An East Asian scorpion venom polypeptide with anti-EV71 virus activity, its preparation method and application

Abstract

This invention discloses an anti-EV71 virus venom polypeptide from the Asian scorpion, its preparation method, and its application, belonging to the field of antiviral formulation technology. Crude venom from the Asian scorpion was obtained through scorpion tail electrostimulation, and then lyophilized and freeze-dried to obtain lyophilized powder. The lyophilized powder was dissolved, filtered, sediment removed, and ultrafiltered and centrifuged to separate the EV71 virus-resistant venom polypeptide. The venom polypeptides S-Ⅰ (<3 kDa) and S-Ⅱ (3~10 kDa) obtained from the Asian scorpion in this invention exhibit good anti-EV71 virus activity. At the same concentration, the venom polypeptide with a relative molecular mass <3 kDa shows better anti-EV71 virus activity and is non-toxic to cells, demonstrating potential as an anti-EV71 virus drug and providing a theoretical basis for its application in biomedical research and clinical practice.

Description

Technical Field

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

[0002] Hand, foot, and mouth disease (HFMD) is one of the key infectious diseases requiring strengthened prevention and control. It is an acute intestinal infectious disease caused by various enteroviruses. Some cases can rapidly induce complications in the central nervous system and respiratory system, resulting in high mortality and disability rates in severe cases. Enterovirus 71 (EV71), belonging to the Picornaviridae family and Enterovirus genus, is one of the main pathogenic microorganisms of HFMD and a major cause of severe cases. Currently, clinical treatment for HFMD often involves combination therapy with interferon, ribavirin, and methylprednisolone. However, the efficacy against severe HFMD caused by EV71 is poor, making the search for new, specific anti-EV71 drugs crucial for the prevention and treatment of HFMD.

[0003] Whole scorpion refers to the dried body of *Butkus martensii* Karsch, an animal of the family Scorpionidae. According to the pharmacopoeia, whole scorpion possesses the effects of "relieving wind and spasms, attacking toxins and dispersing nodules, unblocking meridians and relieving pain," and is used for infantile convulsions, spasms, stroke with facial paralysis, hemiplegia, tetanus, stubborn rheumatism, migraines, sores, scrofula, etc. Modern research shows that scorpion venom polypeptides derived from various scorpions have inhibitory effects on various viruses, including influenza, dengue fever, hepatitis C, herpes simplex virus, and HIV. Zhiqiang Xia et al. (Zhiqiang Xia, Huijuan Wang, Weilie Chen, et al. 2024. Scorpion Venom Antimicrobial Peptide Derivative BmKn2-T5 Inhibits Enterovirus 71 in the Early Stages of the Viral Life Cycle In Vitro[J]. Biomolecules, 14,545) studied the antiviral activity of scorpion venom antimicrobial peptide BmKn2 and its five derivatives derived from *M. martensii*. They found that BmKn2 and its derivative BmKn2-T5 had a significant inhibitory effect on EV71. They also studied the inhibitory activity of other scorpion-derived antimicrobial peptides against EV71 infection using the CPE reduction method. Compared with BmKn2 and its derivative BmKn2-T5, no other scorpion-derived antimicrobial peptides showed significant antiviral activity against EV71 infection, indicating that scorpion-derived antimicrobial peptides with different structures or properties exhibit different pharmacological activities in EV71 infection. There are no existing reports on the anti-EV71 virus activity of polypeptide components prepared from East Asian scorpion. Summary of the Invention

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

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

[0006] A method for preparing an East Asian scorpion venom polypeptide with anti-EV71 virus activity includes the following steps:

[0007] (1) Crude venom of East Asian scorpion was obtained by scorpion tail electrostimulation method, and freeze-dried powder of East Asian scorpion toxin was obtained by centrifugation and freeze-drying.

[0008] (2) The freeze-dried East Asian scorpion toxin powder in step (1) is dissolved, filtered, sludge is removed, and ultrafiltration centrifugation is performed to separate East Asian scorpion toxin polypeptide with anti-EV71 virus activity.

[0009] Furthermore, in step (2), ultrafiltration is performed using an ultrafiltration centrifuge tube with a relative molecular mass cutoff of 10 kDa to obtain East Asian scorpion venom polypeptide with a relative molecular mass <10 kDa.

