CN115252771A - Cypress pollen allergen composition and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a cypress pollen allergen composition and a preparation method and application thereof. The method extracts protein from sabina chinensis pollen, purifies and enriches main allergen protein in the extract, purifies main enzymolysis peptide fragments, performs proteomics analysis on the extract by using an HPLC-MS/MS method, identifies the main allergen protein in the extract, preliminarily evaluates the sensitization of the extract, provides a raw material and a method for developing a prick liquid of the cypress pollen extract, and also provides ideas and references for carrying out separation and identification of the cypress pollen allergen protein, a related detection method and development and establishment of an allergen vaccine.

Description

Cypress pollen allergen composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a cypress pollen allergen composition and a preparation method and application thereof.

Background

The recognition of allergic diseases begins with "hay fever", also known as allergic rhinitis. At present, allergic diseases are one of the major health problems worldwide. Over 25% of the population in industrialized countries is afflicted by allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, with allergic asthma being the most common. The inhalation of allergic pollen is the most important factor for the induction of allergic asthma and other respiratory allergic diseases, and approximately 50% of patients with allergic diseases are allergic to grass pollen.

The Cupressaceae plants belong to 29 species in China, are traditional garden greening tree species in China, are most common in Juniperus chinensis L, and are widely distributed in China. Meanwhile, cypress pollen is also one of the main causes of allergic diseases such as seasonal allergic rhinitis and asthma. With the improvement of ecological greening level and the wide application of cypress such as sabina chinensis and the like, the cypress pollen concentration in the air of various places in China also has the characteristics of higher level and strong seasonality. Research shows that the cypress pollen with high content is reported in air monitoring of a plurality of regions and cities in China, and particularly in spring pollen seasons, the concentration of the cypress pollen floating in the atmosphere of some regions can be as high as 294g/m ³ . Meanwhile, a large number of pollen allergic patients are clinically and correspondingly shown and show a trend of increasing year by year, particularly in some cells or green lands with large proportion of cypress or cypress greening tree species and surrounding areasPeople or groups often have high seasonal allergic rhinitis and/or allergic asthma during the middle and late March to the middle of April every year. The clinical common symptoms mainly comprise continuous sneezing and watery nasal discharge, nasal blockage even affecting respiration, eye secretion increase, red swelling and itching and the like, some serious patients have symptoms of asthma, suffocation, hearing loss, blurred vision and the like, and some allergic patients have symptoms of rash and dermatitis such as skin pruritus, red swelling and the like. In recent years, researches on epidemiology of cypress pollen allergic diseases, pollen allergen and sensitization evaluation, sensitization mechanism and the like have attracted wide attention of scholars at home and abroad.

The cypress pollen allergen protein is the key basis for causing anaphylactic reaction, so the obtained high-purity cypress pollen allergen protein can be used for diagnosis and clinical application of spring arbor pollen allergic diseases, can be used for preparing high-purity cypress pollen pricking liquid, is used for development and application of a pollen vaccine for specific treatment, and can provide necessary materials for researches in aspects of structure, epitope, biological characteristics, sensibility evaluation and measurement method and the like.

However, cypress pollen allergens currently used clinically have problems of poor stability, uncertain components, low positive rate and low accuracy when used for diagnosing allergic diseases, and the like, in various degrees. The invention extracts protein from sabina chinensis pollen, purifies and enriches main allergen protein in the sabina chinensis pollen, purifies main enzymolysis peptide fragments, performs proteomics analysis on the extract by using an HPLC-MS/MS method, identifies the main allergen protein in the extract, preliminarily evaluates the sensitization of the extract, provides a raw material and a method for developing a prick liquid of the cypress pollen extract, and also provides ideas and references for carrying out separation and identification of the cypress pollen allergen protein, a related detection method and development and establishment of an allergen vaccine.

Disclosure of Invention

In order to solve the technical problems, the invention provides a novel cypress pollen allergen composition and a preparation method and application thereof.

