East asian bithoracuspis scorpion polypeptide with anti-ev71 virus activity and application thereof

Abstract

The application discloses a Buthus mathaii polypeptide with anti-EV71 virus activity and application thereof, and belongs to the technical field of traditional Chinese medicinal material polypeptides. The Buthus mathaii polypeptide with anti-EV71 virus activity is Pep 15 or Pep 16; the amino acid sequence of Pep 15 is shown as SEQ ID NO. 1; and the amino acid sequence of Pep 16 is shown as SEQ ID NO. 2. The Buthus mathaii polypeptide obtained by the application has significant anti-EV71 virus activity, can inhibit the cytopathic effect of EV71 virus on host cells Vero, increase the survival rate of infected cells, inhibit the replication and proliferation of EV71 virus in cells, has no toxic effect on cells when the concentration of the Buthus mathaii polypeptide reaches 200 muM, has the potential to be used as an anti-EV71 virus drug, and has a good application prospect in the development of a new drug for preventing and treating infantile hand-foot-mouth disease induced by EV71 virus.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology of traditional Chinese medicine, specifically relating to an East Asian scorpion polypeptide with anti-EV71 virus activity and its application. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a global infectious disease caused by various enteroviruses, primarily affecting children under 5 years old, and has become a significant public health challenge. Some severe cases can involve the nervous, circulatory, and respiratory systems, posing a serious threat to the lives of infected children. Enterovirus 71 (EV71), belonging to the genus Enterovirus in the family Picornaviridae, is a representative of non-enveloped RNA viruses. Its viral particles consist of an icosahedral capsid composed of VP1, VP2, and VP3 proteins, enclosing a 7.5kb positive-sense RNA chain. It is the main pathogen causing HFMD. EV71 infection is highly contagious and rapidly progressing clinically, exhibiting significant neurotropism. Infection easily induces severe illness, and severe cases may lead to complications such as brainstem encephalitis, aseptic meningitis, and pulmonary complications. The case fatality rate is approximately 3.1%. Currently, there are no specific antiviral drugs for EV71 in clinical practice. Treatment often involves combination therapy with interferon, ribavirin, and methylprednisolone, but these methods have limited efficacy against severe HFMD caused by EV71. Therefore, developing novel specific anti-EV71 drugs is of great significance for the prevention and treatment of HFMD.

[0003] East Asian pincers ( Buthus martensii The dried whole scorpion (Karsch. scorpion) is the base material of the traditional animal medicine "whole scorpion." The 2025 edition of the Chinese Pharmacopoeia records its effects of "relieving wind and spasms, attacking toxins and dispersing nodules, and unblocking meridians and relieving pain." Recent studies have shown that venomous peptides derived from scorpion species have inhibitory activity against various viruses, including influenza virus, dengue virus, hepatitis C virus, herpes simplex virus, and human immunodeficiency virus. Research indicates that the antimicrobial peptide BmKn2 and its derivative BmKn2-T5, derived from the East Asian scorpion, can significantly inhibit EV71 virus replication in vitro. This inhibitory effect mainly occurs in the early stages of the viral life cycle. Antimicrobial peptides from scorpions with different structures or properties exhibit different pharmacological activities in EV71 virus infection. Currently, there are no reports on the screening, identification, and preparation of single active peptide compounds based on East Asian scorpion peptide-omics strategies, and on the systematic evaluation of their anti-EV71 virus activity. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an East Asian scorpion polypeptide with anti-EV71 virus activity and its application. The East Asian scorpion polypeptide has a good inhibitory effect on EV71 virus replication, providing a theoretical basis for its biomedical research and clinical application.

[0005] This invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides an East Asian scorpion polypeptide with anti-EV71 virus activity, wherein the East Asian scorpion polypeptide with anti-EV71 virus activity is Pep 15 or Pep 16.

[0007] The amino acid sequence of Pep 15 is shown in SEQ ID NO.1;

[0008] The amino acid sequence of Pep 16 is shown in SEQ ID NO.2.

[0009] Furthermore, the East Asian scorpion polypeptide with anti-EV71 virus activity was synthesized using a solid-phase synthesis method.

[0010] In a second aspect, the present invention provides the application of the East Asian scorpion polypeptide with anti-EV71 virus activity in the preparation of anti-EV71 virus drugs.

