Spray for treating radioactive dry mouth of nasopharyngeal carcinoma patient after radiotherapy as well as preparation method and application of spray

Abstract

The invention relates to spray for treating radioactive dry mouth of nasopharynx cancer patients after radiotherapy as well as a preparation method and application of the spray, and belongs to the technical field of biological medicines. The fusion protein comprises a lamin recombinant protein, an FGF8 recombinant protein and an Eda recombinant protein which are mutually fused and connected, the fusion protein is high in purity and relatively small in molecular weight after being purified in multiple steps, the fusion protein has the function of promoting salivary gland somatic cell regeneration in vitro, and the effect of the fusion protein is better than that of the fusion protein obtained from the lamin recombinant protein, the FGF8 full-length protein and the Eda full-length protein. The spray for treating radioactive xerostomia after radiotherapy of nasopharynx cancer patients is prepared from the fusion protein LFE, has the functions of promoting salivary gland cell regeneration and salivary secretion in vivo, has a treatment effect on radioactive xerostomia, and can be widely applied clinically.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a spray for treating radiation-induced dry mouth in nasopharyngeal carcinoma patients after radiotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] Nasopharyngeal carcinoma is one of the common head and neck malignant tumors. Radiotherapy or comprehensive treatment mainly based on radiotherapy is currently the main treatment method for nasopharyngeal carcinoma. Due to the particularity of the nasopharyngeal location, radiotherapy for nasopharyngeal carcinoma will inevitably irradiate normal tissues and adjacent organs in the head and neck. Common acute side effects of radiotherapy for nasopharyngeal carcinoma include radiation dermatitis, radiation-induced oral mucositis, otitis media, pain, taste change, weight loss, and radiation-induced dry mouth, among which radiation-induced dry mouth is one of the most common side effects. More than 80% of nasopharyngeal carcinoma patients receiving intensity-modulated radiotherapy have varying degrees of radiation-induced dry mouth. After the salivary glands are irradiated, the glandular function is damaged, the salivary secretion volume decreases, the lips are dry and peeling; there is bad breath in the mouth; when thirsty at night or when waking up, severe patients need to drink water to relieve when eating or speaking; there is a burning sensation in the throat, tongue surface, lips, buccal mucosa and other parts; the tongue coating is relatively white; the taste changes, and only bitter and astringent tastes can be tasted; the appetite decreases, not wanting to eat, it is difficult to chew loose, sticky, and dry foods, and food boluses cannot be formed, and there is a feeling of choking when swallowing. Severe cases can aggravate oral mucositis and increase the possibility of Candida infection. Radiation-induced dry mouth reduces the oral comfort of patients and easily generates adverse psychological emotions. Although this series of side effects do not endanger life, they seriously affect the quality of life of nasopharyngeal carcinoma patients and lead to a decrease in treatment compliance. Therefore, radiation-induced dry mouth is a major difficulty in clinical nursing and prognosis rehabilitation, and is a problem that clinical nursing staff urgently need to solve.

