Biregulin recombinant vaccine and application thereof in pulmonary fibrosis treatment

Abstract

The invention relates to the technical field of immunotherapy, in particular to a regulatory protein recombinant vaccine and application thereof in pulmonary fibrosis treatment. The vaccine contains polypeptide of amphiregulin antigen epitopes, and the amino acid sequence of the vaccine is shown as SEQ ID No. 1. A research result shows that the recombinant vaccine can effectively induce generation of a specific antibody aiming at the AREG protein in a mouse body, so that secretion of the AREG protein and activation of downstream receptors of the AREG protein in the pulmonary fibrosis process are inhibited. By reducing collagen deposition in lung tissue and reducing tissue structure damage, the recombinant vaccine significantly reduces gene expression of fibrosis-related protein, recovers lung functions of pulmonary fibrosis model mice, and inhibits further development of diseases. In addition, the recombinant vaccine can be administered in an aerosol inhalation mode, and has broad-spectrum, efficient and long-term stable expression and good safety. The invention provides a brand-new targeted therapy means for clinical prevention and treatment of pulmonary fibrosis, and shows a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and in particular to a recombinant amphiregulin vaccine and an application thereof in the treatment of pulmonary fibrosis. Background Art

[0002] Pathological fibrosis is a common outcome of many chronic inflammatory diseases. It is estimated that in developed countries, almost half of all deaths are related to pathological fibrosis. Among them, pulmonary fibrosis is a progressive and irreversible disease, and its prognosis remains fatal despite the availability of anti-fibrotic treatments. Pulmonary fibrosis consists of a group of heterogeneous lung diseases, characterized by progressive and irreversible destruction of lung structure caused by scarring, which ultimately causes organ dysfunction, gas exchange disorders, and respiratory failure. At present, the incidence and mortality of pulmonary fibrosis are increasing year by year, significantly increasing the burden of the disease. However, existing treatments mainly rely on two drugs approved by the US FDA: nintedanib and pirfenidone, which can only alleviate but not reverse pulmonary fibrosis. Therefore, early diagnosis and exploration of new treatment pathways are crucial for the clinical management of pulmonary fibrosis.

[0003] Amphiregulin (AREG) is found in the epidermal growth factor family gene cluster on chromosome band 4q13.3, and contains two gene copies (AREG and AREGB) about 160 kb apart and spanning about 10 kb of genomic DNA. The AREG gene is transcribed to produce an mRNA with a length of 1.4 kb containing 6 exons, which ultimately encodes a transmembrane glycoprotein precursor (Pro-AREG) consisting of 252 amino acids. Under the action of metalloproteinases TACE or ADAM-17, the protein precursor is hydrolyzed and cleaved to release mature soluble AREG containing EGF motifs, which then induces autocrine or paracrine activation of its downstream epidermal growth factor receptor (EGFR). AREG-EGFR signaling can activate multiple pathways such as Ras-Raf / MAPK, PI3K / Akt or STAT, thereby regulating various cellular processes such as cell proliferation, apoptosis and migration. Studies have shown that a variety of immune cells can produce AREG under different inflammatory stimuli, including basophils, mast cells, type 2 innate lymphocytes, dendritic cells, neutrophils, CD8+T cells, regulatory T cells (Treg), and activated CD4+T cells. This suggests that AREG may play a role in immune-related resistance and tolerance mechanisms. Increased AREG expression is associated with the development of fibrosis in multiple organs (such as the lungs, liver, and heart), and has become a key player in immunity, inflammation, and tissue repair. However, the specific mechanism of the AREG-EGFR axis in the development of pulmonary fibrosis is still unclear. Some studies have shown that knocking out AREG can reduce pulmonary fibrosis induced by bleomycin (BLM) and TGF-β1 transgenic mice. However, there are also literatures indicating that intratracheal instillation of AREG recombinant protein can reduce BLM-induced pulmonary fibrosis. Considering the high expression of AREG in patients with idiopathic pulmonary fibrosis, elucidating the pathogenesis of the AREG-EGFR axis in the process of pulmonary fibrosis has important public health significance and clinical application value.

