Melittin analogue containing hydroxylated phosphorylated unnatural amino acid and preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and particularly relates to a melittin analogue containing a hydroxylated non-natural amino acid. The melittin analogue is obtained by replacing the amino acids at the pepsin cleavage site and / or the amino acids at the trypsin cleavage site in a natural melittin peptide segment with a hydroxylated non-natural amino acid. Compared with melittin, the melittin analogue of the application has better inhibitory effect on pet-derived gram-positive and gram-negative pathogenic bacteria, has lower hemolytic activity, and has better stability in pepsin, trypsin and plasma in vitro. The melittin analogue has very broad application prospects in the preparation of pet antibacterial infection drugs, feed additives and antibacterial in-vitro sprays.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a bee venom peptide analog containing hydroxyl phosphorylated non-natural amino acids, its preparation method, and its application. Background Technology

[0002] With rapid socio-economic development and a significant improvement in living standards, the role of pets in modern families has undergone a fundamental transformation, evolving from traditional functional animals into important emotional companions and family members. However, while enjoying the psychological comfort pets provide, the potential health risks they pose urgently require systematic research and scientific solutions. Epidemiological surveys show that pets' unique behavioral habits and activity patterns make them ideal carriers of various pathogens; more than 200 zoonotic pathogens have been identified. Notably, the widespread cohabitation and close contact between humans and pets in modern households significantly increases the risk of infection through direct contact, aerosol transmission, and other routes. Even more serious is the problem of antibiotic overuse in veterinary clinical treatment. This inappropriate medication not only leads to decreased treatment effectiveness but also accelerates the evolution of multidrug-resistant strains, triggering a series of public health crises, including increased infection rates and treatment costs, posing a persistent threat to global health security.

[0003] Antimicrobial peptides (AMPs), a class of small-molecule active substances (typically composed of 12-50 amino acids) widely found in the biological world, are core defense molecules of the innate immune system. Their unique antibacterial mechanisms enable them to exhibit significant inhibitory effects not only against common pathogens but also remarkable bactericidal activity against multidrug-resistant strains. Of particular note is the characteristic of antimicrobial peptides to disrupt bacterial membrane structures through physical mechanisms, making it extremely difficult to induce bacterial resistance. Among numerous antimicrobial peptides, melittin, due to its unique structural characteristics, exhibits broad-spectrum and highly effective antimicrobial properties, making it a key focus of antimicrobial peptide research. However, in-depth research has revealed significant limitations in the application of natural melittin: its protease sensitivity leads to a short in vivo half-life, and its significant hemolytic activity further restricts its clinical application value. Therefore, developing melittin analogs with high antimicrobial activity, good in vitro and in vivo stability, and low hemolytic toxicity through structural modification strategies is of great significance. Summary of the Invention

[0004] The present invention aims to provide a bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids to solve the problems of instability and strong hemolytic activity of natural bee venom peptides in vivo and in vitro.

[0005] To achieve the above objectives, the present invention provides a bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids, the structure of which is as follows:

[0006] GIGAVLKVLTTGLPALIS-(5-HTP-H2PO3)-IKRKRQQ;

[0007] or, GIGAVLKVLTTGLPALISWIK-(5-HTP-H2PO3)-K-(5-HTP-H2PO3)-QQ;

[0008] or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALISWI-(5-HTP-H2PO3)-R-(5-HTP-H2PO3)-RQQ;

[0009] Or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALISWI-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-QQ;

[0010] Or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALIS-(5-HTP-H2PO3)-I-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-QQ.

[0011] The working principle and beneficial effects of this scheme are as follows: The inventors discovered through experiments that bee venom peptide analogs obtained by replacing the pepsin cleavage site amino acids and / or trypsin cleavage site amino acids of bee venom peptide with hydroxyphosphorylated non-natural amino acids exhibit superior stability in pepsin and trypsin environments compared to bee venom peptides. Furthermore, these bee venom peptide analogs show lower hemolytic activity and better antibacterial effects than bee venom peptides. Among these, the bee venom peptide analogs obtained by simultaneously replacing the pepsin and trypsin cleavage site amino acids with hydroxyphosphorylated 5-hydroxytryptophan exhibit the best stability and antibacterial effect, with a MIC value of 2–8 μg / mL.

