NL-1 polypeptide for inhibiting tight reaction and application thereof

Abstract

The invention relates to the field of polypeptides, in particular to an NL-1 polypeptide capable of inhibiting a tight reaction and application of the NL-1 polypeptide. The NL-1 polypeptide is screened from transcripts of spiders, and the NL-1 polypeptide is predicted and verified to have antibacterial activity through antibacterial screening. An enzyme activity experiment is utilized to prove that synthesis of a tight reaction signal small molecule ppGpp can be reduced by inhibiting activity of key enzymes PaRelA and PaSpoT in a tight reaction of pseudomonas aeruginosa, so that the tight reaction is inhibited. Meanwhile, a bacteriostatic experiment and a bactericidal dynamics experiment prove that the NL-1 polypeptide grows in a concentration-dependent manner through three hospital-sourced multidrug-resistant pseudomonas aeruginosa strain; meanwhile, the NL-1 can effectively kill multidrug-resistant pseudomonas aeruginosa, in addition, the NL-1 also effectively reduces the generation of a quorum sensing key molecule PQS of the pseudomonas aeruginosa, and the component is proved to be related to antibiotic tolerance. In conclusion, the NL-1 polypeptide disclosed by the invention has a good antibacterial effect, can be used as a novel antibiotic, and has a huge application value for treatment of multidrug-resistant bacteria.

Description

Technical Field

[0001] This invention relates to the field of peptides, and more particularly to an NL-1 peptide that inhibits tight reactions and its applications. Background Technology

[0002] Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen, particularly prevalent in hospital-acquired infections, burn wound infections, and cystic fibrosis-related lung infections. Due to its excellent biofilm-forming ability and multidrug resistance, it often develops resistance to various antibiotics, including β-lactams, aminoglycosides, and polymyxins, leading to difficult clinical treatment, high recurrence rates, and a strong tendency to form chronic foci of infection.

[0003] When faced with environmental stress or antibiotic pressure, bacteria activate a so-called stringent response (SR), mediated by RelA / SpoT superfamily enzymes. This response converts GTP / GDP into the signaling molecule (pp)ppGpp, thereby reprogramming cellular metabolism, inhibiting growth, initiating stress defenses, and promoting biofilm maturation and tolerance gene expression. The stringent response is not only a crucial pathway for bacterial regulation of survival and persistence but is also closely related to biofilm stability, virulence factor secretion, and drug resistance formation. It is considered a key mechanism for persistent infection and treatment failure in multidrug-resistant Pseudomonas aeruginosa.

[0004] Current research on inhibitors targeting strict responses mainly focuses on small molecule compounds or synthetic peptides, such as Relacin, which can block RelA activity, and anti-biomembrane peptide 1018, which can induce (pp)ppGpp degradation. However, the cell permeability, specificity, or antibacterial spectrum of these inhibitors in Pseudomonas aeruginosa are limited, making it difficult to meet the needs of both highly effective inhibition of SR and low-toxicity bactericidal action.

[0005] Spider venom is rich in diverse and highly active natural peptides, possessing unique structural diversity and the potential to target and regulate microbial signaling pathways, and has shown broad application prospects in antibacterial, antitumor, and immunomodulatory fields. However, there are no systematic reports on inhibitors of the stringent response from spiders. Using spider peptides to target and block the stringent response of *Pseudomonas aeruginosa* not only holds promise for efficiently inhibiting (pp)ppGpp signaling and disintegrating biofilm barriers, but also for maintaining the low cytotoxicity and excellent biocompatibility of natural peptides, providing a novel strategy for overcoming multidrug-resistant *Pseudomonas aeruginosa* infections. Summary of the Invention

[0006] To address the problems existing in the background technology, an NL-1 peptide that inhibits tight reactions and its applications are proposed.

[0007] This invention proposes an NL-1 polypeptide that inhibits tightness reactions. The NL-1 polypeptide is derived from spiders and its amino acid sequence is shown in SEQ ID NO:1.