[0004] Vaccines are common drugs for preventing infectious diseases. In recent years, they have also been developed for the treatment of autoantigen-related diseases such as hypertension, dyslipidemia, Alzheimer's disease, cancer and inflammatory diseases. If the efficacy and safety of vaccines can be comparable to existing drug treatments, vaccines may become a potential alternative for the treatment of these lifestyle diseases. At present, vaccine treatment for pulmonary fibrosis is not yet mature, and some key disease-related molecules (such as TGF-β, IL-13, CTGF, etc.) have been regarded as potential targets. However, these molecules also play an important role in normal tissue repair and other physiological functions. Treatment against them may cause side effects or interfere with normal immune responses and repair processes. In contrast, AREG is expressed at a low level in normal tissues, reducing the risk of off-target effects. In addition, AREG knockout experiments and siRNA silencing experiments did not cause obvious side effects or shortened lifespan in animal models. Therefore, as a key promoter of pulmonary fibrosis, AREG is expected to become an ideal target for vaccine development. The construction of a vaccine against AREG is not only expected to replace existing drugs for the treatment of pulmonary fibrosis, but also may effectively reduce medical costs and the disease burden of pulmonary fibrosis, which has important clinical and public health significance. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide a recombinant amphiregulin vaccine and its application in the treatment of pulmonary fibrosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] A recombinant amphiregulin vaccine comprises a polypeptide of an amphiregulin antigen epitope, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] The vaccine comprises a polypeptide conjugate comprising the polypeptide according to claim 1.

[0009] The polypeptide conjugate is the polypeptide and the coupling portion according to claim 1; wherein the coupling portion is connected to the polypeptide portion, and the coupling portion is selected from at least one of keyhole limpet hemocyanin, ovalbumin and bovine serum albumin.

[0010] The mass ratio of the polypeptide to the coupling part is 1-10:1, preferably 1-5:1.

[0011] The polypeptide conjugate is prepared by incubating the polypeptide according to claim 1 with a coupling part at room temperature for 8-12 hours; wherein the mass ratio of the polypeptide, the coupling part (KLH) and the protein cross-linking agent (Sulfo-SMCC) is 1-10:1-10:1; preferably 1-5:1-5:1.

[0012] The vaccine is the polypeptide conjugate and a physiologically acceptable excipient thereof, wherein the mass ratio of the polypeptide conjugate to the excipient is 1-10:25, preferably 1-5:25.

[0013] During the use of the vaccine, an adjuvant is added, and the volume ratio of the immune adjuvant to the vaccine is 1:1-5, preferably 1:1-2. The adjuvant is Freund's adjuvant, Montan oil adjuvant, RIBI adjuvant, MF59 emulsion adjuvant, QS-21 adjuvant.

[0014] An application of the recombinant vaccine, and application of the vaccine in the treatment of pulmonary fibrosis.

[0015] The recombinant vaccine for treating pulmonary fibrosis is to induce the production of neutralizing antibodies against AREG protein in vivo through the polypeptide conjugate, thereby reducing the secretion of AREG protein and the activation of downstream signaling pathways, and reducing the deposition of collagen fibers in lung tissue and the destruction of tissue structure during fibrosis.

[0016] The administration method of the recombinant vaccine is intravenous injection, arterial injection, inhalation administration, intramuscular injection, subcutaneous injection, organ injection and intrathoracic and peritoneal injection.

[0017] The present invention discloses the following technical effects:

[0018] The recombinant vaccine targeting AREG protein of the present invention can effectively induce the production of specific antibodies against AREG protein in vivo, thereby reducing the secretion of AREG protein and inhibiting the activation of its downstream signaling pathway. By reducing the collagen fiber deposition and tissue structure damage in the lung tissue of mice with pulmonary fibrosis, the vaccine significantly reduces the gene expression of fibrosis-related proteins, restores lung function, and thus achieves the effect of treating pulmonary fibrosis. In a specific implementation, a humidifier atomization inhalation of polyhexamethylene guanidine (PHMG) and tracheal instillation of bleomycin (BLM) were used to construct a mouse model of pulmonary fibrosis, and the vaccine was administered by atomization inhalation. The results showed that the recombinant vaccine effectively reduced the collagen deposition in the lung tissue and significantly restored lung function in both pulmonary fibrosis mouse models. The vaccine can be used as a candidate drug for the treatment of pulmonary fibrosis, and has no obvious adverse effects, showing a good prospect for promotion and application. The present invention has important clinical application value and research significance by developing or screening drugs for the treatment of pulmonary fibrosis with AREG protein as a target. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a measurement of serum antibody titer in the blood of mice after tracheal instillation of the recombinant vaccine.

