CN110064051B - Birch pollen allergen extract, extract and preparation method thereof - Google Patents

Birch pollen allergen extract, extract and preparation method thereof Download PDF

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CN110064051B
CN110064051B CN201910201921.7A CN201910201921A CN110064051B CN 110064051 B CN110064051 B CN 110064051B CN 201910201921 A CN201910201921 A CN 201910201921A CN 110064051 B CN110064051 B CN 110064051B
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尹佳
顾建青
周俊雄
高冬敖
王子熹
路蒨
党秀
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Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Abstract

The invention provides a birch pollen allergen extract, an immersion liquid thereof and a preparation method thereof, wherein the birch pollen allergen immersion liquid contains an amino acid sequence shown in SEQ ID NO.4-SEQ ID NO. 11. The birch pollen allergen extractive solution can also be prepared by collecting birch pollen, drying, defatting, extracting, ultrafiltering, concentrating, freeze drying, and redissolving. The birch pollen allergen immersion liquid has the characteristics of high specificity, sufficient extraction of the birch allergenic protein component, stable total biological value, long effective period and good aseptic effect; can be effectively used for skin prick test diagnosis, in-vitro basophil activation test diagnosis and specific immunotherapy of allergic diseases, and can effectively diagnose the allergic diseases induced by the birch pollen and carry out the specific immunotherapy.