[0011] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0012] The East Asian scorpion polypeptide (Pep 15 or Pep 16) obtained in this invention has significant anti-EV71 virus activity, inhibits the cytopathic effect (CPE) produced by EV71 virus on host cells Vero, increases the survival rate of infected cells, inhibits the replication and proliferation of EV71 virus in cells, and has no cytotoxic effect on cells at a concentration of 200 μM. It has the potential to be used as an anti-EV71 virus drug and has good application prospects in the development of new drugs for the prevention and treatment of hand-foot-mouth disease induced by EV71 virus in infants and young children. Attached Figure Description

[0013] Figure 1 The graph shows the inhibition rate of different East Asian scorpion peptides against EV71 virus.

[0014] Figure 2 HPLC and MS spectra of Pep 15, a polypeptide from the East Asian scorpion: (a) HPLC spectrum, (b) MS spectrum.

[0015] Figure 3 HPLC and MS spectra of Pep 16, a polypeptide from the East Asian scorpion: (a) HPLC spectrum, (b) MS spectrum.

[0016] Figure 4 The results of different concentrations of East Asian scorpion peptides Pep 15 and Pep 16 on Vero cells are shown in the figure. (a) Pep 15, (b) Pep 16.

[0017] Figure 5 The results of the inhibition rate of different concentrations of East Asian scorpion peptides Pep 15 and Pep 16 against EV71 virus are shown in the figure. (a) Pep 15, (b) Pep 16.

[0018] Figure 6 The images show the inhibitory effects of Pep 15 and Pep 16 peptides from the East Asian scorpion on EV71 virus under a light microscope. (a) Normal group, (b) EV71 virus group, (c) Pep 15, (d) Pep 16.

[0019] Figure 7 This is a plaque observation image showing the inhibitory effect of East Asian scorpion polypeptides Pep 15 and Pep 16 on EV71 virus. Detailed Implementation

[0020] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0022] In a specific embodiment of the present invention, the EV71 virus was donated by Shandong First Medical University (Shandong Academy of Medical Sciences), with GenBank accession number JN200804.

[0023] Example 1

[0024] Preparation of East Asian scorpion polypeptide:

[0025] (1) Preparation of East Asian scorpion polypeptide extract

[0026] 100 mg of East Asian scorpion slices were ground evenly with liquid nitrogen, and then lysis buffer (10% glacial acetic acid aqueous solution (V:V)) was added and ground. Protein was extracted by ultrasonic centrifugation, filtered through a 0.22 μm aqueous phase filter membrane, desalted by SPE column, and lyophilized to obtain East Asian scorpion polypeptide extract.

[0027] The desalting process is as follows: activate the desalting column with 100% acetonitrile, equilibrate the column with 0.1% TFA aqueous solution, load the column with 10% glacial acetic acid aqueous solution containing peptides, wash the residual salts with 0.1% TFA aqueous solution, and finally elute with 70% acetonitrile. Collect the eluent to complete the desalting process.

[0028] (2) Identification and analysis of peptides from East Asian scorpion venom

[0029] 1) Take the East Asian scorpion polypeptide extract from step (1), and use nanoElute 2 nanoliter liquid chromatography, timsTOFPro2 mass spectrometry, and BPS Novor library search analysis to detect the polypeptide. Based on Local Confidence ≥ 85% and Score ≥ 90, the polypeptides are screened. NCBI and the online website PeptideRanker are used to predict the bioactivity probability of the peptides, ToxIBTL is used to predict the toxicity of the peptides, and Algpred2.0 is used to predict the sensitization. The polypeptides are further screened to obtain candidate polypeptides.

[0030] The nanoElute 2-nanometer liquid chromatography column was a C18 column (20 cm x 75 μm, 1.3 μm). The chromatographic conditions were as follows: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution; flow rate: 300 nL / min; gradient elution; elution program: 0–45 min 2–22% mobile phase B, 45–50 min 22–37% mobile phase B, 50–55 min 37–80% mobile phase B, 55–60 min 80% mobile phase B.

[0031] The mass spectrometry conditions for the timsTOFPro2 mass spectrometry analysis were as follows: timsTOFPro2 mass spectrometer; CaptiveSpray nano-spray ion source, ion spray voltage of 1.5 kV, full scan range of m / z 100-1700, and ion mobility of 0.60-1.60 V·s / cm. 2 The standard 10 ddaPASEF scanning protocol is run in dda-PASEF mode;

[0032] The search parameters for BPS Novor are as follows: Enzyme: Non-specific cleavage; Peptide Mass Tolerance: 20 ppm; Fragment Mass Tolerance: 0.02 Da; Missing cutting number: 2.