Specifically, the invention is realized by the following technical schemes:

in a first aspect, the present invention provides a cypress pollen allergen composition, which is a cypress pollen protein extract, prepared by a method comprising the steps of:

step 1: collecting and pretreating pollen:

screening the collected sequence of Sabina chinensis and pollen collected by pollen collector by natural falling method and pollen collector to remove impurities, drying at low temperature, and storing at 4 deg.C;

and 2, step: preparing a pollen crude protein extract:

adding the dry pollen collected after removing impurities into PBS, performing ultrasonic crushing in ice bath, continuously stirring the obtained pollen suspension at 4 ℃ for 24h, centrifuging at 4 ℃, taking the supernatant, filtering the supernatant by using a 0.45-micrometer filter, and storing at 4 ℃;

and step 3: extracting and enriching pollen protein:

concentrating the filtrate obtained in the step 2 by using an ultrafiltration tube, centrifuging at 4 ℃ to obtain ultrafiltration concentrated solution after ultrafiltration, and storing at 4 ℃;

and 4, step 4: enzymolysis:

adding a proper amount of the ultrafiltration concentrated solution prepared in the step 3 into DTT with the final concentration of 5mM, incubating for 1h at 37 ℃, then recovering the room temperature, adding iodoacetamide with the final concentration of 10mM, incubating for 45min at the room temperature in a dark place, diluting a sample with 25mM ammonium bicarbonate, adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added in the next day to adjust the pH value, and the enzyme digestion is stopped;

and 5: desalting and purifying:

use of C ₁₈ Desalting the sample prepared in step 4 with desalting column, treating the eluate with SCX-tip to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

Alternatively, in the cypress pollen allergen composition described above, the cypress pollen allergen composition comprises the following three specific cypress pollen allergenic proteins: Q93X51, A0a808QQB, and A0A7R6H3R2.

Preferably, in the above cypress pollen allergen composition, the cypress pollen allergen composition consists of the following three specific cypress pollen allergenic proteins: Q93X51, A0a808QQB and A0A7R6H3R2.

Alternatively, in the cypress pollen allergen composition described above, the amino acid sequence of Q93X51 is DKSMKVTVAFNQFGPNAGQRMPRARYGL, the amino acid sequence of A0a808QQB is AKFIDTQNGLRIKTWQGGSGLAS, and the amino acid sequence of A0A7R6H3R2 is DDKSMKVTVAFNQFGPNAGQRMPRARYGL.

In a second aspect, the present invention provides a method for preparing a cypress pollen allergen composition according to the first aspect, comprising the steps of:

step 1: collecting and pretreating pollen:

screening the collected sequence of Sabina chinensis and pollen collected by pollen collector by natural falling method and pollen collector to remove impurities, drying at low temperature, and storing at 4 deg.C;

step 2: pollen crude protein preparation of the immersion liquid:

adding the dry pollen collected after removing impurities into PBS, performing ultrasonic crushing in ice bath, continuously stirring the obtained pollen suspension at 4 ℃ for 24h, centrifuging at 4 ℃, taking the supernatant, filtering the supernatant by using a 0.45-micrometer filter, and storing at 4 ℃;

and step 3: extracting and enriching pollen protein:

concentrating the filtrate obtained in the step 2 by using an ultrafiltration tube, centrifuging at 4 ℃ to obtain an ultrafiltration concentrated solution after ultrafiltration, and storing at 4 ℃;

and 4, step 4: enzymolysis:

adding a proper amount of the ultrafiltration concentrated solution prepared in the step 3 into DTT with the final concentration of 5mM, incubating for 1h at 37 ℃, then recovering the room temperature, adding iodoacetamide with the final concentration of 10mM, incubating for 45min at the room temperature in a dark place, diluting a sample with 25mM ammonium bicarbonate, adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added in the next day to adjust the pH value, and the enzyme digestion is stopped;

and 5: desalting and purifying:

use of C ₁₈ Desalting the sample prepared in step 4 with desalting column, treating the eluate with SCX-tip to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

Alternatively, in step 2 of the preparation method, the dried pollen collected after removing the impurities is mixed in a weight-to-volume ratio of 1: adding 0.01mol/L PBS solution into 20, performing ultrasonic wave disruption in ice bath for 15min, continuously stirring the obtained pollen suspension at 4 deg.C for 24h, centrifuging at 4 deg.C under centrifugal force of 10000 Xg for 20min, collecting supernatant, filtering the supernatant with 0.45 μm filter, and storing at 4 deg.C.

Alternatively, in step 3 of the above production method, the filtrate obtained in step 2 is concentrated with a PALL 3kD-10kD ultrafiltration tube, centrifuged at 4500 Xg at 4 ℃ to obtain an ultrafiltrated concentrate, and stored at 4 ℃.