Description

Birch pollen allergen extract, extract and preparation method thereof
Technical Field
The invention relates to the technical field of biology, and in particular relates to a standardized birch pollen allergen extract, an extract thereof and a preparation method thereof.
Background
The recognition of allergic diseases begins with "hay fever", also known as allergic rhinitis. Noon and Freeman used pollen extract for the first time in 1911 to treat hay fever, and the history of treatment of allergic diseases began. At present, allergic diseases are one of the major health problems worldwide. Over 25% of the population in industrialized countries is disturbed by allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, with allergic asthma being the most common. Inhalation of allergenic pollen is the most important factor in the development of allergic asthma and other respiratory allergic diseases. The investigation of VRTA-L.A and the like shows that nearly 50% of patients with allergic diseases are allergic to grass pollen.
The white birch (Betula platyphylla Suk) belongs to Betulaceae of Betulales, has large adaptability and wide distribution in hillside or forest with elevation of 400-4100 m, is distributed in northeast, north China, northwest China, southwest China and Russian far east, mongolia, korean and Japan, and has flowering period of 5-6 months. According to the monitoring data of the air pollen concentration, birch pollen accounts for about 7 percent of the air-borne pollen in spring. Birch pollinosis is very common in northern areas of china.
Zhouyun application of serum specific IgE detection in diagnosis of causes of allergic rhinitis indicated that birch pollen allergy accounts for 6.51% in 261 cases of AR patients; the research of 'value of allergen skin prick test in adult chronic cough etiology diagnosis' of Dihuikouqiang shows that the positive rate result of different allergen prick tests, the positive rate of birch pollen accounts for 2.27%; research on the spreading rule of pollen in the air of the central area of Taiyuan city and the detection of serum-specific IgE antibodies of bronchial asthma patients in Liufang shows that the birch pollen positive rate is 36.4 percent in patients with positive serum-specific IgE antibodies; the oral allergy syndrome of Chao Guang Si indicates that 12.3%, 6.5% and 1% of birch pollen allergic patients with skin symptoms, asthma and abdominal symptoms respectively; wangling's research on epidemiological investigation of asthma allergens in Haikou City indicated that birch pollen positive rate was 8.6% in 324 cases of skin prick results of asthma children.
The research on the epidemiological investigation of the birch pollen shows that the birch pollen allergy is widely distributed in various places in China, and the birch pollen is a main allergen of the pollen allergy in northern areas. The current methods of allergen-specific immunotherapy are largely divided into subcutaneous and sublingual immunotherapy. Subcutaneous injection-administered immunotherapy has been known for over 100 years and its safety and effectiveness have been demonstrated. In the early 90 s of the last century, allergen sublingual drop vaccines were born, 1998, WHO announced that allergen sublingual drop vaccines are safe and effective.
However, compared with subcutaneous injection immunization, the sublingual administration immunization desensitization mode has long treatment period, insignificant curative effect in short time and average desensitization period of 3-5 years. In one treatment cycle, the sublingual administration of an immune desensitizer is 100 times the amount administered by subcutaneous injection. Therefore, compared with sublingual administration, the mode of hyposensitization by subcutaneous injection administration has the advantages of short treatment period, quick response and low treatment cost although the compliance of patients is poor. At present, the stability of the subcutaneous injection immune preparation used for the birch pollen allergen is poor, the protein content and the biological value can be reduced along with time, and when the reagent with uncertain components is stored for a certain time and is used for diagnosing the birch pollen-induced allergic diseases, the positive rate is reduced, and the accuracy is low.
Disclosure of Invention
In order to solve the technical problems, the invention provides the birch pollen allergen-containing extract which has the characteristics of high specificity, sufficient and constant extraction of the birch allergenic protein component, stable total biological potency and long validity period. It can be used for the diagnosis of skin prick test and specific immunotherapy of allergic diseases, and can be used for the diagnosis and specific immunotherapy of allergic diseases induced by birch pollen.
In order to achieve the purpose, the invention adopts the following technical scheme that:
a birch pollen allergen extract comprising birch pollen allergen proteins Bet P1.0106, bet P1.0201, bet P1.0301, bet P2, bet P3, bet P4, bet P6 and Bet P7; wherein, the Bet P1.0106 contains an amino acid sequence shown as SEQ ID NO.4, the Bet P1.0201 contains an amino acid sequence shown as SEQ ID NO.5, the Bet P1.0301 contains an amino acid sequence shown as SEQ ID NO.6, the Bet P2 contains an amino acid sequence shown as SEQ ID NO.7, the Bet P3 contains an amino acid sequence shown as SEQ ID NO.8, the Bet P4 contains an amino acid sequence shown as SEQ ID NO.9, the Bet P6 contains an amino acid sequence shown as SEQ ID NO.10, and the Bet P7 contains an amino acid sequence shown as SEQ ID NO. 11.
The birch pollen allergen extract is prepared from the birch pollen allergen extract, phenol with the volume ratio of 0.2-0.4%, glycerol with the volume ratio of 45-55% and NaCl with the volume ratio of 4.5-5.5 g/L, and the pH value of the birch pollen allergen extract is 6.0-8.0.
Preferably, the birch pollen allergen immersion liquid has an active concentration of the birch pollen allergen of 5000 to 20000BAU/ml and a total protein concentration of 0.27mg/ml to 1.08mg/ml, wherein the content of the main allergenic protein is 0.10mg/ml to 0.60mg/ml.
Further, the protein distribution of the birch pollen allergen is mainly in bands of 7-8kDa, 15kDa, 17-18kDa, 24kDa, 27kDa, 35kDa and 92kDa, and the isoelectric point of the protein of the birch pollen allergen is distributed between 3.5-7, detected by SDS-PAGE and Western Blotting.
Further, the birch pollen allergen extract mainly comprises and is not limited to characteristic peptide fragments as shown in SEQ ID NO.20-SEQ ID NO.97 by total protein mass spectrometry or by SDS-PAGE separation and then respectively sampling corresponding molecular weight gel strips for mass spectrometry detection.
The preparation method of the birch pollen allergen immersion liquid comprises the following steps:
s1, collecting birch pollen, and drying at normal temperature in vacuum or in a fluidized bed;
s2, degreasing and drying the dried pollen;
s3, adding phosphate-saline buffer solution with the pH value of 7.9-8.2 into the degreased and dried birch pollen according to the weight g volume ml ratio of 1;
s4, collecting the leaching liquor obtained in the step S3, and centrifuging to obtain a supernatant; filtering the supernatant, sterilizing and filtering;
s5, carrying out ultrafiltration concentration on the filtered supernatant to obtain an ultrafiltration concentrated solution;
s6, after secondary filtration and sterilization filtration are carried out on the ultrafiltration concentrated solution, vacuum freeze-drying is carried out to obtain a freeze-dried birch pollen allergen product;
s7, re-dissolving the freeze-dried birch pollen allergen product by using a phosphate-saline buffer solution with the pH value of 6.5-7.5 to prepare a birch pollen allergen solution, placing the birch pollen allergen solution at the temperature of 2-8 ℃, uniformly mixing the birch pollen allergen solution with glycerol sterilized by the same volume, and adjusting the pH value of the solution to 6.0-8.0. In the preparation method, in step S2, the pollen is defatted by mixing pollen with acetone in a ratio of 1: and (3) degreasing treatment is carried out according to the weight g volume ml ratio of 5-1.
In the preparation method, in step S4, the centrifugation condition is preferably 8000-12000 g, the centrifugation temperature is 2-8 ℃, and the time is 15-20 min; the filtration and the sterilization filtration are sequentially carried out by filtering with 4000-mesh filter cloth, then filtering with a paperboard and 0.45-micron and 0.22-micron filter membranes.
In the preparation method as described above, preferably, in step S5, the ultrafiltration concentration is performed by using a 3KD ultrafiltration membrane, and when the total protein content of the ultrafiltration permeate is less than or equal to 0.02mg/ml, the ultrafiltration is stopped; when the total protein content of the ultrafiltration permeate is more than 0.02mg/ml, the ultrafiltration membrane is replaced, and then the ultrafiltration is repeated on the ultrafiltration permeate until the total protein content of the ultrafiltration permeate is less than or equal to 0.02 mg/ml.
The preparation method as described above, preferably, in step S6, the vacuum freeze-drying is performed under the conditions of freezing at-50 to-35 ℃, drying at-25 ℃ under the vacuum pressure of 2 to 8mbar, and the water content is controlled to be less than or equal to 3%.
Preferably, the preparation method as described above, the step S1 further comprises the step of performing microscopic examination identification and/or DNA identification on the raw material birch pollen, wherein the DNA identification method is to perform PCR amplification on the raw material birch pollen by using SEQ ID No.1 and SEQ ID No.2 as primers, and detect the amplification product.
In the above-mentioned preparation method, preferably, the phosphate-saline buffer solution comprises 4.5 to 5.5g/L of sodium chloride, 0.03 to 0.05% of monopotassium phosphate, 1.5 to 2% of disodium phosphate dodecahydrate, and 0.2 to 0.4% of phenol.
In preparing birch pollen allergens for extraction, the inventors tried various leaching solutions and compared their leaching efficiency. The leaching liquor used included: 0.8 percent of sodium chloride, a pH5.0 disodium hydrogen phosphate-citric acid buffer solution, a pH6.0 disodium hydrogen phosphate-citric acid buffer solution, a pH7.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a pH8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a pH8.2 coca's solution, a pH8.9 Tris-hydrochloric acid buffer solution, a pH 7.9-8.2 solution containing 4.5-5.5 g/L of sodium chloride, 0.03-0.05 percent of potassium dihydrogen phosphate, 1.5-2 percent of disodium hydrogen phosphate dodecahydrate, 0.2-0.4 percent of phenol and the like. The experiment result proves that the leaching efficiency of the sodium chloride with pH of 7.9-8.2 and the content of 4.5-5.5 g/L, 0.03-0.05% of monopotassium phosphate, 1.5-2% of disodium hydrogen phosphate dodecahydrate and 0.2-0.4% of phenol is high, and the prepared birch pollen allergen extract can be effectively stored for a long time and has high stability.
The invention also provides a white birch pollen allergen freeze-dried product, which is prepared by the steps of white birch pollen collection, drying, degreasing, extraction, ultrafiltration concentration and freeze-drying:
(1) Collecting: collecting birch pollen by natural falling off method;
(2) And (3) drying: drying at normal temperature in vacuum or fluidized bed until pollen is not adhered, and sieving the dried pollen with 150-250 mesh sieve;
(3) Degreasing: mixing the pollen obtained in the step (2) and acetone respectively according to g and ml, and mixing the mixture according to the ratio of 1: degreasing at the weight-volume ratio of 5-1, shaking for 30 minutes, standing for layering, pouring out supernatant, adding new acetone, repeatedly degreasing until the supernatant is clear, uniformly spreading the degreased pollen, and drying at room temperature in vacuum or by a fluidized bed for 48-72 hours;
(4) Extraction: adding 10L of phosphate-saline buffer solution with the pH value of 7.9-8.2 into degreased and dried birch pollen according to the weight volume ratio of 1-1; taking the extracted leaching liquor, centrifuging at the centrifugal force of 8000-12000 g and the centrifugal temperature of 2-8 ℃ for 15-20 minutes, and collecting the supernatant; filtering with 4000-mesh filter cloth, sequentially filtering the filtrate with cardboard, 0.45 μm and 0.22 μm filter membranes;
(5) And (3) ultrafiltration and concentration: ultrafiltering the filtered leaching solution with 3KD ultrafiltration membrane, sampling the ultrafiltration concentrated solution and ultrafiltration permeate, and detecting total protein content; wherein, when the total protein content in the ultrafiltration permeate is less than or equal to 0.02mg/ml, the permeate is directly discarded; if the total protein content of the ultrafiltration permeate is greater than 0.02mg/ml, performing integrity test on the ultrafiltration membrane, and if the ultrafiltration membrane is not damaged, discarding the permeate; if the ultrafiltration membrane is damaged, replacing the ultrafiltration membrane and then repeatedly ultrafiltering the permeate;
(6) Freeze-drying: after secondary filtration and sterilization filtration, the ultrafiltration concentrated solution is frozen at minus 50 to minus 35 ℃ and dried at minus 25 ℃ under the vacuum pressure of 2 to 8mbar according to the freeze-drying process conditions, and the water content is controlled to be less than or equal to 3 percent, so that the freeze-dried birch pollen allergen product is obtained.