[0012] This invention also provides a method for preparing the above-mentioned bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids, wherein the amino acids at the pepsin cleavage sites and / or trypsin cleavage sites of the bee venom peptide are replaced with hydroxyphosphorylated non-natural amino acids. Specifically, this invention uses Rink-MBHA Resin as the starting material and employs the Fmoc solid-phase synthesis method. According to the designed substitution sites, the amino acids at corresponding positions of the natural bee venom peptide fragment are replaced with hydroxyphosphorylated non-natural amino acids, and then the peptide chain is extended sequentially to obtain the target analog. This method features mild reaction conditions, few side reactions, high yield, and the Fmoc group itself has characteristic ultraviolet absorption, making it easy to monitor and control the reaction.

[0013] Optionally, the hydroxyphosphorylated non-natural amino acids at the pepsin cleavage site and the trypsin cleavage site of melittin can be replaced with hydroxyphosphorylated 5-hydroxytryptophan. In this scheme, when hydroxyphosphorylated 5-hydroxytryptophan is used to replace the pepsin cleavage site amino acids and / or trypsin cleavage site amino acids on melittin, the trypsin stability and antibacterial activity of the melittin analog are significantly improved, and the hemolytic activity is significantly reduced.

[0014] This invention also provides the use of the aforementioned melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in the preparation of antibacterial drugs or antibacterial compositions. Compared with melitoxic peptides and antibiotics, the melitoxic peptide analogs in this invention exhibit broad-spectrum antibacterial activity and superior antibacterial effect, especially against Pasteurella canis and Staphylococcus pseudointermediate. Therefore, the melitoxic peptide analogs in this invention can be used in antibacterial drugs or antibacterial compositions to obtain broad-spectrum antibacterial drugs.

[0015] Optionally, the bacteria include pet-derived Gram-positive and Gram-negative bacteria. The melitin analogues in this protocol exhibit good antibacterial effects against both pet-derived Gram-positive and Gram-negative bacteria. In particular, the melitin analogues that use hydroxyphosphorylated 5-hydroxytryptophan to simultaneously replace the amino acids at the pepsin and trypsin cleavage sites of melitin have the lowest MIC values ​​(MIC of 2–8 μg / mL) against some experimental strains, indicating that these melitin analogues have good broad-spectrum antibacterial effects.

[0016] Optionally, the bacteria include *Escherichia coli*, *Pasteurella canis*, *Haemophilus feli*, *Campylobacter*, *Streptococcus canis*, and *Staphylococcus pseudointermediate*. These bacteria are common pathogens that harm pet health, and the melitoxin analogue in this formulation has good antibacterial effects against them.

[0017] This invention also provides the application of the above-mentioned melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in the preparation of feed additives or feed additive compositions. The melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids possess broad-spectrum antibacterial properties, strong enzymatic stability, and low cytotoxicity, making them suitable for preparing pet feed additives or feed additive compositions, thereby imparting antibacterial functionality to the feed.

[0018] This invention also provides the use of the above-mentioned melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in the preparation of pet eye drops or pet eye drop compositions. The melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids have broad-spectrum antibacterial properties and low cytotoxicity, making them suitable for the preparation of pet eye drops or pet eye drop compositions.

