCR反应条件
Figure PCTCN2019088605-appb-000002
对PCR扩增后的反应液进行1％琼脂糖电泳，100mA 20min电泳观察，电泳结果如图1所示。其中L1、L2、L3为3批葎草花粉PCR产物，L mix是上述3批葎草花粉混合原料PCR产物，DNA Marker如图1中所示。
5、测序
利用PCR产物纯化回收试剂盒(生工SK1141)，回收PCR产物进行测序，测序结果编号Hum s ITS2，如SEQ ID NO.3所示。
5、葎草的显微镜检
葎草花粉显微镜下形态学鉴别：花粉粒球形，极面轮廓圆形。直径26.7×27.5微米。具3孔，偶有4孔者，均匀地排列于赤道上。在孔处，外壁外部略加厚，形成孔环。外壁层次不清，表面具细颗粒状纹饰。
实施例2 葎草花粉脱脂及浸提关健工艺步重要参数的确定
1、葎草花粉脱脂工艺步参数确定
(1)将葎草花粉(g)与丙酮(ml)以1：2的重量体积比(w/v)进行脱脂处理。对不同批次(不同采集时间)的葎草花粉不同脱脂时间样品进行脂肪含量进行检测，以确定最优的脱脂时间。
采用海能SOX500脂肪测定仪测定葎草花粉中脂肪含量，通过索式萃取及干燥称重的方法，对比萃前后花粉重量的变化，得到相对应的脂肪含量，并根据脂肪含量结果对之后脱脂后花粉进行质量控制。
结果见图2，可见丙酮脱脂时间为30min后，脂肪含量几乎不随脱脂时间延长而下降，因此脱脂时间确定为30min。
(2)脱脂参数的验证：对不同批次(不同采集时间)的葎草花粉进行30min脱脂后检测脂肪含量，结果如表3所示。
表3.不同批次葎草花粉脱脂30min后脂肪含量降低百分比
Figure PCTCN2019088605-appb-000003
从结果得知，葎草花粉中脂肪含量在0.5％～3％，故在之后脱脂过程中，脱脂后的葎草花粉在脂肪含量上应该下降0.5％～3％，也就是花粉总重同比也应该下降0.5％～3％。
2、葎草花粉蛋白浸提工艺步重要参数确定
(1)将葎草花粉(g)与丙酮(ml)以1：2的重量体积比(w/v)进行搅拌或振荡脱脂30分钟处理，静置分层，观察上层液体澄清情况，重复脱脂至上层液体澄清后，将脱脂后的花粉均匀摊开，室温下干燥48小时以上。
(2)将脱脂干燥后的葎草花粉按重量体积比1:10加入10L，pH 7.9～8.2磷酸盐—盐水缓冲液，2～8℃，在搅拌速度为250rpm条件下，在搅拌时间分别为3h、6h、9h、12h、18h、24h、48h、72h等提取点时，将提取后的提取液，将浸提液全部加入到离心桶内，调节平衡，离心力8000～12000g，离心温度4℃，时间设定为15～20分钟，进行离心，收取上清。
(3)将离心分离后的上清液先用4000目滤布过滤后，通过纸板过滤，0.45μm和0.22μm滤膜依次过滤；得到葎草花粉粗提取液。
取样以Bradford法测定蛋白含量，结果见“图3、葎草花粉浸液总蛋白含量与浸提时间的关系”。
如图3结果表明，在蛋白提取过程中，随搅拌提取时间延长，蛋白提取含量增大，提取时间24h含量达到最高982±10μg/ml，可见蛋白类物质提取时间过长会影响其蛋白含量，故提取时间优选为22～26h左右。
施例3 葎草花粉原料工艺
1、花粉采集
自然脱落法，收集葎草花粉。常温或真空或流化床干燥，直至花粉不再粘附。将干燥后的花粉过150～250目分样筛，本实施例中优选为180目分样筛。
2、干燥
将花粉摊放于通风干燥处自然干燥，或真空干燥、或流化床干燥6-48h，直至花粉不再粘附。
3、脱脂
将上述所得花粉(g)与丙酮(ml)以1：5～1:1的重量体积比(w/v)进行脱脂处理。本实施例中优选为1：2。搅拌或振荡脱脂30分钟后，静置分层，观察上层液体澄清情况。倒出上层液，加入新的丙酮，重复脱脂至上层液体澄清。
4、再干燥
将脱脂后的花粉均匀摊开，室温干燥、或真空干燥、或流化床干燥6-48h。