[0008] This invention proposes a gene encoding an NL-1 polypeptide that inhibits the severe response, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0009] This invention proposes a precursor peptide of the NL-1 polypeptide that inhibits the tight reaction. The amino acid sequence of the precursor peptide is shown in SEQ ID NO:3, and the corresponding encoding gene is shown in SEQ ID NO:4.

[0010] This invention proposes the application of the aforementioned polypeptide NL-1, the encoding gene, or biological materials containing the encoding gene, as well as the mature peptide formed by processing the NL-1 polypeptide or precursor peptide prepared by genetic engineering methods, in the preparation of antibacterial drugs.

[0011] This invention proposes the application of NL-1 polypeptides, encoding genes, or biological materials containing the encoding genes prepared using genetic engineering techniques, or mature peptides formed by processing NL-1 polypeptides or precursor peptides, in the preparation of biofilm removal and anti-biofouling agents.

[0012] This invention proposes the application of NL-1 peptides, encoding genes, or biological materials containing the encoding genes, prepared using genetic engineering techniques, or mature peptides formed by processing NL-1 peptides or precursor peptides, in the preparation of severe reaction inhibitors.

[0013] Preferably, the biological material containing the coding gene includes at least one of the following biological materials: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene.

[0014] Preferably, the drug includes injectable powder and / or injectable solution.

[0015] Compared with existing technologies, this invention has the following beneficial technical effects: This invention screened an NL-1 polypeptide from spider transcripts, and verified its antibacterial activity through antibacterial screening prediction. Furthermore, this invention used enzyme activity experiments to demonstrate that it can inhibit the tightness reaction by suppressing the activities of key enzymes PaRelA and PaSpoT in the Pseudomonas aeruginosa tightness reaction, thereby reducing the synthesis of the tightness reaction signaling molecule ppGpp, with half-inhibition constants (IC50) of 4.86 μM and 1.08 μM, respectively. Simultaneously, through antibacterial and bactericidal kinetic experiments, it was confirmed that the NL-1 peptide inhibited the growth of multidrug-resistant Pseudomonas aeruginosa from three hospitals in a concentration-dependent manner, with MICs of 2.0 μM, 4.0 μM, and 4.0 μM. It also effectively killed multidrug-resistant Pseudomonas aeruginosa, with MBCs of 2.0 × MIC (4.0 μM), 2.0 × MIC (8.0 μM), and 2.0 × MIC (8.0 μM) against the multidrug-resistant Pseudomonas aeruginosa from three hospitals. Furthermore, NL-1 effectively reduced the formation of PQS, a key quorum sensing molecule in Pseudomonas aeruginosa, which has been shown to be associated with antibiotic resistance. In summary, the NL-1 peptide described in this invention has good antibacterial activity and can be used as a novel antibiotic, possessing significant application value for the treatment of multidrug-resistant bacteria. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the inhibitory effect of NL-1 peptide on the PaRelA and PaSpoT enzyme activities of Pseudomonas aeruginosa. Figure 2 A schematic diagram illustrating the difference in growth inhibition of NL-1 peptide on wild-type Pseudomonas aeruginosa and the strict response mutant. Figure 3 This is a schematic diagram illustrating the bactericidal effect of NL-1 peptide on three strains of multidrug-resistant Pseudomonas aeruginosa from a hospital. Figure 4 This is a schematic diagram illustrating the effect of the P-1 polypeptide on PQS, a key signaling molecule in the quorum sensing of Pseudomonas aeruginosa. Detailed Implementation

[0017] This invention provides a spider-derived NL-1 polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 (CDCNNGFIFLKGQSGKCVKPEKCPK). Specifically, the NL-1 polypeptide is derived from a common Chinese spider: Cyrtophora guangxiensis, belonging to the family Araneidae and the genus Cyrtophora, with a molecular weight of 2744.26 Daltons. The NL-1 polypeptide can be obtained through chemical synthesis or recombinant expression via genetic engineering.

[0018] The present invention provides a gene encoding the spider-derived NL-1 polypeptide, the nucleotide sequence of which is shown in SEQ ID NO:2 (5'-TGTGATTGCAATAATGGATTCATCTTTTTGAAAGGACAAAGCGGCAAATGCGTGAAACCTGAAAAATGTCCGAAATAG-3').