[0020] Figure 2 The expression of AREG protein in the bronchoalveolar lavage fluid of mice after tracheal instillation of recombinant vaccine in a mouse model of pulmonary fibrosis induced by aerosol inhalation of PHMG.

[0021] Figure 3 This is the change of lung function after tracheal instillation of recombinant vaccine in the mouse model of pulmonary fibrosis induced by aerosol inhalation of PHMG.

[0022] Figure 4 The deposition of collagen fibers in lung tissue after tracheal instillation of recombinant vaccine in a mouse model of pulmonary fibrosis induced by aerosol inhalation of PHMG.

[0023] Figure 5 The expression levels of type 1 collagen (Col1a1), fibronectin (Fn1) and α-smooth muscle actin (Acta2) in lung tissue of a mouse model of pulmonary fibrosis induced by aerosol inhalation of PHMG were evaluated by tracheal instillation of recombinant vaccine.

[0024] Figure 6 The expression of AREG protein in bronchoalveolar lavage fluid of mice after tracheal instillation of recombinant vaccine in BLM-induced pulmonary fibrosis mouse model.

[0025] Figure 7 This is the change in lung function after tracheal instillation of recombinant vaccine in the BLM-induced pulmonary fibrosis mouse model.

[0026] Figure 8 This is the deposition of collagen fibers in lung tissue after tracheal instillation of recombinant vaccine in the BLM-induced pulmonary fibrosis mouse model.

[0027] Fig. 9 The expression levels of type 1 collagen (Col1a1), fibronectin (Fn1) and α-smooth muscle actin (Acta2) in lung tissue of BLM-induced pulmonary fibrosis mouse model after tracheal instillation of recombinant vaccine.

[0028] Fig.10 This is a schematic diagram of the recombinant vaccine entering the body to produce antibodies.

[0029] In the figure, FVC stands for forced vital capacity, IC stands for deep inspiratory capacity, Cchord stands for quasi-static compliance, Control stands for the control group, and Vaccine stands for the experimental group; GAPDH is widely distributed in cells of various tissues and is an internal reference gene. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Unless otherwise specified, all technical and scientific terms used herein should be interpreted according to the understanding of those skilled in the art. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing process.

[0032] Without departing from the scope and spirit of the present invention, the specific embodiments of this specification may be subject to various modifications and variations, which will be apparent to those skilled in the art. The description of the present invention may lead the skilled person to other obvious embodiments. These descriptions and examples are for illustrative purposes only.

[0033] The recombinant vaccine of the present invention can effectively induce the production of specific antibodies against AREG protein in vivo, neutralize AREG protein and reduce its secretion, and inhibit the activation of downstream signaling pathways, thereby reducing the deposition of collagen fibers and damage to tissue structure during pulmonary fibrosis. At the same time, the vaccine can reduce the gene expression of fibrosis-related proteins, restore lung function, and ultimately achieve the effect of treating pulmonary fibrosis.

[0034] At the same time, the recombinant vaccine can effectively induce the production of specific antibodies against AREG protein in mice, thereby inhibiting the secretion of AREG protein and the activation of its downstream receptors during pulmonary fibrosis. By reducing collagen deposition in lung tissue and alleviating tissue structural damage, the recombinant vaccine significantly reduced the gene expression of fibrosis-related proteins, restored the lung function of pulmonary fibrosis model mice, and inhibited the further development of the disease. In addition, the recombinant vaccine can be administered by aerosol inhalation, with broad-spectrum, high-efficiency, long-term stable expression and good safety. This provides a new targeted treatment method for the clinical prevention and treatment of pulmonary fibrosis, showing broad application prospects.