Alternatively, in step 4 of the above preparation method, an appropriate amount of the ultrafiltration concentrate prepared in step 3 is added to DTT with a final concentration of 5mM, incubated at 37 ℃ for 1h, then returned to room temperature, added with iodoacetamide with a final concentration of 10mM, incubated at room temperature in the dark for 45min, and the sample is diluted 4-fold with 25mM ammonium bicarbonate in a ratio of 50:1 adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added the next day to adjust the pH to less than 3, and the digestion is stopped.

Alternatively, in step 5 of the above-mentioned production method, C is used ₁₈ Desalting the sample prepared in the step 4 by using a desalting column, activating the desalting column by using 100% acetonitrile, loading the sample on the column by using a 0.1% formic acid balance column, washing impurities by using 0.1% formic acid, eluting by using 70% acetonitrile, and collecting and eluting; treating the eluate with SCX-tip, activating SCX membrane with methanol 20 μ L for 2 times, 1min each time, balancing for 2 times, 1min each time, adding eluate sample, eluting with EDTA of pH 4.0 for 2min to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

In a third aspect, the present invention provides the use of the cypress pollen allergen composition according to the first aspect or the cypress pollen allergen composition prepared by the preparation method according to the second aspect in the preparation of a reagent or a medicament for diagnosing or treating allergic diseases.

Alternatively, in the above use, the allergic disease is a type I allergic disease.

Alternatively, in the above use, the allergic disease includes allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.

Alternatively, in the above use, the agent or medicament is a tablet, sublingual drop, injection, allergen spot patch or allergen prick liquid.

Compared with the prior art, the invention has the following beneficial effects:

(1) The inventor carries out protein extraction on sabina chinensis pollen, purifies and enriches main allergen protein in the sabina chinensis pollen, purifies main enzymolysis peptide fragments, carries out proteomics analysis on the extract by using an HPLC-MS/MS method, identifies the main allergen protein in the extract, preliminarily evaluates the sensitization effect of the extract, provides a raw material and a method for developing a pricking liquid of the cypress pollen extract, and also provides ideas and references for developing separation and identification of the cypress pollen allergen protein, a related detection method and development and establishment of an allergen vaccine.

(2) The extraction method of the sabina chinensis pollen protein is based on a solvent extraction method and a protein extraction method, innovatively introduces the SCX-Tip technology used in proteomics sample treatment, enriches and purifies the main protein in the cypress pollen, and has the characteristics that: (1) the efficiency of the preparation process of protein enrichment and purification is greatly improved, and the purification and enrichment process can be about 10 minutes. (2) The purity of the sample is improved, and by utilizing the isoelectric point difference of various proteins in the mixture in an acidic reaction system, the proteins are fully retained on an SCX membrane when SCX tip (Strong position exchange tip) is passed, and other substrates are not retained and transferred into the solution in a centrifugal mode, so that various impurities are removed. The characteristic obtains better response on mass spectrum detection analysis, 37 proteins are obtained from the pollen protein extract extracted by the method of the invention through mass spectrum identification, wherein 3 of the proteins are specific cypress pollen allergenic proteins and are the maximum allergenic proteome data which can be obtained by one-time detection in the same cypress pollen protein extract at present.

Drawings

FIG. 1: and (4) performing a total ion current chromatogram of the protease enzymatic hydrolysis product of the sabina chinensis pollen.

FIG. 2: characteristic peptide fragment maps of 3 proteins Q93X51, A0A808QQB and A0A7R6H3R2. Wherein A is a Q93X51 peptide, B is an A0A808QQB peptide and C is an A0A7R6H3R2 peptide.

FIG. 3: representative microscopic photographs of the blank group of experimental animals.

FIG. 4: representative microscopic photograph of experimental animals in the pollen low dose group.

FIG. 5: representative microscopic photographs of experimental animals in the high dose group of pollen.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: preparation of cypress pollen protein extract

1. Materials and reagents

Sabina chinensis pollen, collected in the university school of Central nation in 2022, release period of 4-month cypress pollen, and stored at 4 deg.C after drying at low temperature.