5、杂质控制
(1)显微镜下用颗粒计数法测定，其中的杂质颗粒含量应满足如下标准：孢子含量≤1％，无关花粉含量≤2％，其它杂质含量≤10％。
(2)重金属及有害元素
总重金属≤50mg/kg；砷≤5mg/kg。
(3)丙酮残留
丙酮残留量≤0.5％(体积分数)。
实施例4 葎草花粉变应原原液(冻干品)制备工艺
1、按实施例3葎草花粉原料工艺制备葎草花粉原料。
2、浸提
将脱脂干燥后的葎草花粉按重量体积比1:50～1:10加入10L，本实施例中优选为1：20，以pH 7.9～8.2磷酸盐—盐水缓冲液，2-8℃搅拌22～26h进行浸提。其中以配制1000ml磷酸盐—盐水缓冲液(pH 8.2)为例的配方如表4。磷酸盐—盐水缓冲液充分溶解后除菌过滤，2～8℃放置，保存期不超过30天。
表4 磷酸盐—盐水缓冲液(pH 8.2)配方
Figure PCTCN2019088605-appb-000004
3、固液分离
取提取后的浸提液，将浸提液全部加入到离心桶内，调节平衡，离心力8000～12000g，离心温度2～8℃，时间设定为15～20分钟，进行离心，收取上清；
4、澄清
将离心分离后的上清液先用4000目滤布过滤后，将滤液依次通过纸板过滤、0.45μm和0.22μm滤膜依次过滤；
5、超滤、透析、浓缩
过滤后的浸提液，用3KD或1KD超滤膜切向流超滤，优选地，本实施例中用3KD超滤膜超滤。透析液选用25-125mM的NH 4HCO 3，本实施例中优选为50mM的NH 4HCO 3。根据蛋白含量质量标准，超滤浓缩至适当体积，检测蛋白含量至质量标准的总蛋白含量区间。取样超滤浓缩液、超滤透过液，检测其总蛋白含量。当超滤透过液中总蛋白含量≤0.02mg/ml，则直接排弃透过液；超滤透过液总蛋白含量>0.02mg/ml，则对超滤膜进行完整性测试，超滤膜未破损，则排弃透过液；若超滤膜破损，则更换超滤膜之后对透过液重复超滤。
6、无菌过滤
用0.22μm膜除菌过滤。
7、冻干
按照冻干工艺条件，-50～-35℃冻结，2～8mbar真空压力下，-25℃干燥，控制水分含量≤3％；获得葎草花粉变应原冻干品。
实施例5 葎草花粉变应原浸液成品制备工艺
1、复溶
将实施例4制备的葎草花粉变应原冻干品用磷酸盐—盐水缓冲液(pH 6.5～7.5，配方见表5)复溶，至总蛋白含量位于质量标准2倍范围区间。2～8℃放置。
表5.磷酸盐—盐水缓冲液(pH值[6.5，7.5])配制
Figure PCTCN2019088605-appb-000005
2、半成品配制
原液复溶后，在净化车间内A级洁净条件下，将复溶液与等体积预先湿热灭菌(121℃、30分钟)并冷却的甘油混匀，调节溶液pH值至6.0～8.0。
3、半成品除菌过滤、无菌分装为成品
在净化车间内A级洁净条件下，将半成品通过0.22μm滤膜除菌过滤，无菌分装为成品，得到pH6.0～8.0浅黄色至棕色液体即为葎草花粉标准化变应原浸液成品。
实施例6 葎草花粉变应原浸液成品质量控制
1、葎草花粉标准化变应原浸液总蛋白成分SDS-PAGE法鉴别
采用还原型SDS-PAGE法，分离胶浓度为4～12％，考马斯亮蓝染色。结果如图3所示。M为Genstar M223-01 Marker，R为冻干内部参考品(即冻干的葎草花粉变应原原液)，L 1，L 2和L 3为不同批次葎草花粉标准化变应原浸液，其蛋白分布主要在6.3-76kDa之间，具体分布在10kDa(具体分子量6.3kDa、7.0kDa、8.09kDa、8.8kDa、9.6kDa、10.03kDa、10.22kDa)、11.8kDa、12.3kDa、13.5kDa、15.2kDa、15.32kDa、16.45kDa、16.61kDa、19kDa(具体为19.04kDa、19.18kDa、19.99kDa)、28kDa、37kDa、42kDa、45kDa、52kDa、66kDa、76kDa。
