CN106749674A - A kind of new asthma polypeptide vaccine and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and in particular relates to a novel polypeptide asthma vaccine and a preparation method thereof. The technical problem to be solved by the present invention is to provide a new effective option for the treatment of asthma in this field. The technical solution of the present invention to solve the technical problem is to connect the nitrogen end of the E3 polypeptide with a DiTOX polypeptide or a PADRE polypeptide to obtain a new fusion polypeptide, which can be used as the main active ingredient to prepare an asthma vaccine. The polypeptide vaccine of the invention can effectively induce specific humoral immunity, can break immune tolerance, and has good application prospects as a therapeutic polypeptide vaccine for asthma.

Description

A novel asthma polypeptide vaccine and its preparation method

technical field

The invention belongs to the field of biomedicine, and in particular relates to a novel asthma polypeptide vaccine and a preparation method thereof.

Background technique

According to the World Health Organization, there are currently about 300 million people worldwide suffering from asthma. Asthma is the most common non-communicable disease in children. Asthma is not just a public health problem in high-income countries; it occurs in all countries, regardless of their level of development. Most asthma-related deaths occur in low- and lower-middle-income countries.

Bronchial asthma (Bronchial asthma, referred to as asthma) is a major non-communicable disease, characterized by dyspnea and recurrent wheezing, it is composed of a variety of cells (such as eosinophils, mast cells, T lymphocytes, middle Chronic inflammatory diseases of the airway involving granulocytes, airway epithelial cells, etc.) and cellular components. This chronic inflammation is associated with airway hyperresponsiveness, usually with widespread and variable reversible airflow limitation, and causes recurrent symptoms such as wheezing, shortness of breath, chest tightness, or coughing, often at night and/or in the morning, Intensified, most patients can be relieved spontaneously or after treatment. If bronchial asthma is not diagnosed and treated in time, irreversible airway narrowing and airway remodeling may occur with the prolongation of the disease course. At present, the genes related to asthma have not been fully clarified, but studies have shown that there are genes related to airway hyperresponsiveness, IgE regulation and atopic response, and these genes play an important role in the pathogenesis of asthma. Environmental factors mainly include certain triggering factors, such as dust mites, pollen, fungi, animal dander, sulfur dioxide, ammonia and other specific and non-specific inhalants; infections, such as bacteria, viruses, protozoa, parasites, etc.; food , such as fish, shrimp, crab, eggs, milk, etc.; drugs, such as propranolol (propranolol), aspirin, etc.; climate change, exercise, pregnancy, etc. may be trigger factors for asthma.

IL-13 is a multifunctional cytokine that regulates inflammation and immune response produced by Th2 cells. It activates eosinophils, reduces their apoptosis, promotes the generation of B cells and the secretion of cytokine IgE, etc. It is involved in asthma, etc. The occurrence of various allergic diseases is also related to the increased expression of various adhesion molecules and chemokines, increased mucus secretion by bronchial goblet cells, and airway subepithelial fibrosis. Therefore, inhibiting the function of IL-13 has become an ideal target for asthma therapy. At present, monoclonal antibodies to IL-13 and soluble IL-13 receptors have entered clinical phase II and III, but there is no IL-13 peptide-related vaccine yet.

Contents of the invention

The purpose of the present invention is to provide a novel polypeptide vaccine for treating asthma and its preparation method, so as to provide a new effective option for the treatment of asthma in this field.

In order to solve the above technical problems, the technical solution adopted by the present invention is to provide a fusion polypeptide, which is formed by linking the polypeptide fragment E3 derived from IL-13 and the helper T cell antigen polypeptide fragment.

The fusion polypeptide of the present invention is formed by linking DiTOX polypeptide or PADRE polypeptide at the nitrogen end of the E3 polypeptide; the amino acid sequence of the E3 polypeptide is LTLKELIEELSNITQ; the amino acid sequence of the DiTOX polypeptide is AYNFVESIINLFQVVHNSY; the amino acid sequence of the PADRE polypeptide is aK-Cha-VAaWTLKAa.