The application of the birch pollen allergen extract, the birch pollen allergen extract and the birch pollen allergen freeze-dried product is used for preparing preparations for diagnosing allergic diseases and specifically immunizing and treating the allergic diseases, wherein the allergic diseases comprise allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.
Further, the freeze-dried birch pollen allergen extract product is used for preparing tablets and orally disintegrating tablets, and the birch pollen allergen extract is used for preparing injection, sublingual drops, allergen spot patches and allergen extract diluent.
The birch pollen allergen extract provided by the invention is used for preparing a medicament for treating birch pollen allergic diseases.
Specifically, the birch pollen allergen extract or the freeze-dried birch pollen allergen product provided by the invention can be used for preparing a medicament for treating the birch pollen allergic diseases according to a therapeutically effective amount or a diagnostically effective amount of the birch pollen allergen and a pharmaceutically acceptable carrier.
The medicament for treating allergic diseases of birch pollen can be prepared into various pharmaceutically acceptable dosage forms, and can be administered by a physician in a dosage amount beneficial to the patient, which depends on the age, weight and general disease condition of the patient. The preparation form of the medicine for treating the birch pollen allergic diseases is selected from liquid preparation forms such as oral preparation, (subcutaneous) injection, sublingual buccal preparation, aerosol, nasal agent, skin prick agent and the like; preferably (subcutaneous) injection, sublingual buccal, or skin prick. Among them, subcutaneous injections and sublingual preparations are generally used as a formulation for specific immunotherapy, and skin prickers are used as a formulation for in vivo allergen detection.
When the birch pollen allergen immersion liquid is used for diagnosing allergic diseases caused by birch pollen (namely, allergen skin prick diagnosis test), besides the birch pollen allergen immersion liquid, a negative control liquid, a positive control liquid and a prick needle are generally included. The negative control solution is generally a liquid (e.g., a mixture of glycerol and a salt solution) that does not cause allergic reactions to a human body, and the positive control solution is generally a histamine phosphate/histamine hydrochloride solution of 1.0 to 5.0 mg/ml.
The birch pollen allergen immersion liquid prepared by the invention can be applied to a patch test. The principle is as follows: suspected sensitizers (allergens) are applied to the skin of a patient, and after entering the body through the skin or mucosa, antigens are presented to T lymphocytes by antigen presenting cells, activating specific T lymphocytes, and inducing an inflammatory response.
The invention has the beneficial effects that:
1. the birch pollen standardized allergen extract provided by the invention can effectively diagnose the allergic diseases induced by the birch pollen and carry out specific immunotherapy on the allergic diseases.
2. The stability and the sustained-release effect are improved by adding the stabilizing agent glycerol, and the effectiveness and the safety of the medicine are improved.
3. The administration mode of hypodermal injection for immune desensitization of allergic diseases has quick response and short period compared with the administration mode of sublingual buccal drop. The dosage of the medicine is far less than that of sublingual drop (about 100 times) in the whole treatment period, and the treatment cost is far less than that of the sublingual drop for immune desensitization, thereby reducing the medical burden of patients.
4. The prepared strain original immersion liquid is used for pricking test by the original liquid to compare with clinical comprehensive specificity diagnosis and serum specific IgE (specific IgE, sIgE) diagnosis of an allergy specialist doctor respectively, and the evaluation result of the intradermal test is consistent with the results of the clinical comprehensive specificity diagnosis and the serum sIgE diagnosis, so that the intradermal test has better sensitivity and specificity and good safety. The birch pollen allergen immersion liquid provided by the invention has the characteristic of high specificity, the birch allergenic protein component is fully extracted, the total biological value is stable, the period of validity is long, and the sterility is ensured; can be effectively used for skin prick test diagnosis and specific immunotherapy of allergic diseases, and can effectively diagnose the allergic diseases induced by the birch pollen and carry out the specific immunotherapy. Can realize standardized control, effectively prolong the service validity and bring better economic benefit.
5. The present invention is prepared by using the allergen immersion liquid prepared in the ratio of 1:10 3 ～10 8 After dilution, an in vitro basophilic granulocyte activation test is carried out, so that the white birch allergic patients can be specifically diagnosed clinically, false positive detection of partial in vitro sIgE can be avoided, and the risk of anaphylactic shock brought to partial white birch pollen allergic patients by an allergen skin test (prick or intradermal) can also be avoided.
Compared with the birch pollen allergen injection stock solution developed by Beijing cooperative hospital, the extract obtained by freeze-drying and redissolving of the method of the invention is more stable, the biological potency and the protein content are more stable, and the effective period is 3 years.
Drawings
FIG. 1 shows the result of PCR electrophoresis of ITS2 sequence of birch pollen material.
FIG. 2 shows the variation of fat content of birch pollen in different batches at different defatting times.
FIG. 3 is the relationship between total protein content of birch pollen extract and extraction time.
FIG. 4 shows the result of SDS-PAGE protein electrophoresis of birch pollen allergen dip.
FIG. 5 shows the result of Western Blotting mixed serum pool reaction detection of birch pollen allergen immersion liquid and white birch allergy patient serum.
FIG. 6 shows the results of two-dimensional electrophoresis of the sensitizing protein of birch pollen.
Figure 7 is the pH test results of birch pollen allergen immersion product in the long term stability test.
Fig. 8 shows the results of the phenol content of the birch pollen allergen immersion product in the long-term stability test.
Fig. 9 is a measurement result of the glycerin content of the birch pollen allergen immersion liquid product in the long-term stability test.
Figure 10 is the results of the sodium chloride content test of the birch pollen allergen infusion product in the long term stability test.
Figure 11 is the results of the protein content test of the total protein content of the birch pollen allergen dip product in the long term stability test.
Fig. 12 is a graph of the results of the measurement of total allergen activity of birch pollen allergen infusion products in the long term stability test.
Figure 13 is the results of the total allergen activity of the main allergenic protein content of birch pollen allergen infusion products in the long-term stability test.
FIG. 14 is a Western Blotting comparative study of the stability of birch allergen immersion liquid and birch allergen injection preparation in medical institutions.
FIG. 15 shows an example of the results of a birch pollen allergen extract product applied to in vitro basophil activation tests of patients with clinical birch pollen allergy.
Detailed Description
The following examples are intended to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 identification of birch pollen in this example, DNA specific sequence detection plus microscopy was used for dual identification and quality control of birch pollen, since identification and purity of the material are of great importance for subsequent allergen diagnosis and therapeutic preparations.
1. DNA extraction of birch pollen
A Tiangen rapid DNA extraction and amplification kit (Tiangen biochemical KG 203) is adopted.
The extraction method comprises the following steps: weighing 5mg of birch pollen sample, placing the birch pollen sample into a 1.5mL centrifuge tube, adding 1. Mu.l of Buffer, thoroughly grinding the sample with a disposable grinding pestle, adding 2. Mu.l of Buffer, shaking, mixing uniformly, centrifuging at 12000r/min for 5min by a centrifuge, and taking the supernatant as a DNA template for standby.
2. Primer design and Synthesis
Bet p ITS2-F(SEQ ID NO.1)：5'-CTGAGAAACGGCTACCACATC-3'
Bet p ITS2-R(SEQ ID NO.2)：5'-GTCGGCCAAGGCTATAAACTC-3'
3. PCR reaction system
PCR amplification kit (tiangen biochemistry). A prepared PCR reaction system using the following 50. Mu.l system for the extracted DNA is shown in Table 1:
TABLE 1. Birch pollen ITS2 sequence PCR reaction System
Figure BDA0001997747360000081
4. The PCR reaction conditions are shown in Table 2.
TABLE 2 PCR reaction conditions for the ITS2 sequence of birch pollen
Figure BDA0001997747360000082
Electrophoresis was carried out on 1% agarose, 150V, 100mA, 20min. The electrophoresis results are shown in FIG. 1. Wherein L1, L2 and L3 are PCR products of 3 batches of birch pollen, L mix Is PCR product of the mixed raw material of the 3 batches of birch pollen, and DNA Marker is shown in figure 1.
5. Sequencing
And (3) recovering the PCR product by using a PCR product purification recovery kit (worker SK 1141) for sequencing, wherein the sequence result number is Bet p ITS2, and is shown as SEQ ID NO. 3.
SEQ ID NO.3：
AGTGACAATAAATAACAATACCGGGCTCTTTAGAGTCTGGTAATTGGA ATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTC TGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAG TTGTTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGTTGGGCAGATCG GTCCGCCCCTGGTGTGCACCGGTCCGCTCGTCCCTTCTACCGGCGATAC GCTCCTGGTCTTAATTGGCCGGGTCGTGCCTCCGGTGCTGTTACTTTGA AGAAATTAGAGTGCTCAAAGCAAGCCTACGCTCTGGATACATTAGCAT GGGATAACATCATAGGATTTCGGTCCTATTGTGTTGGCCTTCGGGATC GGAGTAATGATTAACAGGAACAGTCGGGGGCATTCGTATTTCATAGTC AGAGGTGAAATTCTTGGATTTATGAAAGACGAACAACTGCGAAAGCA TTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGA AGACGATCAGATACCGTCCTAGTCTCAACCATAAACGATGCCGACCAG GGATCGGCGGATGTTGCTTTCAGGACTCCGCCGGCACCTTATGAGAAA TCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTT AAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTA ATTTGACTCAACACGGGGAAACTTACCAGGTCCAGACATAGTAAGGAT TGACAGACTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGC CGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGTTAACGAACG AGACCTCAGCCTGTTAACTAGCTATGCGGAGGATCCCCTCCGCGGCCA GCTTCTTAGAGGGACTATGGCCGCTTAGGCCACGGAAGTTTGAGGCAA TAACAGGTCT
5. Microscopic examination of white birch
Morphological identification of white birch pollen under a microscope: birch pollen is oblate spheroid. The polar surface is usually provided with edges, and the number of the edges is shifted by the number of the holes; the equatorial plane is oblong with an average size of 29X 38 microns. The number of pores is 2-5 (8), which is the largest mutation in this genus. The surface is grainy ornamentation.
Example 2 determination of important parameters of the relevant Process steps for degreasing and leaching birch pollen
1. Betula alba pollen degreasing process step parameter determination
(1) Birch pollen (g) was mixed with acetone (ml) at a ratio of 1:2 (w/v) was subjected to degreasing treatment. And (3) detecting the fat content of the birch pollen samples of different batches (different collection times) at different degreasing times to determine the optimal degreasing time.
The fat content in the birch pollen is measured by a sea energy SOX500 fat measuring instrument, the change of the weight of the pollen before and after extraction is compared by a Soxhlet extraction and drying weighing method to obtain the corresponding fat content, and the quality of the defatted pollen is controlled according to the fat content result.
As a result, as shown in fig. 2, it can be seen that the fat content hardly decreased with the increase of the degreasing time after the acetone degreasing time was 30min, and thus the degreasing time was determined to be 30min.
(2) And (3) verification of degreasing parameters: the fat content was measured after 30min of defatting for different batches (different collection times) of birch pollen, and the results are shown in table 3.
TABLE 3 percentage reduction of fat content after 30min degreasing of birch pollen in different batches
Figure BDA0001997747360000101
As a result, the fat content of the birch pollen is about 0.5-5%, so that the fat content of the defatted birch pollen is reduced by about 0.5-5% in the later defatting process, namely the total weight of the pollen is reduced by about 0.5-5% by the same ratio.
2. Determination of important parameters of white birch pollen protein extraction process step
(1) Birch pollen (g) was mixed with acetone (ml) at a ratio of 1:2 (w/v), stirring or shaking for degreasing for 30 minutes, standing for layering, observing the clarification condition of the upper layer liquid, repeatedly degreasing until the upper layer liquid is clarified, uniformly spreading the degreased pollen, and drying at room temperature for more than 48 hours.