[0010] Further, in step (2), the East Asian scorpion venom peptide with a relative molecular mass <10 kDa is ultrafiltered using an ultrafiltration centrifuge tube with a relative molecular mass cutoff of 3 kDa to obtain East Asian scorpion venom peptide with a relative molecular mass <3 kDa and East Asian scorpion venom peptide with a relative molecular mass between 3 and 10 kDa (excluding 10 kDa).

[0011] Furthermore, the conditions for ultrafiltration centrifugation in step (2) are: 8000~10000 rpm, 4 ℃, 15~30 min.

[0012] Furthermore, the centrifugation conditions in step (1) are 4 ℃, 2500~4000 rpm, and centrifugation for 30~40 min.

[0013] Further, in step (2), the freeze-dried powder of East Asian scorpion venom was dissolved in PBS buffer and filtered through a 0.22 μm filter membrane to remove sediment.

[0014] In this invention, the preparation method described herein yields an East Asian scorpion venom polypeptide with anti-EV71 virus activity.

[0015] Furthermore, the relative molecular mass of the East Asian scorpion venom polypeptide is <10 kDa.

[0016] Furthermore, the East Asian scorpion venom polypeptide is an East Asian scorpion venom polypeptide with a relative molecular mass <3 kDa or an East Asian scorpion venom polypeptide with a relative molecular mass of 3~10 kDa (excluding 10 kDa).

[0017] In this invention, the East Asian scorpion venom polypeptide with anti-EV71 virus activity is used in the preparation of anti-EV71 virus drugs.

[0018] Beneficial effects

[0019] The Scorpion venom polypeptides S-Ⅰ (<3 kDa) and S-Ⅱ (3 ~10 kDa) obtained from the Scorpion scorpion in this invention have good anti-EV71 virus activity. At the same concentration, the Scorpion venom polypeptide with a relative molecular mass of <3 kDa has better anti-EV71 virus activity and no cellular toxicity, and has the potential to be used as an anti-EV71 virus drug, providing a theoretical basis for its biomedical research and clinical application. Attached Figure Description

[0020] Figure 1 The figure shows the results of the Vero cell toxicity experiment of the East Asian scorpion venom polypeptide S-Ⅰ (<3 kDa);

[0021] Figure 2 The figure shows the results of the Vero cell toxicity experiment of East Asian scorpion venom polypeptide S-II (3 ~10 kDa);

[0022] Figure 3 A light microscope image showing the inhibitory effect of East Asian scorpion venom polypeptide S-Ⅰ (<3 kDa) on EV71 virus;

[0023] Figure 4 The light microscope image shows the inhibitory effect of East Asian scorpion venom polypeptide S-II (3-10 kDa) on EV71 virus.

[0024] Figure 5 The graph shows the inhibition rate of the East Asian scorpion venom polypeptide S-Ⅰ (<3 kDa) against EV71 virus.

[0025] Figure 6 The graph shows the inhibition rate of EV71 virus by East Asian scorpion venom polypeptide S-II (3 ~ 10 kDa). Detailed Implementation

[0026] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0027] Example 1

[0028] Preparation of East Asian scorpion venom peptides with anti-EV71 virus activity:

[0029] (1) Collection of crude venom from the East Asian scorpion and preparation of freeze-dried powder:

[0030] The venom was obtained using scorpion tail electrostimulation (Xu X, Duan Z, Di Z, et al. 2014. Proteomic analysis of the venom from the scorpion Mesobuthus martensii [J]. JProteomics, 106: 162-180), specifically as follows:

[0031] Take 6-7 year old scorpions and place them in a basin at a room temperature above 25 ℃. Operating steps for the electro-pulse venom extraction device: Turn on the device, adjust the voltage to 8-12 volts and the frequency to 128 Hz, insert the electrode wire and the harvesting frame wire into the corresponding sockets, and connect the power supply; moisten the tip of the electrode wire on the harvesting frame with physiological saline, grasp the last two segments of the scorpion's tail, and continuously contact the scorpion's pincers with the electrode to stimulate the scorpion to expel venom, collecting it in a 50 ml centrifuge tube placed on ice; centrifuge the collected venom at 4 ℃, 3000 rpm for 40 minutes to separate impurities such as dust and calcium, obtaining pure scorpion venom; freeze-dry under vacuum to obtain East Asian pincer scorpion venom freeze-dried powder, and store at low temperature for later use.