其中鉴定的致敏蛋白分子量、序列、过敏血清阳性率、对应全蛋白质谱鉴定肽段概览见表6。
2、葎草花粉标准化变应原浸总变应原成分Western Blotting法鉴别
采用还原型SDS-PAG电泳，分离胶浓度为4～12％，0.2μm PVDF ImmobilonR-PSQ转印膜，一抗血清反应稀释度1：3，室温杂交2h，随后置于4℃冰箱杂交过夜，1×PBST洗3次，10min/次。二抗Ms mAb to Hu IgE(ab99806)1：1000室温杂交2h，1×PBST洗3次，10min/次。将ECL显影液A液和B液1:1混匀后曝光显色。
结果见图4所示。其中，M为Genstar M223-01 Marker，R为内部参考品，N为浸液与健康受试者血清反应条带，P 1-24分别为浸液与24例临床确诊的葎草过敏阳性患者(sIgE≥3级，皮试≥+++，典型的夏秋季花粉症病史)血清反应条带结果，其中致敏Hum sc 1-Hum sc 11的分子量及过敏血清阳性率汇总如表6，由此确定血清阳性率最高的Hum s 3(9.6kDa)为葎草花粉的主要致敏蛋白(血清阳性率最高，为68.9％)。
3、致敏蛋白序列测定
本发明产品对相应致敏蛋白(见表6)进行了全蛋白序列测定，并对葎草花粉原料中每一致敏蛋白对应mRNA进行了核苷酸全长测序，致敏蛋白代码、序列标识及氨基酸全长序列、对应mRNA全长序列结果详见表6。其中Hum s 3是最为主要的致敏蛋白。
其中本发明所含葎草致敏蛋白的全序列依次为：
Hum sc 1致敏蛋白全序列如SEQ ID NO.4所示；
Hum sx 2致敏蛋白全序列如SEQ ID NO.5所示；
Hum sc 2致敏蛋白全序列如SEQ ID NO.6所示；Hum sc 3致敏蛋白全序列为SEQ ID NO.7所示；
Hum s 3致敏蛋白全序列为SEQ ID NO.8所示(及本申请人此前所发现的NCBI数据库中Hum s 3：ADB97919.1蛋白序列完全一致，但mRNA在第15位发生同义替换)；
Hum sc 4致敏蛋白全序列为SEQ ID NO.9所示；
Hum sc 5致敏蛋白全序列为SEQ ID NO.10所示；
Hum sc 6致敏蛋白全序列为SEQ ID NO.11所示；
Hum sc 7致敏蛋白全序列为SEQ ID NO.12所示；
Hum sc 8致敏蛋白全序列为SEQ ID NO.13所示；
Hum sc 9致敏蛋白全序列为SEQ ID NO.14所示；
Hum sc 10致敏蛋白全序列为SEQ ID NO.15所示；
Hum sc 11致敏蛋白全序列为SEQ ID NO.16所示；
Hum sc 12致敏蛋白全序列为SEQ ID NO.17所示；
Hum sc 13致敏蛋白全序列为SEQ ID NO.18所示；
Hum sc 14致敏蛋白全序列为SEQ ID NO.19所示；
Hum sc 15致敏蛋白全序列为SEQ ID NO.20所示；
通过对葎草花粉原料中上述每一致敏蛋白对应mRNA进行了核苷酸全长测序后，其mRNA对应序列如下
Hum sc 1致敏蛋白基因全序列为SEQ ID NO.21所示；
Hum sx 2致敏蛋白基因全序列为SEQ ID NO.22所示；
Hum sc 2致敏蛋白基因全序列为SEQ ID NO.23所示；
Hum sc 3致敏蛋白基因全序列为SEQ ID NO.24所示；
Hum s3致敏蛋白基因全序列为SEQ ID NO.25所示；(NCBI编号FJ967117.1相比，第15位核苷酸T→C同义替换)
Hum sC 4致敏蛋白基因全序列为SEQ ID NO.26所示；
Hum sC5致敏蛋白基因全序列为SEQ ID NO.27所示；
Hum sC6致敏蛋白基因全序列为SEQ ID NO.28所示；
Hum sC7致敏蛋白基因全序列为SEQ ID NO.29所示；