[0035] Example 1 Synthesis of polypeptide

[0036] In this study, the target polypeptide NH2-CDCRVNLCY-CONH2 (SEQ ID No. 1) was constructed by solid phase synthesis technology. The specific process covered the chemical synthesis, purification and final identification and analysis of the polypeptide.

[0037] 1. Peptide solid phase synthesis process

[0038] The peptides were synthesized using solid phase peptide synthesis (SPPS) based on the Fmoc (9-fluorenylmethoxycarbonyl) protecting group strategy. The main steps of the synthesis process include:

[0039] 1) Covalently bond the carboxyl group of the C-terminal amino acid to the solid phase resin carrier, laying the foundation for subsequent peptide chain extension.

[0040] 2) Before each reaction step, the Fmoc protecting group of the amino acid was removed using a piperidine solution to expose the free N-terminal amino group.

[0041] 3) Introduce activated amino acids (activated by HBTU or HATU) to combine with the free amino group of the previous residue and gradually extend the peptide chain. After each round of coupling, multiple washing and deprotection operations are performed to ensure the accuracy of the synthesis.

[0042] 4) After the sequence connection of all amino acid residues is completed, a TFA solution (containing an appropriate amount of capture agent) is used to cleave and remove the protecting groups from the resin to obtain a crude polypeptide.

[0043] 2. Peptide Purification

[0044] The crude peptides were separated and purified by reverse phase high performance liquid chromatography (RP-HPLC) under the following specific operating conditions:

[0045] Mobile phase A: 0.1% trifluoroacetic acid (TFA) in water

[0046] Mobile phase B: 0.1% TFA in acetonitrile

[0047] Gradient program: Starting from the initial condition of 90% mobile phase A and 10% mobile phase B, the gradient transition to 40% A and 60% B was performed within 30 minutes. The separation flow rate was set at 1 mL / min and the operating temperature was room temperature (about 23° C.).

[0048] Detection method: UV detector with a wavelength of 214nm.

[0049] The synthesized crude polypeptide is dissolved in mobile phase A, and an appropriate amount (about 20-30 mg or 2-2.5 mL) of the sample is injected into the liquid chromatography system, and eluted and separated according to the set gradient conditions, and the main peak portion is collected, and then freeze-dried to obtain a purified polypeptide sample, as shown in the amino acids in SEQ ID No. 1.

[0050] 3. Peptide purity analysis

[0051] The purified peptide was identified for purity by liquid chromatography-mass spectrometry (LC-MS) technology, and the analysis conditions were as follows:

[0052] Mobile phase A: 0.05% TFA in water

[0053] Mobile phase B: 0.1% TFA in acetonitrile

[0054] Gradient elution: starting from an initial ratio of 90% A and 10% B, gradually transitioning to 40% A and 60% B within 10 minutes, the flow rate was maintained at 1 mL / min, and the temperature was room temperature (23°C).

[0055] Detection method: Use 214nm UV detector.

[0056] Mass spectrometry detection: The molecular weight and purity of the peptides were analyzed by atmospheric pressure electrospray ionization (API-ESI) mass spectrometry.

[0057] This identification method combines the high resolution of HPLC and the precise molecular weight determination of mass spectrometry, which can effectively verify the purity and molecular structure of the synthetic peptide and ensure the accuracy of the experimental results. The purity of the synthetic peptide was greater than 95%, reaching the purity for vaccine use.

[0058] Example 2 Preparation of polypeptide conjugates

[0059] In this experiment, the polypeptide synthesized in Example 1 was subjected to a coupling reaction with keyhole limpet hemocyanin (KLH). The specific process includes column bed preparation, polypeptide mixture preparation, coupling reaction, and storage of the final product. The specific steps are as follows:

[0060] 1. Column Bed Preparation

[0061] First, the column bed was pretreated with pure water and coupling buffer. The coupling buffer was AH solution, which contained Na2HPO4, NaH2PO4, NaCl and EDTA, and the pH value of the solution was adjusted to 7.2 to ensure the suitability of the reaction system.

[0062] 2. Preparation of Peptide Mixture

[0063] The polypeptide synthesized in Example 1 was dissolved in N,N-dimethylformamide (DMF) and allowed to stand at room temperature for 30 minutes until no visible particles were present in the solution. AH solution was then added to obtain a polypeptide mixture, in which the final concentration of the polypeptide in the mixture was 10 mg / mL. This step ensured that the polypeptide was fully dissolved and evenly distributed before the coupling reaction.