[0033] A total of 121 candidate peptides were obtained by simultaneously meeting the criteria of Local Confidence ≥ 85% and Score ≥ 90. Further comparison using NCBI identified 39 free peptides. Using online platforms (Peptide Ranker to predict peptide bioactivity probability, Tox IBTL to predict peptide toxicity, and Algpred 2.0 to predict sensitization), 25 candidate peptides were selected. Information on these 25 candidate peptides is shown in Table 1 below.

[0034] Table 1. Information on candidate peptides obtained from screening in Example 1

[0035] .

[0036] Example 2

[0037] The 25 candidate peptides screened in Example 1 were synthesized using a solid-phase synthesis method:

[0038] The solid-phase synthesis method described above is as follows:

[0039] (1) Solvent treatment: DMF and methanol were soaked overnight in G3 pore molecular sieve before use to remove impurities and water;

[0040] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for 30 min;

[0041] (3) Adding the first amino acid: At room temperature, filter out the solvent from step (2) through a sand core filter, add 1 mmol of 5 times the molar excess of the first C-terminal amino acid, 5 times the molar excess of DMAP, 5 times the molar excess of DIC, and DMF as the solvent. React at room temperature for 3 hours. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time. Then add a pyridine and acetic anhydride mixture with a volume ratio of 1:1 and react for 30 minutes. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time.

[0042] (4) Removal of Fmoc protecting group: Remove the solvent in step (3) by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, then stir with DMF 4 times and wash with methanol 2 times, 5-6 mL each time.

[0043] (5) Detection of ninhydrin removal effect: Take 15 mg of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105-110 ℃ for 5 min. If it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.

[0044] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second C-terminal amino acid, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, then add 10 times the molar excess of DIEA. React at room temperature for 40 min, and wash with DMF 5 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns blue, proceed to the next step of the reaction.

[0045] (7) Repeat step (6) in this manner until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry the mixture.

[0046] (8) Resin shedding and pure product separation detection: The peptide was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 hours. The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysate was dried as much as possible with nitrogen gas, then precipitated with ether, centrifuged, and then washed with ether 4 times to obtain a white solid. After being dissolved in pure water, it was desalted and purified by HPLC. After freeze-drying, crystals were precipitated to obtain the target peptide.

[0047] The 25 candidate peptides in Table 1 were all synthesized using the methods described in steps (1) to (8) above.

[0048] Example 3

[0049] Detection of the anti-EV71 virus activity of East Asian scorpion peptides

[0050] (1) The East Asian scorpion peptides Pep 1 to Pep 25 synthesized in Example 2 were dissolved in a small amount of DMSO, and PBS buffer was added to prepare a 10 mM East Asian scorpion peptide solution. The solution was filtered through a 0.22 μm microporous membrane for sterilization, dispensed, and stored at -80 ℃.

[0051] (2) Vero cells were cultured in DMEM high glucose culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 ℃ and 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for subsequent experiments.

[0052] (3) Vero cells in the logarithmic growth phase were digested with 0.25% trypsin and then seeded into 96-well plates at a density of 1×10⁶ cells / well. 4 Cells were cultured per well until a monolayer was formed. The culture medium was discarded, and the cells were washed three times with PBS. 100 μL of cell maintenance medium containing East Asian scorpion peptides (DMEM high-glucose medium containing 2% fetal bovine serum and 1% penicillin-streptomycin) was added to each well, followed by 100 μL of 100 TCID50 solution. 50 The final concentration of EV71 virus suspension containing Pep 1 to Pep 25 of the East Asian scorpion peptide was 2.5 μM. Each candidate peptide was repeated in triplicate and cultured for 48 h. A normal cell control group (normal group, without East Asian scorpion peptide and EV71 virus), an EV71 virus control group (without East Asian scorpion peptide), and a blank control group were also included. 96-well plates were incubated at 37 ℃ and 5% CO2 for 48 h. Cell CPE (cytopathic effect) was continuously observed under a microscope. OD values ​​were measured using a CCK-8 staining assay and the EV71 virus inhibition rate was calculated using the following formula.