[0019] This invention provides a precursor peptide of the spider-derived NL-1 polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3 (DDNQYFNNCGTACPLNCQNRNNPPKICTLQCVRR), and the corresponding encoding gene is shown in SEQ ID NO:4 (5'-GACGATAACCAATATTTTAATAATTGCGGAACTGCTTGTCCTCTGAATTGCCAGAACCGCAACAACCCTCCAAAGATATGTACTTTGCAATGCGTCAGGCGA-3').

[0020] In this embodiment of the invention, the antibacterial effect of the NL-1 peptide was analyzed using growth inhibition and bactericidal kinetic experiments. The results showed that the NL-1 peptide inhibited the growth of *Pseudomonas aeruginosa* in a concentration-dependent manner; simultaneously, it reduced the production of the *P. aeruginosa* quorum sensing signal molecule PQS. In summary, the NL-1 peptide of this invention has potential application value in the treatment of multidrug-resistant *P. aeruginosa* infections.

[0021] Based on the excellent antibacterial activity of the NL-1 polypeptide, the present invention provides an antibacterial drug lead molecule, the active ingredient of which includes the spider-derived NL-1 polypeptide, the coding gene or biological material containing the coding gene prepared by genetic engineering technology, or a mature peptide formed by processing the precursor peptide.

[0022] In this invention, the biological material containing the coding gene preferably includes at least one of the following biological materials: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene. There are no particular limitations on the type of backbone vector in the recombinant vector containing the coding gene; common backbone vectors known in the art can be used, such as prokaryotic expression vectors, eukaryotic expression vectors, viral vectors, and mammalian cell expression vectors. The recombinant vector containing the coding gene is constructed by selecting a suitable backbone vector according to the type of host, and subsequently enters the host through transformation or transfection methods to obtain recombinant bacterial strains containing the coding gene, recombinant viral strains containing the coding gene, and recombinant cell lines containing the coding gene. The host preferably includes a prokaryotic expression system, a eukaryotic expression system, a virus, or mammalian cells. The prokaryotic expression system preferably includes *Escherichia coli*, lactic acid bacteria, etc. The eukaryotic expression system preferably includes yeast and / or insect cells. The virus is preferably adeno-associated virus and / or lentivirus. This invention does not impose any special limitation on the type of mammalian cell used; any mammalian cell well-known in the art can be used, such as Chinese hamster ovary cells (CHO cells). This invention also does not impose any special limitation on the preparation method of the biological material containing the encoding gene; any genetic engineering method well-known in the art can be used.

[0023] In this invention, the mature peptide formed after processing the precursor peptide is the spider-derived NL-1 polypeptide converted into an active precursor peptide by enzymatic hydrolysis of the precursor peptide under the action of a specific protease. The hydrolysis sites are after CVRR and before CDCN.

[0024] Based on the NL-1 polypeptide's ability to inhibit bacterial ppGpp synthesis, this invention provides a tight reaction inhibitor, the active ingredient of which includes the spider-derived NL-1 polypeptide, the coding gene or NL-1 polypeptide prepared by genetic engineering technology from biological materials containing the coding gene, or a mature peptide formed by processing the precursor peptide.

[0025] The present invention does not impose any special restrictions on the effective content of the active ingredient in the inhibitor, and the effective concentration of a tight-response inhibitor well known in the art can be used.

[0026] This invention provides the application of the spider-derived NL-1 polypeptide, the NL-1 polypeptide prepared by genetic engineering using the coding gene or biological material containing the coding gene, or the mature peptide formed by processing the precursor peptide in the preparation of antibacterial drugs.

[0027] This invention provides the application of the spider-derived NL-1 polypeptide, encoding gene, or biological material containing the encoding gene, prepared using genetic engineering techniques, or the mature peptide formed by processing the NL-1 polypeptide or precursor peptide, in the preparation of biofilm removal and anti-biofouling agents.

[0028] This invention provides the application of the spider-derived NL-1 polypeptide, encoding gene, or biological material containing the encoding gene, prepared using genetic engineering techniques, or the mature peptide formed by processing the NL-1 polypeptide or precursor peptide, in the preparation of a severe reaction inhibitor.