DTT, EDTA, iodoacetamide, ammonium bicarbonate, trypsin (sequencing grade), PBS solution (Sigma), methanol, acetonitrile and formic acid for chromatographic and mass spectrometric grade (Merck KGaA company), hydrochloric acid for super grade purity (Merck KGaA company), distilled water (drochen company). 3kD ultrafilter (PALL Co.), SCX tip (Thermo Co.).

The pollen collector (Chinese Xianbishen electronic technology limited), the RIGOL L-3000 high performance liquid chromatography system (Beijing general source smart electronic technology limited), the CV100-DNA vacuum centrifugal concentrator (Beijing Ji Ai science limited), the MX-S vortex oscillator (SCILOGEX), the 5804R centrifuge (Eppendorf), the enzyme marker (DR 200B), the JY96-IIN ultrasonic crusher (Shanghai Kakun industry limited), the XMTD-7000 electric heating constant temperature water bath (Beijing Guangming medical instrument limited), the KQ-500E ultrasonic cleaner (Chinese Kunshan City ultrasonic instrument limited).

2. Preparation of protein extract

2.1 pollen Collection and Pre-treatment

Screening the collected inflorescence of the sabina chinensis and the pollen collected by the pollen collector by adopting a natural falling method and using a pollen collector to remove impurities, drying at low temperature and storing at 4 ℃.

2.2 preparation of pollen crude protein extract

Removing impurities, and collecting dry pollen according to the weight volume ratio of 1: adding 0.01mol/L PBS into 20, performing ultrasonic crushing in ice bath for 15min, continuously stirring the obtained pollen suspension at 4 ℃ for 24h, centrifuging at 4 ℃ under the condition of centrifugal force of 10000 Xg for 20min, and taking supernatant. The supernatant was filtered through a 0.45 μm filter and stored at 4 ℃.

2.3 pollen protein extraction enrichment

Concentrating the filtrate with PALL 3kD-10kD ultrafilter tube, centrifuging at 4500 Xg at 4 deg.C to obtain ultrafiltered concentrate, and storing at 4 deg.C.

2.4 enzymatic hydrolysis

Adding appropriate amount of ultrafiltration concentrated solution into DTT with final concentration of 5mM, incubating at 37 deg.C for 1h, and recovering to room temperature. Iodoacetamide was added to a final concentration of 10mM and incubated at room temperature in the dark for 45min. The samples were diluted 4-fold with 25mM ammonium bicarbonate in a protein to pancreatin ratio of 50:1 adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added the next day to adjust the pH to less than 3, and the digestion is stopped.

2.5 desalination and purification

Use of C ₁₈ Desalting the sample by a desalting column, activating the desalting column by 100% acetonitrile, balancing the column by 0.1% formic acid, loading the sample on the column, washing impurities by 0.1% formic acid, eluting by 70% acetonitrile, and collecting and eluting; treating the eluate with SCX-tip, activating SCX membrane with methanol 20 μ L for 2 times, 1min each time, balancing for 2 times, 1min each time, adding eluate sample, eluting with EDTA of pH 4.0 for 2min to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

Example 2: component analysis of cypress pollen protein extract

1. Analytical methods and conditions

Performing solution enzyme digestion on the extracted cypress pollen protein by adopting a high performance liquid chromatography-composite quadrupole rod orbitrap mass spectrometry, performing pretreatment and chromatographic separation on a peptide segment, analyzing by adopting a composite quadrupole rod orbitrap mass spectrometry, and identifying and analyzing the allergen protein by utilizing an SDAP protein database and the software of protein Discover 2.1. And re-dissolving the freeze-dried enzymolysis sample to 0.4 mu g/mu L by using 0.1% (V/V) formic acid solution, taking 20 mu L, and performing liquid chromatography-mass spectrometry combined analysis for 3 times.

1.1 chromatographic conditions:

RIGOL L-3000LC, reproxil-Pur C18-AQ chromatographic column (1.9 μm, 1.5X 250 mm), mobile phase A water (0.1% formic acid), B acetonitrile (0.1% formic acid) gradient elution, the procedure is 0-5min, 8% B; 5-35min, 12 percent of B;35 to 44min,30 percent by weight B; 44-45min, 40% by weight B; 45-59min, 95 percent B; 59-60min, 99% B. Flow rate: 0.6mL/min; column temperature: 40 ℃; the sample size was 5. Mu.L.