sHum sC8致敏蛋白基因全序列为SEQ ID NO.30所示；
Hum sC 9致敏蛋白基因全序列为SEQ ID NO.31所示；
Hum sC 10致敏蛋白基因全序列为SEQ ID NO.32所示；
Hum sC 12致敏蛋白基因全序列为SEQ ID NO.34所示；
Hum sC 13致敏蛋白基因全序列为SEQ ID NO.35所示；
Hum sC 14致敏蛋白基因全序列为SEQ ID NO.36所示；
Hum sC 15致敏蛋白基因全序列为SEQ ID NO.37所示；
对于本发明中新鉴定的葎草花粉蛋白(Hum sc 1-15)，本发明以高分辨率的双向电泳(2D)结合Western Blotting技术(与15例葎草花粉过敏患者阳性血清池反应)，证实上述蛋白与葎草花粉过敏患者外周血中的sIgE能结合，因此为致敏蛋白。2D的胶图如图6(a)所示及Western Blotting结果如图6(b)所示。
4、质谱检测
对葎草变应原浸液进行全蛋白质谱分析以及对葎草变应原经SDS-PAGE分离后对应的胶条分别取样进行质谱鉴定，其主要包含且不限于以下肽段如：
Hum sc 3质谱鉴定的肽段：
SEQ ID NO.38：TADAAHSAADSVQEGK
Hum S 3质谱鉴定的肽段：
SEQ ID NO.39：ACTSLYDK
SEQ ID NO.40：ACTSLYDKYYQNCVMK
SEQ ID NO.41：LPPGACIDSENYR
SEQ ID NO.42：LPPGACIDSENYRK
SEQ ID NO.43：SIYPSNYAECATTHHNICGDLQG
SEQ ID NO.44：YYQNCVMK
Hum sc 4质谱鉴定的肽段：
SEQ ID NO.45：YESCLLSHISCDFDSCK
SEQ ID NO.46：ADCFTTCDSYYPK
SEQ ID NO.47：IKHDDICNGLP
Hum sc 7质谱鉴定的肽段：
SEQ ID NO.48：ACGSLYDK
SEQ ID NO.49：ACGSLYDKYYQNCVMK
SEQ ID NO.50：YYQNCVMK
SEQ ID NO.51：LPHGACIDDENYQNCLK
SEQ ID NO.52：NNIGSCNINTCFEDTSK
SEQ ID NO.53：SIYPNNYGDCATTHHDICGDLQG
Hum sc 9质谱鉴定的肽段：
SEQ ID NO.54：IISHHYNEK
SEQ ID NO.55：IISHHYNEKR
SEQ ID NO.56：LVKIISHHYNEK
SEQ ID NO.57：NVPTLIDAR
SEQ ID NO.58：RNVPTLIDAR
SEQ ID NO.59：VIGPTTLR
Hum sc 11质谱鉴定的肽段：
SEQ ID NO.60：TLEQVEIK
SEQ ID NO.61：GTTLIECGFSWDWGK
SEQ ID NO.62：QSHHTDIYTPEGPNCR
Hum sc 12质谱鉴定的肽段：
SEQ ID NO.63：IEQVDVR
SEQ ID NO.64：NVLDGKQDLSVYCK
SEQ ID NO.65：QDLSVYCK
SEQ ID NO.66：NNEFNTQILSYK
SEQ ID NO.67：EFHNVQIFMPESLNCR
SEQ ID NO.68：SFTFDCFDWGK
Hum sc 13质谱鉴定的肽段：
SEQ ID NO.69：AFLDAHNAIR
Hum sc 15质谱鉴定的肽段：
SEQ ID NO.70：AYLDAHNAVR
SEQ ID NO.71：TLEDYAQK
表6 葎草花粉致敏蛋白分子量、序列、过敏血清阳性率情况概览
Figure PCTCN2019088605-appb-000006