[0029] In this invention, the inhibitory effect of the severe reaction is manifested in the in vitro inhibition of the activity of PaRelA and PaSpoT, key enzymes of the severe reaction in Pseudomonas aeruginosa, by the NL-1 polypeptide, IC50. 50 The values ​​were 4.86 and 1.08 μM, respectively.

[0030] In this invention, the antibacterial effect of the NL-1 peptide was analyzed using a growth inhibition experiment and a bactericidal kinetic experiment of Pseudomonas aeruginosa. The results showed that the NL-1 peptide reduced the growth of multidrug-resistant Pseudomonas aeruginosa in a concentration-dependent manner and could inhibit the production of the population sensing signal molecule PQS of this bacterium.

[0031] The following detailed description, in conjunction with embodiments, illustrates a spider-derived NL-1 polypeptide, its encoding gene, and its antibacterial applications, but these should not be construed as limiting the scope of protection of this invention.

[0032] I. Screening of NL-1 peptide and its encoding gene A spider-derived tight response inhibitor was identified from the transcript sequence of spider (TRINITY_DN10823_c0_g1) by Blast annotation and Pfam analysis, and obtained by antimicrobial peptide library AMP3.0 prediction and enzyme activity screening. The screening identified an NL-1 peptide with antibacterial activity. NL-1 is a spider-derived polypeptide with the mature peptide sequence shown in SEQ ID NO: 1 (CDCNNGFIFLKGQSGKCVKPEKCPK), and the corresponding coding gene sequence is shown in SEQ ID NO: 2 (TGTGATTGCAATAATGGATTCATCTTTTTGAAAGGACAAAGCGGCAAATGCGTGAAACCTGAAAAATGTCCGAAATAG). The amino acid sequence of the NL-1 precursor peptide is shown in SEQ ID NO: 3 (DDNQYFNNCGTACPLNCQNRNNPPKICTLQCVRR), and the corresponding coding gene is shown in SEQ ID NO: 4 (5'-GACGATAACCAATATTTTAATAATTGCGGAACTGCTTGTCCTCTGAATTGCCAGAACCGCAACAACCCTCCAAAGATATGTACTTTGCAATGCGTCAGGCGA-3').

[0033] II. Effects of NL-1 peptide on the activity of PAO1 tight reaction synthase in E. coli and P. aeruginosa pET-32a-EcRelA and pET-32a-PaRelA vectors were constructed and transformed into BL21 cells to express Pseudomonas aeruginosa PaRelA and PaSpoT enzymes, respectively. Bacteria were collected, and the cells were re-vortexed using an extraction buffer, sonicated, and the supernatant was purified by nickel affinity chromatography or other methods to obtain PaRelA and PaSpoT proteins. In vitro enzyme activity assays were performed in 50 mM HEPE (pH 7.2) buffer, containing 250 mM NaCl, 2 mM EDTA, 1 mM β-mercaptoethanol, 14 mM MgSO4, 8 mM ATP, 6 mM GDP, and 1.0 μg of recombinant protein protease and different concentrations (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16 μM NL-1 peptide). After incubation at 37℃ for 30 min, the reaction was terminated by liquid nitrogen freezing. The ppGpp content was detected by UHPLC-MS / MS. The ppGpp produced by the enzyme reaction without NL-1 was taken as 100% enzyme activity. The ratio of ppGpp yield in other inhibitor groups to ppGpp yield in the enzyme-free group was used as an indicator of enzyme activity. The results are as follows Figure 1 As shown, the NL-1 peptide significantly inhibited the activities of key enzymes (PaRelA and PaSpoT) in the tight reaction of two strains within a certain concentration range, exhibiting a concentration-dependent relationship. The half-maximal inhibitory concentration (IC50) was calculated. 50It was found that the NL-1 peptide inhibited the activity of PaRelA and PaSpoT enzymes by an IC50 value. 50 The concentrations were 4.86 μM and 1.08 μM, respectively, indicating that the polypeptide described in this invention can effectively interfere with the bacterial reaction process and has good potential for anti-infection applications. To verify the target of the NL-1 peptide described in this invention, it was applied at a sublethal concentration (1 μM) to both wild-type (WT) and tightly reactive double gene knockout mutant strains (RelA). - SpoT - The strain was revived and amplified in LB liquid medium, and its growth curve was monitored. 600 After approximately 0.05 μL, the culture was inoculated into 96-well plates with or without NL-1 peptide, and incubated at 37°C with shaking. OD values ​​were measured at 0, 4, 8, 12, 16, 20, and 24 hours. 600 The value is used to assess growth. The results are as follows: Figure 2 As shown, the NL-1 polypeptide has a significant inhibitory effect on wild-type strains, reducing OD200 by 24 hours. 600 It reduced by approximately 55%; however, the inhibitory effect on knockout mutants was weaker, with OD... 600 The decrease was less than 10%. These results indicate that the NL-1 peptide has good antibacterial activity against Pseudomonas aeruginosa, and its effect is closely related to the integrity of the strain's strict response pathway.