[0003] So far, there are many clinical methods for treating xerostomia and certain achievements have been made. Most of them are palliative treatments, lacking specificity and high efficiency. A number of studies have shown that pilocarpine and cevimeline can increase salivary flow, but the symptoms of dry mouth still persist after drug withdrawal, and adverse reactions such as night sweats, increased urine, and irritable bowel will occur, making them not suitable for long-term use. DAVID N. et al. (David LA, Sàndor GK, Evans AW, Brown DH. Hyperbaric oxygen therapy and mandibular osteoradionecrosis: a retrospective study and analysis of treatment outcomes. J Can Dent Assoc. 2001 Jul-Aug; 67(7):384. PMID: 11468095.) showed that hyperbaric oxygen therapy has the effects of anti-inflammatory, eliminating edema, and stimulating phagocytes, and can significantly improve the condition of radiation-induced xerostomia, but it requires more than 20 treatment courses and the cost is relatively expensive. Philip Riley (Riley P, Glenny AM, Hua F, Worthington HV. Pharmacological interventions for preventing dry mouth and salivary gland dysfunction following radiotherapy. Cochrane Database Syst Rev. 2017 Jul 31; 7(7):CD012744. doi: 10.1002 / 14651858.CD012744. PMID: 28759701; PMCID: PMC6483146.) believes that the use of amifostine before or during radiotherapy can relieve dry mouth in a short time, but its effect is limited to 3 months after radiotherapy, and it is prone to side effects such as hypotension, nausea, and vomiting. The long-term efficacy is unknown. Scholars at home and abroad have tried to displace the patient's salivary glands before radiotherapy, moving one submandibular gland to the submental space to protect the salivary gland from radiation, but the surgical risk is relatively large, postoperative complications need to be considered, and the clinical application specifications also need to be improved. In addition, saliva substitutes such as dry mouth gels, dry mouth-relieving mouthwashes, transcutaneous electrical nerve stimulation, and laser therapy have also played an important role, but most of the literature reports have problems such as small sample size and intervention bias, and the treatment effect needs to be further clarified. In recent years, traditional Chinese medicine treatments such as herbs, traditional Chinese medicine, and acupuncture have shown unique advantages in xerostomia, but the safety of their application in the treatment process of cancer patients still needs in-depth clinical verification. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies of the prior art and provide a spray for treating radiation-induced xerostomia in nasopharyngeal carcinoma patients after radiotherapy, and its preparation method and application.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a fusion protein, which comprises a laminin recombinant protein, an FGF8 recombinant protein and an Eda recombinant protein that are fused and connected to each other. The amino acid sequence of the laminin recombinant protein is as shown in SEQ ID NO: 3, the amino acid sequence of the FGF8 recombinant protein is as shown in SEQ ID NO: 4, and the amino acid sequence of the Eda recombinant protein is as shown in SEQ ID NO: 5.

[0007] By fusing the laminin recombinant protein, the FGF8 recombinant protein and the Eda recombinant protein, the present invention constructs a fusion protein, which has the function of promoting the regeneration of salivary gland cells, and the effect is better than that of the fusion protein obtained by laminin recombinant protein, full-length FGF8 protein and full-length Eda protein.

[0008] Further, the C-terminus of the laminin recombinant protein is located at the N-terminus of the FGF8 recombinant protein, and the N-terminus of the Eda recombinant protein is located at the C-terminus of the FGF8 recombinant protein.

[0009] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the coding gene of the fusion protein is as shown in SEQ ID NO: 2.

[0010] In the second aspect, the present invention provides the application of the fusion protein in the preparation of a product for treating xerostomia.

[0011] Further, the xerostomia includes radiation-induced xerostomia in nasopharyngeal carcinoma patients after radiotherapy.

[0012] In the third aspect, the present invention provides a drug for treating xerostomia, and the drug comprises the fusion protein.

[0013] Further, the drug also contains pharmaceutically acceptable excipients.

[0014] Further, the drug also contains water, a humectant, a surfactant, licorice, menthol and vitamin C.

[0015] In a specific embodiment of the present invention, the humectant includes trehalose, and the surfactant includes lecithin.

[0016] Further, the drug comprises the following components in parts by weight: 60-64 parts of water, 3-7 parts of trehalose, 0.5-1.5 parts of lecithin, 3-7 parts of liquorice, 3-7 parts of menthol, 3-7 parts of vitamin C, and 8-12 parts of the fusion protein.

[0017] In a specific embodiment of the present invention, the drug comprises the following components in parts by weight: 62 parts of water, 5 parts of trehalose, 1 part of lecithin, 5 parts of liquorice, 5 parts of menthol, 5 parts of vitamin C, and 10 parts of the fusion protein.

[0018] Fourthly, the present invention provides a method for preparing the drug, comprising the following steps:

[0019] S1: Thoroughly stir and homogenize water, a humectant, and a surfactant at 55-65 °C to obtain liquid A;

[0020] S2: Adjust the temperature of liquid A in step S1 to 45-55 °C, add liquorice, menthol, and vitamin C, and stir and mix until dissolved uniformly to obtain liquid B;

[0021] S3: Adjust the temperature of liquid B in step S2 to 25-35 °C, add the fusion protein according to any one of claims 1-3, and stir and mix until dissolved uniformly to obtain the drug.