1.2 Mass Spectrometry conditions:

q active HF-X high resolution Mass spectrometer (Thermo Co., USA), nanospray Flex ^TM (NSI) a source of ions; temperature of ion transport tube 275 deg.C, sheath gas (N) ₂ ) Flow rate 40L/min, auxiliary gas (N) ₂ ) The flow rate is 10L/min; ion spray voltage 2.4kV, lens voltage 50V; positive ion scan mode. Using Full MS/dd-MS ² The mode is that the mass spectrum full scan range is 350-1500 m/z, the primary mass spectrum full scan resolution is 120000 (200 m/z), the maximum injection time of C-trap is 80ms, the AGC target is 3 multiplied by 10 ⁶ (ii) a Selecting parent ions with ion intensity TOPN =40 in full scan, fragmenting by high energy collision fragmentation (HCD) method, setting full scan resolution of secondary ions at 15000 (200 m/z), and setting AGC at 5 × 10 ⁴ Maximum injection time is 45ms, and secondary minimum threshold 104; opening a Peptide match; and (4) performing top excitation for 2-8s, setting NEC/step as the fragmentation collision energy of the peptide fragment to be 27, and generating mass spectrum detection raw data (. Raw). Mass number calibration was performed every 7 days using Pierce LTQ Velos ESI positive ion calibration solution (Thermo corporation, usa).

1.3 data acquisition, analysis and verification:

sequence data for cypress pollen proteins are from a database website and downloaded as Fasta files. The collected data information of peptide mass spectrum (PMF) and secondary mass spectrum (MS/MS) is searched, calculated and matched by introducing protein Discover 2.1 software (Thermo company in America), a search engine is a request HT, and parameters are set as follows: the maximum number of the missed cutting sites is 2, the range of the peptide fragment is 5-120, the tolerance of parent ions is 10ppm, the tolerance of fragment ions is 0.02Da, the weights of y and b ions are both 1, and the confidence coefficient of the identified peptide fragment is more than 95 percent; the sequence HT score value is used for scoring the protein matching degree by adopting a search algorithm, and the higher the score is, the higher the credibility is. Matching related Proteins are retrieved in the SDAP protein Database (Structural Database of Allergic Proteins, https:// fermi. Utmb. Edu/SDAP/SDAP _ src. Html) and information about the matched Proteins and peptide fragments is extracted. The verification method adopts a Parallel Reaction Monitoring (PRM) mode, targets and screens proteins with the peptide segment coverage rate and the sequence HT score ranking at the top, and completes the confirmation and identification.

2. Analysis of results

(1) Identification of major allergen proteins and peptide fragments

After the juniper pollen protein extract is subjected to solution enzyme digestion treatment by trypsin, the product is analyzed by LC-MS/MS, and the total ion current chromatogram is shown in figure 1, wherein the peak type of the spectrum is good, the reproducibility of the spectrum is good, and the data acquisition is stable. Using Sequest HT as a search engine, 37 pollen proteins were matched by searching the SDAP protein database in PMF + MS/MS mode (see Table 1).

Table 1: protein identification in PMF + MS/MS mode

Peptide fragment sequence data obtained by HPLC-MS/MS analysis are searched, matched and compared in an SDAP protein Database (structured Database of organic Proteins, https:// fermi. Utmb. Edu/SDAP/SDAP _ src. Html) to obtain protein identification information (see Table 2), including the sequence number, theoretical molecular weight, isoelectric point, protein name and source species name of the protein. The 37 proteins identified had a theoretical molecular weight range of 13.1-105kD and their sources included Juniperus chinensis L., juniperus Juniperus formosana Hayata, platycladus orientalis (L.) -Franco, cryptomeria japonica D.Don, and the like.