[0019] This invention also provides the application of the above-mentioned bee venom peptide analogs containing hydroxyphosphorylated non-natural amino acids in the preparation of pet topical antibacterial sprays or pet topical antibacterial compositions. The bee venom peptide analogs containing hydroxyphosphorylated non-natural amino acids possess broad-spectrum antibacterial properties, strong enzymatic stability, and low cytotoxicity, making them suitable for the preparation of pet topical antibacterial sprays or pet topical antibacterial compositions. Attached Figure Description

[0020] Figure 1 This is the RP-HPLC analysis chromatogram of MEL-H19 in Example 1 of the present invention;

[0021] Figure 2 This is the RP-HPLC analysis chromatogram of MEL-H22,24 in Example 2 of the present invention;

[0022] Figure 3 The RP-HPLC analysis chromatograms of MEL-H7,21,23 in Example 3 of this invention are shown below.

[0023] Figure 4 The RP-HPLC analysis chromatograms of MEL-H7, 21, 22, 23, and 24 in Example 4 of this invention are shown below.

[0024] Figure 5 The RP-HPLC analysis chromatograms of MEL-H7, 19, 21, 22, 23, and 24 in Example 5 of this invention are shown.

[0025] Figure 6 This is the RP-HPLC analysis chromatogram of MEL in Comparative Example 1 of this invention;

[0026] Figure 7 This is a graph showing the hemolytic evaluation results of the melitoxin analogue in Experimental Example 2 of the present invention;

[0027] Figure 8 This is a graph showing the stability evaluation results of the pepsin solution of the melitoxin analogue in Experimental Example 3 of this invention;

[0028] Figure 9 This is a graph showing the stability evaluation results of the melitoxin analogue in the trypsin solution in Experiment Example 4 of this invention;

[0029] Figure 10 This is a graph showing the plasma stability evaluation results of the melitoxin analogue in Experimental Example 5 of this invention. Detailed Implementation

[0030] The following detailed description provides further details through specific implementation methods. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] The experimental materials used in the following examples—melitonite, amino acids required for the synthesis of melitonite analogs containing hydroxyphosphorylated non-natural amino acids, trypsin, pepsin, and Triton X-100 enzyme—were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Other conventional reagents were either imported and repackaged or domestically produced analytical grade. The sources of the antibacterial experimental strains are shown in Table 1.

[0032] Table 1. Strains and their sources

[0033] Escherichiacoli CVCC3034 Purchased from CVCC PasteurellacanisCAU0082 Purchased from CVCC Haemophilusfelis BNCC364403 Purchased from Beina Bio CampylobactercoliCICC23925 Purchased from CICC Streptococcus canis ATCC43496 Purchased from ATCC StaphylococcuspseudintermediusATCC49444 Purchased from ATCC

[0034] Example 1: Synthesis of bee venom peptide analogs containing hydroxyphosphorylated non-natural amino acids

[0035] 2.0 g of Rink Amide MBHA resin was weighed into a peptide synthesis tube, and N,N-dimethylformamide (DMF) was added to the synthesis tube to swell the resin. After the resin swelled, the DMF in the synthesis tube was removed by vacuum filtration, and 4 × 0.8 mmol of 20% (v / v) piperidine solution (prepared with DMF) was added to remove the Fmoc protecting groups of the amino groups on the resin. The molar amount of active groups on the resin was calculated based on the resin loading and weight. Fmoc-amino acids, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and 1-hydroxybenzotriazole (HOBt) were weighed into a dry beaker, dissolved in DMF, and then N,N-diisopropylethylamine (DIEA) was transferred into the beaker, activated, and then transferred to the synthesis tube. Amino acid coupling was performed at room temperature in the dark. The peptide chain was further elongated according to the designed amino acid sequence Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-(5-HTP-H2PO3)-Ile-Lys-Arg-Lys-Arg-Gln-Gln(GIGAVLK VLTTGLPALIS-(5-HTP-H2PO3)-IKRKRQQ), and amino acid coupling was repeated until the last amino acid was coupled, and the Fmoc protecting group of the last amino acid was removed. The resin was washed alternately with DMF and anhydrous methanol, and then dried by vacuum filtration. It was then transferred to a round-bottom flask, and a cleavage reagent (trifluoroacetic acid: β-mercaptoethanol: methyl phenyl sulfide: water: phenol in a volume ratio of 82.5:5:5:5:2.5) was added. The mixture was magnetically stirred at room temperature in the dark for 3-4 hours to complete the cleavage process. The peptide solution in the flask was filtered through a sintered glass funnel into an Erlenmeyer flask. Ice-cold diethyl ether was added, and the mixture was allowed to stand at -20°C for 24 hours to precipitate the peptides. The solution was transferred to a centrifuge tube, the precipitate was collected by centrifugation, and the precipitate was washed three times thoroughly with diethyl ether. Distilled water was added to dissolve the precipitate, and the mixture was freeze-dried to obtain the crude peptide product. The crude peptide product was purified using preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain a pure peptide, designated MEL-H19. The purity was determined by HPLC, and the RP-HPLC chromatogram of MEL-H19 is shown below. Figure 1 As shown.