[0022] In a specific embodiment of the present invention, the temperature in step S1 is 60 °C.

[0023] Further, in step S1, the homogenization time is 5-10 min.

[0024] Further, in step S1, the water can be deionized water, and the deionized water is sterilized by heat preservation at 85-90 °C for 30 min and then used for preparing the drug.

[0025] In a specific embodiment of the present invention, the temperature in step S2 is 50 °C.

[0026] In a specific embodiment of the present invention, the temperature in step S3 is 30 °C.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The present invention constructs a fusion protein LFE by using recombinant laminin recombinant protein, FGF8 recombinant protein, and Eda recombinant protein. The fusion protein has high purity and relatively small molecular weight after multi-step purification, and has the function of promoting the regeneration of salivary gland cells in vitro, and the effect is better than that of the fusion protein obtained from laminin recombinant protein, FGF8 full-length protein, and Eda full-length protein.

[0029] (2) The spray for treating radiation-induced xerostomia in nasopharyngeal carcinoma patients after radiotherapy is prepared from the fusion protein LFE. This spray has the function of promoting the regeneration of salivary gland cells and saliva secretion in vivo, has a therapeutic effect on radiation-induced xerostomia, and can be widely applied clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the fusion protein structure.

[0031] Figure 2 It is the Western Blot graph of SOX2 protein in in vitro cell experiments.

[0032] Figure 3 It is the saliva flow rate in in vivo animal experiments.

[0033] Figure 4 It is the Western Blot graph of SOX2 protein in in vivo animal experiments. DETAILED DESCRIPTION OF THE INVENTION

[0034] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0035] SPF-grade mice or rats purchased from the Guangdong Provincial Medical Experimental Animal Center.

[0036] Example 1 Preparation of the fusion protein LFE

[0037] I. Experimental method

[0038] 1. Design of the fusion protein

[0039] (1) As Figure 1 shown, the fusion protein LFE1: consists of laminin recombinant protein, FGF8 recombinant protein and Eda recombinant protein. The N-terminus of the FGF8 recombinant protein is connected to the C-terminus of the laminin recombinant protein, and the C-terminus of the FGF8 recombinant protein is connected to the N-terminus of the Eda recombinant protein.

[0040] The amino acid sequence of the fusion protein LFE1 is as shown in SEQ ID NO: 1, and the nucleotide sequence is as shown in SEQ ID NO: 2.

[0041] The amino acid sequence of the laminin recombinant protein is as shown in SEQ ID NO: 3, the amino acid sequence of the FGF8 recombinant protein is as shown in SEQ ID NO: 4, and the amino acid sequence of the Eda recombinant protein is as shown in SEQ ID NO: 5.

[0042] Fusion protein LFE2: It is composed of a laminin recombinant protein, the full-length FGF8 protein (SEQ ID NO: 6), and the full-length Eda protein (SEQ ID NO: 7). The N-terminus of the full-length FGF8 protein is connected to the C-terminus of the laminin recombinant protein, and the C-terminus of the full-length FGF8 protein is connected to the N-terminus of the full-length Eda protein.

[0043] The amino acid sequence of the fusion protein LFE2 is shown in SEQ ID NO: 8, and the nucleotide sequence is shown in SEQ ID NO: 9.

[0044] (2) Constructing vectors

[0045] After connecting a 6×His tag to the N-terminus of the fusion proteins LFE1 and LFE2, the fusion proteins 6×His-LFE1 and 6×His-LFE2 with a 6×His tag are obtained. Then, an NdeⅠ restriction site is added to the 5’ end of their corresponding coding genes, and an XhoⅠ restriction site is added to the 3’ end, resulting in a 6×His-LFE1 gene fragment containing the restriction sites and a 6×His-LFE2 gene fragment containing the restriction sites.

[0046] The 6×His-LFE1 gene fragment containing the restriction sites is ligated between the NdeⅠ and XhoⅠ restriction sites of the pET-28a(+) vector to obtain the 6×His-laminin-FGF8-Eda-1 recombinant plasmid.

