CN117567649A

CN117567649A - Fusion protein, DNA fragment, expression vector and oral immunotherapeutic medicine

Info

Publication number: CN117567649A
Application number: CN202311631774.XA
Authority: CN
Inventors: 魏欣; 曹珂
Original assignee: Shaanxi Chang'an Huake Development Co ltd; Xi'an Baoshiter Scientific Research Co ltd
Current assignee: Shaanxi Chang'an Huake Development Co ltd; Xi'an Baoshiter Scientific Research Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2024-02-20
Anticipated expiration: 2043-11-30
Also published as: CN117567649B

Abstract

The invention discloses a fusion protein, a DNA fragment thereof, an expression vector and an oral immunotherapeutic medicine. The amino acid sequence of the fusion protein is shown as SEQ ID NO: 1. The fusion protein is the fusion protein of the dendritic cell targeting peptide and the yellow spar allergen, the dendritic cell targeting peptide can specifically and efficiently deliver the protein macromolecular yellow spar allergen into dendritic cells, so that corresponding immune response is induced, specific IgG of the yellow spar allergen is induced and generated, and the immune effect is improved; meanwhile, the dendritic cell targeting peptide greatly reduces the pollen allergic reaction induced by IgE by inhibiting a giant cell immune channel, thereby realizing the immunotherapy of pollen allergy and improving the curative effect. The invention can realize effective immunotherapy of pollen allergy without separating and purifying pollen extract containing allergen.

Description

Fusion protein, DNA fragment, expression vector and oral immunotherapeutic medicine

Technical Field

The invention relates to the technical field of biological medicines, in particular to a fusion protein, a DNA fragment, an expression vector and an oral immunotherapeutic medicine.

Background

Allergy is an increasingly serious disease of the immune system, and the associated risk of allergic reactions and occasional death have a significant impact on the well-being and quality of life of patients and their families. Avoiding allergens, although the best management of allergic diseases, is not generally feasible and anti-allergic drugs are only partially effective symptomatic. Thus, allergen immunotherapy has been developed.

Pollen from Artemisia plants, commonly known as Artemisia annua or Salvia officinalis, is considered to be the primary cause of seasonal allergic respiratory diseases in summer and autumn. Currently, there are several reported allergen immunotherapies developed based on artemisia pollen by subcutaneous injection and sublingual administration of natural artemisia pollen extracts containing allergens. Although these methods have achieved a certain effect, these methods have problems of complicated procedures, high cost, and unsustainable procedures for separating and purifying pollen extract containing allergen, and also have problems of limited therapeutic effect, long treatment course, and the like, and further need to be solved.

Accordingly, the prior art is still in need of improvement and development.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a fusion protein, a DNA fragment, an expression vector and an oral immunotherapeutic agent, which aims to solve the problems that the preparation process of the existing allergen immunotherapeutic agent is complex, the pollen extract containing the allergen needs to be separated and purified, and the curative effect needs to be further improved.

The technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a fusion protein, wherein the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 1.

In a second aspect of the present invention, there is provided a DNA fragment comprising a nucleotide sequence encoding the fusion protein of claim 1.

Optionally, the nucleotide sequence is as set forth in SEQ ID NO: 2.

In a third aspect of the present invention, there is provided an expression vector, wherein the expression vector comprises a vector and the DNA fragment of the present invention as described above contained in the vector.

Optionally, the vector is a cabbage chloroplast.

In a fourth aspect of the present invention, there is provided a method for producing an expression vector, comprising the steps of:

the amino acid sequence of the fusion protein disclosed by the invention is subjected to codon optimization according to lettuce chloroplast specific codons, so that an optimized nucleotide sequence for encoding the fusion protein is obtained, and the nucleotide sequence is shown as SEQ ID NO:2 is shown in the figure;

cloning the optimized nucleotide sequence for encoding the fusion protein into lettuce chloroplast specific vector MoChlo by introducing FspI/HindIII restriction enzyme site to obtain the expression vector.