[0036] The preparation methods of Examples 2-5 and Comparative Example 1 are basically the same as those described in Example 1, and the differences in their peptide chains are shown in Table 2.

[0037] Table 2. Differences in peptide chains between Examples 1-5 and Comparative Example 1.

[0038]

[0039] In Table 2, the three-letter amino acid sequences of MEL-H22 and 24 are Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-(5-HTP-H2PO3)-Lys-(5-HTP-H2PO3)-Gln-Gln. The three-letter amino acid sequences of MEL-H7, 21, and 23 are Gly-Ile-Gly-Ala-Val-Leu-(5-HTP-H2PO3)-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-(5-HTP-H2PO3)-Arg-(5-HTP-H2PO3)-Arg-Gln-Gln. The three-letter amino acid sequence of MEL-H7,21,22,23,24 is Gly-Ile-Gly-Ala-Val-Leu-(5-HTP-H2PO3)-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-(5-HTP-H2PO3)-(5-HT P-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-Gln-Gln. The three-letter amino acid sequences of MEL-H7,19,21,22,23,24 are Gly-Ile-Gly-Ala-Val-Leu-(5-HTP-H2PO3)-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-(5-HTP-H2PO3)-Ile-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-Gln-Gln. Furthermore, the RP-HPLC chromatograms of MEL-H22,24 are as follows: Figure 2 As shown, the RP-HPLC chromatograms of MEL-H7,21,23 are as follows: Figure 3 As shown, the RP-HPLC chromatograms of MEL-H7, 21, 22, 23, and 24 are as follows: Figure 4 As shown, the RP-HPLC chromatograms of MEL-H7, 19, 21, 22, 23, and 24 are as follows: Figure 5 As shown, the RP-HPLC chromatogram of MEL is as follows: Figure 6 As shown.

[0040] Depend on Figures 1-6 It can be seen that the products obtained in Examples 1-5 and Comparative Example 1, after purification, all have a purity > 95%.

[0041] Example 1: Evaluation of the antibacterial activity of melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids

[0042] Antimicrobial assays were performed using a broth-based microdilution method, in accordance with the CLSI Guidelines for Antimicrobial Susceptibility Testing. *Escherichia coli* CVCC 3034, *Pasteurella canis* CAU0082, *Haemophilus felis* BNCC364403, *Campylobacter coli* CICC 23925, *Streptococcus canis* ATCC 43496, and *Staphylococcus pseudintermedius* ATCC 49444 were cultured in broth and grown to the logarithmic growth phase. The bacterial suspensions were then diluted with broth to a concentration of 10. 5 ~10 6 CFU / mL. Melitoxin and its analogues were dissolved in sterile broth to obtain a stock solution with a concentration of 512 μg / mL. Test concentrations were prepared by two-fold dilution. 50 μL of each of the continuously diluted melitoxin and its analogue solutions and 50 μL of indicator bacteria dilution were added to sterile 96-well plates, with three replicates for each concentration. The plates were incubated at 37°C for 18 h. The clarity of the solution was observed visually; if indicator bacteria were present, the solution would become turbid. The concentration of melitoxin and its analogues corresponding to the clear well at the boundary between turbidity and clarity was the minimum inhibitory concentration (MIC). Sterile MH broth was used as a negative control, and polymyxin and vancomycin were used as positive controls. The results are shown in Table 3.