[0047] The 6×His-LFE2 gene fragment containing the restriction sites is ligated between the NdeⅠ and XhoⅠ restriction sites of the pET-28a(+) vector to obtain the 6×His-laminin-FGF8-Eda-2 recombinant plasmid.

[0048] (3) Culturing and inducing expression

[0049] The above-constructed 6×His-laminin-FGF8-Eda-1 recombinant plasmid and 6×His-laminin-FGF8-Eda-2 recombinant plasmid are respectively transformed into BL21(DE3) chemically competent cells. After ice-bathing for 30 min, heat shock for 90 s and then continue ice-bathing, add pre-warmed LB medium (lysogeny broth medium), culture at 37℃ and 220 rpm for 40 - 60 min, centrifuge at low speed, discard part of the supernatant in the laminar flow hood, resuspend the cells with the remaining supernatant, spread on an LB solid plate, and select monoclonal colonies for sequencing the next day to screen positive monoclonal strains.

[0050] After determining the positive clone strains, verify the induced expression. Transfer the positive monoclonal strains to an LB liquid medium. Wait until OD 600When it is 0.6 - 0.8, add 0.5 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG) for induction culture. Incubate at 28 °C for 4 h. Resuspend the cells with PBS buffer (phosphate buffer, pH = 7.4), and disrupt the cells by high-pressure homogenization. Centrifuge at 12,000 rpm for 30 min, and collect the supernatant, which is the soluble fusion proteins 6×His-LFE1 and 6×His-LFE2.

[0051] (4) Purification

[0052] Use cation exchange chromatography and Ni-NTA affinity chromatography to remove the 6×His-tagged proteins and other miscellaneous proteins in the soluble fusion proteins 6×His-LFE1 and 6×His-LFE2. Concentrate by ultrafiltration membrane package to obtain the fusion proteins LFE1 and LFE2, and perform purity analysis.

[0053] The eluent for cation exchange chromatography is: 20 mM PB (phosphate buffer), 350 mM NaCl, pH 7.5.

[0054] The eluent for affinity chromatography is: 20 mM PB, 350 mM NaCl, 300 mM imidazole, pH 7.5.

[0055] II. Experimental Results

[0056] The purity of the fusion protein LFE1 is 99%, and the purity of the fusion protein LFE2 is 96%.

[0057] It can be seen that the recombinant fusion protein LFE1 constructed after truncation has a relatively small molecular weight and higher purity after multiple steps of purification.

[0058] Example 2 In vitro cell experiment of the fusion protein

[0059] I. Experimental methods

[0060] 1. Primary culture

[0061] (1) Take the salivary gland tissue of SPF mice, soak it in 75% (v / v) ethanol for 15 s, rinse it twice with pre-cooled 1×PBS, and cut the tissue into minced meat-like to obtain minced tissue.

[0062] (2) Transfer the minced tissue to a 15 mL centrifuge tube, add 50 units / mL Dispase, and incubate at 37 °C for 1 h to obtain enzymatically digested tissue.

[0063] (3) Rinse the enzymatically digested tissue twice with pre-cooled 1×PBS, add 10 mL 1×PBS, stir on ice for 10 min, and filter through a 40 μm filter membrane to obtain filtered cells.

[0064] (4) Collect the cell count after filtration, and inoculate 1 - 2×10 4 cells / well / every 50 μL into a 24-well plate, add conventional cell culture medium (using DMEM / F12 as the basal medium, supplemented with 10% v / v FBS and 0.1 mg / mL penicillin-streptomycin double antibody), and culture in a cell incubator (37 °C, 5% CO2) for 48 h before subculture.

[0065] 2. Subculture

[0066] (1) Add 1 mL of 1×PBS to each well in the 24-well plate of primary culture, place it in a centrifuge tube, centrifuge at 1200 rpm for 5 min, and collect the precipitate, which is the primary cultured cells.