[0064] 3. Reaction of coupling moiety with Sulfo-SMCC

[0065] Next, the coupling part (KLH) was mixed with the AH solution to obtain a first mixture, and the final concentration of the coupling part was 20 mg / mL. At the same time, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt (Sulfo-SMCC) was dissolved in DMSO to prepare a second mixture with a concentration of 150 mg / mL. The two were mixed in proportion and reacted at 4°C for 12 hours. After the reaction was completed, the reaction product was separated by a chromatography column to obtain an intermediate.

[0066] 4. Generation of Peptide Conjugates

[0067] After obtaining the intermediate, it was fully mixed with the peptide mixture prepared in step 2, and a vertical mixer was used to ensure that the reactants were evenly dispersed. Thereafter, the mixture continued to react overnight at room temperature to ensure the completion of the coupling reaction. During the reaction, the mass ratio of the intermediate to the peptide, coupling part (KLH) and Sulfo-SMCC in the peptide mixture was 10:10:1.

[0068] The final product is a peptide conjugate, which is stored at -20°C after production to ensure the stability and biological activity of the peptide.

[0069] Example 3 Mouse Immunization Experiment

[0070] The polypeptide conjugate prepared in Example 2 was diluted in physiological saline and emulsified with an equal volume of adjuvant for an immunization experiment. The experimental subjects were eight-week-old C57BL / 6 mice, which were randomly divided into groups for immunization.

[0071] 1. Immunization dose and strategy

[0072] The immunization process was administered by tracheal instillation using a pulmonary liquid quantitative nebulizer, and each mouse received an immunization dose on days 0, 14, 28, and 56. The two groups received 2 μg or 20 μg of peptide conjugate each time, and the injection volume was determined by the experimental design. The control group mice were injected with an equal amount of KLH, which was mixed with an equal volume of Freund's adjuvant to eliminate the effect of the adjuvant on the immune response.

[0073] 2. Serum collection and antibody testing

[0074] During the immunization experiment, serum samples of mice were collected through the tail vein at the same time as the immunization dose. Subsequently, the antibody titer against the immunization peptide in the serum was measured using an enzyme-linked immunosorbent assay (ELISA) to evaluate the immune response of the mice.

[0075] Example 4 ELISA test of serum after mouse immunization

[0076] The mouse serum prepared in Example 3 was analyzed by ELISA method to determine the specific antibody level against AREG protein. The specific experimental steps are as follows:

[0077] 1. ELISA plate coating

[0078] First, add 200 ng of purified AREG protein (R&D) to each ELISA plate well, and use 50 mM Na2CO3 / NaHCO3 buffer (pH 9.6) as the coating solution. Place the ELISA plate at 4°C overnight for protein coating. After coating, discard the coating solution.

[0079] 2. Blocking and washing

[0080] Add 150 μL of 5% skim milk powder to each well and block for 1 hour at room temperature to prevent nonspecific binding. After blocking, wash the ELISA plate twice with PBS solution containing 0.05% Tween-20 to ensure that the residue is fully removed.

[0081] 3. Serum Incubation

[0082] Subsequently, 100 μL of serially diluted antigen-immunized serum or PBS-immunized serum as a control was added to each well and incubated for 1 hour at room temperature. Afterwards, the supernatant was discarded and the ELISA plate was washed five times again with PBS solution containing 0.05% Tween-20 to remove unbound antibodies.

[0083] 4. Secondary Antibody Incubation and Color Development

[0084] 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG antibody (HRP secondary antibody) diluted 1:3000 (v / v) was added to each well and incubated for 1 hour at room temperature. After incubation, the secondary antibody solution was discarded and the plate was washed five times with PBS containing 0.05% Tween-20. Subsequently, 50 μL of ELISA color development solution was added to each well and color was developed for 15 minutes.

[0085] 5. Reaction termination and OD value reading

[0086] To terminate the reaction, 50 μL of 2M H2SO4 was added to each well, and the optical density (OD) at 450 nm was read by a microplate reader. 450 ) value.

