Application of protease-related active preparations in intervening in the course of respiratory inflammatory diseases

Abstract

The present invention discloses the application of protease-related active preparations in intervening in the course of respiratory inflammatory diseases, belongs to the field of biomedical technology, and provides a method for preventing and treating allergic asthma. The present invention provides the application of cysteine ​​protease inhibitors in the preparation of drugs for preventing or treating allergic asthma, and provides new ideas for the treatment and prevention of allergic asthma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of protease-related active preparations in intervening in the course of respiratory inflammatory diseases. Background Art

[0002] Asthma is a chronic inflammatory disease of the lungs that can cause patients to have airway hyperresponsiveness, increased mucus secretion, and airflow obstruction. When affected by a variety of environmental allergens, exercise, respiratory viral infections, or certain irritants, asthma may develop from initially benign intermittent attacks to severe asthma. Allergic asthma is a type of pulmonary asthma mediated by allergic reactions, an inflammatory response of the respiratory tract caused by an excessive type 2 immune response, characterized by eosinophil infiltration in the lung tissue, the formation of specific IgE, swelling of the bronchial mucosa, increased secretions, infiltration of inflammatory cells in the airways, and spasm of airway smooth muscles. At present, the main treatments for asthma are β-adrenergic receptor agonists, leukotriene modifiers, inhaled glucocorticoids, avoidance of allergens and irritants, or immunotherapy, but the above methods have a long cycle and short-lived efficacy, and have not yet been able to suppress the occurrence of asthma or completely cure it.

[0003] The hygiene hypothesis and the old friend hypothesis suggest that the increase in the incidence of allergic diseases may be related to the decrease in helminth infection rates. The development of urbanization and the improvement of sanitary environment have reduced the probability of people being exposed to and infected with parasites, thus causing intolerance of the host immune system, making it more likely to produce excessive immune responses to allergens, causing inflammatory responses in the body. An effective method for preventing and treating allergic asthma is urgently needed. Summary of the invention

[0004] The object of the present invention is to provide a method for preventing and treating allergic asthma.

[0005] The present invention provides an application of a cysteine ​​protease inhibitor in preparing a medicine for preventing or treating allergic asthma.

[0006] The present invention provides an application of a cysteine ​​protease inhibitor in preparing a drug for auxiliary treatment of allergic asthma.

[0007] It is further defined that the dosage form of the drug is any one of tablets, capsules, granules, powders, and liquid preparations.

[0008] It is further defined that the amino acid sequence of the cysteine ​​protease inhibitor is shown as SEQ ID NO.4.

[0009] It is further defined that allergic asthma is induced by OVA.

[0010] The invention provides a method for obtaining a cysteine ​​protease inhibitor. The gene sequence of the cysteine ​​protease inhibitor is obtained and inserted into a Pet-28a vector, which is transformed into a competent state. After being screened by kanamycin, a monoclonal strain is selected, and the plasmid is extracted and transformed into BL21 (DE3). The recombinant protein is obtained through expanded culture and IPTG induction.

[0011] Beneficial effects: Cysteine ​​protease inhibitor molecules derived from Trichinella spiralis can significantly inhibit allergic inflammatory responses in lung tissue, such as reduced inflammatory cell infiltration in lung tissue, thinning of bronchiolar walls, and reduced mucus secretion in the bronchioles. Cysteine ​​protease inhibitors are most effective in preventing allergic asthma. Trichinella spiralis-derived cysteine ​​protease inhibitor molecules can induce higher levels of anti-inflammatory factors and lower levels of type 2 immune-related cytokines when intervened before the sensitization stage, with better results. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the electrophoresis result of cDNA amplification for constructing cysteine ​​protease inhibitor;

[0013] Figure 2 The result of plasmid restriction enzyme electrophoresis for constructing cysteine ​​protease inhibitor;

[0014] Figure 3 The results of protein polyacrylamide electrophoresis for constructing cysteine ​​protease inhibitors;

[0015] Figure 4 This is a grouped result diagram for the evaluation of inflammation in the allergic asthma model;

[0016] Figure 5 Pathological results of the evaluation of inflammation in the allergic asthma model