[0067] (2) Add 10×TE (Tris-EDTA buffer, Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer) to digest the primary cultured cells, incubate at 37 °C for 30 min to obtain digested cells;

[0068] (3) Rinse the digested cells twice with 1×PBS, and inoculate 0.5 - 1×10 4 cells / well / every 50 μL into a 24-well plate, add conventional cell culture medium (using DMEM / F12 as the basal medium, supplemented with 10% v / v FBS and 0.1 mg / mL penicillin-streptomycin double antibody), and culture in a cell incubator (37 °C, 5% CO2).

[0069] 3. Radiation

[0070] Divide the 24-well plate of primary culture into 4 groups, namely the normal culture group (Group A), the radiation + PBS intervention group (Group B), the radiation + fusion protein LFE1 intervention group (Group C), and the radiation + fusion protein LFE2 intervention group (Group D), with 6 replicate wells in each group. Radiation is carried out in a sterile environment, and the normal group is protected from radiation exposure. Only Groups B, C, and D are irradiated with a single dose of 15 Gy. After radiation, culture according to the method in Step 2 for 1 d.

[0071] 4. Intervention

[0072] On the second day after radiation, change the medium normally for Group A. After changing the medium normally for Group B, add 50 μL of PBS for intervention. After changing the medium normally for Group C, add 50 μL of fusion protein LFE1 (1 mg / mL) for intervention. After changing the medium normally for Group D, add 50 μL of fusion protein LFE2 (1 mg / mL) for intervention. Change the medium after 3 days of intervention in the incubator, perform the same intervention again, continue to culture for 3 days, then observe the cell morphology under a microscope and collect the cells to harvest the total cell protein.

[0073] 5. Detect the expression of SOX2 protein (sex-determining region Y-box protein 2)

[0074] Analyze the expression of SOX2 protein in total protein by Western Blot (immunoblotting).

[0075] II. Experimental Results

[0076] Through microscopic observation, the salivary gland cells in the radiation + PBS intervention group had the smallest morphology. The morphology of the radiation + fusion protein intervention groups (groups C and D) was smaller than that of the normal group but better than that of the radiation + PBS intervention group. Moreover, the radiation + fusion protein LFE1 intervention group was better than the radiation + fusion protein LFE2 intervention group.

[0077] Through Western Blot analysis, the expression of SOX2 was as follows: normal group > radiation + fusion protein LFE1 intervention group > radiation + fusion protein LFE2 intervention group > radiation + PBS intervention group. Among them, the radiation + fusion protein intervention groups were significantly higher than the radiation + PBS intervention group, showing the same conclusion as the cell morphology under microscopic observation, that is, the fusion proteins LFE1 and LFE2 could effectively promote the regeneration of salivary gland cells, and the effect of the truncated recombinant fusion protein LFE1 was better than that of the full-length recombinant fusion protein LFE2( Figure 2 ).

[0078] Example 3 Preparation of Spray

[0079] 1. Take 62 parts by mass of deionized water, keep it at 85 - 90 °C for 30 min for heat preservation and sterilization, cool it to 60 °C, add 5 parts by mass of humectant and 1 part by mass of surfactant, stir well and homogenize for 5 - 10 min to obtain solution A; the humectant is trehalose and the surfactant is lecithin.

[0080] 2. Cool solution A to 50 °C, add 5 parts by mass of licorice, 5 parts by mass of menthol and 5 parts by mass of vitamin C, stir and mix until dissolved evenly to obtain solution B.

[0081] 3. Cool solution B to below 30 °C, add 10 parts by mass of fusion protein LFE1, continue to stir until dissolved evenly to obtain the spray.

[0082] Example 4 In Vivo Animal Experiment of Spray

[0083] I. Experimental Method

[0084] Divide SPF rats into 3 groups, with 6 rats in each group, namely the normal group (group a), the radiation + PBS intervention group (group b), and the radiation + spray intervention group (group c).

[0085] (1) Construction of Radiation Model

[0086] Rats were anesthetized with 2.5% (v / v) isoflurane and shielded with an aluminum shield to prevent radiation exposure. The shield had a 1-cm long slit that allowed targeted radiation exposure to the neck area while protecting other parts. Next, a single dose of 15 GY of radiation was administered to the head and neck area using a biological irradiator, Multi-Rad 225.