In a fifth aspect of the invention there is provided the use of a fusion protein according to the invention as described above, a DNA fragment according to the invention as described above, an expression vector according to the invention as described above or an expression vector prepared by a preparation method according to the invention as described above in the manufacture of a medicament for the immunotherapy of pollen allergy.

In a sixth aspect of the present invention, there is provided an oral immunotherapeutic agent comprising the fusion protein of the present invention as described above, the DNA fragment of the present invention as described above, the expression vector of the present invention as described above or an expression vector prepared by the preparation method of the present invention as described above.

Optionally, the oral immunotherapeutic agent further comprises a pharmaceutically acceptable carrier.

Optionally, the oral immunotherapeutic agent is a plant-derived oral immunotherapeutic agent, which is prepared by freeze-drying lettuce expressing the fusion protein of the invention as described above, grinding the lettuce into powder and adding the powder into capsules, or is prepared by freeze-drying lettuce expressing the fusion protein of the invention as described above, grinding the lettuce into powder and mixing the powder with a pharmaceutically acceptable carrier.

The beneficial effects are that: the fusion protein is the fusion protein of the dendritic cell targeting peptide and the yellow spar allergen, the dendritic cell targeting peptide can specifically and efficiently deliver the protein macromolecular yellow spar allergen into dendritic cells, so that corresponding immune response is induced, specific IgG of the yellow spar allergen is induced and generated, and the immune effect is improved; meanwhile, the dendritic cell targeting peptide greatly reduces the pollen allergic reaction induced by IgE by inhibiting a giant cell immune channel, thereby realizing the immunotherapy of pollen allergy and improving the curative effect. The invention can realize effective immunotherapy of pollen allergy without separating and purifying pollen extract containing allergen.

Drawings

FIG. 1 is a map of MoChlo-DCpep-Art an1 expression vector in example 1 of the present invention.

FIG. 2 is a graph showing the results of PCR detection of 4 seedlings in example 3 of the present invention.

FIG. 3 is a graph showing Southern immunoblotting of 4 seedlings in example 3 according to the present invention.

FIG. 4 is a diagram showing the transgenic shoots obtained in example 3 of the present invention.

FIG. 5 is a physical view of a plant obtained in example 4 of the present invention.

FIG. 6 is a graph showing Western blot immunoblotting of total protein extracted in example 5 of the present invention.

FIG. 7 is a schematic diagram of the process of establishing a model of Artemisia annua pollen allergy mouse asthma.

FIG. 8 is a graph showing the results of measurement of IgE in serum specific to Artemisia annua pollen in different mice groups in example 6 of the present invention.

FIG. 9 is a graph showing the results of measurement of the serum IgG2a specific for Artemisia annua pollen in different mice groups in example 6 of the present invention.

Detailed Description

The invention provides a fusion protein, a DNA fragment, an expression vector and an oral immunotherapeutic agent, which are used for making the purposes, the technical scheme and the effects of the invention clearer and more definite, and are further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The embodiment provides a fusion protein, wherein the fusion protein is a fusion protein of dendritic cell targeting peptide and a yellow spar allergen, and the amino acid sequence of the fusion protein is shown as SEQ ID NO:1, specifically, the amino acid sequence of the fusion protein is formed by connecting the amino terminal of an artemisinin allergen and the carboxyl terminal of a dendritic cell targeting peptide through a linker GGGGS. Wherein the amino acid sequence of the Artemisia annua allergen is derived from Genbank NO.: ANC85006.1; the amino acid sequence of the dendritic cell targeting peptide is derived from (Matsuda Tet al.,2022, see reference 1 for details).