Wherein, the amino acid sequence of the above-mentioned polypeptide is:

AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ;

Or aK-Cha-VAaWTLKAaLTLKELIEELSNITQ. Wherein a represents D-alanine; -Cha- represents L-cyclohexylalanine.

Wherein, the N-terminal amino group of the fusion polypeptide is either acetylated or acetylated.

Wherein, the C-terminal carboxyl group of the fusion polypeptide is amidated.

Further, the amino acid sequence of the above-mentioned fusion polypeptide is:

Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2;

Or Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.

Wherein Ac- represents that the amino group of the connected amino acid is acetylated; -NH ₂ represents that the carboxyl group of the connected amino acid is amidated; a represents D-alanine; -Cha- represents L-cyclohexylalanine;

The present invention also provides the use of the above-mentioned polypeptide in preparing an asthma vaccine.

The present invention also provides an asthma vaccine. The asthma vaccine is prepared with the above-mentioned polypeptide as the main active ingredient.

Wherein, the above-mentioned asthma vaccine further contains an adjuvant.

Wherein, the adjuvant described in the above-mentioned asthma vaccine is at least one of aluminum hydroxide adjuvant, cationic liposome adjuvant, GM-CSF (granulocyte-macrophage colony-stimulating factor), and γ-interferon . In addition, the present invention also provides a method for preparing the above-mentioned fusion polypeptide and a method for preparing the above-mentioned asthma vaccine.

The beneficial effect of the present invention is that: the fusion polypeptide of the present invention has small molecular weight and is easy to synthesize, and can be synthesized by Fmoc solid-phase polypeptide synthesis method conveniently, and adjuvants such as aluminum hydroxide, cationic liposome, GM-CSF, γ -Interferon etc. are made into various clinically applicable vaccine dosage forms; more importantly, the polypeptide vaccine of the present invention can effectively induce specific humoral immunity, can break immune tolerance, and has excellent curative effect on animal models of asthma , which has a good application prospect in the preparation of asthma therapeutic polypeptide vaccines.

Description of drawings

Fig. 1. Trend chart of serum antibody titers of mice after animal immunization with three IL-13 antigen short peptide-related fusion polypeptides.

Fig. 2. Trend chart of serum antibody titer of mice after immunization of animals treated with four kinds of fusion polypeptides for asthma.

Fig. 3. Cell count results of alveolar lavage fluid (BALF) of four kinds of fusion polypeptides for the treatment of asthma in animals.

Fig. 4 is a graph showing the relevant cytokine detection results of the animal immunization experiment of four kinds of fusion polypeptides treating asthma.

Fig. 5 is a diagram of detection results of OVA-specific antibody titers in animal immunization experiments of four polypeptides for treating asthma.

Figure 6: The results of pathological analysis of animal immunological experiments for four polypeptides for treating asthma.

detailed description

The fusion polypeptide of the present invention is formed by connecting the polypeptide fragment E3 derived from IL-13 and the helper T cell antigen polypeptide fragment. Specifically, the present invention connects DiTOX polypeptide or PADRE polypeptide at the nitrogen end of E3 polypeptide to obtain a new fusion polypeptide.

The amino acid sequence of the E3 polypeptide is LTLKELIEELSNITQ; the amino acid sequence of the DiTOX polypeptide is AYNFVESIINLFQVVHNSY; the amino acid sequence of the PADRE polypeptide is aK-Cha-VAaWTLKAa.

Wherein, the amino group of the above-mentioned fusion polypeptide is or acetylated. Carboxyl groups are acetylated to increase peptide stability.

Wherein, the C-terminal carboxyl group of the fusion polypeptide is amidated. Carboxyl groups are amidated to increase peptide stability.

Further, the amino acid sequence of the above-mentioned fusion polypeptide is:

Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2;

Or Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.

Wherein Ac- represents that the amino group of the connected amino acid is acetylated; -NH ₂ represents that the carboxyl group of the connected amino acid is amidated; a represents D-alanine; -Cha- represents L-cyclohexylalanine;

The present invention also provides the use of the above-mentioned polypeptide in preparing an asthma vaccine.