(2) Peptide fragments consistent with Cupressaceae pollen allergens

At present, there are 10 cypress pollen allergen proteins recorded in the Uniprot Protein database (Uniersal Protein), and the proteins have molecular weight of about 40kD, and have strong allergenicity and sensitization, and can be divided into two groups according to their activities. Wherein group 1 found 7, all having pectin lyase activity, including Cha o 1, cryj 1, cup a1, cup s 1, jun a1, jun s 1, and Jun v 1; group 2 found 3 species, generally having polygalacturonase activity, including Cha o 2, cryj 2, and Jun a 2. In the experiment, 3 proteins (Q93X 51, A0A808QQB and A0A7R6H3R 2) can meet the related confirmation identification requirements in the PRM mode, namely, the sequences have high similarity, the characteristic peptide segment with rich y and b ions can be extracted, the response is high, and the characteristic peptide segment maps of the 3 proteins are shown in a figure 2A-a figure 2C. The results of proteomic analysis and identification of the experiment on the protein enzymolysis products of the sabina chinensis pollen show that the sabina chinensis pollen contains peptide fragments which are respectively consistent with the allergens of the 1 group and the 2 groups of the cypress pollen, and the peptide fragments with consistent sequences, the molecular weights and the consistency conditions are shown in a table 3.

Table 3: peptide fragments corresponding to group 1 and 2 allergens of Cupressaceae pollen

Example 3: sensitization evaluation of cypress pollen protein extract

A BALB/c mouse model is applied to carry out pollen allergenicity evaluation research, the extracted arborvitae pollen extract is subjected to intraperitoneal injection, allergic nasal drip excitation is carried out to establish an animal allergy evaluation model, experimental animal behaviours and scores thereof, serum IgE detection and lung tissue pathological section are observed, and the allergenicity of the arborvitae pollen is evaluated.

1. Laboratory animals and materials

BALB/c female mice, weighing 18. + -.2 g, purchased from SPF (Beijing) biotechnology co., LTD; mouse immunoglobulin E (IgE) kit, purchased from shanghai enzyme-linked organisms; aluminium hydroxide gel, self-made in this laboratory; cypress pollen extract injection, which is prepared by the laboratory.

2. Preparation of pollen extract injection

2.4g of the pollen extract prepared in example 1 was suspended in physiological saline containing 5% aluminum hydroxide gel to give concentrations of 0.5g/mL (low dose) and 1g/mL, respectively

(high dose) solution for intraperitoneal injection, suspending pollen extract 1.5g in physiological saline to obtain 1g/mL solution for nasal drip excitation, and storing at 4 deg.C.

3. Preparation of pollen allergenicity evaluation model

10 mice were used for preliminary experiments, and the methods and conditions were investigated and improved.

30 mice are randomly divided into a normal group, a low dose group and a high dose according to the body weight, the intraperitoneal injection method is adopted for mouse sensitization, each mouse in an administration group is injected with 300 mu L of pollen injection by a 1mL injector, and the intraperitoneal injection is carried out on the mice in a normal control group by using the physiological saline with the same dose. Once daily, injections were continued for one week and mice were observed for signs. Antigen priming: the next day after the last intraperitoneal injection, nasal drip excitation was performed, 40. Mu.L of pollen extract suspension with the concentration of 1g/mL was administered to each mouse in the administration group, and the same amount of physiological saline was administered to the normal control group. The challenge was continued once daily for four days and the response of the mice was observed within 10min after challenge.

4. Overall symptom observation and scoring results

The BALB/c mouse model is applied to the evaluation of the allergenicity of cypress pollen, and the mouse mainly shows various symptoms of allergic rhinitis. After the mice are stimulated by antigen, the mice are subjected to characteristic and typical symptom observation for about 30min, and the degree of allergic reaction of the animals is generally evaluated by mainly observing respiratory systems and other typical symptom performances of sensitized animals to score. Specific symptoms include allergic rhinitis such as nasal mucus, watery nasal discharge and sneeze in mice, negative symptoms are asymptomatic, and evaluation can be performed according to the frequency and degree of various symptoms (see table 4).

Table 4: allergic symptoms of each group of animals

Note: "-": normal diet and activity;

"+": the phenomena of silence, low mood, reduced activity and dark hair appear;

"++": slight sneezing and nasal scratching appear;

"+++": obvious sneezing, nasal scratching and nasal discharge phenomena appear, and the frequency is increased;

"++++": obvious symptoms and death.

5. Pulmonary symptom observation and scoring results

And (4) observing with naked eyes: normal group 10 mice had normal surface color of lung tissue. The lung tissues of 10 mice in the pollen low dose group and 9 mice in the high dose group have heavy gray color and dark color in 6 lung tissues, and the surfaces of partial lung tissues have small particles with different thicknesses, so that the section is fragile.

