Application of recombinant protein of Echinococcus granulosus muscle antigen

Abstract

The present invention discloses the use of a recombinant protein of a muscle-attached antigen of Echinococcus granulosus in the preparation of a drug for treating asthma. The present invention uses a recombinant protein of a muscle-attached antigen of a strain of Echinococcus granulosus from mainland China (NCBI gene sequence number Z29075) to intervene in an asthma mouse model caused by ovalbumin (OVA). The results show that the muscle-attached antigen recombinant protein of the present invention is used to prepare a drug for treating asthma. The drug can effectively increase the number of Th1 and Treg cells, reduce the number of Th2 and Th17 cells, reduce inflammatory factors in asthma, and increase the IL-10 type cytokine that is reduced in asthma. It can have a good preventive, intervention and / or relief effect on asthma. In addition, the drug also has the characteristics of few side effects and low drug resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to the use of a recombinant protein of muscle antigen of Echinococcus granulosus (mainland China strain) in the preparation of a drug for treating asthma. Background Art

[0002] Asthma is one of the most common chronic inflammatory diseases in the world and poses a serious threat to public health. The pathogenesis of asthma is very complex. It is generally believed that asthma is related to genetic factors, acute respiratory infections, increased airway reactivity, mental factors and unstable environment. Allergic asthma is the most common type of asthma and is usually defined by the presence of environmental allergens. In recent decades, allergic asthma has become increasingly common in children and adults around the world. Airway secretions and CD4 + Increased levels of Th2 lymphocytes and their associated cytokines (IL-4) are hallmarks of allergic asthma. Increased eosinophilia may be associated with airway obstruction and predict asthma exacerbations. Current asthma treatments primarily involve bronchodilator, anti-inflammatory, and anti-allergic medications, such as glucocorticoids, long-acting β2-receptor agonists, anti-IgE monoclonal antibodies, and some emergency medications. These treatments can achieve temporary control of allergic asthma through effective management, but issues such as side effects and drug resistance remain largely unresolved. Summary of the Invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a use of a recombinant protein of Echinococcus granulosus muscle-tropic antigen in the preparation of a drug for treating asthma.

[0004] The present invention is achieved by using a recombinant protein of Echinococcus granulosus muscle antigen in the preparation of a drug for treating asthma. The amino acid sequence of the recombinant protein is disclosed in NCBI and its gene sequence number is Z29075.

[0005] Preferably, the recombinant protein treats asthma by reducing the number of Th2 and Th17 cells and the expression of inflammatory factors in asthmatic mice, and by increasing the number of Th1 and Treg cells and elevating the IL-10 cytokine level that is reduced in asthma.

[0006] Preferably, the asthma is airway inflammation caused by ovalbumin.

[0007] Preferably, said treatment includes prevention, intervention and / or alleviation.

[0008] Preferably, the drug further contains one or at least two pharmaceutically acceptable carriers; the carrier is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant and a lubricant.

[0009] Preferably, the muscle antigen recombinant protein is derived from the Chinese mainland strain of Echinococcus granulosus.

[0010] The present invention overcomes the shortcomings of the prior art and provides a recombinant protein containing a muscle-tropic antigen from Echinococcus granulosus for use in the preparation of a drug for the treatment of asthma. Based on the "hygiene hypothesis," the present invention posits that the relatively clean environment created by modern health care and medical measures reduces exposure to pathogens such as bacteria, viruses, and fungi, thereby causing an imbalance in the immune system and ultimately leading to an increase in allergic diseases. It has been found that infection with Echinococcus granulosus can significantly inhibit OVA-induced airway inflammation in mice. [1～2] However, infection through adult worms has certain biosafety and side effects. Therefore, the present invention uses the recombinant antigen muscle antigen recombinant protein of the fine-grained echinococcus cyst derived from the Chinese mainland strain (NCBI gene sequence number is Z29075) to intervene in the asthma mouse model caused by OVA, and uses hematoxylin-eosin (HE) staining of lung tissue to reflect lung tissue inflammation, Masson staining to detect the degree of lung tissue fibrosis, and peroxidase (MPO) histochemical staining to observe the expression level of neutrophils in lung tissue; flow cytometry is used to detect the level of eosinophils in lung tissue to reflect the degree of inflammatory cell infiltration; lung tissue flow cytometry is used to detect the levels of mouse lung helper T cells (Th1, Th2, Th17 cells) and regulatory T cells (Treg cells), and multiple liquid phase protein quantification technology is used to detect the changes in the levels of cytokines in mouse plasma. The results showed that in asthmatic mice, the infiltration of inflammatory cells and the degree of fibrosis in the lung tissue were significantly reduced, the levels of Th1 and Treg cells were upregulated, and the levels of Th2 and Th17 cells were downregulated, thereby effectively alleviating airway inflammation in mice. Therefore, it can be used as one of the effective candidate drugs for the treatment of asthma.