The present invention also provides an asthma vaccine. The asthma vaccine is prepared with the above-mentioned polypeptide as the main active ingredient.

Wherein, the above-mentioned asthma vaccine further contains an adjuvant.

Wherein, the adjuvant described in the above-mentioned asthma vaccine is at least one of aluminum hydroxide adjuvant, cationic liposome adjuvant, GM-CSF (granulocyte-macrophage colony-stimulating factor), and γ-interferon .

The routes of use of the above polypeptide vaccines include but not limited to intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection and nasal mucosal instillation.

That is, the dosage form of the above-mentioned polypeptide vaccine can be injection, drip or spray. Further, the injection is any one of intramuscular injection, subcutaneous injection, intradermal injection or intravenous injection. Of course, the injection can be a lyophilized preparation or an injection.

In addition, the present invention also provides a method for preparing the above-mentioned fusion polypeptide and a method for preparing the above-mentioned asthma vaccine.

The polypeptide in the present invention can be prepared by artificial synthesis and other methods. One of the methods that can be selected is the Fmoc solid-phase peptide synthesis method,

The method comprises the steps of:

1) Using chloromethyl polystyrene resin as an insoluble solid-phase carrier, first, an amino acid whose amino group is protected by a blocking group Fmoc-, is added with an appropriate amount of condensing agent, catalyst and base, and the amino acid is covalently linked to the solid-phase carrier Then remove the blocking group of the amino group with 10-50% piperidine solution, so that the first amino acid is connected to the solid-phase support.

2) Another amino acid whose amino group is protected by the blocking group Fmoc-, adding an appropriate amount of condensing agent, catalyst and base, covalently linking the amino acid to the solid phase carrier to which the amino acid has been connected, and then adding 10 to 50% piperidine The solution removes the blocking group of the amino group, so that a dipeptide is attached to the solid phase carrier.

3) Repeat the above-mentioned peptide bond generation reaction, so that the peptide chain of the polypeptide to be synthesized is grown from the C-terminal to the N-terminal in sequence until the required length of the peptide chain is reached, and the ester bond between the peptide chain and the solid-phase carrier is hydrolyzed, The synthesized polypeptide can be obtained by purifying by high performance liquid chromatography.

Wherein, the condensing agent in the above method is DCC (dicyclohexylcarbodiimide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), PyBOP (1H -Benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate), HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU ( At least one of 2-(1H-benzotrisazo-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate); the catalyst is DMAP (N, N- At least one of lutidine), HOBT (1-hydroxybenzotriazole); the base is triethylamine, DIEA (diisopropylethylamine), N-methylmorpholine at least one.

Wherein, the catalyst in the method is at least one of DMAP (N,N-lutidine) and HOBT (1-hydroxybenzotriazole).

Wherein the base in the above method is at least one of triethylamine, DIEA (diisopropylethylamine), N-methylmorpholine.

More specifically, the synthesis steps are as follows:

1. Fmoc-amino acid raw materials with side chain protecting groups can refer to the following raw materials

2. Solid phase synthesis

Amino acids are activated by the HBTU/HOBT method and connected to the amino resin according to the sequence:

Taking the scale of 10mmol as an example, weigh 20 grams of solid-phase synthetic resin (resin loading rate is 0.5mmol/g), pour it into the reactor of the polypeptide synthesizer, and weigh it from the C-terminal to the N-terminal according to the amino acid sequence of the target polypeptide. 40mmol of the corresponding protected amino acid and arrayed in the synthesizer. Under the condition of room temperature, the synthesis reactions are automatically completed respectively according to the computer program. After the synthesis is finished, a polypeptide resin with a side chain protecting group is obtained. Take out the polypeptide resin, put it into a vacuum desiccator and dry it for 2 hours, then weigh it.