[0043] Table 3. MIC values ​​of melitoxin and melitoxin analogues

[0044]

[0045] As shown in Table 3, vancomycin has a significant antibacterial effect against Gram-positive bacteria (MIC value of 1-2 μg / mL), but its antibacterial effect against Gram-negative bacteria is not obvious (MIC value of 64-512 μg / mL); colistin sulfate has the opposite antibacterial effect, with a significant antibacterial effect against Gram-negative bacteria (MIC value of 1-8 μg / mL). Neither of them has broad-spectrum antibacterial activity.

[0046] Melitoxin analogues exhibited broad-spectrum antibacterial activity. Among them, MEL-H7,19,21,22,23,24 showed significantly better antibacterial effects (MIC values ​​of 2–8 μg / mL) against Gram-negative bacteria such as Escherichia coli, Pasteurella canis, Haemophilus felis, and Campylobacter coli than the parent melitoxin (MIC value of 32–64 μg / mL). Their antibacterial effects against Gram-positive bacteria such as Streptococcus canis and Staphylococcus pseudintermedius (MIC values ​​of 2–4 μg / mL) were also significantly better than the parent melitoxin (MIC value of 32–128 μg / mL). This indicates that hydroxyphosphorylation of 5-hydroxytryptophan, by simultaneously replacing amino acids at the pepsin and trypsin cleavage sites on bee venom peptides, can significantly improve the antibacterial activity of the peptides and exhibit good antibacterial effects against both Gram-positive and Gram-negative bacteria. In particular, MEL-H7,19,21,22,23,24 showed the best broad-spectrum antibacterial activity (MIC values ​​of 2–8 μg / mL), making them suitable for the preparation of antibacterial drugs for pets and possessing very broad application prospects.

[0047] Example 2: Evaluation of the hemolytic activity of melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids

[0048] Red blood cells were isolated from beagle blood and washed 4–5 times with phosphate-buffered saline (PBS, pH 7.4). The washed red blood cells were dispersed in PBS to prepare a 0.25% (v / v) red blood cell suspension. Meliostein and meliostein analogues were dissolved in PBS and diluted to a stock solution of 512 μg / mL. The stock solution was then diluted 2-fold with PBS to obtain the working solution. Equal volumes of red blood cell suspension and different concentrations of meliostein and meliostein analogue working solutions were added to 96-well plates. Triton X-100 solution (1%, v / v 20 μL) was used as a positive control, and the red blood cell PBS solution served as a negative control. The 96-well plates were incubated at 37°C for 1 hour and then centrifuged at 3000 rpm at 4°C for 10 minutes. The supernatant was carefully transferred to new wells, and the absorbance was measured at 490 nm. The hemolysis rate was calculated using the following formula:

[0049]

[0050] All experiments were repeated three times, and the results were as follows: Figure 7 As shown.

[0051] Melipotine itself exhibits high hemolytic activity; the MEL prepared in Comparative Example 1 showed a hemolytic activity of 99.63%, which is unfavorable for the clinical application of melipotine. Replacing the amino acids in the melipotine peptide with hydroxyphosphorylated 5-hydroxytryptophan alters the peptide's conformation, making it difficult for the melipotine analog to bind to the lipid membrane of erythrocytes, thus reducing hemolytic activity. Experimental results showed that within a concentration range of 256–0.5 g / mL, the hemolytic activity of the parent melipotine ranged from 11.41% to 99.63%, revealing that melipotine possesses strong hemolytic activity. The hemolytic activities of melivexin analogues are as follows: within the concentration range of 256–0.5 g / mL, the hemolytic activity of melivexin analogue MEL-H19 is 10.49–99.97%, that of MEL-H22,24 is 7.94–84.58%, that of MEL-H7,21,23 is 2.68–78.33%, that of MEL-H7,21,22,23,24 is 0–43.53%, and that of MEL-H7,19,21,22,23,24 is 0–32.26%. These results show that the analogues MEL-H7,19,21,22,23,24 have lower hemolytic activity and less cytotoxicity, indicating broad application prospects.