(2) Adding degreased and dried birch pollen into 10L of phosphate-saline buffer solution with the pH value of 7.9-8.2 according to the weight-volume ratio of 1 to 10, adding all extracted liquid into a centrifugal barrel when the stirring speed is 250rpm and the extraction points are respectively 3h, 6h, 9h, 12h, 18h, 24h, 48h, 72h and the like, adjusting the balance, setting the centrifugal force to be 8000-12000 g, the centrifugal temperature to be 4 ℃ and the time to be 15-20 minutes, centrifuging and collecting supernatant.
(3) Filtering the supernatant after centrifugal separation with 4000 mesh filter cloth, filtering with cardboard, and sequentially filtering with 0.45 μm and 0.22 μm filter membranes; to obtain a birch pollen crude extract.
Samples were taken and protein content was determined by the Bradford method, and the results are shown in FIG. 3.
As shown in the results of FIG. 3, in the process of protein extraction, the extraction content of protein increases with the extension of stirring extraction time, the extraction time 24h reaches the maximum content of 1374 +/-11 mug/ml, and the extraction time is preferably about 22-26 h as the extraction time is too long to influence the protein content.
Example 3 birch pollen raw material Process
1. Pollen collection
And (4) collecting the birch pollen by a natural falling method. Drying at normal temperature or in vacuum or fluidized bed until pollen is no longer adhered. The dried pollen is screened through 150-250 mesh sieve, preferably 180 mesh sieve in this example.
2. Drying
Spreading the pollen in a ventilated drying place, and naturally drying, or vacuum drying, or fluidized bed drying for 6-48 hr until the pollen no longer adheres.
3. Degreasing
Mixing the pollen (g) obtained above with acetone (ml) at a ratio of 1: and (2) degreasing at a weight-to-volume ratio (w/v) of 5-1. In the present embodiment, the ratio of 1:2. stirring or shaking for degreasing for 30 minutes, standing for layering, and observing the clarification condition of the upper layer liquid. Pouring out the upper liquid, adding new acetone, and repeatedly degreasing until the upper liquid is clear.
4. Drying again
Spreading the defatted pollen uniformly, and drying at room temperature, or vacuum drying, or fluidized bed drying for 6-48h.
5. Impurity control
(1) The content of impurity particles in the particles is determined by a particle counting method under a microscope, and the following standards are met: the content of spores is less than or equal to 1 percent, the content of unrelated pollen is less than or equal to 2 percent, and the content of other impurities is less than or equal to 10 percent.
(2) Heavy metals and harmful elements
The total weight of metal is less than or equal to 50mg/kg; arsenic is less than or equal to 5mg/kg.
(3) Acetone residue
The residual quantity of acetone is less than or equal to 0.5 percent (volume fraction).
Example 4 preparation of lyophilized birch pollen allergen product
1. Birch pollen material was prepared according to the birch pollen material process of example 3.
2. Leaching
Adding 10L of degreased and dried birch pollen into the mixture according to the weight volume ratio of 1:20, leaching with phosphate-saline buffer solution with pH of 7.9-8.2 and stirring for 22-26 h at 2-8 ℃. The formulation is shown in Table 4, which is an example of preparing 1000ml of phosphate-saline buffer (pH 8.2). After the phosphate-saline buffer solution is fully dissolved, the mixture is sterilized, filtered and placed at the temperature of 2-8 ℃, and the storage life is not more than 30 days.
TABLE 4 formulation of phosphate-saline buffer (pH 8.2)
Figure BDA0001997747360000121
3. Solid-liquid separation
Taking the extracted leaching liquor, adding the leaching liquor into a centrifugal barrel, adjusting the balance, carrying out centrifugation at 8000-12000 g of centrifugal force at 2-8 ℃ for 15-20 minutes, and collecting the supernatant;
4. clarification
Filtering the supernatant after centrifugal separation with 4000-mesh filter cloth, and sequentially filtering the filtrate with paperboard filter and 0.45-micron and 0.22-micron filter membranes;
5. ultrafiltering, dialyzing, and concentrating
The filtered leach liquor is subjected to tangential flow ultrafiltration by using a 3KD or 1KD ultrafiltration membrane, preferably, the 3KD ultrafiltration membrane is used in the embodiment. The dialysate is 25-125mM NH 4 HCO 3 In this embodiment, 50mM NH is preferred 4 HCO 3 . According to the quality standard of the protein content, carrying out ultrafiltration concentration to a proper volume, and detecting the total protein content interval from the protein content to the quality standard. Sampling the ultrafiltration concentrated solution and the ultrafiltration permeating solution, and detecting the total protein content. Directly discharging the permeated liquid when the total protein content in the ultrafiltration permeated liquid is less than or equal to 0.02 mg/ml; total protein content of ultrafiltration permeate>0.02mg/ml, performing integrity test on the ultrafiltration membrane, and discarding the permeation solution if the ultrafiltration membrane is not damaged; if the ultrafiltration membrane is damaged, the ultrafiltration membrane is replaced and then the ultrafiltration of the permeate is repeated.
6. Sterile filtration
Sterile filtration through a 0.22 μm membrane.
7. Freeze-drying
Freezing at-50-35 deg.c and drying at-25 deg.c under 2-8 mbar vacuum pressure, and controlling the water content to be less than or equal to 3%; obtaining the freeze-dried birch pollen allergen product.
Example 5 preparation of birch pollen allergen extract product
1. Redissolution
The lyophilized birch pollen allergen product prepared in example 4 was reconstituted with phosphate-saline buffer (pH 6.5-7.5, formulation see table 5) until the total protein content was within the range of 2 times the mass standard. Standing at 2-8 ℃.
TABLE 5 preparation of phosphate-saline buffer (pH 6.5-7.5)
Figure BDA0001997747360000131
2. Preparation of semi-finished product
After the stock solution is redissolved, the redissolved solution is mixed with glycerol which is subjected to equal volume pre-moist heat sterilization (at 121 ℃ for 30 minutes) and cooling in a purification workshop under the grade A clean condition, and the pH value of the solution is adjusted to 6.0-8.0.
3. Sterilizing, filtering, and packaging
And (3) under the grade-A clean condition in a purification workshop, sterilizing and filtering the semi-finished product by a 0.22 mu m filter membrane, and subpackaging into finished products in an aseptic manner to obtain pale yellow to brown liquid with the pH value of 6.0-8.0, namely the finished product of the standardized strain original extract of the birch pollen.
Example 6 quality control of finished birch pollen allergen infusions
1. Identification of total protein component of standardized allergen steep of birch pollen by SDS-PAGE method
The reduced SDS-PAGE method is adopted, the concentration of the separation gel is 4-12%, and Coomassie brilliant blue staining is carried out. The result is shown in fig. 4. M is Genstar Marker, R is lyophilized internal reference (lyophilized birch pollen allergen solution), L 1 ，L 2 And L 3 The protein distribution of the standardized allergen steep of the birch pollen in different batches is mainly about 7-8kDa, 15kDa, 17-18kDa, 24kDa, 27kDa, 35kDa and 92kDa, wherein the summary of the identified sensitizing protein molecular weight, the allergic seropositivity and the corresponding whole protein mass spectrum identification peptide segment is shown in Table 6.
2. Western Blotting method for identifying total allergen components in standardized allergen leaching of birch pollen
Reduced SDS-PAG electrophoresis is adopted, the concentration of separation gel is 4-12%, 0.2 mu m PVDF Immobilon R-PSQ transfer printing film, antiserum reaction dilution is 1:3, hybridization at room temperature for 2h, followed by hybridization in a refrigerator at 4 ℃ overnight, 3 times with 1 XPBST, 10 min/time. Secondary antibody Ms mAb to Hu IgE (ab 99806) 1: hybridization at 1000 ℃ for 2h,1 XPBST for 3 times, 10 min/time. And mixing the ECL developing solution A and the solution B1 uniformly, and then exposing and developing the mixture.
The results are shown in FIG. 5. Wherein M is Genstar Marker, R is internal reference, N is immersion fluid and serum reaction strip of healthy subject, and P 1 -P 26 The results of the immersion liquid and the serum reaction bands of 26 clinically confirmed positive birch allergy patients (sIgE is more than or equal to grade 3, skin test is more than or equal to + + +, typical pollen disease history in summer and autumn) are shown in FIG. 5 (a) as P 1 -P 15 FIG. 5 (b) shows the result of the test of (1) 16 -P 26 The test result of (1), wherein the Bet P is sensitized 1 -Bet P 8 ，Bet X 1 The molecular weights and the serum positivity rates of allergy are summarized in Table 6, and the Bet P with the highest serum positivity rate is determined 1 (17 kDa, depending on the gel strip) is the major allergenic protein of birch pollen (highest seropositivity, 76.9%).
3. Allergenic protein sequencing
The product of the invention performs whole protein sequence determination on corresponding allergenic proteins (shown in table 6), performs full-length nucleotide sequencing on mRNA corresponding to each allergenic protein in the birch pollen raw material, and details of the allergenic protein codes, sequence identifiers, full-length amino acid sequences and the corresponding mRNA sequence results are shown in table 6.
Wherein, the complete sequence of the white birch sensitization protein contained in the invention is as follows in sequence:
the complete sequence of the Bet P1.0106 sensitizing protein is SEQ ID NO.4:
MGVFNYEIEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGN GGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYSYSVIEGGPVGDTLEKI SNEIKIVATPNGGSILKINNKYHTKGDHEVKAEQIKASKEMGETLLRAVES YLLAHSDAYN
the full sequence of the Bet P1.0201 sensitizing protein is SEQ ID NO.5:
MGVFNYETETTSVIPAARLFKAFILEGDTLIPKVAPQAISSVENIEGNG GPGTIKKITFPEGSPFKYVKERVDEVDHANFKYSYSLIEGGPVGDTLEKIC NEIKIVATPDGGSILKISNKYHTKGDHEMKAEHMKAIKEKGEALLRAVES YLLAHSDAYN
the full sequence of the Bet P1.0301 allergenic protein is SEQ ID NO.6:
MGVFNYEDEATSVIAPARLFKSFVLDADNLIPKVAPENVSSAENIEGN GGPGTIKKITFPEGSHFKYMKHRVDEIDHANFKYCYSIIEGGPLGDTLEKIS YEIKIVAAPGGGSILKITSKYHTKGDISLNEEEIKAGKEKGAGLFKAVENY LVAHPNAYN
the full sequence of the Bet P2 sensitizing protein is SEQ ID NO.7:
MSWQTYVDEHLMCDIDGQGQQLAASAIVGHDGSVWAQSSSFPQFK PQEITGIMKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITI KKTGQALVFGIYEEPVTPGQCNMVVERLGDYLIDQGL
the full sequence of the Bet P3 allergenic protein is SEQ ID NO.8:
MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLF DKNSDGIITVDELSRALNLLGLETDLSELESTVKSFTREGNIGLQFEDFISLH QSLNDSYFAYGGEDEDDNEEDMRKSILSQEEADLSEAFKVFDEDGDGYIS ARELQMVLGKLGFSEGSEIDRVEKMIVSVDSNRDGRVDFFEFKDMMRSV LVRSS
the complete sequence of the Bet P4 sensitizing protein is SEQ ID NO.9:
MADDHPQDKAERERIFKRFDANGDGKISAAELGEALKTLGSITPDEV KHMMAEIDTDGDGFISFQEFTDFGRANRGLLKDVAKIF
the complete sequence of the Bet P6 sensitizing protein is SEQ ID NO.10:
MAHKSKILIIGGTGYIGKFIVEASAKSGHPTFALVRESTVSDPVKGKL VEKFKGLGVTLLHGDLYDHESLVKAFKQVDVVISTVGHLQLADQVKIIA AIKEAGNIKRFFPSEFGNDVDRVHAVEPAKTAFATKAEIRRKTEAEGIPYT YVSSNFFAGYFLPTLAQPGLTSPPREKVVIFGDGNARAVFNKEDDIGTYTI RAVDDPRTLNKIVYIKPAKNIYSFNEIVALWEKKIGKTLEKIYVPEEKLLK DIQESPIPINVILAINHSVFVKGDHTNFEIEASFGVEASELYPDVKYTTVEEY LQQFV
the complete sequence of the Bet P7 sensitizing protein is SEQ ID NO.11:
MASNPKVFFDMEVGGQPVGRIVMELYADTTPRTAENFRALCTGEKG NGRSGKPLHYKKSSFHRVIPGFMCQGGDFTAGNGTGGESIYGAKFADENF IKKHTGPGILSMANAGPGTNGSQFFICTAKTEWLDGKHVVFGQVVEGLDI VKAIEKVGSSSGRTSKPVVVADCGQLS
after the mRNA corresponding to each allergenic protein in the birch pollen raw material is subjected to nucleotide full-length sequencing, the corresponding sequence of the mRNA is as follows
The complete gene sequence of the Bet P1.0106 sensitizing protein is SEQ ID NO.12:
ATGGGTGTTTTCAATTACGAGATAGAGGCCACCTCTGTTATCCCA GCGGCTAGGCTATTCAAGGCCTTCATCCTTGATGGAGATAATCTCTTTC CAAAGGTTGCACCCCAAGCCATTAGCAGTGTTGAAAACATTGAAGGA AATGGAGGGCCTGGAACCATCAAGAAGATCAGCTTTCCCGAAGGCTTC CCTTTCAAGTACGTGAAGGACAGGGTTGATGAGGTGGACCACACAAA TTTCAAATACAGTTACAGCGTGATCGAGGGCGGTCCCGTGGGGGACAC ATTGGAGAAGATCTCTAACGAGATAAAGATAGTGGCAACCCCTAATG GCGGATCCATCTTGAAGATCAACAACAAGTACCATACCAAAGGAGAC CATGAGGTGAAGGCAGAGCAGATTAAGGCAAGTAAAGAAATGGGAG AGACACTTTTGAGGGCCGTTGAGAGCTACCTCTTGGCACACTCCGATG CCTACAACTAA
the full sequence of the Bet P1.0201 allergenic protein gene is SEQ ID NO.13:
ATGGGTGTTTTCAATTACGAAACTGAGACCACCTCTGTTATCCCA GCAGCTCGACTGTTCAAGGCCTTTATCCTCGAGGGTGATACTCTCATTC CAAAGGTTGCACCCCAAGCCATTAGCAGTGTTGAAAACATTGAAGGA AATGGAGGGCCTGGAACCATCAAGAAGATCACATTTCCCGAAGGCAG CCCTTTCAAGTACGTGAAGGAGAGGGTTGATGAGGTGGACCACGCAA ACTTCAAATATAGCTACAGCCTAATCGAGGGAGGGCCCGTGGGCGAC ACATTGGAGAAGATTTGCAACGAGATAAAGATAGTGGCAACCCCTGA TGGAGGATCCATCTTGAAGATCAGCAACAAGTACCACACCAAAGGCG ACCATGAGATGAAGGCAGAGCATATGAAGGCTATTAAAGAAAAGGGC GAGGCACTTTTGAGGGCCGTTGAGAGCTACCTCTTGGCACACTCTGAT GCCTACAACTAA
the full sequence of the Bet P1.0301 sensitizing protein gene is SEQ ID NO.14:
ATGGGTGTTTTCAACTACGAGGATGAGGCCACCTCCGTTATTGCT CCGGCTAGGCTCTTCAAGTCCTTTGTCCTTGACGCCGACAACCTCATTC CCAAGGTTGCACCTGAGAACGTTAGCAGTGCCGAAAATATTGAAGGA AACGGAGGACCTGGAACCATCAAGAAGATCACCTTTCCCGAAGGAAG CCATTTCAAGTACATGAAGCACAGGGTTGATGAGATCGACCACGCAA ACTTCAAATACTGCTACAGCATAATCGAGGGCGGTCCATTGGGCGACA CACTGGAGAAGATCTCTTACGAGATCAAGATTGTGGCCGCCCCTGGTG GAGGATCCATCTTGAAGATCACCAGCAAGTACCACACCAAAGGCGAC ATTTCGCTCAATGAGGAGGAGATTAAGGCCGGCAAAGAAAAGGGCGC CGGACTTTTCAAGGCTGTTGAGAACTACCTCGTGGCACACCCAAATGC CTACAACTAA
the complete sequence of the Bet P2 allergenic protein gene is SEQ ID NO.15:
ATGTCGTGGCAAACGTACGTGGATGAACATTTGATGTGCGATATC GACGGGCAAGGCCAGCAACTCGCTGCATCTGCGATCGTCGGTCACGAT GGCTCTGTGTGGGCCCAGAGCTCTTCCTTCCCACAGTTTAAGCCTCAG GAAATCACTGGTATCATGAAGGACTTTGAGGAGCCGGGTCATCTTGCT CCGACGGGCTTACACCTTGGGGGCATAAAATACATGGTCATCCAGGGA GAGGCTGGTGCTGTCATCCGTGGAAAGAAGGGATCTGGAGGTATTACT ATAAAGAAGACTGGTCAAGCTCTCGTTTTTGGCATCTATGAAGAGCCT GTGACACCAGGACAGTGCAACATGGTTGTTGAGAGGTTGGGGGATTA CCTTATTGACCAGGGCCTGTAG
the complete sequence of the Bet P3 allergenic protein gene is SEQ ID NO.16:
ATGCCCTGTTCCACAGAAGCCATGGAAAAAGCAGGGCATGGGCA TGCAAGTACACCTCGCAAGCGTAGCCTCAGCAACTCGTCCTTCCGCCT CCGCTCCGAGAGTCTGAATACCCTCCGCCTCCGACGCATATTCGATCT ATTTGACAAGAACAGCGATGGCATCATCACCGTCGATGAACTCAGCCG AGCCCTCAACCTTCTTGGCCTCGAAACCGACCTCTCGGAGCTCGAATC CACCGTCAAATCATTCACTCGAGAGGGCAACATTGGGCTTCAATTCGA AGACTTCATATCGCTGCACCAATCCCTAAACGACAGCTACTTTGCTTA CGGCGGCGAGGATGAGGATGATAATGAGGAGGATATGAGAAAGAGC ATATTGTCGCAAGAGGAAGCAGATCTTTCGGAGGCGTTCAAGGTGTTC GACGAGGACGGGGATGGTTACATATCGGCCAGAGAACTGCAAATGGT ACTGGGGAAGCTGGGATTCTCTGAAGGGAGCGAGATTGACAGAGTTG AGAAGATGATCGTGTCCGTTGACAGCAACCGAGATGGCCGGGTTGATT TCTTTGAGTTCAAGGATATGATGCGTAGCGTTCTCGTGCGGAGCTCTTG A
the complete sequence of the Bet P4 sensitizing protein gene is SEQ ID NO.17:
ATGGCTGATGATCATCCACAGGACAAGGCTGAACGCGAGCGCAT TTTCAAGCGCTTTGACGCCAATGGCGATGGTAAAATCTCTGCAGCAGA GCTTGGGGAGGCCTTGAAAACACTTGGCTCCATCACACCGGATGAGGT GAAACATATGATGGCCGAGATTGACACCGATGGCGACGGCTTCATTTC GTTCCAAGAGTTCACGGATTTTGGTCGTGCTAATCGTGGTTTACTAAA GGATGTTGCCAAGATATTTTAA
the full sequence of the Bet P6 allergenic protein gene is SEQ ID NO.18:
ATGGCTCACAAAAGCAAGATTCTGATCATCGGAGGCACCGGCTAC ATCGGAAAATTCATCGTGGAAGCAAGTGCAAAGTCTGGCCATCCCACC TTTGCTTTGGTCAGAGAGAGTACGGTCTCTGATCCCGTTAAGGGAAAA CTTGTCGAGAAATTCAAGGGCTTAGGCGTCACTTTGCTCCATGGAGAT CTGTATGACCATGAGAGTTTGGTAAAGGCGTTTAAGCAGGTGGACGTG GTGATATCGACGGTAGGCCACCTGCAGTTAGCAGATCAGGTCAAGATT ATTGCTGCCATTAAAGAGGCTGGTAATATTAAGAGATTCTTCCCTTCG GAATTCGGAAACGACGTAGACCGTGTGCATGCTGTTGAGCCAGCAAA GACTGCATTTGCTACCAAGGCCGAAATCCGCCGCAAGACTGAGGCGG AAGGCATCCCCTACACTTATGTGTCATCCAATTTCTTCGCTGGATATTT TCTTCCTACGTTGGCACAACCAGGACTCACTTCTCCTCCTAGAGAGAA AGTCGTTATCTTCGGAGATGGAAATGCCAGGGCTGTTTTTAACAAGGA AGACGACATAGGAACCTACACAATTAGAGCTGTGGATGACCCAAGAA CACTGAATAAGATAGTCTACATTAAGCCTGCCAAGAACATTTACTCAT TCAATGAGATTGTTGCCCTTTGGGAGAAAAAGATTGGCAAAACCCTTG AGAAAATCTATGTTCCAGAGGAGAAACTTTTGAAGGACATCCAAGAG TCCCCAATTCCAATCAACGTGATATTAGCAATCAACCACTCAGTTTTTG TGAAGGGAGATCATACCAACTTCGAGATTGAGGCATCCTTCGGTGTGG AGGCCTCCGAGCTATACCCAGATGTCAAATACACCACAGTGGAAGAG TACCTTCAGCAGTTTGTTTGA
the complete sequence of the Bet P7 allergenic protein gene is SEQ ID NO.19:
ATGGCGTCAAACCCTAAGGTCTTCTTCGACATGGAGGTCGGTGGC CAGCCCGTTGGGCGGATCGTGATGGAGCTCTACGCCGACACCACTCCC CGCACGGCCGAGAACTTCCGGGCCCTCTGCACCGGTGAGAAGGGCAA CGGCCGCTCCGGCAAGCCCCTCCACTACAAGAAATCGTCCTTCCACAG GGTGATCCCCGGGTTCATGTGCCAGGGAGGCGACTTCACTGCCGGAAA CGGCACCGGCGGCGAGTCCATCTACGGCGCCAAGTTCGCCGATGAGA ACTTCATCAAGAAGCACACCGGCCCCGGCATCCTCTCCATGGCTAATG CCGGCCCCGGCACCAATGGATCTCAGTTCTTCATCTGTACCGCCAAGA CCGAGTGGCTCGACGGCAAGCACGTGGTGTTCGGCCAGGTCGTGGAG GGTCTGGACATCGTGAAAGCCATCGAGAAGGTCGGGTCCAGCTCCGG CAGGACTTCCAAGCCCGTGGTCGTCGCCGACTGTGGTCAACTCTCTTA G
4. mass spectrometric detection
Performing whole protein mass spectrometry on the birch allergen immersion liquid, and respectively sampling corresponding adhesive strips after the birch allergen is separated by SDS-PAGE to perform mass spectrometry identification, wherein the adhesive strips mainly comprise the following peptide fragments:
detecting corresponding peptide fragments by Bet P1.0106 sensitized protein mass spectrum:
SEQ ID NO.20：AFILDGDNLFPK
SEQ ID NO.21：AFILDGDNLFPKVAPQAISSVENIEGNGGPGTIK
SEQ ID NO.22：ISFPEGFPFK
SEQ ID NO.23：DRVDEVDHTNFK
SEQ ID NO.24：VDEVDHTNFK
SEQ ID NO.25：ASKEMGETLLR
SEQ ID NO.26：EMGETLLR
SEQ ID NO.27：KISFPEGFPFK
SEQ ID NO.28：GVFNYEIEATSVIPAAR
SEQ ID NO.29：YSYSVIEGGPVGDTLEK
SEQ ID NO.30：IVATPNGGSILK
SEQ ID NO.31: vapqassississingengiggpgtik (Bet P1.0201 also contains the peptide)
SEQ ID NO.32: AVESYLLAAHSDAYN (Bet P1.0201 also contains the peptide)
SEQ ID NO.33: VAPQAISSVEENIEGNGGPGTIKK (Bet P1.0201 also contains the peptide)
Mass spectrum detection of the Bet P1.0201 allergenic protein on the corresponding peptide fragment:
SEQ ID NO.31: VAPQAISSVEENIEGNGGPGTIK (Bet P1.0106 also contains the peptide)
SEQ ID NO.32: AVESYLLAAHSDAYN (Bet P1.0106 also contains the peptide)
SEQ ID NO.33: VAPQAISSVEENIEGNGGPGTIKK (Bet P1.0106 also contains the peptide)
SEQ ID NO.34：KITFPEGSPFK
SEQ ID NO.35：ITFPEGSPFK
SEQ ID NO.36：ITFPEGSPFKYVK
SEQ ID NO.37：AFILEGDTLIPK
SEQ ID NO.38：ERVDEVDHANFK
SEQ ID NO.39：VDEVDHANFK
SEQ ID NO.40：IVATPDGGSILKISNK
SEQ ID NO.41：IVATPDGGSILK
SEQ ID NO.42：YHTKGDHEMKAEHMKAIKEKGEALLR
SEQ ID NO.43：EKGEALLR
SEQ ID NO.44：GVFNYETETTSVIPAAR
SEQ ID NO.45：GDHEMKAEHMK
SEQ ID NO.46：ISNKYHTK
SEQ ID NO.47：YSYSLIEGGPVGDTLEK
Mass spectrum detection of beta P1.0301 sensitized protein on corresponding peptide fragments: SEQ ID No.48: AVENYLVAHPNAYN
SEQ ID NO.49：GDISLNEEEIK
SEQ ID NO.50：GVFNYEDEATSVIAPAR
SEQ ID NO.51：ITFPEGSHFK
SEQ ID NO.52：KITFPEGSHFK
SEQ ID NO.53：IVAAPGGGSILK
SEQ ID NO.54：SFVLDADNLIPK
SEQ ID NO.55：VAPENVSSAENIEGNGGPGTIKK
SEQ ID NO.56：HRVDEIDHANFK
SEQ ID NO.57：VDEIDHANFK
Mass spectrum detection of beta P2 allergenic protein of corresponding peptide fragments:
SEQ ID NO.58：DFEEPGHLAPTGLHLGGIK
SEQ ID NO.59：YMVIQGEAGAVIR
SEQ ID NO.60：DFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIR
SEQ ID NO.61：KTGQALVFGIYEEPVTPGQCNMVVER
SEQ ID NO.62：TGQALVFGIYEEPVTPGQCNMVVER
SEQ ID NO.63：KGSGGITIK
SEQ ID NO.64：GSGGITIK
SEQ ID NO.65：LGDYLIDQGL
mass spectrum detection of beta P3 allergenic protein of corresponding peptide fragments:
SEQ ID NO.66：NSDGIITVDELSR
mass spectrum detection of beta P4 sensitized protein on corresponding peptide fragments:
SEQ ID NO.67：ISAAELGEALK
SEQ ID NO.68：TLGSITPDEVK
mass spectrum detection of beta P6 allergenic protein of the corresponding peptide fragment:
SEQ ID NO.69：AVFNKEDDIGTYTIR
SEQ ID NO.70：EDDIGTYTIR
SEQ ID NO.71：DIQESPIPINVILAINHSVFVK
SEQ ID NO.72：EKVVIFGDGNAR
SEQ ID NO.73：ESTVSDPVK
SEQ ID NO.74：ESTVSDPVKGK
SEQ ID NO.75：RFFPSEFGNDVDR
SEQ ID NO.76：FFPSEFGNDVDR
SEQ ID NO.77：FFPSEFGNDVDRVHAVEPAK
SEQ ID NO.78：FIVEASAK
SEQ ID NO.79：GLGVTLLHGDLYDHESLVK
SEQ ID NO.80：ILIIGGTGYIGK
SEQ ID NO.81：IVYIKPAK
SEQ ID NO.82：NIYSFNEIVALWEK
SEQ ID NO.83：QVDVVISTVGHLQLADQVK
SEQ ID NO.84：SGHPTFALVR
SEQ ID NO.85：VHAVEPAK
SEQ ID NO.86：VHAVEPAKTAFATK
SEQ ID NO.87：VVIFGDGNAR
mass spectrum detection of beta P7 allergenic protein of the corresponding peptide fragment:
SEQ ID NO.88：FADENFIK
SEQ ID NO.89：FADENFIKK
SEQ ID NO.90：HVVFGQVVEGLDIVK
SEQ ID NO.91：IVMELYADTTPR
SEQ ID NO.92：SGKPLHYK
SEQ ID NO.93：SGKPLHYKK
SEQ ID NO.94：VFFDMEVGGQPVGR
SEQ ID NO.95：AIEKVGSSSGR
SEQ ID NO.96：TSKPVVVADCGQLS
SEQ ID NO.97：VIPGFMCQGGDFTAGNGTGGESIYGAK
in addition, when the peptide segment of the sequence shown in SEQ ID NO.20-97 is artificially synthesized to treat allergic diseases such as allergic asthma, allergic rhinitis, allergic dermatitis and chronic urticaria patients, the therapeutic effect is obvious.
The summary of the identification peptide segments of the identified sensitized protein molecular weight, the allergic seropositivity and the corresponding whole protein mass spectrum is shown in a table 6.
TABLE 6 overview of molecular weight of birch pollen allergenic protein and allergic seropositivity
Figure BDA0001997747360000231
5. Two-dimensional electrophoresis determination of isoelectric point of sensitized protein
The two-dimensional electrophoresis result shows that the isoelectric point of the main allergenic protein of the birch pollen is distributed between 3.5 and 7, as shown in figure 6.
6. Physical and chemical property detection
The birch allergen steep has the quality standard shown in Table 7 after physical and chemical properties test:
TABLE 7 quality standards for physicochemical properties of birch pollen allergen infusions
Figure BDA0001997747360000241
7. Total allergen Activity assay
When the allergen steep comprises a therapeutically or diagnostically effective amount of birch pollen allergen, the relative bio-potency is 5000-20000 BAU/ml as determined by competitive ELISA.
8. Sterility testing
No bacteria can grow.
Example 7 stability test
The birch pollen standardized allergen immersion finished product prepared in example 5 was subjected to a long-term stability study test at 2-8 ℃, and the pH change, the phenol content, the glycerin content, the sodium chloride content, the protein content, the total allergen activity and the main allergenic protein content were respectively measured at 0 month, 3 months, 6 months, 9 months and 12 months for three batches of samples, and the results are shown in fig. 7 to fig. 13.
From the above stability results, it can be found that in the long-term test of 12 months, the quality control parameters of the standardized allergen extract of birch pollen are irregular and may be caused by experimental errors, but all are within the quality control range of the standard, and the variation trend is basically consistent among different batches, which shows that the standardized allergen extract of birch pollen according to the present invention can be stably stored for 12 months at 2-8 ℃.
Example 8 Western Blotting comparative study on the stability of the birch allergen immersion liquid and the birch allergen injection preparation in medical institutions (Beijing coordination Hospital)
The allergen immersion liquid composition of the present inventionThe method adds 50% of glycerol as a stabilizer, optimizes the pH value and the formula of a phosphate buffer solution, and makes the phosphate buffer solution more suitable for leaching and storing pollen allergen, the raw materials are identified by a DNA bar code technology, the purity is ensured, the stock solution adopts an optimized freeze-drying process, and the design and the process realize that the finished product of the allergen immersion solution obtained according to the invention is good in stability and the allergen activity is well stored, so that the effectiveness and the safety of clinical use are improved. In order to verify that the preservation of the birch allergen activity of the formula and the process under the invention is better than that of the birch allergen crude extract of the same variety prepared by the existing process and the formula, the present example is directed to a birch allergen immersion liquid (the invention is a product A below) and a birch allergen injection medical institution preparation (prepared in Beijing coordination and hospitals, and extracted by Coca's liquid with the pH of 8.2, and mainly comprises 0.5% of NaCl, 0.275% of NaHCO 3 0.4% phenol and water for injection, i.e. the product described below as B) were compared for stability.
In the embodiment, the product A and the product B are compared and placed for 12 months at 4 ℃ (the temperature is controlled to be 2-6 ℃, and the product B is stored in a dark place) and then sampled at 0 point and 12 months respectively, and the stability difference is evaluated by Western Blotting reaction between the product and a white birch allergy patient serum pool (15 white birch allergy patients constitute a serum pool, sIgE is not less than 3 grade, skin test is not less than + + +, and typical summer and autumn pollen disease history people are selected).
The test adopts reduced SDS-PAG electrophoresis, the concentration of separation gel is 4-12%, 0.2 mu m PVDF Immobilon R-PSQ transfer printing film, the first antiserum reaction dilution is 1:3, hybridization at room temperature for 2h, followed by hybridization in a refrigerator at 4 ℃ overnight, 3 times with 1 XPBST, 10 min/time. . Secondary antibody Ms mAb to Hu IgE (ab 99806) 1: hybridization at 1000 ℃ for 2h,1 XPBST for 3 times, 10 min/time. And mixing ECL developing solution 1 uniformly and then exposing and developing.
The results are shown in FIG. 14, in which M is protein molecular weight Marker (Genstar), N is serum reaction band of the immersion fluid and healthy subjects, L1 and L2 are respectively the reaction result of the product of the present invention at 0 (A0) and 12 months (A12) with serum pool, and L3 and L4 are respectively the reaction band result of the original hospital preparation at 0 (B0) and 12 months (B12) with clinical serum pool. The result shows that after the product is placed for 12 months at 4 ℃ compared with the original hospital preparation product, the main sensitization protein band is clear, and the reaction of other sensitization bands is still more obvious, which shows that the white birch sensitization protein in the product is more abundant, better in preservation and higher in activity, thereby proving that the product is more stable and effective.
Example 9 evaluation application 1