And (4) observing under a mirror: the lung tissue cells of 10 mice in the normal control group are arranged regularly, inflammatory infiltration and swelling lesions are not seen, hyperplasia and inflammation are not seen in interstitium, and the structure is normal. Pollen low and high dose groups were analyzed as follows, and the statistics are shown in table 5.

Table 5: lung tissue pathology analysis result statistics

Note: "-": normal lung tissue, orderly arranged lung cells and normal structure;

"+": the lung tissue cells are slightly swollen, the interstitium has a small amount of fat granules, and the inflammatory cells are limited;

"++": lymphocytes, neutrophils and macrophages can infiltrate, and the alveolar wall is thickened;

"+++": inflammatory infiltration can be seen, and necrotic cell debris can be seen locally;

"++++": pulmonary edema is seen locally, and a small amount of eosinophilic seroid substance is exuded from the alveolar space.

Microscopic observation results show that the lungs of the blank experimental animals are shown in FIG. 3

The tissue surface is coated with a layer of smooth serous membrane without obvious abnormality; the bronchial structures of all levels of the lung are not obviously abnormal, and the alveolar wall structure is clear; the interstitium comprises connective tissues in the lung, blood vessels and the like, and has no obvious abnormality; no significant inflammatory changes were seen.

As shown in fig. 4, the low-dose group of pollen showed mild inflammation, with scattered lymphocyte and neutrophil infiltration (black arrows) on the alveolar wall widely seen in lung tissue; eosinophilic mucus secretion was seen in the local bronchial lumen (yellow arrows); inflammatory cell infiltration is not seen around the trachea, the bronchus and the blood vessel, the structure of the tracheal mucosa is complete, and the injury of the epithelial cells is not seen.

As shown in fig. 5, in the high-dose group of pollen, severe inflammation occurs, severe infiltration of lymphocytes, neutrophils and macrophages is observed in lung tissue (black arrows), infiltration of inflammatory cells is observed around a small amount of blood vessels to form a loop to form a blood vessel sleeve (yellow arrows), and the alveolar wall of a plurality of positions is thickened; more necrotic cell debris was visible locally (red arrows); pulmonary edema is seen locally, with a small amount of eosinophilic seroid exudation in the alveolar space (blue arrows); inflammatory cell aggregation is not seen around the trachea and the bronchus, the structure of the tracheal mucosa is complete, and the damage of the epithelial cells is not seen.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. A cypress pollen allergen composition characterized by: the cypress pollen allergen composition is a cypress pollen protein extract, and the preparation method of the cypress pollen protein extract comprises the following steps:

step 1: collecting and pretreating pollen:

screening the collected inflorescence of the juniperus chinensis and the pollen collected by the pollen collector by adopting a natural falling method and using a pollen collector to remove impurities, drying at a low temperature and storing at 4 ℃;

step 2: preparing a pollen crude protein extract:

adding the dry pollen collected after removing impurities into PBS, performing ultrasonic wave crushing in ice bath, continuously stirring the obtained pollen suspension for 24h at 4 ℃, then centrifuging at 4 ℃, taking supernatant, filtering the supernatant by using a filter of 0.45 mu m, and storing at 4 ℃;

and 3, step 3: extracting and enriching pollen protein:

concentrating the filtrate obtained in the step 2 by using an ultrafiltration tube, centrifuging at 4 ℃ to obtain ultrafiltration concentrated solution after ultrafiltration, and storing at 4 ℃;

and 4, step 4: enzymolysis:

adding a proper amount of the ultrafiltration concentrated solution prepared in the step 3 into DTT with the final concentration of 5mM, incubating for 1h at 37 ℃, then recovering to room temperature, adding iodoacetamide with the final concentration of 10mM, incubating for 45min at room temperature in a dark place, diluting a sample with 25mM ammonium bicarbonate, adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added in the next day to adjust the pH value, and the enzyme digestion is stopped;

and 5: desalting and purifying:

use of C ₁₈ Desalting the sample prepared in step 4 with desalting column, treating the eluate with SCX-tip to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

2. The cypress pollen allergen composition of claim 1, wherein: the cypress pollen allergen composition comprises the following three specific cypress pollen allergenic proteins: Q93X51, A0a808QQB, and A0A7R6H3R2.