3. Deprotection group and precipitation:

Put the target polypeptide resin with protective groups into a stoppered Erlenmeyer flask, and add the cleavage reagent as shown in the table below:

Stir the reaction at a constant temperature of 25° C. for 2 hours; filter and collect the filtrate, wash the resin with a small amount of trifluoroacetic acid, filter and collect the filtrate. With stirring, 3000 mL of glacial diethyl ether (-10°C) was added dropwise to obtain a white precipitate, which was filtered, washed with a small amount of glacial diethyl ether, and dried in a vacuum desiccator overnight.

4. HPLC purification

A pure trifluoroacetate solution of the target polypeptide (purity>95%) was prepared by HPLC reverse phase purification.

① Chromatographic column:

50mm*250mm Kromasil RP-18 10μm can be used Preparative column

②Mobile phase:

A: 0.1% trifluoroacetic acid aqueous solution

B: 0.1% trifluoroacetic acid in acetonitrile

③ Loading solution:

The crude polypeptide (purity about 40%-50%) was formulated with 0.1% trifluoroacetic acid aqueous solution to a concentration of 8.0 mg/mL (calculated as the crude product), and filtered through a 0.22 μm membrane filter.

④ Elution conditions:

Using linear gradient elution, the flow rate is 100mL/min, ultraviolet 254nm detection, the elution gradient is shown in Table 1:

Table 1

time (min) mobile phase B 0-0.5 6% 0.5-5 6%-20% 5-15 20%-40% 15-20 40%

⑤Sample collection:

During the period of 5-20 minutes, the elution main peak was collected according to the distribution of 50mL/bottle, and each sample liquid was analyzed and detected. Combine all sample solutions with a purity greater than 98%.

(4) Change the salt

The purified sample solution was subjected to reverse-phase salt exchange by HPLC to obtain a pure polypeptide acetate solution (purity>98%). ① Chromatographic column:

50mm*250mm Kromasil RP-18 10μm Preparative column

②Mobile phase:

A: 0.5% acetic acid in water

B: 0.5% acetic acid in acetonitrile

③ Loading solution:

Add an equal volume of ultrapure water to the pure peptide solution

④ Elution conditions:

Using linear gradient elution, the flow rate is 100mL/min, ultraviolet 254nm detection, the elution gradient is shown in Table 2:

Table 2

⑤Sample collection:

Collect all eluting main peak solutions

⑥Removal of acetonitrile:

Pour the peptide solution into a round-bottomed flask, and rotate it to evaporate at 25 °C and -0.1 MPa to remove all acetonitrile, and filter the remaining liquid through a 0.22 μm filter membrane and leave it for freeze-drying.

5. Freeze drying:

Pour the acetic acid aqueous solution of the target polypeptide into the sample tray of the vacuum freeze dryer, and freeze-dry according to the computer program. The obtained peptides were verified to be correct by mass spectrometry.

Obviously, those skilled in the art can also use other methods to synthesize the polypeptide involved in the present invention.

The content of the present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

Example 1 Screening and Determination of Asthma IL-13 Short Peptide in the Present Invention

By consulting the library and detecting the binding affinity of IL-13 antigen short peptides to MHC, as well as the interaction between IL-13 antigen short peptides and IL-13 receptors, the best of the new asthma vaccines can be established. IL-13 antigen short peptide.

Specific method: From the Allele Frequency Net Database (AFND, http://www.allelefrequencies.net/) database, the coverage of asthma-related screening ranges from 13.5% (DRB1*09:01) to 1% (DRB1*14:05) DR epitopes of human leukocytes, covering 95.9% of the Chinese population in total, obtained 20 epitopes. As a vaccine candidate antigen polypeptide, it needs to have high affinity with MHC, so 20 cases of Chinese HLA-DRB1 epitopes were predicted by six commonly used Immune Epitope Database (IEDB, http://www.iedb.org) method to filter. Among the 20 epitopes, the epitopes that can bind to MHC class I molecules were first screened out; secondly, in considering the affinity of HLA-DRB1 epitopes, the top 95 and the top 95 epitopes were established by Accelrys Discovery Studio software. 5% or less of the epitopes are able to dock on and interact with the IL-13 receptor.