Figure PCTCN2019088605-appb-000007
备注：SDS-PAGE分辨率有限，10kDa蛋白总体血清阳性率66.7％，Hum sc 3与Hum S 3为同一蛋白；19kDa蛋白总体血清阳性率24.1％。
5、理化性质检测
该葎草变应原浸液经理化性质检验后，其质量标准如表7：
表7 葎草花粉变应原浸液理化性质质量标准
Figure PCTCN2019088605-appb-000008
6、总变应原活性测定
当变应原浸液包含治疗有效量或诊断有效量的葎草花粉变应原时，用竞争性ELISA法测定相对生物效价为50000BAU/ml-200000BAU/ml，蛋白浓度0.40～1.60mg/ml。
7、主要致敏蛋白Hum s 3含量检测
采用双单抗夹心ELISA法检测葎草花粉变应原浸液中主要致敏蛋白含量。经多批次检测，确定其波动范围0.08～0.32mg/ml。
8、无菌检查
不得有菌生长。
实施例7 葎草花粉变应原浸液成品稳定性试验
对实施例5制备的葎草花粉标准化变应原浸液成品在2-8℃下进行长期稳定性研究试验，分别对三个批次样品分别检测0时，3个月，6个月，9个月和12个月的pH值变化，苯酚含量，氯化钠含量，蛋白含量，总变应原活性，主要致敏蛋白Hum s 3含量的变化情况，结果如图7至图12所示。
从上述稳定性结果中，可以发现在12个月长期试验中，葎草花粉标准化变应原浸液各质控参数虽有不规则波动，可能是实验误差导致，但均在质量标准质控范围内，且不同批次间变化趋势基本一致，这说明按照本发明所述的葎草花粉标准化变应原浸液，在2-8℃条件下，至少12月内能稳定地保存。因此，葎草花粉变应原浸液成品的效期合理预计在36个月。
实施例8 评价应用1
本发明的制备的葎草花粉变应原浸液对葎草花粉过敏进行临床特异性皮试诊断的有效性和安全性。方法：选择2015-08-24日至2016-09-21间，在北京协和医院变态反应科门诊就诊的、患有过敏性结膜炎、过敏性鼻炎、过敏性哮喘、荨麻疹、特应性皮炎等过敏性疾病的门诊患者1026例。对受试者进行葎草花粉变应原皮肤点刺试验(Skin prick test，SPT)，采用原液进行，以平均风团直径(Mean wheal diameter，MWD)为判读标准；以花粉sIgE为标准，做ROC分析，分析不同诊断界值下，采用葎草花粉变应原浸液用于诊断葎草花粉过敏的准确性。同时记录不良事件，评价安全性。结果(Results)共入组门诊患者1026例(4～70岁)，无脱落病例，剔除率22.1％。安全集(Safety Set，SS)1026例，全分析集(Full Analysis Set，FAS)990例，符合方案集(Per protocol set，PPS)799例。年龄最小6岁，最大67岁。分析PPS集的ROC曲线，估算葎草花粉SPT的最佳诊断阈值为MWD 6.75mm；特异度达95％时的诊断阈值为MWD 8.25mm。分别以MWD 3mm(国际通用阈值)、6.75mm、8.25mm为诊断阈值，本发明的葎草花粉变应原浸液用于诊断葎草花粉过敏的灵敏度依次降低，分别为0.9366(95％CI：0.9159～0.9572)、0.5485(95％CI：0.5064～0.5906)、0.4216(95％CI：0.3798～0.4634)；特异度依次升高，分别为0.0913(95％CI：0.0565～0.1261)、0.8517(95％CI：0.8088～0.8947)、0.9696(95％CI：0.9488～0.9903)。1026例患者中有7例出现8次不良事件，不良事件发生率0.68％(7/1026)，主要表现为流涕，喷嚏，鼻痒、呼吸不畅、眼痒、胸闷、点刺局部皮肤反应等。无严重不良事件。结论：本发明葎草花粉变应原浸液用于诊断葎草花粉过敏，诊断价值较高，安全性好，可作为葎草花粉过敏原特异性体内诊断的临床检查方法。
实施例9 评价应用2