The effectiveness and safety of birch pollen allergen dip in diagnosing birch pollen allergy were evaluated by Skin Prick Test (SPT). The method comprises the following steps: selecting outpatients with allergic diseases such as allergic rhinitis, allergic asthma, allergic conjunctivitis, atopic dermatitis, urticaria, etc. in Beijing cooperative hospital allergy (anaphylaxis) department from 8 months and 10 days in 2015 to 10 months and 20 days in 2016. All subjects were subjected to the synergistic birch pollen allergen SPT and after 15min the Mean Wheal Diameter (MWD) was determined. Taking birch pollen serum specific immunoglobulin E (sIgE) as a diagnosis standard for confirming birch pollen allergy, performing receiver operating characteristic curve (ROC curve) analysis on the birch pollen allergy, and judging the accuracy of clinical diagnosis of birch pollen allergy by the birch pollen allergen immersion liquid under different diagnosis threshold values; adverse events were also recorded and the safety of the synergistic birch pollen allergen was evaluated. Results A total of 1029 cases were included in the study, with no shedding cases. The complete analysis set (FAS) 1 case 007, average age (32.14. + -. 13.69) years. The area under the ROC curve (AUC) of FAS was 0.909 (95% confidence interval 0.891-0.928). According to the ROC estimation of FAS, the MWD optimal diagnosis threshold value of SPT for diagnosing birch pollen allergy is 4.25mm, and the MWD diagnosis threshold value when the specificity reaches 95% is 5.75mm. The sensitivity of the synergistic birch pollen allergen SPT for diagnosing birch pollen allergy is sequentially reduced by taking MWD (measured molecular weight) of 3.00, MWD of 4.25 and MWD of 5.75mm as diagnosis threshold values, and is respectively 0.061 (95% confidence interval of 0.8840-0.9282), 0.7973 (95% confidence interval of 0.7669-0.827) and 0.6706 (95% confidence interval of 0.6351-0.7062), and the specificity is sequentially increased by 0.690 5 (95% confidence interval of 0.6410-0.7399), 0.9018 (95% confidence interval of 0.8700-0.9336) and 0.9524 (95% confidence interval of 0.9296-0.9752). 1029 cases and 6 cases of Safe Set (SS) have 7 adverse events, the occurrence rate of the adverse events is 0.58% (6/1029), and the adverse events are mainly manifested by nasal itching, sneezing, watery nasal discharge, nasal obstruction and itching of eyes after SPT and large wheal reaction of local skin at the pricked part. All subjects did not develop serious adverse events. And (4) conclusion: the birch pollen allergen extract has good diagnostic value and safety for birch pollen allergy. The accuracy of the diagnosis can be improved by combining the medical history with different SPT diagnosis threshold values.
Example 10 evaluation application 2
The birch pollen allergen immersion liquid prepared by the invention can be used for carrying out basophilic granulocyte activation test on the whole blood of a birch pollen allergic patient, and can be used for carrying out clinical specific allergy in vitro diagnosis. The detection method is applicable to IgE or non-IgE mediated anaphylactic reaction, and can be used for the in-vitro diagnosis of partial sIgE patients with false negative and false positive and the diagnosis of partial anaphylactic shock patients not suitable for skin test diagnosis.
The test principle is as follows: the reaction of allergens with patient's whole blood cells can mimic the allergic process in humans: i.e. specific IgE antibodies bind to the cell surface via bridging with the corresponding allergen, activating the intracellular signaling cascade leads to activated degranulation of basophils (CCR 3 is persistently expressed on basophils, being its specific marker). During this degranulation, the intracellular complex affects the transmembrane protein CD63 (gp 53), making it expressed on the cell surface and exposed to the extracellular matrix, and thus can rely on flow cytometry principles (labeling of basophils with anti-human chemokine receptor CCR 3-phycoerythrin (anti-CCR 3-PE), labeling of basophils in the activated state with anti-human CD63 monoclonal antibody-fluorescein isothiocyanate (anti-CD 63-FITC), non-specific cell activator fMLP as a positive quality control), and determining whether a subject is allergic to a particular allergen as a percentage change in degranulation of basophils. The method comprises the following steps: selecting healthy subject and birch allergic patient, collecting EDTA anticoagulated whole blood sample, adding stimulation buffer (negative control), and birch allergen steep (1: 10 for each) 3 ～10 10 The dilution ratio was optimized, in this example 1:10 8 ) And fMLP stimulating solution (positive quality control) is used as an activator of basophils and added into whole blood, then anti-CD63-FITC and anti-CCR3-PE are added for staining, and detection is carried out by an up-flow cytometer within 48h. The results are shown in fig. 15 and show that: when the negative control, the birch allergen immersion liquid and the positive control are respectively basophil activators, the basophil activation rates of healthy subjects are respectively less than 15 percent, less than 15 percent and more than or equal to 15 percent, and when the negative control, the birch allergen immersion liquid and the positive control are respectively basophil activators, the basophil activation rates of white birch pollen allergic patients are respectively less than 15 percent, more than or equal to 15 percent and more than or equal to 15 percent. And (4) conclusion: the birch allergen extract can be effectively used as an activator for basophilic granulocyte activation test, and can effectively make clinical diagnosis according to the judgment standard.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing coordination hospital of Chinese academy of medical sciences
<120> birch pollen allergen extract, extract thereof and preparation method thereof
<160> 97
<170> SIPOSequenceListing 1.0
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctgagaaacg gctaccacat c 21
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gtcggccaag gctataaact c 21
<210> 29
<211> 971
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
agtgacaata aataacaata ccgggctctt tagagtctgg taattggaat gagtacaatc 60
taaatccctt aacgaggatc cattggaggg caagtctggt gccagcagcc gcggtaattc 120
cagctccaat agcgtatatt taagttgttg cagttaaaaa gctcgtagtt ggatcttggg 180
ttgggcagat cggtccgccc ctggtgtgca ccggtccgct cgtcccttct accggcgata 240
cgctcctggt cttaattggc cgggtcgtgc ctccggtgct gttactttga agaaattaga 300
gtgctcaaag caagcctacg ctctggatac attagcatgg gataacatca taggatttcg 360
gtcctattgt gttggccttc gggatcggag taatgattaa caggaacagt cgggggcatt 420
cgtatttcat agtcagaggt gaaattcttg gatttatgaa agacgaacaa ctgcgaaagc 480
atttgccaag gatgttttca ttaatcaaga acgaaagttg ggggctcgaa gacgatcaga 540
taccgtccta gtctcaacca taaacgatgc cgaccaggga tcggcggatg ttgctttcag 600
gactccgccg gcaccttatg agaaatcaaa gtctttgggt tccgggggga gtatggtcgc 660
aaggctgaaa cttaaaggaa ttgacggaag ggcaccacca ggagtggagc ctgcggctta 720
atttgactca acacggggaa acttaccagg tccagacata gtaaggattg acagactgag 780
agctctttct tgattctatg ggtggtggtg catggccgtt cttagttggt ggagcgattt 840
gtctggttaa ttccgttaac gaacgagacc tcagcctgtt aactagctat gcggaggatc 900
ccctccgcgg ccagcttctt agagggacta tggccgctta ggccacggaa gtttgaggca 960
ataacaggtc t 971
<210> 5
<211> 160
<212> PRT
<213> amino acid
<400> 5
Met Gly Val Phe Asn Tyr Glu Ile Glu Ala Thr Ser Val Ile Pro Ala
1 5 10 15
Ala Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro
20 25 30
Lys Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn
35 40 45
Gly Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro
50 55 60
Phe Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe
65 70 75 80
Lys Tyr Ser Tyr Ser Val Ile Glu Gly Gly Pro Val Gly Asp Thr Leu
85 90 95
Glu Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asn Gly Gly
100 105 110
Ser Ile Leu Lys Ile Asn Asn Lys Tyr His Thr Lys Gly Asp His Glu
115 120 125
Val Lys Ala Glu Gln Ile Lys Ala Ser Lys Glu Met Gly Glu Thr Leu
130 135 140
Leu Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155 160
<210> 5
<211> 160
<212> PRT
<213> amino acid
<400> 5
Met Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala
1 5 10 15
Ala Arg Leu Phe Lys Ala Phe Ile Leu Glu Gly Asp Thr Leu Ile Pro
20 25 30
Lys Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn
35 40 45
Gly Gly Pro Gly Thr Ile Lys Lys Ile Thr Phe Pro Glu Gly Ser Pro
50 55 60
Phe Lys Tyr Val Lys Glu Arg Val Asp Glu Val Asp His Ala Asn Phe
65 70 75 80
Lys Tyr Ser Tyr Ser Leu Ile Glu Gly Gly Pro Val Gly Asp Thr Leu
85 90 95
Glu Lys Ile Cys Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly
100 105 110
Ser Ile Leu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asp His Glu
115 120 125
Met Lys Ala Glu His Met Lys Ala Ile Lys Glu Lys Gly Glu Ala Leu
130 135 140
Leu Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155 160
<210> 6
<211> 160
<212> PRT
<213> amino acid
<400> 6
Met Gly Val Phe Asn Tyr Glu Asp Glu Ala Thr Ser Val Ile Ala Pro
1 5 10 15
Ala Arg Leu Phe Lys Ser Phe Val Leu Asp Ala Asp Asn Leu Ile Pro
20 25 30
Lys Val Ala Pro Glu Asn Val Ser Ser Ala Glu Asn Ile Glu Gly Asn
35 40 45
Gly Gly Pro Gly Thr Ile Lys Lys Ile Thr Phe Pro Glu Gly Ser His
50 55 60
Phe Lys Tyr Met Lys His Arg Val Asp Glu Ile Asp His Ala Asn Phe
65 70 75 80
Lys Tyr Cys Tyr Ser Ile Ile Glu Gly Gly Pro Leu Gly Asp Thr Leu
85 90 95
Glu Lys Ile Ser Tyr Glu Ile Lys Ile Val Ala Ala Pro Gly Gly Gly
100 105 110
Ser Ile Leu Lys Ile Thr Ser Lys Tyr His Thr Lys Gly Asp Ile Ser
115 120 125
Leu Asn Glu Glu Glu Ile Lys Ala Gly Lys Glu Lys Gly Ala Gly Leu
130 135 140
Phe Lys Ala Val Glu Asn Tyr Leu Val Ala His Pro Asn Ala Tyr Asn
145 150 155 160
<210> 7
<211> 133
<212> PRT
<213> amino acid
<400> 7
Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp
1 5 10 15
Gly Gln Gly Gln Gln Leu Ala Ala Ser Ala Ile Val Gly His Asp Gly
20 25 30
Ser Val Trp Ala Gln Ser Ser Ser Phe Pro Gln Phe Lys Pro Gln Glu
35 40 45
Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro
50 55 60
Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu
65 70 75 80
Ala Gly Ala Val Ile Arg Gly Lys Lys Gly Ser Gly Gly Ile Thr Ile
85 90 95
Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val
100 105 110
Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu
115 120 125
Ile Asp Gln Gly Leu
130
<210> 8
<211> 205
<212> PRT
<213> amino acid
<400> 8
Met Pro Cys Ser Thr Glu Ala Met Glu Lys Ala Gly His Gly His Ala
1 5 10 15
Ser Thr Pro Arg Lys Arg Ser Leu Ser Asn Ser Ser Phe Arg Leu Arg
20 25 30
Ser Glu Ser Leu Asn Thr Leu Arg Leu Arg Arg Ile Phe Asp Leu Phe
35 40 45
Asp Lys Asn Ser Asp Gly Ile Ile Thr Val Asp Glu Leu Ser Arg Ala
50 55 60
Leu Asn Leu Leu Gly Leu Glu Thr Asp Leu Ser Glu Leu Glu Ser Thr
65 70 75 80
Val Lys Ser Phe Thr Arg Glu Gly Asn Ile Gly Leu Gln Phe Glu Asp
85 90 95
Phe Ile Ser Leu His Gln Ser Leu Asn Asp Ser Tyr Phe Ala Tyr Gly
100 105 110
Gly Glu Asp Glu Asp Asp Asn Glu Glu Asp Met Arg Lys Ser Ile Leu
115 120 125
Ser Gln Glu Glu Ala Asp Leu Ser Glu Ala Phe Lys Val Phe Asp Glu
130 135 140
Asp Gly Asp Gly Tyr Ile Ser Ala Arg Glu Leu Gln Met Val Leu Gly
145 150 155 160
Lys Leu Gly Phe Ser Glu Gly Ser Glu Ile Asp Arg Val Glu Lys Met
165 170 175
Ile Val Ser Val Asp Ser Asn Arg Asp Gly Arg Val Asp Phe Phe Glu
180 185 190
Phe Lys Asp Met Met Arg Ser Val Leu Val Arg Ser Ser
195 200 205
<210> 9
<211> 85
<212> PRT
<213> amino acid
<400> 9
Met Ala Asp Asp His Pro Gln Asp Lys Ala Glu Arg Glu Arg Ile Phe
1 5 10 15
Lys Arg Phe Asp Ala Asn Gly Asp Gly Lys Ile Ser Ala Ala Glu Leu
20 25 30
Gly Glu Ala Leu Lys Thr Leu Gly Ser Ile Thr Pro Asp Glu Val Lys
35 40 45
His Met Met Ala Glu Ile Asp Thr Asp Gly Asp Gly Phe Ile Ser Phe
50 55 60
Gln Glu Phe Thr Asp Phe Gly Arg Ala Asn Arg Gly Leu Leu Lys Asp
65 70 75 80
Val Ala Lys Ile Phe
85
<210> 10
<211> 308
<212> PRT
<213> amino acid
<400> 10
Met Ala His Lys Ser Lys Ile Leu Ile Ile Gly Gly Thr Gly Tyr Ile
1 5 10 15
Gly Lys Phe Ile Val Glu Ala Ser Ala Lys Ser Gly His Pro Thr Phe
20 25 30
Ala Leu Val Arg Glu Ser Thr Val Ser Asp Pro Val Lys Gly Lys Leu
35 40 45
Val Glu Lys Phe Lys Gly Leu Gly Val Thr Leu Leu His Gly Asp Leu
50 55 60
Tyr Asp His Glu Ser Leu Val Lys Ala Phe Lys Gln Val Asp Val Val
65 70 75 80
Ile Ser Thr Val Gly His Leu Gln Leu Ala Asp Gln Val Lys Ile Ile
85 90 95
Ala Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe Phe Pro Ser Glu
100 105 110
Phe Gly Asn Asp Val Asp Arg Val His Ala Val Glu Pro Ala Lys Thr
115 120 125
Ala Phe Ala Thr Lys Ala Glu Ile Arg Arg Lys Thr Glu Ala Glu Gly
130 135 140
Ile Pro Tyr Thr Tyr Val Ser Ser Asn Phe Phe Ala Gly Tyr Phe Leu
145 150 155 160
Pro Thr Leu Ala Gln Pro Gly Leu Thr Ser Pro Pro Arg Glu Lys Val
165 170 175
Val Ile Phe Gly Asp Gly Asn Ala Arg Ala Val Phe Asn Lys Glu Asp
180 185 190
Asp Ile Gly Thr Tyr Thr Ile Arg Ala Val Asp Asp Pro Arg Thr Leu
195 200 205
Asn Lys Ile Val Tyr Ile Lys Pro Ala Lys Asn Ile Tyr Ser Phe Asn
210 215 220
Glu Ile Val Ala Leu Trp Glu Lys Lys Ile Gly Lys Thr Leu Glu Lys
225 230 235 240
Ile Tyr Val Pro Glu Glu Lys Leu Leu Lys Asp Ile Gln Glu Ser Pro
245 250 255
Ile Pro Ile Asn Val Ile Leu Ala Ile Asn His Ser Val Phe Val Lys
260 265 270
Gly Asp His Thr Asn Phe Glu Ile Glu Ala Ser Phe Gly Val Glu Ala
275 280 285
Ser Glu Leu Tyr Pro Asp Val Lys Tyr Thr Thr Val Glu Glu Tyr Leu
290 295 300
Gln Gln Phe Val
305
<210> 11
<211> 173
<212> PRT
<213> amino acid
<400> 11
Met Ala Ser Asn Pro Lys Val Phe Phe Asp Met Glu Val Gly Gly Gln
1 5 10 15
Pro Val Gly Arg Ile Val Met Glu Leu Tyr Ala Asp Thr Thr Pro Arg
20 25 30
Thr Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly Asn Gly
35 40 45
Arg Ser Gly Lys Pro Leu His Tyr Lys Lys Ser Ser Phe His Arg Val
50 55 60
Ile Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr Ala Gly Asn Gly
65 70 75 80
Thr Gly Gly Glu Ser Ile Tyr Gly Ala Lys Phe Ala Asp Glu Asn Phe
85 90 95
Ile Lys Lys His Thr Gly Pro Gly Ile Leu Ser Met Ala Asn Ala Gly
100 105 110
Pro Gly Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Ala Lys Thr Glu
115 120 125
Trp Leu Asp Gly Lys His Val Val Phe Gly Gln Val Val Glu Gly Leu
130 135 140
Asp Ile Val Lys Ala Ile Glu Lys Val Gly Ser Ser Ser Gly Arg Thr
145 150 155 160
Ser Lys Pro Val Val Val Ala Asp Cys Gly Gln Leu Ser
165 170
<210> 12
<211> 483
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggtgttt tcaattacga gatagaggcc acctctgtta tcccagcggc taggctattc 60
aaggccttca tccttgatgg agataatctc tttccaaagg ttgcacccca agccattagc 120
agtgttgaaa acattgaagg aaatggaggg cctggaacca tcaagaagat cagctttccc 180
gaaggcttcc ctttcaagta cgtgaaggac agggttgatg aggtggacca cacaaatttc 240
aaatacagtt acagcgtgat cgagggcggt cccgtggggg acacattgga gaagatctct 300
aacgagataa agatagtggc aacccctaat ggcggatcca tcttgaagat caacaacaag 360