Then, the high-affinity HLA-DR epitope capable of interacting with the IL-13 receptor was simulated by molecular dynamics to determine the stability of its molecular conformation in the IL-13 protein and polypeptide vaccine. However, it is difficult to experimentally simulate the interaction between a candidate IL-13 polypeptide vaccine and the IL-13 receptor at the atomic level. Therefore further work is required.

The IL-13/IL-13R complex structure was downloaded from the PDB database, and the candidate IL-13 antigen short peptide structure was simulated by Accelrys Discovery Studio software and the Amber 12package module. Before molecular dynamics simulations, each simulated system requires energy minimization to avoid conflicts between steric complexity and solvents. In the equilibrium simulation, the PME (Particle Mesh Ewald) method is used to deal with the long-range electrostatic interaction between spatial structures, and the electrostatic cut-off distance and van der Waals force are set as 10 angstrom units. In the network termination system, the simulated system is gradually heated from 0 to 300K at a rate of 100ps. Finally, the products of this long-term molecular dynamics simulation were placed in an NPT environment under periodic boundary conditions for detection for 500 ns. At the same time, use the SHAKE method to control the tolerance of all covalent bonds to a unit value of 10 ^-5 angstroms. The coupling constant of temperature and pressure value are set at 1.0 ps. For the sampling parameters, the coordinates of the total complex were saved at a time interval of 0.1 ps.

Results: Based on the above method, three kinds of short peptide fragments of IL-13 antigen capable of complexing with IL-13 receptor were simulated by molecular dynamics for 500 ns, which have the prospect of being used as a vaccine.

The fragments of the three antigen short peptides obtained were E1 (DTKIEVAHF), E2 (NSYTKQLFRHGPF), and E1 (LTLKELIEELSNITQ).

In order to improve the immunogenicity, we considered to couple the helper T cell antigen polypeptide Ditox (AYNFVESIINLFQVVHNSYN) to the nitrogen-terminus of the above-mentioned short antigen peptide, and the RMSD values of the complexes with IL-13R were 0.17, 0.19 and 0.23nm, respectively. Through artificial chemical synthesis, the Fmoc solid-phase peptide synthesis method was adopted, and the amino group at the nitrogen end was acetylated, and the carboxyl group at the carbon end was amidated, and the polypeptides Ditox-E1 (V1), Ditox-E2 (V2) and Ditox-E3 were obtained. (V3), the amino acid sequences of V1, V2, and V3 and the modifications made are shown in Table 3.

The peptide vaccines were prepared according to the following schemes. The total dosage of each mouse was 200 μl system, and those less than 200 μl were supplemented with physiological saline to make up to 200 μl. Since the same T cell helper epitope was coupled, each mouse had the same molecular weight. For immunization, the dosage of 5 mice in each group was prepared, and the dosage of each mouse was as follows:

1) NS group: 200 μl PBS

2) DiTOX-E1(V1) group: 200μg DiTOX-E1+1000μg Al(OH)3;

3) DiTOX-E2(V2) group: 225μg DiTOX-E2+1125μg Al(OH)3;

4) DiTOX-E3 (V3) group: 244μg DiTOX-E3+1220μg Al(OH)3;

② Immunization program

Female Balb/c mice aged 6-8 weeks and weighing about 18-20 g were selected, randomly divided into the above five groups, and administered subcutaneously at multiple points. The drug was administered once at 0, 2, 4, 6, 8, and 10 weeks, and blood was collected from the orbit at 1, 3, 5, 7, 9, and 11 weeks. The serum was separated and the humoral immune response induced by it was detected.

Detection index and method: ELISA method was used to detect the generation of antibody titer in the serum of mice in each group.