本发明的制备的葎草花粉变应原浸液对葎草花粉过敏患者全血进行嗜碱性粒细胞活化试验，能进行临床特异性过敏体外诊断。这个检测方法在IgE或非IgE介导的过敏反应中均适用，可以用于部分sIgE体外诊断存在假阴性、假阳性患者的确诊及部分过敏性休克患者不适于进行皮试诊断的情况。
试验原理：变应原与患者全血细胞反应可以模拟人体内变态反应过程：即特异性IgE抗体通过与相应的变应原桥联结合到细胞表面，激活细胞内信号级联导致嗜碱性粒细胞(CCR3持续表达于嗜碱性粒细胞，是其特异性标记)的活化脱颗粒。在这个脱颗粒的过程中，细胞内复合物影响跨膜蛋白CD63(gp53)，使其外表达于细胞表面，并暴露于细胞外基质中，因此可以依赖流式细胞术原理(使用抗人趋化因子受体CCR3-藻红蛋白(anti-CCR3-PE)对嗜碱性粒细胞进行标记，使用抗人CD63单克隆抗体-异硫氰酸荧光素(anti-CD63-FITC)对活化状态的嗜碱性粒细胞进行标记，非特异性细胞活化剂fMLP作为一种阳性质控)，并以嗜碱性粒细胞脱颗粒的百分数变化来判断受试者是否对特定变应原过敏。方法：选择健康受试者、葎草过敏患者，取其EDTA抗凝全血样本，以刺激缓冲液(阴性对照)、葎草变应原浸液(对1：10 3～10 10稀释比例进行优化，本实施例中取1：10 8)、fMLP刺激液(阳性质控)作为嗜碱性粒细胞的激活物，加入到全血中，然后加入anti-CD63-FITC、anti-CCR3-PE染色，48h内上流式细胞仪进行检测。结果：见图13。健康受试者以阴性对照、葎草变应原浸液、阳性对照为嗜碱性粒细胞活化物时，其嗜碱性粒细胞活化率分别应＜15％、＜15％、≥15％，葎草花粉过敏患者以阴性对照、葎草变应原浸液、阳性对照为嗜碱性粒细胞活化物时，其嗜碱性粒细胞活化率分别应＜15％、≥15％、≥15％。结论：该葎草变应原浸液能有效作为活化物运用于嗜碱性粒细胞活化试验，并依据其判断标准有效做出临床诊断。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (29)

  1. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 1，含有如SEQ ID NO.4所示的氨基酸序列。
  2. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 2，含有如SEQ ID NO.6所示的氨基酸序列。
  3. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 3，含有如SEQ ID NO.7所示的氨基酸序列。
  4. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 4，含有如SEQ ID NO.9所示的氨基酸序列。
  5. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 5，含有如SEQ ID NO.10所示的氨基酸序列。
  6. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 6，含有如SEQ ID NO.11所示的氨基酸序列。
  7. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 7，含有如SEQ ID NO.12所示的氨基酸序列。
  8. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 8，含有如SEQ ID NO.13所示的氨基酸序列。
  9. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 9，含有如SEQ ID NO.14所示的氨基酸序列。