taccatacca aaggagacca tgaggtgaag gcagagcaga ttaaggcaag taaagaaatg 420
ggagagacac ttttgagggc cgttgagagc tacctcttgg cacactccga tgcctacaac 480
taa 483
<210> 13
<211> 483
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgggtgttt tcaattacga aactgagacc acctctgtta tcccagcagc tcgactgttc 60
aaggccttta tcctcgaggg tgatactctc attccaaagg ttgcacccca agccattagc 120
agtgttgaaa acattgaagg aaatggaggg cctggaacca tcaagaagat cacatttccc 180
gaaggcagcc ctttcaagta cgtgaaggag agggttgatg aggtggacca cgcaaacttc 240
aaatatagct acagcctaat cgagggaggg cccgtgggcg acacattgga gaagatttgc 300
aacgagataa agatagtggc aacccctgat ggaggatcca tcttgaagat cagcaacaag 360
taccacacca aaggcgacca tgagatgaag gcagagcata tgaaggctat taaagaaaag 420
ggcgaggcac ttttgagggc cgttgagagc tacctcttgg cacactctga tgcctacaac 480
taa 483
<210> 14
<211> 483
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgggtgttt tcaactacga ggatgaggcc acctccgtta ttgctccggc taggctcttc 60
aagtcctttg tccttgacgc cgacaacctc attcccaagg ttgcacctga gaacgttagc 120
agtgccgaaa atattgaagg aaacggagga cctggaacca tcaagaagat cacctttccc 180
gaaggaagcc atttcaagta catgaagcac agggttgatg agatcgacca cgcaaacttc 240
aaatactgct acagcataat cgagggcggt ccattgggcg acacactgga gaagatctct 300
tacgagatca agattgtggc cgcccctggt ggaggatcca tcttgaagat caccagcaag 360
taccacacca aaggcgacat ttcgctcaat gaggaggaga ttaaggccgg caaagaaaag 420
ggcgccggac ttttcaaggc tgttgagaac tacctcgtgg cacacccaaa tgcctacaac 480
taa 483
<210> 15
<211> 402
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaaggccag 60
caactcgctg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120
ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210> 16
<211> 618
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgccctgtt ccacagaagc catggaaaaa gcagggcatg ggcatgcaag tacacctcgc 60
aagcgtagcc tcagcaactc gtccttccgc ctccgctccg agagtctgaa taccctccgc 120
ctccgacgca tattcgatct atttgacaag aacagcgatg gcatcatcac cgtcgatgaa 180
ctcagccgag ccctcaacct tcttggcctc gaaaccgacc tctcggagct cgaatccacc 240
gtcaaatcat tcactcgaga gggcaacatt gggcttcaat tcgaagactt catatcgctg 300
caccaatccc taaacgacag ctactttgct tacggcggcg aggatgagga tgataatgag 360
gaggatatga gaaagagcat attgtcgcaa gaggaagcag atctttcgga ggcgttcaag 420
gtgttcgacg aggacgggga tggttacata tcggccagag aactgcaaat ggtactgggg 480
aagctgggat tctctgaagg gagcgagatt gacagagttg agaagatgat cgtgtccgtt 540
gacagcaacc gagatggccg ggttgatttc tttgagttca aggatatgat gcgtagcgtt 600
ctcgtgcgga gctcttga 618
<210> 17
<211> 258
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atggctgatg atcatccaca ggacaaggct gaacgcgagc gcattttcaa gcgctttgac 60
gccaatggcg atggtaaaat ctctgcagca gagcttgggg aggccttgaa aacacttggc 120
tccatcacac cggatgaggt gaaacatatg atggccgaga ttgacaccga tggcgacggc 180
ttcatttcgt tccaagagtt cacggatttt ggtcgtgcta atcgtggttt actaaaggat 240
gttgccaaga tattttaa 258
<210> 18
<211> 927
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atggctcaca aaagcaagat tctgatcatc ggaggcaccg gctacatcgg aaaattcatc 60
gtggaagcaa gtgcaaagtc tggccatccc acctttgctt tggtcagaga gagtacggtc 120
tctgatcccg ttaagggaaa acttgtcgag aaattcaagg gcttaggcgt cactttgctc 180
catggagatc tgtatgacca tgagagtttg gtaaaggcgt ttaagcaggt ggacgtggtg 240
atatcgacgg taggccacct gcagttagca gatcaggtca agattattgc tgccattaaa 300
gaggctggta atattaagag attcttccct tcggaattcg gaaacgacgt agaccgtgtg 360
catgctgttg agccagcaaa gactgcattt gctaccaagg ccgaaatccg ccgcaagact 420
gaggcggaag gcatccccta cacttatgtg tcatccaatt tcttcgctgg atattttctt 480
cctacgttgg cacaaccagg actcacttct cctcctagag agaaagtcgt tatcttcgga 540
gatggaaatg ccagggctgt ttttaacaag gaagacgaca taggaaccta cacaattaga 600
gctgtggatg acccaagaac actgaataag atagtctaca ttaagcctgc caagaacatt 660
tactcattca atgagattgt tgccctttgg gagaaaaaga ttggcaaaac ccttgagaaa 720
atctatgttc cagaggagaa acttttgaag gacatccaag agtccccaat tccaatcaac 780
gtgatattag caatcaacca ctcagttttt gtgaagggag atcataccaa cttcgagatt 840
gaggcatcct tcggtgtgga ggcctccgag ctatacccag atgtcaaata caccacagtg 900
gaagagtacc ttcagcagtt tgtttga 927
<210> 19
<211> 522
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atggcgtcaa accctaaggt cttcttcgac atggaggtcg gtggccagcc cgttgggcgg 60
atcgtgatgg agctctacgc cgacaccact ccccgcacgg ccgagaactt ccgggccctc 120
tgcaccggtg agaagggcaa cggccgctcc ggcaagcccc tccactacaa gaaatcgtcc 180
ttccacaggg tgatccccgg gttcatgtgc cagggaggcg acttcactgc cggaaacggc 240
accggcggcg agtccatcta cggcgccaag ttcgccgatg agaacttcat caagaagcac 300
accggccccg gcatcctctc catggctaat gccggccccg gcaccaatgg atctcagttc 360
ttcatctgta ccgccaagac cgagtggctc gacggcaagc acgtggtgtt cggccaggtc 420
gtggagggtc tggacatcgt gaaagccatc gagaaggtcg ggtccagctc cggcaggact 480
tccaagcccg tggtcgtcgc cgactgtggt caactctctt ag 522
<210> 20
<211> 12
<212> PRT
<213> amino acid
<400> 20
Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
1 5 10
<210> 21
<211> 34
<212> PRT
<213> amino acid
<400> 21
Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys Val Ala Pro Gln
1 5 10 15
Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly Gly Pro Gly Thr
20 25 30
Ile Lys
<210> 22
<211> 10
<212> PRT
<213> amino acid
<400> 22
Ile Ser Phe Pro Glu Gly Phe Pro Phe Lys
1 5 10
<210> 23
<211> 12
<212> PRT
<213> amino acid
<400> 23
Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys
1 5 10
<210> 24
<211> 10
<212> PRT
<213> amino acid
<400> 24
Val Asp Glu Val Asp His Thr Asn Phe Lys
1 5 10
<210> 25
<211> 11
<212> PRT
<213> amino acid
<400> 25
Ala Ser Lys Glu Met Gly Glu Thr Leu Leu Arg
1 5 10
<210> 26
<211> 8
<212> PRT
<213> amino acid
<400> 26
Glu Met Gly Glu Thr Leu Leu Arg
1 5
<210> 27
<211> 11
<212> PRT
<213> amino acid
<400> 27
Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe Lys
1 5 10
<210> 28
<211> 17
<212> PRT
<213> amino acid
<400> 28
Gly Val Phe Asn Tyr Glu Ile Glu Ala Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg
<210> 29
<211> 17
<212> PRT
<213> amino acid
<400> 29
Tyr Ser Tyr Ser Val Ile Glu Gly Gly Pro Val Gly Asp Thr Leu Glu
1 5 10 15
Lys
<210> 30
<211> 12
<212> PRT
<213> amino acid
<400> 30
Ile Val Ala Thr Pro Asn Gly Gly Ser Ile Leu Lys
1 5 10
<210> 31
<211> 22
<212> PRT
<213> amino acid
<400> 31
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
1 5 10 15
Gly Pro Gly Thr Ile Lys
20
<210> 32
<211> 14
<212> PRT
<213> amino acid
<400> 32
Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
1 5 10
<210> 33
<211> 23
<212> PRT
<213> amino acid
<400> 33
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
1 5 10 15
Gly Pro Gly Thr Ile Lys Lys
20
<210> 34
<211> 11
<212> PRT
<213> amino acid
<400> 34
Lys Ile Thr Phe Pro Glu Gly Ser Pro Phe Lys
1 5 10
<210> 35
<211> 10
<212> PRT
<213> amino acid
<400> 35
Ile Thr Phe Pro Glu Gly Ser Pro Phe Lys
1 5 10
<210> 36
<211> 13
<212> PRT
<213> amino acid
<400> 36
Ile Thr Phe Pro Glu Gly Ser Pro Phe Lys Tyr Val Lys
1 5 10
<210> 37
<211> 12
<212> PRT
<213> amino acid
<400> 37
Ala Phe Ile Leu Glu Gly Asp Thr Leu Ile Pro Lys
1 5 10
<210> 38
<211> 12
<212> PRT
<213> amino acid
<400> 38
Glu Arg Val Asp Glu Val Asp His Ala Asn Phe Lys
1 5 10
<210> 39
<211> 10
<212> PRT
<213> amino acid
<400> 39
Val Asp Glu Val Asp His Ala Asn Phe Lys
1 5 10
<210> 40
<211> 16
<212> PRT
<213> amino acid
<400> 40
Ile Val Ala Thr Pro Asp Gly Gly Ser Ile Leu Lys Ile Ser Asn Lys
1 5 10 15
<210> 41
<211> 12
<212> PRT
<213> amino acid
<400> 41
Ile Val Ala Thr Pro Asp Gly Gly Ser Ile Leu Lys
1 5 10
<210> 42
<211> 26
<212> PRT
<213> amino acid
<400> 42
Tyr His Thr Lys Gly Asp His Glu Met Lys Ala Glu His Met Lys Ala
1 5 10 15
Ile Lys Glu Lys Gly Glu Ala Leu Leu Arg
20 25
<210> 43
<211> 8
<212> PRT
<213> amino acid
<400> 43
Glu Lys Gly Glu Ala Leu Leu Arg
1 5
<210> 44
<211> 17
<212> PRT
<213> amino acid
<400> 44
Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg
<210> 45
<211> 11
<212> PRT
<213> amino acid
<400> 45
Gly Asp His Glu Met Lys Ala Glu His Met Lys
1 5 10
<210> 46
<211> 8
<212> PRT
<213> amino acid
<400> 46
Ile Ser Asn Lys Tyr His Thr Lys
1 5
<210> 47
<211> 17
<212> PRT
<213> amino acid
<400> 47
Tyr Ser Tyr Ser Leu Ile Glu Gly Gly Pro Val Gly Asp Thr Leu Glu
1 5 10 15
Lys
<210> 48
<211> 14
<212> PRT
<213> amino acid
<400> 48
Ala Val Glu Asn Tyr Leu Val Ala His Pro Asn Ala Tyr Asn
1 5 10
<210> 49
<211> 11
<212> PRT
<213> amino acid
<400> 49
Gly Asp Ile Ser Leu Asn Glu Glu Glu Ile Lys
1 5 10
<210> 50
<211> 17
<212> PRT
<213> amino acid
<400> 50
Gly Val Phe Asn Tyr Glu Asp Glu Ala Thr Ser Val Ile Ala Pro Ala
1 5 10 15
Arg
<210> 51
<211> 10
<212> PRT
<213> amino acid
<400> 51
Ile Thr Phe Pro Glu Gly Ser His Phe Lys
1 5 10
<210> 52
<211> 11
<212> PRT
<213> amino acid
<400> 52
Lys Ile Thr Phe Pro Glu Gly Ser His Phe Lys
1 5 10
<210> 53
<211> 12
<212> PRT
<213> amino acid
<400> 53
Ile Val Ala Ala Pro Gly Gly Gly Ser Ile Leu Lys
1 5 10
<210> 54
<211> 12
<212> PRT
<213> amino acid
<400> 54
Ser Phe Val Leu Asp Ala Asp Asn Leu Ile Pro Lys
1 5 10
<210> 55
<211> 23
<212> PRT
<213> amino acid
<400> 55
Val Ala Pro Glu Asn Val Ser Ser Ala Glu Asn Ile Glu Gly Asn Gly
1 5 10 15
Gly Pro Gly Thr Ile Lys Lys
20
<210> 56
<211> 12
<212> PRT
<213> amino acid
<400> 56
His Arg Val Asp Glu Ile Asp His Ala Asn Phe Lys
1 5 10
<210> 57
<211> 10
<212> PRT
<213> amino acid
<400> 57
Val Asp Glu Ile Asp His Ala Asn Phe Lys
1 5 10
<210> 58
<211> 19
<212> PRT
<213> amino acid
<400> 58
Asp Phe Glu Glu Pro Gly His Leu Ala Pro Thr Gly Leu His Leu Gly
1 5 10 15
Gly Ile Lys
<210> 59
<211> 13
<212> PRT
<213> amino acid
<400> 59
Tyr Met Val Ile Gln Gly Glu Ala Gly Ala Val Ile Arg
1 5 10
<210> 60
<211> 32
<212> PRT
<213> amino acid
<400> 60
Asp Phe Glu Glu Pro Gly His Leu Ala Pro Thr Gly Leu His Leu Gly
1 5 10 15
Gly Ile Lys Tyr Met Val Ile Gln Gly Glu Ala Gly Ala Val Ile Arg
20 25 30
<210> 61
<211> 26
<212> PRT
<213> amino acid
<400> 61
Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val Thr
1 5 10 15
Pro Gly Gln Cys Asn Met Val Val Glu Arg
20 25
<210> 62
<211> 25
<212> PRT
<213> amino acid
<400> 62
Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val Thr Pro
1 5 10 15
Gly Gln Cys Asn Met Val Val Glu Arg
20 25
<210> 63
<211> 9
<212> PRT
<213> amino acid
<400> 63
Lys Gly Ser Gly Gly Ile Thr Ile Lys
1 5
<210> 64
<211> 8
<212> PRT
<213> amino acid
<400> 64
Gly Ser Gly Gly Ile Thr Ile Lys
1 5
<210> 65
<211> 10
<212> PRT
<213> amino acid
<400> 65
Leu Gly Asp Tyr Leu Ile Asp Gln Gly Leu
1 5 10
<210> 66
<211> 13
<212> PRT
<213> amino acid
<400> 66
Asn Ser Asp Gly Ile Ile Thr Val Asp Glu Leu Ser Arg
1 5 10
<210> 67
<211> 11
<212> PRT
<213> amino acid
<400> 67
Ile Ser Ala Ala Glu Leu Gly Glu Ala Leu Lys
1 5 10
<210> 68
<211> 11
<212> PRT
<213> amino acid
<400> 68
Thr Leu Gly Ser Ile Thr Pro Asp Glu Val Lys
1 5 10
<210> 69
<211> 15
<212> PRT
<213> amino acid
<400> 69
Ala Val Phe Asn Lys Glu Asp Asp Ile Gly Thr Tyr Thr Ile Arg
1 5 10 15
<210> 70
<211> 10
<212> PRT
<213> amino acid
<400> 70
Glu Asp Asp Ile Gly Thr Tyr Thr Ile Arg
1 5 10
<210> 71
<211> 22
<212> PRT
<213> amino acid
<400> 71
Asp Ile Gln Glu Ser Pro Ile Pro Ile Asn Val Ile Leu Ala Ile Asn
1 5 10 15
His Ser Val Phe Val Lys
20
<210> 72
<211> 12
<212> PRT
<213> amino acid
<400> 72
Glu Lys Val Val Ile Phe Gly Asp Gly Asn Ala Arg
1 5 10
<210> 73
<211> 9
<212> PRT
<213> amino acid
<400> 73
Glu Ser Thr Val Ser Asp Pro Val Lys
1 5
<210> 74
<211> 11
<212> PRT
<213> amino acid
<400> 74
Glu Ser Thr Val Ser Asp Pro Val Lys Gly Lys
1 5 10
<210> 75
<211> 13
<212> PRT
<213> amino acid
<400> 75
Arg Phe Phe Pro Ser Glu Phe Gly Asn Asp Val Asp Arg
1 5 10
<210> 76
<211> 12
<212> PRT
<213> amino acid
<400> 76
Phe Phe Pro Ser Glu Phe Gly Asn Asp Val Asp Arg
1 5 10
<210> 77
<211> 20
<212> PRT
<213> amino acid
<400> 77
Phe Phe Pro Ser Glu Phe Gly Asn Asp Val Asp Arg Val His Ala Val
1 5 10 15
Glu Pro Ala Lys
20
<210> 78
<211> 8
<212> PRT
<213> amino acid
<400> 78
Phe Ile Val Glu Ala Ser Ala Lys
1 5
<210> 79
<211> 19
<212> PRT
<213> amino acid
<400> 79
Gly Leu Gly Val Thr Leu Leu His Gly Asp Leu Tyr Asp His Glu Ser
1 5 10 15
Leu Val Lys
<210> 80
<211> 12
<212> PRT
<213> amino acid
<400> 80
Ile Leu Ile Ile Gly Gly Thr Gly Tyr Ile Gly Lys
1 5 10
<210> 81
<211> 8
<212> PRT
<213> amino acid
<400> 81
Ile Val Tyr Ile Lys Pro Ala Lys
1 5
<210> 82
<211> 14
<212> PRT
<213> amino acid
<400> 82
Asn Ile Tyr Ser Phe Asn Glu Ile Val Ala Leu Trp Glu Lys
1 5 10
<210> 83
<211> 19
<212> PRT
<213> amino acid
<400> 83
Gln Val Asp Val Val Ile Ser Thr Val Gly His Leu Gln Leu Ala Asp
1 5 10 15
Gln Val Lys
<210> 84
<211> 10
<212> PRT
<213> amino acid
<400> 84
Ser Gly His Pro Thr Phe Ala Leu Val Arg
1 5 10
<210> 85
<211> 8
<212> PRT
<213> amino acid
<400> 85
Val His Ala Val Glu Pro Ala Lys
1 5
<210> 86
<211> 14
<212> PRT
<213> amino acid
<400> 86
Val His Ala Val Glu Pro Ala Lys Thr Ala Phe Ala Thr Lys
1 5 10
<210> 87
<211> 10
<212> PRT
<213> amino acid
<400> 87
Val Val Ile Phe Gly Asp Gly Asn Ala Arg
1 5 10
<210> 88
<211> 8
<212> PRT
<213> amino acid
<400> 88
Phe Ala Asp Glu Asn Phe Ile Lys
1 5
<210> 89
<211> 9
<212> PRT
<213> amino acid
<400> 89
Phe Ala Asp Glu Asn Phe Ile Lys Lys
1 5
<210> 90
<211> 15
<212> PRT
<213> amino acid
<400> 90
His Val Val Phe Gly Gln Val Val Glu Gly Leu Asp Ile Val Lys
1 5 10 15
<210> 91
<211> 12
<212> PRT
<213> amino acid
<400> 91
Ile Val Met Glu Leu Tyr Ala Asp Thr Thr Pro Arg
1 5 10
<210> 92
<211> 8
<212> PRT
<213> amino acid
<400> 92
Ser Gly Lys Pro Leu His Tyr Lys
1 5
<210> 93
<211> 9
<212> PRT
<213> amino acid
<400> 93
Ser Gly Lys Pro Leu His Tyr Lys Lys
1 5
<210> 94
<211> 14
<212> PRT
<213> amino acid
<400> 94
Val Phe Phe Asp Met Glu Val Gly Gly Gln Pro Val Gly Arg
1 5 10
<210> 95
<211> 11
<212> PRT
<213> amino acid
<400> 95
Ala Ile Glu Lys Val Gly Ser Ser Ser Gly Arg
1 5 10
<210> 96
<211> 14
<212> PRT
<213> amino acid
<400> 96
Thr Ser Lys Pro Val Val Val Ala Asp Cys Gly Gln Leu Ser
1 5 10
<210> 97
<211> 27
<212> PRT
<213> amino acid
<400> 97
Val Ile Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr Ala Gly Asn
1 5 10 15
Gly Thr Gly Gly Glu Ser Ile Tyr Gly Ala Lys
20 25