The ELISA detection method is as follows: the polypeptide LTLKELIEELSNITQ-NH2 is diluted to 1 μg/ml with coating buffer (0.05M carbonate buffer, pH9.6), and 100 μl is added to each well of a 96-well plate, overnight at 4°C. The next day, the solution in the well was discarded and washed 3 times with PBST. Block with 5% skimmed milk powder at 37°C for 1 hour, and wash 3 times with PBST. Serum samples were diluted with 5% skimmed milk powder (1:100-1:12,800), 100 μl of diluted serum samples were added to each well, and incubated at 37°C for 1 hour. Wash 5 times with PBST. Thereafter, 100 μl of HRP-Pr.A (1:5000 dilution) was added to each well, and incubated at 37° C. for 1 hour. Wash 5 times with PBST. Add 100 μl of temporarily prepared TMB substrate solution to each reaction well, develop color at room temperature for 20 minutes, add 100 μl of 1N H ₂ SO ₄ to stop the reaction, and read at 450 nm wavelength.

The results are shown in Figure 2. Compared with the normal saline immunization group and the other two experimental groups, DiTOX-E3 (V3), a new vaccine for treating asthma, can induce mice to produce a sustained increase in immune response after injection. Therefore, it is determined that E3 is the optimal short peptide of IL-13 antigen, which will serve as the basis for subsequent experiments.

Example 3 Animal immunization experiments and antibody titer evaluation of four polypeptides constructed on the basis of E3

After determining E3 as the optimal epitope with the function of treating asthma among the three candidate IL-13 antigen short peptides, the present invention considers coupling different helper T cell epitope polypeptides to enhance IL-13 antigen short peptide Immunogenicity, so as to obtain the best candidate polypeptide for the treatment of asthma, to be developed as a vaccine. Because when determining the IL-13 antigen short peptide epitope, the coupled helper T cell DiTOX epitope peptide (AYNFVESIINLFQVVHNSYN). However, it is not sure that this epitope has the best effect of increasing the immunogenicity of short peptides of IL-13 antigen, so we screened three other epitope peptides: PADRE; TT8; TT9; respectively coupled with E3 to obtain TT8- E3(V4), TT9-E3(V5) and PADRE((V6) are used together with DiTOX-E3(V3) to evaluate the therapeutic effect. The synthesis method is the same as that of DiTOX-E3, and it is also carried out at the amino group at the nitrogen terminal Acetylation and amidation of the carbon-terminal carboxyl group.

Table 3 Fusion polypeptide V1～V6 in the present invention

Experimental Design and Grouping:

The peptide vaccines were prepared according to the following schemes. The total dosage of each mouse was 200 μl system, and those less than 200 μl were supplemented with physiological saline to make up to 200 μl. Since different T cell helper epitopes were coupled, each mouse had the same molecular weight For immunization, the dosage of 5 mice in each group was prepared, and the dosage of each mouse was as follows:

1) NS group: 200 μl PBS;

2) DiTOX-E3(V3) group: 244μg DiTOX-E3+1220μg Al(OH)3;

3) TT8-E3(V4) group: 205μg DiTOX-E2+1025μg Al(OH)3;

4) TT9-E3(V5) group: 250μg DiTOX-E3+1250μg Al(OH)3;

6) PADRE-E3(V6) group: 178μg DiTOX-E3+890μg Al(OH)3;

Immunization scheme: female Balb/c mice aged 6 to 8 weeks and weighing about 18 to 20 g were selected, randomly divided into the above five groups, and administered subcutaneously at multiple points. The drug was administered once at 0, 2, 4, 6, 8, and 10 weeks, and blood was collected from the orbit at 1, 3, 5, 7, 9, and 11 weeks. Then the asthma model was constructed.

Asthma model construction: mice were sensitized intraperitoneally with a certain concentration of OVA (ovalbumin) at weeks 0, 1, and 2 for three consecutive weeks, and the intraperitoneal sensitization dose of each mouse was 20 μg OVA+2mg Al(OH)3 In the third week, the mice in each group were aerosolized at a concentration of 1% OVA, once a day, for three consecutive days. After 36-72 hours, the mice were sacrificed, and the alveolar lavage fluid was collected and separated for cell counting and related cytokine detection; eyeballs were removed to collect blood, serum was separated, and related cytokines and OVA-specific antibodies were detected.