[0017] Figure 6 This is a graph showing the pathological change scores for the evaluation of inflammation in the allergic asthma model;

[0018] Figure 7 This is the result graph of ovalbumin-specific IgG concentration in serum;

[0019] Figure 8 This is the result graph of ovalbumin-specific IgG1 concentration in serum;

[0020] Fig. 9 This is the result graph of ovalbumin-specific IgE concentration in serum;

[0021] Fig.10 The result graph is the IL-4 concentration of cytokines in serum;

[0022] Fig.11 The result graph is the IL-5 concentration of the cytokine in the serum;

[0023] Fig.12 The result graph is the IL-13 concentration of cytokines in serum;

[0024] Fig.13 The result graph is the concentration of TGF-β1 cytokine in serum;

[0025] Fig.14 This is the result graph of IL-10 concentration of cytokine in serum. DETAILED DESCRIPTION

[0026] In order to more intuitively and clearly illustrate the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below according to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0028] Example 1. Construction of cysteine ​​protease inhibitors

[0029] A method for preparing a cysteine ​​protease inhibitor provided by one embodiment of the present invention comprises the following steps:

[0030] Step 1) Obtain the cysteine ​​protease inhibitor gene sequence: predict its protein domain, determine the signal peptide region, and then design the front and back primers of the sequences other than the signal peptide, insert the EcoR I and Xho I endonuclease cleavage sites (F: TAAC) into the front and back primer sequences respectively. GAATTC CAGATACTTGGTGA (SEQ ID NO.1); R: GACG CTCGAG TTAACATTCAACA, (SEQ ID NO.2)). Then, the cDNA infected with the third Trichinella spiralis was specifically amplified to obtain the target sequence, such as Figure 1 As shown, the target sequence was successfully amplified with a size of 1167 bp.

[0031] Step 2) Construction of expression vector: Use EcoR I and Xho I two endonucleases to digest the PCR product of step 1) and the Pet-28a vector at 37°C for 2 hours. The digestion products are recovered by nucleic acid electrophoresis and gel cutting to obtain sequences with sticky ends. Then use T4 ligase to connect the two digested sequences at 16 degrees overnight. The ligation product is transformed into Escherichia coli DH5α, and the positive monoclonal strains are screened through LB solid culture plates with kanamycin resistance, and the plasmids of individual strains are sequenced. The plasmid of the target strain is transformed into BL21 (DE3), and the positive monoclonal strains are screened again through LB solid culture plates with kanamycin resistance to obtain the expression bacteria containing the target gene expression vector. The double enzyme digestion identification results of the plasmid are as follows: Figure 2 As shown, the constructed plasmid can be successfully cut by two endonucleases to produce two bands, including the vector sequence and the target gene sequence.

[0032] Step 3) Expression and purification of recombinant protein: The expression bacteria in step 2) were expanded and cultured in an incubator at 37°C. When the absorbance of the bacterial solution at OD600nm was 0.4-0.6, isopropyl-β-D-thiogalactoside with a final concentration of 0.1nmol was added to the bacterial solution to induce the bacteria to express protein. After 6-8 hours of induction culture, the bacteria were collected by centrifugation and resuspended in PBS buffer. The collected bacteria were crushed by ultrasonic crushing, the supernatant was removed by centrifugation, and the precipitate was dissolved in a Binding solution containing 8mol urea. The protein sample was separated and purified using an AKTA purification instrument. First, pumps A and B were placed in 20% alcohol, the machine was run to fill the pipeline with alcohol, a nickel column was installed, and then after the absorption peak of ultraviolet light was stable for 4-6 column volumes, pump A was placed in a Binding solution of 8mol urea, and pump B was placed in a Binding solution without urea. Then run pump A, use 8 mol urea Binding solution to balance the nickel column and then load the protein. Then slowly replace the 8 mol urea Binding solution with a urea-free Binding solution for 2 hours to complete the protein renaturation process. Use an imidazole solution without urea to competitively elute the protein on the nickel column. After dialysis, collect the protein and replace the protein solution with PBS. Use the BCA detection kit to detect the concentration of the renatured protein, then divide it into aliquots and store it at -80°C. The protein is subjected to polyacrylamide electrophoresis and Coomassie Brilliant Blue staining. The results are as follows: Figure 3 The recombinant cysteine ​​protease inhibitor expressed by Escherichia coli mainly exists in the form of inclusion bodies, and the protein size is 45KDa. After nickel column renaturation and purification, a soluble recombinant cysteine ​​protease inhibitor with high purity was obtained.