[0087] 6. Antibody titer calculation

[0088] The calculation of antibody titer was based on the negative control value of the group without serum added multiplied by 2.1 times to obtain the benchmark value, and the logarithm value (log value) of the lowest dilution multiple exceeding the benchmark value was used as the antibody titer. The results showed that the antibody titer binding to AREG antigen in the serum of mice treated with the vaccine was significantly higher than that of the control group ( Figure 1 ), indicating that the vaccine has a good immune effect.

[0089] Example 5 Therapeutic effect of vaccine on mice with pulmonary fibrosis induced by aerosol inhalation of PHMG

[0090] C57BL / 6 mice were used in the experiment and were evenly divided into four experimental groups according to their body weight, with 5 mice in each group. The specific groups are as follows:

[0091] Control group: This group of mice received normal treatment without PHMG exposure or vaccine intervention. It is used to observe the physiological state under normal conditions as a baseline control.

[0092] Simple intervention group (Vaccine): The mice in this group were not exposed to PHMG and only received intervention with the recombinant vaccine during the experiment. The purpose was to evaluate the effect of the vaccine itself on normal mice.

[0093] Fibrosis group (PHMG): Mice in this group were exposed to PHMG aerosol to induce pulmonary fibrosis and simulate the disease model. This group did not receive vaccine intervention and was used to evaluate the effect of PHMG-induced fibrosis.

[0094] Fibrosis intervention group (PHMG-Vaccine): Mice in this group were also exposed to PHMG aerosol to induce pulmonary fibrosis, but received recombinant vaccine intervention starting from the third week of PHMG exposure. This group was used to evaluate the intervention effect of the vaccine in the process of fibrosis.

[0095] All mice were placed in cages, with 5 mice in each cage, corresponding to the four groups of the experiment. The experimental treatment steps of each group were the same, and the only difference was whether PHMG exposure and vaccine intervention were performed. Through this group design, the differences between the control group, vaccine intervention group, fibrosis group and fibrosis intervention group can be effectively compared, and the individual and combined effects of PHMG and vaccine can be clarified.

[0096] The specific method of PHMG-induced pulmonary fibrosis is as follows: First, PHMG powder was dissolved in ultrapure water to prepare a 0.1 mg / mL solution. Then the solution was added to the aerosol generator, and the mice in the fibrosis group and the fibrosis intervention group were exposed; the mice in the control group and the simple intervention group were exposed using ultrapure water. The spray volume was set at 30-40 mL / h, and the exposure was performed once every two days, each lasting 4 hours, and the exposure period was 8 weeks. The intervention group was intervened with recombinant vaccines starting from the third week of PHMG exposure. Each mouse in the simple intervention group and the fibrosis intervention group used 50 μL PBS and an equal volume of Freund's adjuvant, and 20 μg of recombinant vaccine was dissolved and injected into the mouse lungs through tracheal instillation through a lung liquid quantitative nebulizer. The control group and the fibrosis group were injected with an equal amount of KLH and an equal volume of Freund's adjuvant. The intervention was once a week for a total of 5 times.

[0097] Extract the alveolar lavage fluid from mice, and use the AREG ELSIA detection kit to detect the content of AREG protein in the alveolar lavage fluid. Figure 2Compared with the Control group, the AREG protein content in the alveolar lavage fluid of the Vaccine group decreased slightly but there was no statistical difference. The AREG protein content in the alveolar lavage fluid of the PHMG group increased significantly. The AREG protein content in the lavage fluid of the PHMG-Vaccine group was significantly decreased compared with that of the PHMG group, indicating that the vaccine intervention can effectively reduce the secretion of AREG protein in the lung tissue of mice with PHMG-induced pulmonary fibrosis.