[0053] Viral inhibition rate (%) = (OD value of experimental group - OD value of virus control group) / (OD value of normal cell control group - OD value of virus control group) × 100%;

[0054] The inhibition rates of different East Asian scorpion peptides against EV71 virus are shown in the figure below. Figure 1 As shown, by Figure 1 It can be seen that the inhibitory effects of different East Asian scorpion peptides on EV71 virus are significantly different. Among them, Pep 15 and Pep 16 have the highest inhibition rate on EV71 virus, showing good anti-EV71 virus activity. Pep 15 and Pep 16 were selected as East Asian scorpion antiviral peptides for subsequent experiments.

[0055] The amino acid sequence (SEQ ID NO.1) of the East Asian scorpion polypeptide Pep 15 is LNWGNSAGNK, and its HPLC and MS chromatograms are shown below. Figure 2 As shown, (a) is the HPLC chromatogram and (b) is the MS chromatogram; the amino acid sequence (SEQ ID NO.2) of the East Asian scorpion polypeptide Pep 16 is LLFLKDHQY, and its HPLC and MS chromatograms are shown below. Figure 3 As shown, (a) is the HPLC chromatogram and (b) is the MS chromatogram.

[0056] Example 4

[0057] Cytotoxicity assays of East Asian scorpion peptides (Pep 15, Pep 16)

[0058] Vero cells in logarithmic growth phase were digested with 0.25% trypsin and seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells were seeded per well. Once the cells had grown into a confluent monolayer, the culture medium was discarded. Pep 15 and Pep 16 were added to 96-well plates, with 5 wells seeded for each dilution as the experimental group. The final concentrations were 12.5, 25, 50, 100, and 200 μM, respectively. A normal cell control group and a blank control group were also set up. Each well contained 100 μL of culture medium. The plates were incubated at 37°C in a 5% CO2 incubator. After 48 hours of incubation, the OD value was measured using the CCK8 method, and the cell viability was calculated using the following formula.

[0059] Cell viability (%) = (OD value of experimental group - OD value of blank control group) / (OD value of normal cell control group - OD value of blank control group) × 100%;

[0060] The results of the cytotoxicity of different concentrations of East Asian scorpion peptides Pep 15 and Pep 16 on Vero cells are shown in the figure below. Figure 4 As shown, (a) represents Pep 15, and (b) represents Pep 16; by Figure 4 It can be seen that the cell survival rate of each concentration treatment group did not change significantly compared with the normal cell control group, indicating that Pep 15 and Pep 16 have no cytotoxic effect on Vero cells at concentrations of 12.5~200uM.

[0061] Example 5

[0062] Inhibition rate of different concentrations of East Asian scorpion peptides (Pep 15, Pep 16) against EV71 virus

[0063] Vero cells in logarithmic growth phase were digested with 0.25% trypsin and seeded in 96-well plates at a density of 1×10⁶ cells / well. 4 Cells were cultured per well until a confluent monolayer was formed. The culture medium was discarded, and the cells were washed three times with PBS. 100 μL of 2% maintenance medium (DMEM high-glucose medium containing 2% fetal bovine serum and 1% penicillin-streptomycin) containing Pep 15 and Pep 16 were added to each well, followed by 100 μL of 100 TCID50 solution. 50EV71 virus suspension was prepared with Pep 15 and Pep 16 at final concentrations of 2.5, 1.25, and 0.625 μM, respectively, with three replicates per concentration. The cells were cultured for 48 hours, and a normal cell control group, an EV71 virus control group, and a blank control group were also included. The 96-well plates were incubated at 37 °C in a 5% CO2 incubator, and cell cytotoxicity (CPE) was continuously observed under a microscope. Cellular inhibition rate was calculated using CCK-8 staining and an ELISA reader, following the same formula as in Example 4.

[0064] The inhibition rates of different concentrations of East Asian scorpion peptides Pep 15 and Pep 16 against EV71 virus are shown in the figure below. Figure 5 As shown, (a) represents Pep 15, and (b) represents Pep 16; by Figure 5 It can be seen that different concentrations of Pep 15 and Pep 16 have inhibitory effects on EV71 virus, and there are significant differences between low and high concentrations, with the inhibitory trend showing a concentration-dependent effect.