[0033] WN10 gene sequence: (SEQ ID NO.3)

[0034]

[0035] WN10 amino acid sequence: (SEQ ID NO.4)

[0036] SIQKMSFMHCIFVVLFFAVGEAQILGETTHYGRNDPVMLRNAHEALFSSDLKQESGVFHKLLELEESSTMGILTTMKVVMQDTDCPVSFALLSYYDVLVNCQGEGRRKHCTMEYTHRNPSKATVSKCFEEVEEPLIIPQRVKMIGGRAVYIDSNADVEEQMQMLGETTHYGRNDPVMLPKAREALFSSDSKEQSGVLHKLVELEE SSTMGILTTMKVVIQDTECRVSSAYSSYYDVLHYCHGKGPRKHCTLEYRHRTPSTATVSECFEEVEEPLIVPQRVQRVNGRTIYLDSSDDVEEQVVSQRSQML GGTTKYTDSNVHIKEEVKQAIFESDKKKSSGTYLLLDKIVEGFNMGISSRFQVLVKETECGIKEKAFNSYEDVYKNCSGSGDSKVCSVEYKYFDPTKSTVEC.

[0037] Example 2. Establishment of a mouse allergic asthma disease model

[0038] To evaluate the effects of recombinant cysteine ​​protease inhibitors on the prevention and treatment of allergic asthma.

[0039] The concentration of ovalbumin was quantified using a BCA assay kit, and 50 μL of ovalbumin at a concentration of 2 μg / μL was mixed with an equal volume of alum adjuvant for 30 minutes to obtain a mixture, which was then injected intraperitoneally into mice. The immunization time points were day 1, day 7, and day 14, completing the sensitization phase of the allergic asthma model. On days 28-30, a 1% ovalbumin solution was atomized and inhaled by mice for 30 minutes every day using a nebulizer, completing the challenge phase of the allergic asthma model, and the lung tissue of the mice produced severe allergic inflammatory responses.

[0040] According to the development characteristics of allergic diseases, the intervention of recombinant proteins in the allergic asthma model was divided into three groups according to the time points: before the sensitization phase (50 μg of recombinant protein was injected intraperitoneally on days −2, −1, and 0), before the challenge phase (50 μg of recombinant protein was injected intraperitoneally on days 25, 26, and 27), and after the challenge phase (50 μg of recombinant protein was injected intraperitoneally on days 31, 32, and 33). The mice were sacrificed on day 37 for the evaluation of inflammation in the allergic asthma model, e.g. Figure 4 In addition, in order to exclude the influence of other factors on the allergic asthma model, three control groups need to be set up: 1. saline + OVA control group: intraperitoneal injection of saline (1, 7 and 14 days) and OVA nebulization inhalation (28-30 days); 2. adjuvant + OVA control group: intraperitoneal injection of alum adjuvant (1, 7 and 14 days) and OVA nebulization inhalation (28-30 days); 3. OVA + OVA control group: intraperitoneal injection of OVA (1, 7 and 14 days) and OVA nebulization inhalation (28-30 days).

[0041] Pathological changes in lung tissue: The lung tissues of mice were collected and fixed with 4% paraformaldehyde for 24 hours, and finally embedded in paraffin. They were cut into 4μm slices using a microtome and attached to glass slides. After the slices were dewaxed, they were stained with hematoxylin-eosin. Hematoxylin can stain the chromatin in the cell nucleus and the nucleic acid in the cytoplasm into blue-purple, while eosin can stain the components in the cytoplasm and extracellular matrix into red. The stained slices were observed under an optical microscope and their pathological changes were scored, including: normal (0), mild lesions (1), moderate lesions (2), severe lesions (3) and extremely severe lesions (4). The results are as follows: Figure 5 , 6 As shown, the cysteine ​​protease inhibitor molecules derived from Trichinella spiralis can significantly inhibit the allergic inflammatory response of lung tissue, such as reduced inflammatory cell infiltration in lung tissue, thinning of bronchiolar walls and reduced mucus secretion in the bronchioles.