[0098] Small animal pulmonary function instrument was used to detect the changes in lung function of mice. The results are shown in Figure 3 . Forced vital capacity (FVC, Figure 3 A) is used to evaluate the ventilation function of the lungs. The results showed that the FVC of the Vaccine group did not change significantly compared with the Control group, while the FVC value of the PHMG group mice was significantly reduced, indicating that PHMG inhalation exposure caused damage to the pulmonary ventilation function. Compared with the PHMG group, the pulmonary ventilation function of the PHMG-Vaccine group mice was significantly restored. Deep inspiratory capacity (IC, Figure 3 B) is used to evaluate the ability of the lungs to inhale deeply. Compared with the mice in the control group, the IC of the mice in the vaccine group did not change significantly, while the IC of the mice in the PHMG group was significantly reduced, indicating that PHMG inhalation exposure led to a decrease in lung volume, and the lung volume of the mice in the PHMG-Vaccine group was significantly restored. Static compliance (Cchord, Figure 3 C) was used to evaluate the elasticity and expansion capacity of the lung under static conditions. Compared with the mice in the Control group, the Cchord of the mice in the Vaccine group did not change significantly, but the Cchord value of the PHMG group was significantly reduced, indicating that PHMG inhalation exposure caused a decrease in the static compliance of the lung tissue, and the static compliance of the lungs of the mice in the PHMG-Vaccine group was significantly restored. The overall results showed that vaccine intervention can effectively restore the decline in lung function caused by PHMG in mice.

[0099] After the mice were sacrificed, lung tissue sections were taken for Masson staining to evaluate collagen deposition. The results are shown in Figure 4 A. The analysis results showed that compared with the control group, the lung tissue of the mice in the vaccine group did not show obvious collagen deposition or pathological structural destruction of the lung tissue. By quantitatively analyzing the collagen fibers in the Masson staining sections ( Figure 4 B) found that PHMG inhalation exposure caused a significant increase in collagen deposition in the lung tissue of mice. Vaccine intervention was able to effectively reduce collagen deposition.

[0100] The lung tissues of the above mice were taken and ground using TRIzol reagent. RNA was extracted and then verified by qPCR to detect the expression of Col1a1, Fn1 and Acta2 genes in the lung tissues of mice. The results are shown in Figure 5 The analysis results showed that intratracheal instillation of the vaccine could effectively inhibit the expression of fibrosis markers in the lung tissue of mice with pulmonary fibrosis induced by PHMG.

[0101] The above results confirm that the vaccine targeting AREG is highly effective and safe for lung tissue. In addition, AREG protein plays a key role in PHMG-induced pulmonary fibrosis. By injecting mice with PHMG-induced pulmonary fibrosis with a vaccine targeting AREG protein, the lung function of mice can be effectively restored and the progression of pulmonary fibrosis can be alleviated.

[0102] Example 6: Therapeutic effect of vaccine on mice with pulmonary fibrosis induced by tracheal instillation of BLM

[0103] C57BL / 6 mice were used in the experiment and were evenly divided into four experimental groups according to their body weight, with 5 mice in each group. The specific groups are as follows:

[0104] Fibrosis group (BLM): This group of mice was exposed to BLM to induce pulmonary fibrosis and simulate the disease model. This group did not receive vaccine intervention and was used to evaluate the effect of BLM-induced fibrosis.

[0105] Fibrosis intervention group (BLM-Vaccine): This group of mice was also exposed to BLM to induce pulmonary fibrosis, but received recombinant vaccine intervention at the end of the first week of BLM exposure. This group was used to evaluate the intervention effect of the vaccine in the process of fibrosis.

[0106] All mice were placed in cages, with 5 mice in each cage, corresponding to the two groups of the experiment. The experimental treatment steps for each group were the same, the only difference was whether the vaccine intervention was performed.

[0107] The process of BLM-induced pulmonary fibrosis is as follows: BLM powder was dissolved in PBS, and the solution containing BLM was added to the lung liquid quantitative nebulizer. The mice in the fibrosis group, fibrosis intervention group, and fibrosis capsid protein optimization intervention group were exposed at a dose of 2U / kg. The fibrosis intervention group was intervened with recombinant vaccines starting from the first week of BLM exposure. Each mouse in the fibrosis intervention group used 50μL PBS and an equal volume of Freund's adjuvant to dissolve 20μg of recombinant vaccine, and then injected into the mouse lungs through tracheal instillation through a lung liquid quantitative nebulizer. The control group and fibrosis group were injected with an equal amount of KLH and an equal volume of Freund's adjuvant. The intervention was once a week for a total of three times.