Claims (7)

1. The application of a group of birch pollen allergen proteins Bet P1.0106, bet P1.0201, bet P1.0301, bet P2, bet P3, bet P4, bet P6 and Bet P7 in preparation of preparations for diagnosing allergic diseases and performing specific immunotherapy on the allergic diseases is characterized in that the Bet P1.0106 has an amino acid sequence shown in SEQ ID NO.4, the Bet P1.0201 has an amino acid sequence shown in SEQ ID NO.5, the Bet P1.0301 has an amino acid sequence shown in SEQ ID NO.6, the Bet P2 has an amino acid sequence shown in SEQ ID NO.7, the Bet P3 has an amino acid sequence shown in SEQ ID NO.8, the Bet P4 has an amino acid sequence shown in SEQ ID NO.9, the Bet P6 has an amino acid sequence shown in SEQ ID NO.10, and the Bet P7 has an amino acid sequence shown in SEQ ID NO. 11.
2. A method of preparing a birch pollen allergen liquid comprising the birch pollen allergen proteins Bet P1.0106, bet P1.0201, bet P1.0301, bet P2, bet P3, bet P4, bet P6, and Bet P7 of claim 1, comprising the steps of:
s1, collecting birch pollen, and drying at normal temperature or in vacuum or in a fluidized bed;
s2, degreasing and drying the dried pollen; pollen degreasing adopts the steps that pollen and acetone are mixed in a proportion of 1: and (3) degreasing treatment is carried out according to the weight g volume ml ratio of 5-1
S3, adding the degreased and dried birch pollen into a phosphate-saline buffer solution with the pH value of 7.9-8.2 according to the weight g volume ml ratio of 1;
s4, collecting the leaching liquor obtained in the step S3, and centrifuging to obtain a supernatant; filtering the supernatant, sterilizing and filtering;
s5, carrying out ultrafiltration concentration on the filtered supernatant to obtain an ultrafiltration concentrated solution; performing ultrafiltration with 3KD ultrafiltration membrane, and stopping ultrafiltration when total protein content of ultrafiltration permeate is less than or equal to 0.02 mg/ml; when the total protein content of the ultrafiltration permeate is more than 0.02mg/ml, the ultrafiltration membrane is replaced, and then the ultrafiltration is repeated on the ultrafiltration permeate until the total protein content of the ultrafiltration permeate is less than or equal to 0.02 mg/ml;
s6, after secondary filtration and sterilization filtration are carried out on the ultrafiltration concentrated solution, vacuum freeze-drying is carried out to obtain a freeze-dried birch pollen allergen product;
s7, redissolving the freeze-dried birch pollen allergen product by using a phosphate-saline buffer solution with the pH value of 6.5-7.5 to prepare a birch pollen allergen solution, placing the birch pollen allergen solution at the temperature of 2-8 ℃, uniformly mixing the birch pollen allergen solution with glycerol sterilized in the same volume, and adjusting the pH value of the solution to 6.0-8.0;
the step S1 also comprises the step of microscopic examination identification and/or DNA identification of the birch pollen serving as the raw material, wherein the DNA identification method comprises the steps of carrying out PCR amplification on the birch pollen raw material by taking SEQ ID NO.1 and SEQ ID NO.2 as primers and detecting an amplification product.
3. The method according to claim 2, wherein in step S4, the centrifugation is carried out under 8000-12000 g at 2-8 deg.C for 15-20 min; the filtration and the sterilization filtration are sequentially carried out by filtering with 4000-mesh filter cloth, then filtering with a paperboard and 0.45-micron and 0.22-micron filter membranes.
4. The method according to claim 2, wherein;
in step S6, the vacuum freeze-drying condition is freezing at-50 to-35 ℃, drying at-25 ℃ under the vacuum pressure of 2-8 mbar, and controlling the water content to be less than or equal to 3%.
5. A method for the preparation of birch pollen allergen lyophilisates comprising the allergen proteins Bet P1.0106, bet P1.0201, bet P1.0301, bet P2, bet P3, bet P4, bet P6 and Bet P7 as defined in claim 1, characterized in that it is prepared by steps comprising birch pollen collection-drying-defatting-extraction-ultrafiltration concentration-lyophilisation:
(1) Collecting: collecting birch pollen by natural falling method;
(2) And (3) drying: drying at normal temperature or vacuum drying or fluidized bed drying until pollen is not adhered, and sieving the dried pollen with 150-250 mesh sieve;
(3) Degreasing: mixing the pollen obtained in the step (2) and acetone respectively according to g and ml, and mixing the mixture according to the ratio of 1: degreasing at the weight-volume ratio of 5-1, stirring or shaking for 30 minutes, standing for layering, pouring out supernatant, adding new acetone, repeatedly degreasing until the supernatant is clear, uniformly spreading the degreased pollen, and drying at room temperature or in vacuum or in a fluidized bed for 48-72 hours;
(4) Extraction: adding 10L of phosphate-saline buffer solution with the pH value of 7.9-8.2 into degreased and dried birch pollen according to the weight volume ratio of 1; taking the extracted leaching liquor, centrifuging at the centrifugal force of 8000-12000 g and the centrifugal temperature of 2-8 ℃ for 15-20 minutes, centrifuging and collecting the supernatant; filtering with 4000-mesh filter cloth, sequentially filtering the filtrate with cardboard, and 0.45 μm and 0.22 μm filter membranes;
(5) And (3) ultrafiltration and concentration: ultrafiltering the filtered leaching solution with 3KD ultrafiltration membrane, sampling ultrafiltration concentrated solution and ultrafiltration permeate, and detecting total protein content; wherein, when the total protein content in the ultrafiltration permeate is less than or equal to 0.02mg/ml, the permeate is directly discarded; if the total protein content of the ultrafiltration permeate is greater than 0.02mg/ml, performing integrity test on the ultrafiltration membrane, and if the ultrafiltration membrane is not damaged, discarding the permeate; if the ultrafiltration membrane is damaged, replacing the ultrafiltration membrane and then repeatedly ultrafiltering the permeate;
(6) Freeze-drying: after the ultrafiltration concentrated solution is subjected to secondary filtration and sterilization filtration, the freeze-drying process conditions are as follows: freezing at-50 to-35 ℃, drying at-25 ℃ under the vacuum pressure of 2 to 8mbar, and controlling the water content to be less than or equal to 3 percent to obtain the birch pollen allergen freeze-dried product.
6. Use of a birch pollen allergen liquid as defined in claim 2, or a birch pollen allergen lyophilisate as defined in claim 5, for the preparation of a formulation for the diagnosis and specific immunotherapy of allergic diseases, including allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.
7. The use according to claim 6, wherein the lyophilized product is used for preparing a tablet, an orally disintegrating tablet, and the birch pollen allergen solution is used for preparing an injection, a sublingual drop, an allergen spot patch, or an allergen dip diluent.
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Inventor after: Yin Jia

Inventor after: Gu Jianqing

Inventor after: Zhou Junxiong

Inventor after: Gao Dongao

Inventor after: Wang Zixi

Inventor after: Lu San

Inventor after: Dang Xiu

Inventor before: Yin Jia

Inventor before: Zhou Junxiong

Inventor before: Gu Jianqing

Inventor before: Gao Dongao

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