The fusion protein (marked as DCpep-Art an 1) is a fusion protein of a dendritic cell targeting peptide (DCpep) and a yellow flower pole allergen (Art an 1), and the dendritic cell targeting peptide can specifically and efficiently deliver a protein macromolecule yellow flower pole allergen into a Dendritic Cell (DC), so that corresponding immune response is induced, and the immune treatment effect is improved. The fusion protein can efficiently pass through intestinal epidermal cells to enter a blood circulation system, and successfully induces and generates the specific IgG of the yellow spar allergen, and meanwhile, the dendritic cell targeting peptide greatly reduces the pollen allergic reaction induced by IgE by inhibiting a giant cell immune channel, so that the effect of immunotherapy of pollen allergy is further improved. The invention can realize effective immunotherapy of pollen allergy without separating and purifying pollen extract containing allergen.

The embodiment of the invention also provides a DNA fragment, wherein the DNA fragment comprises a nucleotide sequence for encoding the fusion protein as described above. Specifically, the nucleotide sequence is shown as SEQ ID NO: 2.

The embodiment of the invention also provides an expression vector, wherein the expression vector comprises a vector and the DNA fragment which is contained in the vector and is described in the embodiment of the invention.

In some embodiments, the vector is a cabbage chloroplast.

The embodiment of the invention utilizes lettuce chloroplast bioreactor to express fusion protein (DCpep-Art an1, also called fusion antigen protein) efficiently and economically. The fusion protein can pass through stomach to reach intestinal tract under the natural biological wrapping condition of plant cell wall, so as to realize oral administration. The dendritic cell targeting peptide then mediates capture by the dendritic cell, eliciting an immune response. The specific targeting of the fusion protein to dendritic cells greatly enhances the content of IgG, and simultaneously remarkably inhibits IgE-mediated allergic reaction under the condition of allergen stimulation, thereby successfully obtaining the effect of the pollen allergic immunotherapy of the Artemisia annua.

The embodiment of the invention also provides a preparation method of the expression vector, specifically taking lettuce chloroplast specific MoCholo vector as a skeleton, constructing the expression vector of the fusion protein of the dendritic cell targeting peptide and the artemisia annua allergen in lettuce chloroplast, wherein the preparation method comprises the following steps:

the amino acid sequence of the fusion protein disclosed by the embodiment of the invention is subjected to codon optimization according to lettuce chloroplast specific codons to obtain an optimized nucleotide sequence for encoding the fusion protein, wherein the nucleotide sequence is shown in SEQ ID NO:2 is shown in the figure;

The embodiment of the invention also provides application of the fusion protein, the DNA fragment, the expression vector or the expression vector prepared by the preparation method in the embodiment of the invention in preparation of medicaments for immunotherapy of pollen allergy.

Existing immunotherapy developed based on artemisia plant pollen is mainly subcutaneous injection and sublingual administration of natural artemisia plant pollen extract containing allergens. Although the methods achieve a certain effect, the administration mode brings pain to patients, has the characteristics of poor operability, long treatment course and the like, and the compliance of the patients is poor, so that the treatment effect is obviously affected. Moreover, the disadvantages of complex operation process, high cost, unsustainable and the like of separating and purifying pollen extract containing allergen make it necessary to find an alternative method which is simple and efficient, high in patient compliance, low in cost, economical and green. Based on this, the embodiment of the present invention provides an oral immunotherapeutic agent, wherein the fusion protein of the embodiment of the present invention, the DNA fragment of the embodiment of the present invention, the expression vector of the embodiment of the present invention, or the expression vector prepared by the preparation method of the embodiment of the present invention.

In some embodiments, the oral immunotherapeutic agent further comprises a pharmaceutically acceptable carrier. In the present invention, a pharmaceutically acceptable carrier is used to deliver an active ingredient to a mammal (e.g., a human) in order to facilitate administration to a living organism, facilitate absorption of the active ingredient and thereby exert biological activity. In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, at least one of an excipient, glidant, sweetener, diluent, preservative, colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier.