[0011] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the recombinant protein of the muscle-tropic antigen of Echinococcus granulosus of the present invention is used to prepare a drug for treating asthma, which can effectively increase the number of Th1 and Treg cells, reduce the number of Th2 and Th17 cells, reduce inflammatory factors in asthma and increase the IL-10 type cytokine that is reduced in asthma, and can have a good preventive, intervention and / or alleviating effect on asthma. In addition, the drug also has the characteristics of small side effects and low drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 11 is a flow chart of experimental operation of an embodiment of the present invention;

[0013] Figure 2 This is the method for establishing an OVA-induced asthma mouse model in the embodiment of the invention;

[0014] Figure 3 These are lung pathological section staining images; Figure A is a lung tissue HE section staining image, Figure B is a lung tissue MASSON section staining image, Figure C is a lung tissue periodic acid-Schiff PAS section staining image, and Figure D is a lung tissue MPO section staining image;

[0015] Figure 4 is the number of eosinophils in lung tissue;

[0016] Figure 5 is the number of Th2 cells in lung tissue;

[0017] Figure 6 is the number of Th1 cells in lung tissue;

[0018] Figure 7 is the number of Treg cells in lung tissue;

[0019] Figure 8 is the number of Th17 cells in lung tissue;

[0020] Figure 9 is the amount of Th1 / Th2 / Th17 cytokines in serum;

[0021] Figures 3 to 9 In the data, Con was the blank control group, Con+rEg.myo was the blank control group + muscle antigen recombinant protein control group, OVA was the asthma model group, and OVA+rEg.myo was the asthma model group + muscle antigen recombinant protein treatment group. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to 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.

[0023] The overall operation idea of ​​the embodiment of the present invention is as follows: Figure 1 As shown, specifically including the following:

[0024] 1. OVA-induced modeling

[0025] Female BALB / C mice aged 6 to 8 weeks were divided into four groups, with 6 mice in each group. Figure 2 As shown, specifically:

[0026] (1) On days 0, 7, and 14, mice were intraperitoneally injected with 200 μL of sensitization solution, which consisted of 20 μg of OVA and 2 mg of aluminum hydroxide emulsified in PBS.

[0027] (2) On days 21 to 27, the rats were challenged by intranasal dripping of PBS solution containing 100 μg OVA in a total volume of 50 μL.

[0028] 2. Use of the recombinant protein of the Chinese mainland strain of Echinococcus granulosus (NCBI gene sequence number Z29075)

[0029] (1) The asthma mice treated with the recombinant protein of myocardial antigen (OVA+rEg.myo) were subcutaneously injected with 20 μg of the recombinant protein of myocardial antigen (PBS, 1 μg / μL) on days -1, 6, and 13 based on the OVA-induced asthma model.

[0030] (2) Blank control group mice (Con): PBS was used instead of the sensitization solution, and the dosage and administration method were the same as those of the asthma mice treatment group;

[0031] (3) Blank control group + muscle antigen recombinant protein control group (Con+rEg.myo): Mice in the blank control group were subcutaneously injected with 20 μg of muscle antigen recombinant protein on days -1, 6, and 13.

[0032] On the 28th day, mice were killed, and mononuclear cells from lung tissues of each group were extracted for flow cytometry analysis and lung pathological sections were performed. Plasma was retained and the number of Th1, Th2, and Th17 cells was detected by flow cytometry. The relevant cytokines were detected by multiple liquid phase protein quantification technology. For detailed procedures, please refer to the literature. [3] .