[0087] (2) Intervention

[0088] The normal group received no treatment. The radiation + PBS intervention group was intervened by administering PBS, and the radiation + spray intervention group was intervened by administering the spray. The intervention times were the 1st, 3rd, and 7th days after radiation.

[0089] (3) Measurement of salivary flow rate

[0090] On the 15th day after radiation, 3 rats were taken from each group for measurement of salivary flow rate.

[0091] Each group of rats was anesthetized with tribromoethanol (300 mg / kg), and pilocarpine (2.5 mg / kg) was injected intraperitoneally to stimulate salivary secretion. Subsequently, the rats were positioned at a 45-degree angle with their abdomens facing up to collect saliva.

[0092] One minute after administration of pilocarpine, saliva was collected for 20 min using a 200-μL pipette. Then the saliva was centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to a new test tube, and the volume of the collected saliva was measured.

[0093] The salivary flow rate was calculated using the following formula:

[0094] Salivary flow rate = volume of saliva collected by stimulation (μL) / (rat weight (g) × collection time (20 min))

[0095] (4) Detection of SOX2 protein expression

[0096] On the 15th day after radiation, 3 rats were taken from each group to extract total protein, and Western Blot was used to analyze the expression of SOX2 protein.

[0097] II. Experimental results

[0098] Salivary flow rate: group a > group c > group b. Among them, after intervention with the spray containing the fusion protein LFE1, group b showed a good salivary flow rate and was close to group a without radiation ( Figure 3 ).

[0099] SOX2 protein expression: Through Western Blot analysis, group a > group c > group b. Among them, after spray intervention with the fusion protein LFE in group b, the expression of SOX2 was significantly higher than that in group c( Figure 4 ).

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a laminin recombinant protein, an FGF8 recombinant protein and an Eda recombinant protein which are fused and linked to each other. The amino acid sequence of the laminin recombinant protein is as shown in SEQ ID NO:

3. The amino acid sequence of the FGF8 recombinant protein is as shown in SEQ ID NO:

4. The amino acid sequence of the Eda recombinant protein is as shown in SEQ ID NO:

5.

2. The fusion protein according to claim 1, wherein The C-terminus of the laminin recombinant protein is located at the N-terminus of the FGF8 recombinant protein, and the N-terminus of the Eda recombinant protein is located at the C-terminus of the FGF8 recombinant protein.

3. The fusion protein according to claim 2, wherein The amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the coding gene of the fusion protein is as shown in SEQ ID NO:

2.

4. Use of the fusion protein according to any one of claims 1 to 3 in the preparation of a product for treating dry mouth.

5. The application according to claim 4, wherein The dry mouth includes radiation-induced dry mouth in nasopharyngeal carcinoma patients after radiotherapy.

6. A drug for treating dry mouth, characterized in that, The medicament comprises the fusion protein according to any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that, The medicament further contains pharmaceutically acceptable excipients.

8. The drug according to claim 7, wherein, The medicament further contains water, a humectant, a surfactant, licorice, menthol and vitamin C.

9. The medicament according to claim 8, wherein The medicament comprises the following components in parts by weight: 60 to 64 parts of water, 3 to 7 parts of trehalose, 0.5 to 1.5 parts of lecithin, 3 to 7 parts of licorice, 3 to 7 parts of menthol, 3 to 7 parts of vitamin C, and 8 to 12 parts of the fusion protein according to any one of claims 1 to 3.

10. The preparation method of the drug according to any one of claims 6 to 9, characterized in that, Comprising the following steps: S1: Stir water, a humectant and a surfactant evenly at 55 to 65 °C, and homogenize to obtain solution A; S2: Adjust the temperature of solution A in step S1 to 45 to 55 °C, add licorice, menthol and vitamin C, and stir and mix until dissolved evenly to obtain solution B; S3: Adjust the temperature of solution B in step S2 to 25 to 35 °C, add the fusion protein according to any one of claims 1 to 3, and stir and mix until dissolved evenly to obtain the medicament.

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