  10. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 10，含有如SEQ ID NO.15所示的氨基酸序列。
  11. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 11，含有如SEQ ID NO.16所示的氨基酸序列。
  12. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 12，含有如SEQ ID NO.17所示的氨基酸序列。
  13. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原 蛋白Hum sc 13，含有如SEQ ID NO.18所示的氨基酸序列。
  14. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 14，含有如SEQ ID NO.19所示的氨基酸序列。
  15. 一种葎草花粉变应原浸提物，其特征在于，其含有葎草花粉变应原蛋白Hum sc 15，含有如SEQ ID NO.20所示的氨基酸序列。
  16. 用于编码葎草花粉花粉变应原浸提物的基因，其特征在于，所述基因为编码如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.9-20所示的氨基酸序列，分别对应如SEQ ID NO.21、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.26-37所示的序列。
  17. 一种的葎草花粉变应原浸液，其特征在于，所述变应原浸液中含如权利要求1-15所述的葎草花粉变应原浸提物、体积比为0.2～0.4％的苯酚、体积比为45～55％的甘油和4.5～5.5g/L的NaCl，其pH值为6.0～8.0。
  18. 根据权利要求17所述的葎草花粉变应原浸液，其特征在于，所述葎草花粉变应原浸液的活性浓度为50000～200000BAU/ml，蛋白浓度0.40～1.60mg/ml。
  19. [根据细则26改正28.06.2019]　
    根据权利要求17所述的葎草花粉变应原浸液，其特征在于，所述葎草花粉变应原浸液的总蛋白浓度为0.40～1.60mg/ml。
  20. [根据细则26改正28.06.2019]　
    根据权利要求17所述的葎草花粉变应原浸液，其特征在于，通过SDS-PAGE和Western Blotting检测，所述葎草花粉变应原提取物的蛋白主要分布在10kDa、11.8kDa、12.3kDa、13.5kDa、15.2kDa、15.32kDa、16.45kDa、16.61kDa、19kDa、28kDa、37kDa、42kDa、45kDa、52kDa、66kDa、76kDa。
  21. 根据权利要求17所述的葎草花粉变应原浸液，其特征在于，通过全蛋白质谱检测，其主要包含且不限于如SEQ ID NO.38-SEQ ID NO.71的特征性肽段。
  22. 一种根据权利要求17-21中任一项所述葎草花粉变应原浸液的制备方法，其特征在于，其包括如下步骤：
    S1、采集葎草花粉，常温干燥或真空干燥或流化床干燥；
    S2、对干燥后的花粉脱脂、干燥；
    S3、将脱脂干燥后的葎草花粉按重量g体积ml比1:50～1:10加入pH为7.9～8.2磷酸盐—盐水缓冲液，2～8℃搅拌22～26h进行浸提；
    S4、取步骤S3后的浸提液，离心取上清液；将上清液过滤及除菌过滤；
    S5、将过滤后的上清液进行超滤浓缩，获得超滤浓缩液；
    S6、将所述超滤浓缩液经过二次过滤和除菌过滤后，真空冻干获得葎草花粉变应原冻干品；