In some embodiments, the oral immunotherapeutic agent is a botanical oral immunotherapeutic agent prepared from lettuce expressing the fusion protein of the examples of the invention as described above by lyophilization and grinding into powder, or prepared from lettuce expressing the fusion protein of the examples of the invention as described above by lyophilization and grinding into powder and mixing with a pharmaceutically acceptable carrier for later use.

The invention is not limited to the specific dosage form of the plant-derived oral immunotherapeutic agent, and the plant-derived oral immunotherapeutic agent can be, for example, a tablet, a solution, a capsule or the like.

When the dosage form of the plant source oral immunotherapeutic agent is a capsule, the plant source oral immunotherapeutic agent is prepared by adding lettuce expressing the fusion protein disclosed by the embodiment of the invention into the capsule through freeze-drying and grinding into powder, or is prepared by adding lettuce expressing the fusion protein disclosed by the embodiment of the invention into the capsule through freeze-drying and grinding into powder, and mixing with a pharmaceutically acceptable carrier.

The following is a detailed description of specific examples.

Formulation of RMOP medium (1L) in the following examples: 4.33g MS salt (Murashige and Skoog), 100mg inositol, 1mg thiamine hydrochloride, 1mg 6-Benzyladenine (BAP), 0.1mg 1-Naphthalene Acetic Acid (NAA), 30g sucrose and 6g plant agar, pH=5.8, RMOP medium with or without spectinomycin.

EXAMPLE 1 construction of MoChlo-DCpep-Art an1 expression vector

In order to efficiently express foreign proteins in raw vegetable chloroplasts, first, a lettuce chloroplast-specific (or lettuce chloroplast-preferential) codon optimization is performed on a nucleotide sequence corresponding to the amino acid sequence (shown as SEQ ID NO: 1) of a fusion protein (designated as DCpep-Art an 1), and the optimization algorithm is referred to the published method (Kwon et al, 2016, see reference 2 for detailed information). The codon optimized nucleotide sequence (shown as SEQ ID NO: 2) was synthesized in Shanghai and used as a subsequent expression vector construction. The synthesized nucleotide sequence optimized by the codon is amplified from a pUC57 plasmid vector through PCR reaction, and then subjected to homologous recombination by introducing FspI/HindIII restriction endonuclease sites to subclone into lettuce chloroplast specific expression vector MoChlo (purchased from https:// www.addgene.org/Kit #1000000156, the MoChlo vector contains homologous sequences of 1kb at the upstream and downstream of the trnI/trnA sites of chloroplast genome, and homologous recombination mediated gene editing in chloroplasts can be realized to form MoChlo-DCpep-Art an1 expression vector, and the map of the MoChlo-DCpep-Art an1 expression vector is shown in figure 1.

Example 2 Gene gun-mediated genetic transformation of MoCholo-DCpep-Art an1 lettuce chloroplasts

(1) 100-200 lettuce seeds (Simpson elite Simpson Elite) were placed in 1.5mL Eppendorf tubes and washed with 1mL 70% (v/v) ethanol solution for 30s to remove any greasy material.

(2) To the Eppendorf tube described above, 1mL of water diluted commercial bleach, specifically 1.5% (v/v) sodium hypochlorite and 0.1% (v/v) Tween 20, was added. Incubate for 10 minutes, mix gently by inverting the Eppendorf tube.

(3) The seeds were rinsed 5 times with 1mL of sterile deionized water to remove commercial bleach.

(4) Inoculating 40 treated lettuce seeds in MS culture medium (4.33 g MS salt, 30g sucrose and 6g plant agar, pH=5.8), placing in a culture room at 26deg.C, and periodically growing under light (1900 lux) for 16 h/dark 8h (i.e. light for 16h, then dark for 8h, then light for 16h, and then dark for 8h, and so on), and circulating for 7-10 days.

(5) Single germinated seedlings were transferred to plant culture cassettes containing MS medium and stored in culture chambers for 4-7 weeks.

(6) Leaves were harvested at the 5-7 leaf stage of plant growth. A70 mm high pressure Whatman round filter disc was placed on the RMOP medium in a petri dish. The vanes are placed on the filter disc with their front face facing the media.