Detection index and method: detect the antibody titer in each group of mouse serum by ELISA method

The ELISA detection method is as follows: the polypeptide LTLKELIEELSNITQ-NH2 is diluted to 1 μg/ml with coating buffer (0.05M carbonate buffer, pH9.6), and 100 μl is added to each well of a 96-well plate, overnight at 4°C. The next day, the solution in the well was discarded and washed 3 times with PBST. Block with 5% skimmed milk powder at 37°C for 1 hour, and wash 3 times with PBST. Serum samples were diluted with 5% skimmed milk powder (1:100-1:12,800), 100 μl of diluted serum samples were added to each well, and incubated at 37°C for 1 hour. Wash 5 times with PBST. Thereafter, 100 μl of HRP-Pr.A (1:5000 dilution) was added to each well and incubated at 37° C. for 1 hour. Wash 5 times with PBST. Add 100 μl of temporarily prepared TMB substrate solution to each reaction well, develop color at room temperature for 20 minutes, add 100 μl of 1N H ₂ SO ₄ to stop the reaction, and read at 450 nm wavelength.

The results are shown in Figure 2. It can be found that after immunization with the four vaccines, the IgG antibody titers in the mice were stimulated to a certain extent, and with the increase in the number of immunizations, the antibody titers showed a clear upward trend. After the fourth immunization, although the titer has risen to a certain extent, it has tended to be stable. The antibody titers of the V3 and V6 groups reached 10,000, and the data of the V4 and V5 groups also reached 1,000. Comparing the V3 group with the V5 group, there is a significant statistical difference (P﹤0.05); compared with the V6 group, the difference is also statistically significant.

Example 4 The results of the alveolar lavage fluid (BALF) cell count and related cytokine detection results of the animal immunization experiment of four polypeptides constructed on the basis of E3:

See embodiment 3 for grouping experimental treatment.

BALF count detection method is as follows: when the mice are killed, use pre-cooled saline for alveolar lavage, collect about 1mL of alveolar lavage fluid; Resuspended in saline and sent for GLP detection to count eosinophils. The supernatant was detected using the relevant kit detection method.

IFN-γ: The detection kit is R&D Systems Mouse IFN-gamma (Catalog Number: VAL607)

IL-4: The detection kit is R&D Systems Mouse IL-4 (Catalog Number: VAL603)

IL-13: The detection kit is eBioscience Mouse IL-13 (Catalog Number: 88-7137)

At the same time, the eyeballs were picked to collect blood, and the supernatant was collected by centrifugation and stored at -20°C for the detection of Total IgE.

IgE: Kit eBioscience Mouse IL-13 (Catalog Number: 88-50460)

The results showed that compared with the mice in the Normal group, the total cell count (Tcc), eosinophils (Eos) and neutrophils (Neu) in the alveolar lavage fluid after modeling were higher than those in the unmodeled (Fig. 3). Compared with the asthma model NS group, the cell counts Tcc, Eos, and Neu of the mice after vaccine immunization were all down-regulated to a certain extent, and the results of these three cell counts in the V3 group decreased significantly, and the difference was statistically significant ( P﹤0.05), the effect of V6 is also more significant.

At the same time, the test results of various asthma-related cytokines (Figure 4) showed that the contents of the four cytokines in the immune group all decreased significantly, and compared with the NS group, the down-regulation trends of the V3 group and the V6 group were more obvious . The test results of IL-13, IL-4, IFN-γ and Total IgE in group V3 were 1.86pg/ml, 15.34pg/ml, 9.44pg/ml and 5.650ng/ml respectively. Compared with other groups, each cytokine in V3 group has significant statistical difference (P﹤0.05), which has the best effect, and the effect of V6 is also more significant.

Example 5 Detection results of OVA-specific antibody titers in animal immunization experiments of four polypeptides constructed on the basis of E3

In this example, the production of OVA-specific antibody titers in the serum of mice in each group was detected by ELISA method. See embodiment 3 for grouping experimental treatment.