[0032] (2) Isolation of East Asian scorpion venom peptides with anti-EV71 virus activity:

[0033] 1) Take 100 mg of the freeze-dried powder of East Asian scorpion venom from step (1), add 10 ml of PBS to make a solution of 10 mg / mL, pre-filter with a 0.22 μm filter membrane to remove the sediment and keep it for later use, and obtain East Asian scorpion venom solution.

[0034] 2) Add ultrapure water to the 10 kDa ultrafiltration tube (with a molecular weight cutoff of 10 kDa) and the 3 kDa ultrafiltration tube (with a molecular weight cutoff of 3 kDa), ensuring the water completely passes through the membrane, and pre-cool in an ice bath for 5 minutes;

[0035] 3) Pour out the water from the 10 kDa ultrafiltration tube and add the East Asian scorpion venom solution prepared in step 1), ensuring it does not exceed the white line at the top of the ultrafiltration tube. Handle gently. Before adding the East Asian scorpion venom solution, the ultrafiltration tube must be pre-cooled in an ice bath. After adding the East Asian scorpion venom solution, centrifuge at 9000 rpm and 4 ℃ for 15 min. Carefully aspirate the liquid from the inner tube of the 10 kDa ultrafiltration centrifuge tube to obtain the fraction with a relative molecular mass >10 kDa. The liquid at the bottom of the centrifuge tube is the East Asian scorpion venom polypeptide with a relative molecular mass <10 kDa.

[0036] 4) Take the liquid from the bottom of the 10 kDa ultrafiltration centrifuge tube (relative molecular mass <10 kDa) and put it into the inner tube of the 3 kDa ultrafiltration tube. Centrifuge at 9000 rpm and 4 ℃ for 30 min. The liquid in the inner tube of the 3 kDa ultrafiltration tube is 3~10 kDa (excluding 10 kDa) East Asian scorpion venom polypeptide (S-II), and the liquid in the outer tube of the centrifuge tube is East Asian scorpion venom polypeptide (S-I) with a relative molecular mass <3 kDa.

[0037] 5) Store East Asian scorpion venom polypeptides (S-Ⅰ and S-Ⅱ) with different relative molecular weights at -80℃.

[0038] (3) Freeze-drying

[0039] The East Asian scorpion venom polypeptides (S-Ⅰ and S-Ⅱ) obtained in step (2) were concentrated and freeze-dried to obtain East Asian scorpion venom polypeptide freeze-dried powder.

[0040] Example 2

[0041] Detection of the anti-EV71 virus activity of East Asian scorpion venom peptides:

[0042] (1) Prepare a homogeneous solution by adding phosphate buffer to the freeze-dried East Asian scorpion toxin polypeptide powders S-Ⅰ and S-Ⅱ in Example 1, determine their concentration by BCA method, filter sterilize by 0.22 μm microporous membrane, dispense, and store at -80 ℃;

[0043] (2) CCK8 assay for the cytotoxicity of East Asian scorpion venom peptides:

[0044] Vero cells were cultured in DMEM containing 10% FBS at 37°C with 5% CO2. Vero cells in logarithmic growth phase were digested with trypsin and seeded into 96-well plates at 1 × 10⁶ cells / well. 4 Cells were seeded per well. Once the cells had grown into a monolayer, the culture medium was discarded. S-I and S-II were added to 96-well plates, with 3 wells seeded for each dilution as the experimental group. A normal cell control group and a blank control group were also set up. Each well contained 100 μL of culture medium as the final volume. The plates were incubated at 37 °C in a 5% CO2 incubator. After 72 h of incubation, the OD value was measured using the CCK8 method, and the cell viability was calculated.

[0045]

[0046] The cell survival rates of East Asian scorpion venom peptides (S-Ⅰ and S-Ⅱ) are shown in the following figures. Figure 1 and Figure 2As shown, the maximum non-toxic concentration of East Asian scorpion venom polypeptide S-Ⅰ (<3 kDa) is 50 μg / mL, and the maximum non-toxic concentration of East Asian scorpion venom polypeptide S-Ⅱ (3~10 kDa) is 100 μg / mL, which will be used as the maximum concentration for subsequent experiments.