(7) mu.L (about 5. Mu.g) of DNA, 50. Mu.g of gold powder particles, 10. Mu.L of 2.5M calcium chloride solution, 20. Mu.L of 0.1M spermidine solution were applied to a sterile rupture membrane and allowed to dry in a laminar flow hood. DNA delivery was performed using standard particle bombardment (also known as the particle gun method) as directed by the manufacturer.

(8) After 2 days of dark, the bombarded leaves were cut into 5mm pieces ² Is placed in RMOP selective cultureThe first round of selection was performed on the substrate (containing 50mg/mL of spectinomycin) with the bombarded side in contact with the medium. The dishes were sealed with a preservative film.

EXAMPLE 3 screening and identification of Positive plants

Transformants of lettuce were efficiently selected using 50mg/mL spectinomycin. Antibiotics were added when the medium cooled to 45-50 ℃.

Prior to the second round of screening, 100mg of leaf material was harvested from putative positive seedlings. DNA was isolated using the DNeasy Plant Mini kit following the manufacturer's procedure. This procedure produced 20-30mg of DNA.

Two separate 50. Mu.L PCR reactions were performed in two 0.2mL PCR tubes. One for checking whether the antibiotic resistance gene on the transformation vector is integrated into the chloroplast genome and the other for checking whether the gene expression cassette of interest is integrated into the chloroplast genome. At the same time, untransformed wild-type leaf DNA was again detected in a separate PCR tube as a negative control.

mu.L of the PCR product was detected by agarose gel electrophoresis. Amplified PCR products were observed by ethidium bromide staining. Plants that have been confirmed by PCR to be capable of transgene integration will receive the second and third rounds of selection.

As shown in FIG. 2, the transgenic specific PCR reaction carries out molecular identification on 4 positive seedlings (respectively marked as 1, 2, 3 and 4 in the figure) which are screened preliminarily, and other 3 strains except the 3 rd strain obviously show specific 492bp bands, so that the transgenic positive seedlings are proved. Thereafter, as shown in FIG. 3, southern immunoblotting confirmed that plants 1, 2 and 4 were positive, and that only plant 2 contained no 9.1kb wild-type chloroplast genomic fragment, which proved to be homozygous. The positive homozygous plants will be used for the second and third rounds of selection.

A second round of selection is performed: 2mm cut from PCR positive homozygous plants ² Leaves were placed on RMOP selection medium (containing 50mg/mL spectinomycin). Placed in a culture room at 26 ℃, and grown periodically under a white fluorescent lamp (1900 lux) in the light for 16 h/dark for 8h, and leaves generate transgenic buds within 3-4 weeks. As shown in FIG. 4, part of the transformant grew outFresh green shoots, which indicate that they may contain a spectinomycin resistance gene and expression cassette.

A third round of selection was performed: the regenerated shoots were excised and transferred to rooting medium (MS medium+0.5 mg/L auxin IAA) containing 50mg/mL spectinomycin, placed in a culture chamber at 26℃and periodically grown under light of 1900lux for 16 h/dark for 8h in a white fluorescent lamp, and rooted after 3-4 weeks.

EXAMPLE 4 cultivation of Positive homozygous plants

The homoplasmic plants with roots obtained in example 3 (i.e. the second positive homozygous plant, designated mocholo-DCpep-Art an 1-2) were taken and thoroughly washed with water to remove the plant mixture or agar (all the plant mixture or agar must be removed, otherwise the plants may be infected with fungi and eventually die). The jiffy pellet culture soil block (http:// www.jiffypot.com /) was soaked in water for 20min. Plants were transferred to small containers containing jiffy pellet and enough water was added to cover the surface. Covered with a plastic bag to maintain humidity. The temperature was kept in the growth chamber at 26℃and 1900lux light time was 16h and the darkness was 8h. After 4 days, a small hole is formed in the plastic bag, so that air exchange is facilitated. After 3 days more, the bag was taken out. After taking out the bag, the plants were grown in the growth chamber for one week, and watered 1 time every 2 days. The transient granules containing plants are transferred to pots containing high pressure soil in the greenhouse. Plants were watered every 2 days according to manufacturer's instructions, and water-soluble universal plant feed was added weekly, resulting in plants as shown in fig. 5. After 5 weeks, healthy leaves were collected for transgenic protein identification.