The ELISA detection method is as follows: dilute Ovalbumin to 1 μg/ml with coating buffer (0.05M carbonate buffer, pH9.6), add 100 μl to each well of a 96-well plate, and overnight at 4°C. The next day, the solution in the well was discarded and washed 3 times with PBST. Block with 5% skimmed milk powder at 37°C for 1 hour, and wash 3 times with PBST. Serum samples were diluted with 5% skimmed milk powder (1:100-1:12,800), 100 μl of diluted serum samples were added to each well, and incubated at 37°C for 1 hour. Wash 5 times with PBST. Thereafter, 100 μl of HRP-Pr.A/HRP-IgE (1:5000 dilution) was added to each well, and incubated at 37° C. for 1 hour. Wash 5 times with PBST. Add 100 μl of temporarily prepared TMB substrate solution to each reaction well, develop color at room temperature for 20 minutes, add 100 μl of 1N H ₂ SO ₄ to stop the reaction, and read at 450 nm wavelength.

The results are shown in Figure 5, which shows that compared with the mice in the model group, the OVA-specific IgE, IgG and typing of the mice immunized with the new vaccine were significantly down-regulated.

Example 6 The Immunological Experimental Pathological Analysis of the Animal Immunological Experiments of Four Polypeptides Constructed Based on E3

In the immunization cycle of each experimental group mice in Example 3, the body weight of the mice was measured at 0, 2, 4, 6, 8, and 10 weeks, and the heart, liver, spleen, lung, and kidney were collected when the mice were sacrificed. Stored in 4% paraformaldehyde. After embedding and slicing the tissues, the pathological sections were stained according to H&E and PAS staining methods to analyze whether the pathological phenomena such as the infiltration of inflammatory cells and goblet cells had changed after the mice were immunized with new asthma drugs.

The results are shown in Figure 6, which shows the semi-quantitative data analysis of HE and PAS stained pathological sections in the lungs of mice after vaccine immunization, and the inflammatory cell infiltration in the lungs (Figure 6 left, tracheal and alveolar peri-alveolar inflammation method blind tube scores ) and goblet cell proliferation (the right of Figure 6, which is the percentage of alveolar goblet cells) was reduced to a certain extent compared with the mouse lung tissue of the model group, the effect of V6 was more significant, and the effect of V3 group was the most significant .

Claims (10)

1. fused polypeptide, it is characterised in that：Formed in the nitrogen end connection DiTOX polypeptides or PADRE polypeptides of E3 polypeptides；

The amino acid sequence of described E3 polypeptides is LTLKELIEELSNITQ；The amino acid sequence of described DiTOX polypeptides is AYNFVESIINLFQVVHNSY；The amino acid sequence of described PADRE polypeptides is aK-Cha-VAaWTLKAa.

2. fused polypeptide according to claim 1, it is characterised in that：The amino acid sequence of described polypeptide is：

AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ；

Or be aK-Cha-VAaWTLKAaLTLKELIEELSNITQ.

3. fused polypeptide according to claim 1 and 2, it is characterised in that：The N-terminal amino of described polypeptide is acetylation.

4. fused polypeptide according to claim 1 and 2, it is characterised in that：The C-terminal carboxyl of described polypeptide is amidated.

5. the fused polypeptide according to any one of Claims 1 to 4, it is characterised in that：Described polypeptide is：

Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2；

Or be Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.

6. the fused polypeptide described in any one of Claims 1 to 5 prepare asthma vaccine in purposes.

7. asthma vaccine, it is characterised in that：Fused polypeptide described in any one of claim 1~6 is as main active system It is standby to form.

8. asthma vaccine according to claim 7, it is characterised in that：Also contain adjuvant.

9. asthma vaccine according to claim 12, it is characterised in that：Described adjuvant is aluminum hydroxide adjuvant, cation At least one in Liposome Adjuvant, GM-CSF, gamma interferon.

10. the fused polypeptide described in any one of Claims 1 to 4 or the asthma vaccine described in any one of claim 5~8 are prepared Method.