[0108] After 4 weeks of exposure, the alveolar lavage fluid of mice was extracted and the content of AREG protein in the alveolar lavage fluid was detected using the AREG ELSIA detection kit. Figure 6The AREG protein content in the lavage fluid of the BLM-Vaccine group was significantly decreased compared with that of the BLM group, indicating that the vaccine intervention can effectively reduce the secretion of AREG protein in the lung tissue of mice with BLM-induced pulmonary fibrosis.

[0109] Small animal pulmonary function test was used to detect changes in lung function of mice. Figure 6 The results of the lung function tests on mice are shown. Figure 7 A) was used to evaluate the ventilation function of the lungs. The results showed that the pulmonary ventilation function of the mice in the BLM-Vaccine group was significantly restored compared with that in the BLM group. Figure 7 B) was used to evaluate the ability of the lungs to inhale deeply. The results showed that the lung volume of mice in the BLM-Vaccine group was significantly restored compared with that in the BLM group. Figure 7 C) was used to evaluate the elasticity and expansion capacity of the lungs under static conditions. The results showed that the lung compliance of mice in the BLM-Vaccine group was significantly restored compared with that in the BLM group. The overall results showed that vaccine intervention can effectively restore the decreased lung function of mice induced by BLM.

[0110] After the mice were sacrificed, lung tissue sections were taken for MASSON staining to evaluate collagen deposition ( Figure 8 A). Quantitative analysis of collagen fibers in MASSON-stained sections ( Figure 8 B) The results showed that BLM inhalation exposure led to a significant increase in collagen deposition in the lung tissue of mice. Vaccine intervention can effectively reduce collagen deposition.

[0111] The lung tissues of the above mice were taken and ground with TRIzol to extract RNA, and then qPCR was performed to detect the expression of Col1a1, Fn1 and Acta2 genes in the lung tissues of mice ( Fig. 9 ). The results showed that tracheal instillation of the vaccine could effectively inhibit the expression of fibrosis markers in the lung tissue of mice with BLM-induced pulmonary fibrosis.

[0112] The above results confirm that AREG protein plays a key role in BLM-induced pulmonary fibrosis. By injecting mice with BLM-induced pulmonary fibrosis with a vaccine targeting AREG protein, the lung function of mice can be effectively restored and the progression of pulmonary fibrosis can be alleviated.

[0113] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant amphiregulin vaccine, characterized in that: The vaccine contains a polypeptide of the amphiregulin antigen epitope, and its amino acid sequence is shown in SEQ ID No.

1.

2. The amphiregulin recombinant vaccine according to claim 1, characterized in that The vaccine comprises a polypeptide conjugate comprising the polypeptide according to claim 1.

3. The amphiregulin recombinant vaccine according to claim 2, characterized in that The polypeptide conjugate is the polypeptide according to claim 1 and a coupling portion; wherein the coupling portion is connected to the polypeptide portion according to claim 1, and the coupling portion is selected from at least one of keyhole limpet hemocyanin, ovalbumin and bovine serum albumin.

4. The amphiregulin recombinant vaccine according to claim 3, characterized in that The mass ratio of the polypeptide to the coupling part is 1-10:

1.

5. The amphiregulin recombinant vaccine according to claim 3, characterized in that The polypeptide conjugate is prepared by incubating the polypeptide according to claim 1 with a coupling part at room temperature for 8-12 hours; wherein the mass ratio of the polypeptide, the coupling part (KLH) and the protein cross-linking agent (Sulfo-SMCC) is 1-10:1-10:

1.

6. The amphiregulin recombinant vaccine according to any one of claims 1 to 5, characterized in that The vaccine is the polypeptide conjugate and its physiologically acceptable excipient, wherein the mass ratio of the polypeptide conjugate to the excipient is 1-10:

25.

7. The amphiregulin recombinant vaccine according to claim 6, characterized in that During the use of the vaccine, an adjuvant is added, and the volume ratio of the immune adjuvant to the vaccine is 1:1-5.

8. Use of the recombinant vaccine according to claim 1, characterized in that: Use of the vaccine in the treatment of pulmonary fibrosis.

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