When the flower heads appear, the flower heads are covered by waterproof paper bags (moisture in the pods can increase fungal infection), and the openings of the waterproof paper bags are firmly fixed on stems below the flower branches by ropes or rubber bands. When the seed pod is ripe, the bag is taken down, the seed pod is collected and dried in a dryer to obtain the seed. These seeds can be further used to plant transgenic plants. The seeds can be stored in a sealed Eppendorf tube at 24-26deg.C for 2-3 years, and the seeds can be stored at 4deg.C or 70deg.C for a longer time.

Example 5 extraction of soluble Total protein and Western blot detection of fusion protein DCpep-Art an1 expression

(1) Extraction of total soluble proteins

Green and healthy leaves (i.e., the former lettuce leaves obtained in example 4 and the latter wild type leaves) were collected from transformed and untransformed plants grown in the greenhouse. Soil and debris on the leaves are washed off, and the midrib portion is cut off. The blade material was ground to a fine powder in liquid nitrogen. 200. Mu.L of freshly prepared vegetable protein extract (100 mM sodium chloride, 200mM Tris-HCl pH8.0, 14mM beta-mercaptoethanol, 200mM sucrose, 0.05% (v/v) Tween-20, 0.2% (w/v) sodium dodecyl sulfate) was added to each of two powdered vegetable samples (100 mg of powder per sample). The homogenized sample was spun at 15000g at 4℃for 10min and the supernatant (containing soluble proteins) was preserved.

(2) Confirmation of transgene expression by Western blot analysis

Different amounts (e.g., 100mg, 10mg, and 1 mg) of the supernatant prepared in the previous step were diluted with equal amounts (by volume) of sample buffer and boiled for 4-20min. Samples (including the non-boiled control samples) were loaded into wells of a 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel. The proteins were separated by electrophoresis. The initial current was set at 85V in 1x electrode buffer until the proteins migrated into the solubilized gel, then the current was increased to 110V and electrophoresed until the dye reached the bottom of the gel. The separated proteins were transferred to nitrocellulose or PVDF membranes using an electrophoresis track for about 2 hours at 120V. Before use, PVDF was pre-wetted in methanol for 15s, then soaked in water for 2min, then the membrane was carefully equilibrated in transfer buffer for 5min. After transfer, the membrane was immersed in a sufficient volume of PBS-t (0.1% v/v Tween-20 added to PBS) and covered completely at room temperature (25 ℃) for 5min. PBS-T was decanted. To prevent non-specific binding, the membrane was gently shaken in PTM (PBS-T with 3% w/v skim milk added) at room temperature for 1 hour to allow complete coverage of the membrane. The PTM was poured out. In the detection of total protein, the membrane was completely covered with primary antibody diluted with PTM (the dilution ratio depends on the antibody titer). The membrane and primary antibody solution were gently shaken at room temperature for 2h (or overnight at 4 ℃). The membrane was washed once with 1 XPBS-T for 5 minutes at room temperature, then the appropriate dilution of secondary antibody (horseradish peroxidase (HRP) conjugated) was added to the PTM. Incubate with gentle shaking for 1.5h. The membrane was washed 3 times with PBS-t for 15min each, and 1 time with 1 inch PBS for 10min. ECL substrate was added and incubated at room temperature with gentle shaking for 5min. Chemiluminescent signals are generated by exposing the film to x-ray film. The initial exposure time is 1min; depending on the signal obtained, the subsequent exposure time may be extended to 30min.