[0052] Example 3: Stability evaluation of melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in pepsin solution.

[0053] The stability of melittin and its analogues in pepsin solution was determined by high-performance liquid chromatography (HPLC). According to the Pharmacopoeia of the People's Republic of China (2020 edition), 16.4 mL of dilute hydrochloric acid was mixed with approximately 800 mL of water and 10 g of pepsin, then transferred to a 1000 mL volumetric flask and diluted to the mark with ultrapure water to obtain artificial gastric fluid. The melittin and its analogues were dissolved and diluted with the artificial gastric fluid to a concentration of 200 μg / mL, and then placed in a 37℃ water bath. Samples were taken at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. After inactivating the protease by bathing the samples in an 85℃ water bath for 5 min, they were centrifuged at 10000 rpm and 4℃ for 15 min. The peak areas of melittin and its analogues at different time points were detected, and the degradation rate was calculated. The results are shown below. Figure 8 As shown.

[0054] pass Figure 8It can be seen that the degradation rate of melittin in pepsin solution is generally higher than that of most melittin analogs. Specifically, after incubation in pepsin solution for 0.5–6 hours, the degradation rate of melittin ranged from 44.03% to 98.99%; while the degradation rates of MEL-H19 ranged from 1.86% to 9.59%, MEL-H22,24 from 33.80% to 96.83%, MEL-H7,21,23 from 8.33% to 83.36%, MEL-H7,21,22,23,24 from 9.77% to 80.13%, and MEL-H7,19,21,22,23,24 from 0% to 0.82%. This indicates that replacing the amino acid at the pepsin cleavage site with hydroxyphosphorylated 5-hydroxytryptophan can significantly improve the pepsin resistance of melittin.

[0055] Example 4: Stability evaluation of melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in trypsin solution.

[0056] The stability of melittin and its analogues in trypsin solution was determined by high performance liquid chromatography (HPLC). According to the Pharmacopoeia of the People's Republic of China (2020 edition), 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution was dissolved in 118 mL of 0.2 mol / L sodium hydroxide solution. Separately, 10 g of trypsin was dissolved in water. The two solutions were mixed and transferred to a 1000 mL volumetric flask, then diluted to the mark with water to obtain the trypsin solution. The melittin and its analogues were dissolved and diluted with trypsin solution to a concentration of 200 μg / mL. The solutions were placed in a 37℃ water bath, and samples were taken at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. After inactivating the protease by incubating the samples in an 85℃ water bath for 5 min, they were centrifuged at 10000 rpm and 4℃ for 15 min. The peak areas of melittin and its analogues at different time points were detected, and the degradation rate was calculated. The results are shown below. Figure 9 As shown.

[0057] pass Figure 9It can be seen that the degradation rate of melittin in trypsin solution is generally higher than that of most melittin analogs. Specifically, after incubation in pepsin solution for 0.5–6 h, the degradation rate of melittin ranged from 78.03% to 97.78%; while the degradation rates after incubation with MEL-H19 ranged from 68.87% to 96.17%, MEL-H22,24 from 2.86% to 23.74%, MEL-H7,21,23 from 0.73% to 13.56%, MEL-H7,21,22,23,24 from 0% to 3.15%, and MEL-H7,19,21,22,23,24 from 0% to 2.36%. This indicates that replacing the amino acid at the trypsin cleavage site with hydroxyphosphorylated 5-hydroxytryptophan can significantly improve the trypsin resistance of melittin.