3. The cypress pollen allergen composition of claim 2, wherein: the amino acid sequence of Q93X51 is DKSMKVTVAFNQFGPNAGQRMPRARYGL, the amino acid sequence of A0A808QQB is AKFIDTQNGLRIKTWQGGSGLAS, and the amino acid sequence of A0A7R6H3R2 is DDKSMKVTVAFNQFGPNAGQRMPRARYGL.

4. A method of preparing a cypress pollen allergen composition according to any one of claims 1 to 3, characterized in that: the preparation method comprises the following steps:

step 1: collecting and pretreating pollen:

screening the collected inflorescence of the juniperus chinensis and the pollen collected by the pollen collector by adopting a natural falling method and using a pollen collector to remove impurities, drying at a low temperature and storing at 4 ℃;

step 2: preparing a pollen crude protein extract:

adding the dry pollen collected after removing impurities into PBS, performing ultrasonic wave crushing in ice bath, continuously stirring the obtained pollen suspension for 24h at 4 ℃, then centrifuging at 4 ℃, taking supernatant, filtering the supernatant by using a filter of 0.45 mu m, and storing at 4 ℃;

and step 3: extracting and enriching pollen protein:

concentrating the filtrate obtained in the step 2 by using an ultrafiltration tube, centrifuging at 4 ℃ to obtain ultrafiltration concentrated solution after ultrafiltration, and storing at 4 ℃;

and 4, step 4: enzymolysis:

adding a proper amount of the ultrafiltration concentrated solution prepared in the step 3 into DTT with the final concentration of 5mM, incubating for 1h at 37 ℃, then recovering the room temperature, adding iodoacetamide with the final concentration of 10mM, incubating for 45min at the room temperature in a dark place, diluting a sample with 25mM ammonium bicarbonate, adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added in the next day to adjust the pH value, and the enzyme digestion is stopped;

and 5: desalting and purifying:

use of C ₁₈ Desalting the sample prepared in step 4 with desalting column, treating the eluate with SCX-tip to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

5. The method of claim 4, wherein: in the step 2, the dry pollen collected after removing the impurities is mixed according to the weight-volume ratio of 1: adding 0.01mol/L PBS (phosphate buffer solution) into the solution 20, carrying out ultrasonic crushing for 15min in an ice bath, continuously stirring the obtained pollen suspension for 24h at 4 ℃, then centrifuging the pollen suspension for 20min at 4 ℃ under the centrifugal force of 10000 Xg, taking the supernatant, filtering the supernatant by using a 0.45 mu m filter, and storing the supernatant at 4 ℃; and/or, in the step 3, the filtrate obtained in the step 2 is concentrated by using a PALL 3kD-10kD ultrafiltration tube, and is centrifuged at 4500 Xg at 4 ℃ to obtain an ultrafiltration concentrated solution which is stored at 4 ℃.

6. The production method according to claim 4 or claim 5, characterized in that: in step 4, adding a proper amount of the ultrafiltration concentrated solution prepared in step 3 into DTT with the final concentration of 5mM, incubating at 37 ℃ for 1h, then recovering to room temperature, adding iodoacetamide with the final concentration of 10mM, incubating at room temperature in the dark for 45min, diluting the sample with 25mM ammonium bicarbonate by 4 times according to the volume ratio of protein to pancreatin of 50:1 adding pancreatin for solution enzyme digestion, and incubating overnight at 37 ℃; formic acid is added in the next day to adjust the pH value to be less than 3, and the enzyme digestion is stopped; and/or, in step 5, using C ₁₈ Desalting the sample prepared in the step 4 by using a desalting column, activating the desalting column by using 100% acetonitrile, balancing a column by using 0.1% formic acid, loading the sample on the column, washing impurities by using 0.1% formic acid, eluting by using 70% acetonitrile, and collecting and eluting; treating the eluate with SCX-tip to obtain purified cedar pollen allergen composition, freeze drying, and storing at-20 deg.C.

7. Use of the cypress pollen allergen composition according to any one of claims 1 to 3 or prepared by the preparation method according to any one of claims 4 to 6 in the preparation of a reagent or medicament for diagnosing or treating allergic diseases.

8. Use according to claim 7, characterized in that: the allergic disease is a type I allergic disease.

9. Use according to claim 7, characterized in that: the allergic diseases include allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.

10. Use according to claim 7, characterized in that: the agent or medicament is in the form of tablet, sublingual drop, injection, allergen spot patch or allergen prick liquid.

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