[0034] III. Antibacterial and bactericidal effects of NL-1 peptide against multidrug-resistant Pseudomonas aeruginosa from hospital sources. 3.1 Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration (MIC) of the antimicrobial peptides described in this invention against three strains of *Pseudomonas aeruginosa* from a hospital was determined using the microtube dilution method. The specific method is as follows: The test strains were inoculated into the corresponding nutrient media and cultured at 37°C until the logarithmic growth phase. The bacterial suspension was collected by centrifugation (3500 rpm, 5 min), washed twice with PBS buffer (1.5 mmol / L KH₂PO₄, 2.7 mmol / L Na₂HPO₄, 0.15 mol / L NaCl, pH 7.4), resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and diluted to 2 × 10⁻⁶. 5CFU / mL. 100 μL of bacterial suspension was added to a 96-well plate, followed by different concentrations of peptide (final concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μM), bringing the final volume to 200 μL. Kanamycin and levofloxacin were used as positive controls, with different concentrations applied simultaneously. After incubating the mixture at 37°C for 18–24 hours, the absorbance was measured at 600 nm using an Epoch Etock microplate reader to assess bacterial growth. The MIC was defined as the lowest concentration at which no detectable colony growth was observed. The experimental results are as follows: Figure 3 As shown, the three strains of *Pseudomonas aeruginosa* derived from the hospital, containing the polypeptide of this invention, all exhibit significant antibacterial activity, with MICs of 2.0 μM, 4.0 μM, and 4.0 μM, respectively. For comparison, under the same conditions, the MICs of kanamycin and levofloxacin against the three strains of multidrug-resistant *Pseudomonas aeruginosa* derived from the hospital were >16.0 μM, proving that the three strains of *Pseudomonas aeruginosa* derived from the hospital are indeed drug-resistant bacteria, and also demonstrating the antibacterial effect of the NL-1 polypeptide. 3.2 Bactericidal Kinetics Pseudomonas aeruginosa was inoculated into beef extract peptone medium and cultured at 37°C until the logarithmic growth phase (OD600 approximately 0.4–0.6). The viable bacterial suspension was then diluted to 1×10⁻⁶. 6 CFU / mL was added to peptide solutions of different concentrations (final concentrations of 0.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μM). The mixtures were incubated at 37°C with shaking, and samples were taken after 24 hours. Samples at each time point were serially diluted tenfold and plated onto LB agar plates, incubated at 37°C, and samples were taken at 0, 2, 4, 6, 8, 12, and 24 hours. After dilution with PBS, the samples were spread onto LB agar plates and incubated at 37°C for 24 hours to count colonies and calculate logarithmic counts. 10 CFU / mL, starting with log 10 The CFU / mL level was reduced by 3 as the cutoff value to find the MBC (method overload) and to evaluate the bactericidal rate and intensity of the peptide against bacteria. The experiment was conducted in triplicate, with a PBS solvent control group included to eliminate the influence of the solvent on bacterial growth. All data were averaged and the standard deviation was calculated. The experimental results are as follows: Figure 3 As shown, the MBC values ​​of the polypeptide NL-1 described in this invention against three multidrug-resistant bacteria from hospitals were 4.0 μM, 8.0 μM, and 8.0 μM, respectively. The MBC values ​​of the antibiotics levofloxacin and kanamycin, used as controls, against the three multidrug-resistant bacteria were both >16.0 μM, demonstrating the drug resistance of the three multidrug-resistant bacteria from hospitals and the highly effective bactericidal effect of the NL-1 polypeptide against multidrug-resistant Pseudomonas aeruginosa.