[0058] Example 5: Stability evaluation of melitoxic peptide analogs containing hydroxyphosphorylated non-natural amino acids in plasma

[0059] To assess the stability of melitoxic peptides and their analogues in canine plasma, the melitoxic peptides obtained in Comparative Example 1 and the melitoxic peptide analogues obtained in Examples 1-5 were mixed with 10% canine plasma and incubated at 37°C. Samples were taken at 0, 0.5, 1, 2, 3, 4, 5, and 6 hours. After inactivating plasma proteins by bathing in an 85°C water bath for 5 minutes, the samples were centrifuged at 10,000 rpm and 4°C for 15 minutes. The peak areas of the melitoxic peptides and their analogues at different time points were measured, and the degradation rate was calculated. The results are shown below. Figure 10 As shown.

[0060] pass Figure 10 It can be seen that the degradation rate of melittin in plasma is generally higher than that of most melittin analogues. Specifically, after incubation in plasma for 0.5–6 hours, the degradation rate of melittin ranged from 14.37% to 68.45%; while the degradation rates of MEL-H19 ranged from 19.87% to 68.39%, MEL-H22,24 from 3.53% to 49.33%, MEL-H7,21,23 from 3.63% to 48.56%, MEL-H7,21,22,23,24 from 0% to 23.15%, and MEL-H7,19,21,22,23,24 from 0% to 11.69%. This indicates that replacing the amino acids at the pepsin and trypsin cleavage sites with hydroxyphosphorylated 5-hydroxytryptophan can significantly improve the plasma stability of melittin.

[0061] The above are merely embodiments of the present invention. The invention is not limited to the fields covered by these embodiments. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can improve and implement this solution based on the guidance provided in this application and their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness or practicality of the invention. The scope of protection claimed in this application should be determined by the content of its claims. The specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids, characterized in that: The structure of the melitoxin analogue is as follows: GIGAVLKVLTTGLPALIS-(5-HTP-H2PO3)-IKRKRQQ; or, GIGAVLKVLTTGLPALISWIK-(5-HTP-H2PO3)-K-(5-HTP-H2PO3)-QQ; or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALISWI-(5-HTP-H2PO3)-R-(5-HTP-H2PO3)-RQQ; Or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALISWI-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-QQ; Or, GIGAVL-(5-HTP-H2PO3)-VLTTGLPALIS-(5-HTP-H2PO3)-I-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-(5-HTP-H2PO3)-QQ.

2. The method for preparing bee venom peptide analogs containing hydroxyphosphorylated non-natural amino acids as described in claim 1, characterized in that: The amino acids at the pepsin cleavage site and / or trypsin cleavage site of melipotoxins are replaced with hydroxyphosphorylated non-natural amino acids.

3. The preparation method according to claim 2, characterized in that: The non-natural amino acids at the pepsin cleavage site and trypsin cleavage site of melitricein were replaced with hydroxyphosphorylated 5-hydroxytryptophan.

4. The use of the bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids as described in claim 1 in the preparation of antibacterial drugs or antibacterial compositions, wherein the bacteria are one or more of Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter, Streptococcus canis, and Staphylococcus pseudointermediate.

5. The use of the bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids as described in claim 1 in the preparation of pet antibacterial feed additives or antibacterial feed additive compositions, wherein the bacteria are one or more of Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter, Streptococcus canis, and Staphylococcus pseudointermediate.

6. The use of the bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids as described in claim 1 in the preparation of anti-inflammatory eye drops or anti-inflammatory eye drop compositions for pets, wherein the inflammation is caused by one or more of Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter, Streptococcus canis, and Staphylococcus pseudointermediate.

7. The application of the bee venom peptide analog containing hydroxyphosphorylated non-natural amino acids as described in claim 1 in the preparation of pet topical antibacterial sprays or pet topical antibacterial compositions, wherein the bacteria are one or more of Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter, Streptococcus canis, and Staphylococcus pseudointermediate.

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