As a result, as shown in FIG. 6, it was found that the expression of the fusion protein DCpep-Art an1 protein was detected in three independent (A, B, C) positive plants (DCpep-Art an 1-2), and the molecular weight of the fusion protein DCpep-Art an1 was 16.26kDa.

The identified lettuce leaves containing DCpep-Art an1 are picked, freeze-dried, ground to prepare dry plant powder, and mixed with PBS buffer solution according to a certain proportion to prepare a suspension preparation for the downstream animal test.

EXAMPLE 6 in vivo validation of the efficacy of DCpep-Ar t an1 oral immunotherapy

(1) Test mice

BALB/c mice (6-8 weeks old; 18-20 g) were purchased from the national academy of military medicine (Beijing) and bred in the Beijing co-Hospital animal laboratory center for the absence of specific pathogens. All animal feeding and handling procedures were approved by the ethical committee of animal experiments in beijing co-ordinates hospitals.

(2) Allergen and method for producing the same

Artemisia annua (Artemisia annua) pollen is obtained from Beijing office key laboratory for accurate diagnosis and treatment of allergic diseases. The preparation and use method is as follows: briefly, defatting with acetone at 4deg.C for 8 hr, stirring with 0.125M ammonium bicarbonate solution (1:20, w/v) for 24 hr, extracting protein (denoted as AAP protein), filtering, dialyzing on distilled water for 24 hr, packaging, and lyophilizing. The lyophilized AAP proteins were dissolved in Phosphate Buffered Saline (PBS) prior to use and the total protein concentration (AAP protein concentration 0.93. Mu.g/. Mu.L) was determined using the Bradford method.

(3) Design of experiment

Establishing a model of the yellow spar pollen allergic mouse asthma: the 20 mice were randomly divided into asthma group and Control group (Control), 10 each. The modeling process is shown in FIG. 7(wherein SC: subcutaneous sensitization subcutaneous sensitization; IH: inhalation; sac sacrifices sacrifice). Briefly, asthmatic mice were sensitized on days 1, 8, 15, respectively, by subcutaneous injection of a 200. Mu.LAPP in combination with alum (where APP is 25. Mu.g) via the nape of the neck. The asthma group mice were challenged on days 18 to 24 to aerosol AAP aerosol (ddH solvent) in an amount of 0.1% of the mice volume ₂ O) 30min, 1 time daily, 7 consecutive days; the control group was sensitized and stimulated with an equal volume of PBS.

Asthma mice were divided into two groups, the treatment group (designated as DCpep-Art an1 group) and the solvent group (designated as Vehicle group). From day 33, the treated mice were orally gavaged with 150 μl (containing 0.14mg lettuce leaf powder with DCpep-Art an 1) of PBS solution. Solvent group mice were subcutaneously injected with 150 μl PBS. On days 94, 95 and 96, the solvent group and the treatment group mice received again 0.1% aap aerosol challenge. Meanwhile, control mice received PBS only at each step. Mice were tested for Airway Hyperresponsiveness (AHR) on day 97 and sacrificed 1 day later. Serum was used to detect specific IgE and IgG2a antibodies against the pollen of artemisia annua.

Determination of Artemisia annua pollen specific serum IgE (Artan-sIgE)

Enzyme-linked immunosorbent assay (ELISA) detects the levels of Artan-sIgE. A96-well plate (Thermo Fisher Scientific, waltham, MA, USA) was coated with 100. Mu.L/well AAP (50. Mu.g/mL). The next day the plate was washed and 100. Mu.L of serum samples (diluted 1:5 with plate buffer) were taken in the wells. After overnight incubation, 100. Mu.L of anti-mouse IgE monoclonal antibody (1. Mu.g/mL; abcamplc) and horseradish peroxidase (HRP) -labeled secondary antibody (1:1000;BD Biosciences Pharmingen) were each stored at 37℃for 1 hour. Finally, 100. Mu.L/well TMB substrate chromogenic kit (Kangji, beijing, china) was used as substrate and reacted at room temperature for 20min. ELISA plate detectors (ELX 800; bioTek, winioski, VT, USA) were used and reference was made to the IgE standard curve of mice in the control group.