    S7、将所述葎草花粉变应原冻干品用pH 6.5～7.5磷酸盐—盐水缓冲液复溶配置为葎草花粉变应原原液，2～8℃放置，与等体积灭菌的甘油混匀，调节溶液pH值至6.0～8.0。
  23. 根据权利要求22所述的制备方法，其特征在于，在步骤S2中，所述花粉脱脂采用花粉与丙酮以1：5～1:1的重量g体积ml比进行脱脂处理，重复脱脂至上层液体澄清。
  24. 根据权利要求22所述的制备方法，其特征在于，在步骤S4中，所述离心的条件为8000～12000g，离心温度2～8℃，时间为15～20min；所述过滤及除菌过滤为先用4000目滤布过滤后，再通过纸板过滤、0.45μm和0.22μm滤膜依次过滤。
  25. 根据权利要求22所述的制备方法，其特征在于，所述超滤浓缩用3KD超滤膜超滤，当超滤透过液总蛋白含量≤0.02mg/ml，停止超滤；当超滤透过液总蛋白含量>0.02mg/ml，更换超滤膜之后对超滤透过液重复超滤，直至超滤透过液总蛋白含量≤0.02mg/ml为止；
    在步骤S6中，所述真空冻干的条件为-50～-35℃冻结，2～8mbar真空压力下，-25℃干燥，控制水分含量≤3％。
  26. 根据权利要求22所述的制备方法，其特征在于，所述步骤S1还包括对原料葎草花粉进行镜检鉴别和/或DNA鉴定的步骤，其中DNA鉴定方法为，以SEQ ID NO.1、SEQ ID NO.2为引物，对鉴别葎草花粉原料进行PCR扩增，并检测扩增产物。
  27. 一种葎草花粉变应原浸液冻干品，其特征在于，其由包括葎草花粉收集-干燥-脱脂-提取-超滤浓缩-冻干的步骤制得：
    (1)收集：用自然脱落法收集葎草花粉；
    (2)干燥：常温干燥或真空干燥或流化床干燥，直至花粉不再粘附，将干燥后的花粉过150～250目分样筛；
    (3)脱脂：将(2)所得到的花粉与丙酮分别按g与ml计，以1：5～1:1的重量体积比进行脱脂处理，搅拌或振荡脱脂30分钟后，静置分层，倒出上层液，加入新的丙酮，重复脱脂至上层液体澄清，将脱脂后的花粉均匀摊开，室温干燥或真空干燥或流化床干燥48小时至72小时；
    (4)提取：将脱脂干燥后的葎草葎草花粉按重量体积比1:50～1:10加入10L，pH 7.9～8.2磷酸盐—盐水缓冲液，2～8℃搅拌22～26h进行浸提；取提取后的浸提液，在离心力8000～12000g，离心温度2～8℃，时间设定为15～20分钟，进行离心，收取上清；先用4000目滤布过滤后，将滤液依次通过纸板过滤、0.45μm和0.22μm滤膜依次过滤；
    (5)超滤浓缩：将过滤后的浸提液，用3KD超滤膜超滤，取样超滤浓缩液、超滤透过液，检测其总蛋白含量；其中，当超滤透过液中总蛋白含量≤0.02mg/ml，则直接排弃透过液；超滤透过液总蛋白含量>0.02mg/ml，则对超滤膜进行完整性测试，超滤膜未破损，则排弃透过液；若超滤膜破损，则更换超滤膜之后对透过液重复超滤；
    (6)冻干：将超滤浓缩液经过二次过滤和除菌过滤后，按照冻干工艺条件：-50～-35℃冻结，2～8mbar真空压力下，-25℃干燥，控制水分含量≤3％，获得葎草花粉变应原冻干品。
  28. 根据权利要求1-15中任一项所述葎草花粉变应原浸提物、权利要求17-21中任一项所述葎草花粉变应原浸液、权利要求27所述的葎草花粉变应原浸液冻干品的应用，用于在制备诊断变态反应疾病并对其进行特异性免疫治疗制剂中的应用，所述诊断变态反应疾病包括过敏性哮喘、变应性鼻炎、特应性皮炎和慢性荨麻疹。
  29. 根据权利要求28所述的应用，其特征在于，所述葎草花粉变应原浸提物冻干品用于制备成片剂、口崩片，所述葎草花粉变应原浸液用于制备注射液、舌下滴剂、变应原斑贴剂、变应原浸液稀释液。
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