[0035] IV. NL-1 effectively inhibits the generation of PQS, a quorum sensing signaling molecule in Pseudomonas aeruginosa. In the logarithmic phase bacterial culture (OD) 600 Add a sublethal concentration of NL-1 peptide (1 μM) to the culture medium (approximately 0.1%) and incubate at 37°C with shaking at 200 rpm until the logarithmic phase ends (approximately 0.1%). 600 =1.0–1.2), the supernatant was collected (obtained by centrifugation at 10,000 rpm for 10 min), and extracted three times with an equal volume of acidified ethyl acetate (0.1% formic acid). The organic phases were combined, concentrated to dryness by rotary evaporation, and redissolved in methanol (or 60% acetonitrile:water). After filtration through a 0.22 μm filter membrane, the solution was used for UPLC injection. The HPLC detection conditions were as follows: Column: ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 μm); Mobile phase: Phase A: water + 0.1% formic acid, Phase B: acetonitrile + 0.1% formic acid; Elution gradient: Gradient elution (time / A%): 0–1 min: 95%, 1–5 min: 95%→5%, 5–7 min: 5%, 7–8 min: 5%→95%, 8–10 min: 95% equilibration; Detection wavelength: 254 nm. A PQS standard curve was generated using the same method. The PQS content of the bacterial culture was accurately quantified using the standard curve, and normalization was performed using protein concentration. Results are expressed as mean ± standard deviation and were analyzed using Student's test (p < 0.05). Results are as follows: Figure 4 As shown, under 1 μM NL-1 treatment, the PQS content decreased from 826.1±124.6 μg / mg protein to approximately 116.8±24.3 μg / mg protein, from 732.9±16.8 μg / mg protein to 301.2±38.5 μg / mg protein, and from 436.4±25.6 μg / mg protein to 346.2±24.5 μg / mg protein, respectively. These results indicate that the NL-1 peptide significantly reduced the synthesis of the PQS quorum sensing signal in Pseudomonas aeruginosa.

[0036] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. An NL-1 polypeptide that inhibits severe reactions, characterized in that, The NL-1 polypeptide is derived from spiders, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the NL-1 polypeptide that inhibits the severe response according to claim 1, characterized in that, The nucleotide sequence encoding the gene is shown in SEQ ID NO:

2.

3. The precursor peptide of the NL-1 polypeptide for inhibiting severe reactions according to claim 1, characterized in that, The amino acid sequence of the precursor peptide is shown in SEQ ID NO:3, and the corresponding encoding gene is shown in SEQ ID NO:

4.

4. The application of the polypeptide NL-1 of claim 1, the NL-1 polypeptide prepared by genetic engineering based on the encoding gene of claim 2 or biological material containing the encoding gene, or the mature peptide formed by processing the precursor peptide of claim 3 in the preparation of antibacterial drugs.

5. The application of the NL-1 polypeptide of claim 1, the NL-1 polypeptide of claim 2 encoding the gene or biological material containing the encoding gene prepared by genetic engineering technology, or the mature peptide formed by processing the precursor peptide of claim 3 in the preparation of biofilm removal and anti-biofouling.

6. The use of the NL-1 polypeptide of claim 1, the NL-1 polypeptide prepared by genetic engineering technology from the encoding gene of claim 2 or biological material containing the encoding gene, or the mature peptide formed by processing the precursor peptide of claim 3 in the preparation of a severe reaction inhibitor.

7. The application according to any one of claims 4-6, characterized in that, Biological materials containing the coding gene include at least one of the following: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene.

8. The application according to claim 4, characterized in that, The drugs include injectable powders and / or injectable solutions.

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