[0047] (3) The ameliorative effect of East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ on EV71 virus-induced cytopathic effects

[0048] Vero cells in the logarithmic growth phase were digested with trypsin and then seeded into 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells were cultured in one well. Once the cells had grown into a monolayer, the culture medium was discarded, and the cells were washed three times with PBS. Vero cells were then infected with 100 TCID50 EV71 virus for 1 h. 2% FBS DMEM medium containing different concentration gradients of East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ was added, and the cells were cultured for another 72 h. A normal cell control group, an EV71 virus control group, and a blank control group were set up. When about 90% of the virus control wells showed CPE (cytopathic effect), the cytopathic effect was observed under a microscope.

[0049] A lesion rate of 0% indicates a CPE of "-".

[0050] CPE is marked as "+" between 1% and 25%;

[0051] CPE between 25% and 50% is judged as "++";

[0052] CPE levels between 50% and 75% are graded as "+++";

[0053] A CPE greater than 75% is indicated by "++++".

[0054] The light microscopic images of the inhibitory effects of East Asian scorpion venom polypeptide S-Ⅰ (<3 kDa) and East Asian scorpion venom polypeptide S-Ⅱ (3 ~10 kDa) on EV71 virus are shown below. Figure 3 (A: Normal cell control group, B: EV71 virus control group, C: S-I 3.125 μg / mL, D: S-I 6.25 μg / mL, E: S-I 12.5 μg / mL, F: S-I 25 μg / mL, G: S-I 50 μg / mL) and Figure 4As shown in the figure (A: normal cell control group, B: EV71 virus control group, C: S-II 6.25μg / mL, D: S-II 12.5μg / mL, E: S-II 25μg / mL, F: S-II 50μg / mL, G: S-II 100μg / mL), the cells in the normal cell control group showed normal morphology and good growth. The cells in the EV71 virus group were shrunken and rounded, with larger intercellular spaces, and some cells detached and died. Compared with the EV71 virus group, scorpion toxin peptides S-I 50μg / mL and S-II 100μg / mL could effectively inhibit EV71-induced cytopathic effects.

[0055] (4) Inhibitory effect of East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ on EV71 virus

[0056] Vero cells in the logarithmic growth phase were harvested, digested with trypsin, and seeded into 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells were cultured in 96-well plates until a monolayer was formed. The culture medium was discarded, and the cells were washed three times with PBS. Cell maintenance medium containing different concentration gradients of East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ was added, with five replicates for each dilution. Immediately afterward, 100 μL of virus suspension containing 100 TCID50EV71 was added, and the cells were cultured for approximately 72 hours. A normal cell control group, a virus control group, and a blank control group were also included. The plates were incubated at 37 ℃ in a 5% CO2 incubator, and cell cytotoxicity (CPE) was continuously observed under a microscope. Cells were stained with CCK-8, and OD values ​​were measured using a microplate reader. The virus inhibition rate was calculated.

[0057] The results of the inhibitory effects of East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ on EV71 virus are shown in the figures below. Figure 5 and Figure 6 As shown in the figure, both East Asian scorpion venom peptides S-Ⅰ and S-Ⅱ have inhibitory effects on EV71 virus. S-Ⅰ has a better inhibition rate against EV71 virus than S-Ⅱ, showing better anti-EV71 virus activity.

Claims

1. The application of an East Asian scorpion venom polypeptide with anti-EV71 virus activity in the preparation of anti-EV71 virus drugs, wherein the preparation method of the East Asian scorpion venom polypeptide includes the following steps: (1) The crude venom of the East Asian scorpion was obtained by the scorpion tail electrostimulation method, and centrifuged at 4℃ and 2500~4000 rpm for 30~40 min, and then freeze-dried to obtain the freeze-dried powder of East Asian scorpion toxin. (2) The freeze-dried East Asian scorpion venom powder in step (1) was dissolved in PBS buffer, filtered through a 0.22 μm filter membrane to remove sediment, and then subjected to ultrafiltration centrifugation. Ultrafiltration was performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to obtain East Asian scorpion venom peptides with a molecular weight cutoff of <10 kDa. The East Asian scorpion venom peptides with a molecular weight cutoff of <10 kDa were subjected to ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain East Asian scorpion venom peptides with a molecular weight cutoff of <3 kDa and East Asian scorpion venom peptides with a molecular weight cutoff of 3~10 kDa. The ultrafiltration centrifugation conditions were: 8000~10000 rpm, 4℃, 15~30 min.

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