[0065] Example 6

[0066] The ameliorative effect of East Asian scorpion polypeptides (Pep 15, Pep 16) on EV71 virus-induced cytopathic effects

[0067] Vero cells in the logarithmic growth phase were digested with trypsin and then seeded into 6-well plates at a density of 5 × 10⁶ cells / well. 5 Cells were cultured in 2% maintenance medium containing 2.5 μM Pep 15 and Pep 16 for 1 hour, then the medium was discarded and the cells were washed twice with PBS. The drug group (Pep 15 and Pep 16) and the EV71 virus group were incubated in 100 μL of medium containing 100 TCID50. 50 Vero cells were infected with EV71 virus suspension for 1 h, the culture medium was discarded, the cells were washed twice with PBS, and 100 μL of 2% maintenance medium was added. At the same time, a normal cell control group (normal group) was set up. The cells were cultured for another 48 h, and the cell cytopathic effect (CPE) was observed and photographed under a microscope.

[0068] The inhibitory effect of East Asian scorpion polypeptides Pep 15 and Pep 16 on the pathological effects of EV71 virus, such as... Figure 6 As shown, (a) is the normal group, (b) is the EV71 virus group, (c) is Pep 15, and (d) is Pep 16; Figure 6It can be seen that the cells in the normal group are spindle-shaped, triangular or polygonal, adhere well to the wall, and have tight intercellular connections; the cells in the EV71 virus group are shrunken and rounded, the intercellular spaces are enlarged, and some cells detach and die; compared with the EV71 virus group, the Middle Eastern scorpion polypeptides Pep 15 and Pep 16 in the experimental group can effectively reduce the cytopathic effects caused by EV71 virus, and the cell morphology of most cells tends to be normal.

[0069] Example 7

[0070] Inhibitory effect of East Asian scorpion peptides (Pep 15, Pep 16) on EV71 virus replication

[0071] EV71 virus infects cells, replicates, and releases progeny viruses. When a semi-solid coating covers the cell surface, the progeny viruses infect only neighboring cells. After several replication cycles, this forms a localized lesion area, known as a viral plaque or vacuole. Therefore, when drugs inhibit viral replication, they reduce the formation of viral plaques. Vero cells were seeded in 24-well plates at 2 × 10⁶ cells / well. 5 Cells per well, allowed to grow into a confluent monolayer for later use; 2-fold dilution of Pep 15 and Pep 16 and 1000 TCID2. 50 EV71 virus was thoroughly mixed, and 500 μl was added to each well of a 24-well plate. After incubation for 1 h, the drug and virus mixture was removed, and 500 μl of serially diluted Pep 15 and Pep 16 (containing 0.8% agar and 2% FBS) were added to each well. The final concentrations of Pep 15 and Pep 16 were 2.5 and 1.25 μM, respectively. An EV71 virus control group and a normal cell control group were set up, and the cells were incubated for another 48 h. 500 μl of 4% paraformaldehyde fixative was added to each well of the 24-well plate, and the cells were fixed at room temperature for 1 h. The upper covering material in the cell wells was removed, crystal violet staining solution was added, and the cells were incubated at room temperature for 30 min. After rinsing with water, the cells were photographed and the empty spots were counted on an LED light box. The empty spot observation images are shown below. Figure 7 As shown; Figure 7 As shown, the normal cell control group was stained evenly, showing a uniform blue-purple color. The EV71 virus group had the largest blank area and the most empty plaques. After treatment with different concentrations of peptides Pep 15 and Pep 16, the number of empty plaques was significantly reduced compared to the virus group. Pep 15 and Pep 16 had a significant inhibitory effect on viral replication.

Claims

1. A scorpion polypeptide with anti-EV71 virus activity, characterized in that, The East Asian scorpion polypeptide with anti-EV71 virus activity is Pep 16; The amino acid sequence of Pep 16 is shown in SEQ ID NO.

2.

2. The East Asian scorpion polypeptide with anti-EV71 virus activity according to claim 1, characterized in that, The East Asian scorpion polypeptide with anti-EV71 virus activity was synthesized using a solid-phase synthesis method.

3. The application of an East Asian scorpion polypeptide with anti-EV71 virus activity in the preparation of anti-EV71 virus drugs, characterized in that, The East Asian scorpion polypeptide with anti-EV71 virus activity is Pep 15 or Pep 16. The amino acid sequence of Pep 15 is shown in SEQ ID NO.1; The amino acid sequence of Pep 16 is shown in SEQ ID NO.2.

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