[0033] 3. Experimental Results

[0034] 1. Pathological section of the lungs, the results are as follows Figure 3 As shown, Figure 3 A shows the infiltration of inflammatory cells around the airways and alveoli after HE (hematoxylin-eosin) staining. Figure 3 B is the collagen deposition after MASSON staining. Figure 3 C is the degree of bronchial goblet cell metaplasia after PAS (periodic acid-Schiff) staining. Figure 3 D shows the functional markers and activity status of neutrophils after MPO (myeloperoxidase) staining.

[0035] pass Figure 3 The four stainings shown showed that the inflammatory cell infiltration in the lungs of mice in the asthma model group was aggravated, accompanied by collagen deposition and neutrophil activation, but in the asthma model + muscle antigen recombinant protein treatment group, the above pathological changes were improved.

[0036] 2. The number of eosinophils in lung tissue

[0037] like Figure 4 As shown, the increase of eosinophils in the asthma model group can be alleviated by the treatment of muscle antigen recombinant protein.

[0038] 3. The number of Th2 cells in lung tissue

[0039] like Figure 5 As shown, the occurrence of asthma is mainly caused by the increase of Th2 cells, but this result shows that the treatment of muscle antigen recombinant protein can reduce the number of Th2 cells.

[0040] 4. The number of Th1 cells in lung tissue

[0041] like Figure 6 As shown, the number of Th1 cells decreases in asthma, but this result shows that the treatment with muscle antigen recombinant protein can increase the number of Th1 cells.

[0042] 5. The number of Treg cells in lung tissue

[0043] like Figure 7 As shown in the results, the number of Treg cells decreased in asthma patients, but the results showed that the treatment with muscle antigen recombinant protein could increase the number of Treg cells.

[0044] 6. The number of Th17 cells in lung tissue

[0045] like Figure 8 As shown, Th17 cells increased after the onset of asthma, but this result shows that treatment with muscle antigen recombinant protein can reduce the number of Th17 cells.

[0046] 7. The amount of Th1 / Th2 / Th17 cytokines in serum

[0047] like Figure 9 As shown, treatment with the pro-muscle antigen recombinant protein can reduce the elevated Th2 and Th17 cytokines (IL-4, IL-6, Th17) in asthma, and increase the levels of TNF, IFN-γ, and IL-10 cytokines that are decreased in asthma.

[0048] The above results indicate that the recombinant protein of muscle antigen can alleviate asthma induced by OVA.

[0049] References:

[0050] [1]Kim HJ,Kang SA,Yong TS,Shin MH,Lee KJ,Park GM,Suvonkulov U,YuHS.Therapeutic effects of Echinococcus granulosus cystic fluid on allergic airway inflammation.EXP PARASITOL 2019,198:63-70.,13.

[0051] [2]Wang H, Li J, Pu H, Hasan B, Ma J, Jones MK, Zheng K, Zhang X, Ma H, McManus DP, Lin R, Wen H, Zhang W. Echinococcus granulosus infection reduces airway inflammation of mice likely through enhancing IL-10and down-regulation of IL-5and IL-17A. Parasit Vectors 2014,7:522.

[0052] [3] Zhao Jiaqing, Shi Chunli, Wang Liying, Yin Mei, Zheng Caixia, Wu Yu, Zhao Wei, Gao Xiaoping. Effects and mechanisms of Lycium barbarum polysaccharides on inflammatory damage in asthmatic mice [J]. Chinese Journal of Food Hygiene, 2021, 33(01): 23-28. DOI: 10.13590 / j.cjfh.2021.01.005.).

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a recombinant protein of Echinococcus granulosus muscle-tropic antigen in the preparation of a drug for treating allergic asthma. The amino acid sequence of the recombinant protein is disclosed in NCBI and its gene sequence number is Z29075.

2. The use according to claim 1, characterized in that: Said treatment includes prevention, intervention and / or alleviation.

3. The use according to claim 1, characterized in that The medicine further contains one or at least two pharmaceutically acceptable carriers; the carrier is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant and a lubricant.

4. The use according to claim 1, wherein The muscle antigen recombinant protein is derived from the Chinese mainland strain of Echinococcus granulosus.

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