[0042] Changes in serum specific antibody levels: Mouse serum was collected for detection of specific antibodies IgG, IgG1, and IgE. First, 100 μL of 5 μg / mL ovalbumin was added to a high-affinity 96-well plate and incubated overnight at 4°C. Wash three times with 200 μL of PBST washing solution, add 5% skim milk powder solution and incubate at 37°C for 2 hours for blocking. Wash three times with 200 μL of PBST washing solution, add serum diluted 500 times and incubate at 37°C for 1 hour. Wash three times with 200 μL of PBST washing solution, add HRP-coupled anti-mouse IgG, IgG1, and IgE antibody dilutions and incubate at 37°C for 1 hour. Wash three times with 200 μL of PBST washing solution, add 100 μL of TMB colorimetric solution, incubate for 10-15 minutes, add sulfuric acid stop solution, and use an enzyme-labeled detector to detect the absorbance at 450 nm. The concentration of ovalbumin-specific IgE in serum was calculated based on the IgE standard curve. Figure 7 , 8As shown in Figure 9, the three Trichinella spiralis-derived cysteine ​​protease inhibitor molecular intervention groups can reduce the levels of OVA-specific IgG, IgG1, and IgE antibodies in the serum of allergic asthmatic mice. Among them, the intervention group of cysteine ​​protease inhibitors before the sensitization stage induced the lowest IgG, IgG1, and IgE levels; followed by the intervention effect of cysteine ​​protease inhibitors before the challenge stage; the poorest effect was the intervention of cysteine ​​protease inhibitors during the onset stage. This shows that cysteine ​​protease inhibitors are the most effective in preventing the occurrence of allergic asthma.

[0043] Changes in serum cytokines: Use a commercial cytokine detection kit to quantify the concentration of cytokines IL-4, IL-5, IL-13, IL-10, and TGF-β1 in serum. Dilute the serum according to the instructions of the kit. After the serum is incubated, add HRP-labeled antibodies for incubation. Finally, add colorimetric solution and stop solution and detect changes in absorbance. The concentration of cytokines in serum is calculated based on the curve of the standard. The results are as follows: Figure 10-14 As shown, the three Trichinella spiralis-derived cysteine ​​protease inhibitor molecular intervention groups can increase the levels of anti-inflammatory factors (IL-10 and TGF-β1) in serum, among which the levels of IL-10 and TGF-β1 in the protein intervention groups before the sensitization stage and before the challenge stage were significantly higher, indicating that these two groups of proteins can induce stronger immune responses. Compared with the OVA positive control group, the cytokines of IL-4, IL-5 and IL-13 related to type 2 immunity were significantly reduced after protein treatment, among which the protein intervention group before the sensitization stage had the best reduction effect, followed by the protein intervention group before the challenge stage. The inhibitory effect of the protein intervention group in the treatment stage was worse than the first two groups. The results show that protein intervention before the sensitization stage can induce higher levels of anti-inflammatory factors and lower levels of type 2 immune-related cytokines, with a better effect.

[0044] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Use of WN10 in the preparation of a drug for preventing or treating allergic asthma, characterized in that: The amino acid sequence of WN10 is shown in SEQ ID NO.

4.

2. The use of WN10 in the preparation of a drug for the auxiliary treatment of allergic asthma, characterized in that: The amino acid sequence of WN10 is shown in SEQ ID NO.

4.

3. The use according to claim 1, characterized in that: The dosage form of the drug is any one of tablets, capsules, granules, powders and liquid preparations.

4. The use according to any one of claims 1 to 3, characterized in that: Allergic asthma is induced by OVA.

5. The use according to claim 1, characterized in that: The gene sequence of WN10 was obtained and inserted into the Pet-28a vector, transformed into a competent state, selected by kanamycin, and the monoclonal strain was picked. The plasmid was extracted and transformed into BL21 (DE3), and the recombinant protein was obtained by expanded culture and IPTG induction.

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