Artemisia annua pollen specific serum IgG2a (Artan-sIgG 2 a) assay ELISA detects Artan-sIgG2a levels. Plates were coated overnight at 4℃with 100. Mu.L/well AAP (5. Mu.g/mL), the next day the plates were washed, serum samples were again washed 3 times, diluted with blocking buffer (1:2000), 100. Mu.L/well added to the plates and incubated for 1h at 37 ℃. Then washed three times, added with 200. Mu.L of 1:5000 diluted mouse IgG2a monoclonal antibody blocking buffer (5% skim milk in PBS) and incubated for 2h at room temperature (22-25 ℃). The cells were washed three more times and incubated at 37℃with HRP-labeled secondary antibody (1:3000) for 1h. The plates were then read at a wavelength of 450nm using an ELISA plate detector.

Asthma BALB/c model mice were subjected to pollen stimulation by Artemisia annua pollen after oral administration of plant powder containing DCpep-Art an1 (oral immunotherapeutic agent), and then subjected to ELISA quantitative detection of IgE and IgG in their blood. The results are shown in fig. 8 and 9, and demonstrate that the IgE content in the blood of the mice in the treated group is significantly reduced compared with the solvent group, approaching the level of the control group. However, the allergen-specific IgG content of the Artemisia annua pollen was significantly increased compared to both the solvent group and the control group. This suggests that the dendritic cell targeting peptide specifically targets Art an1 to dendritic cells, but not to IgE-dependent post cell pathways, inhibiting IgE formation. Meanwhile, the dendritic cells induce the formation of IgG, thereby having the effect of treating pollen allergy by oral immunotherapy.

It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Reference is made to:

1、Matsuda T,Misato K,Tamiya S,et al.Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice.iScience.2022；25(11):105324.Published 2022Oct 10.doi:10.1016/j.isci.2022.105324。

2、Kwon KC,Chan HT,León IR,Williams-Carrier R,Barkan A,Daniell H.Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation.Plant Physiol.2016；172(1):62-77.doi:10.1104/pp.16.00981。

Claims

1. a fusion protein, characterized in that the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1.

2. A DNA fragment comprising a nucleotide sequence encoding the fusion protein of claim 1.

3. The DNA fragment of claim 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 2.

4. An expression vector comprising a vector and the DNA fragment of any one of claims 2-3 contained in the vector.

5. The expression vector of claim 4, wherein the vector is lettuce chloroplast.

6. A method for preparing an expression vector, comprising the steps of:

optimizing the codon of the amino acid sequence of the fusion protein according to lettuce chloroplast specific codons to obtain an optimized nucleotide sequence for encoding the fusion protein, wherein the nucleotide sequence is shown in SEQ ID NO:2 is shown in the figure;

7. Use of the fusion protein of claim 1, the DNA fragment of any one of claims 2-3, the expression vector of any one of claims 4-5 or the expression vector prepared by the preparation method of claim 6 in the preparation of a medicament for immunotherapy of pollen allergy.

8. An oral immunotherapeutic comprising the fusion protein of claim 1, the DNA fragment of any one of claims 2 to 3, the expression vector of any one of claims 4 to 5 or an expression vector prepared by the preparation method of claim 6.

9. The oral immunotherapeutic agent of claim 8, further comprising a pharmaceutically acceptable carrier.

10. The oral immunotherapeutic agent of claim 8, wherein the oral immunotherapeutic agent is a plant-derived oral immunotherapeutic agent prepared from lettuce expressing the fusion protein of claim 1 by lyophilization, grinding into powder, or prepared from lettuce expressing the fusion protein of claim 1 by lyophilization, grinding into powder, and mixing with a pharmaceutically acceptable carrier.