EV71 virus activity resistant buthus martensii toxin polypeptide as well as preparation method and application thereof

Abstract

The invention discloses an anti-EV71 virus activity buthus martensii toxin polypeptide as well as a preparation method and application thereof, and belongs to the technical field of antiviral preparations. A scorpion tail electricity-saving stimulation method is adopted to obtain crude scorpion venom, and centrifugation and freeze drying are performed to obtain scorpion venom freeze-dried powder; dissolving the scorpion toxin freeze-dried powder, filtering, removing sediment, carrying out ultrafiltration centrifugation, and separating to obtain the anti-EV71 virus activity scorpion toxin polypeptide. According to the present invention, the scorpion martensii toxin polypeptides S-I (lt; 3kDa) and S-II (3-10 kDa) obtained from the scorpion martensii have good EV71 virus resistance; under the same concentration, the relative molecular mass is lt; compared with the prior art, the scorpion margaritae toxin polypeptide with the molecular weight of 3kDa has better anti-EV71 virus activity, has no toxic effect on cells, has the potential of being used as an anti-EV71 virus drug, and provides a theoretical basis for biomedical research and clinical application of the scorpion margaritae toxin polypeptide.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral preparations, and in particular relates to an East Asian scorpion toxin polypeptide with anti-EV71 virus activity, a preparation method and an application thereof. Background Art

[0002] Hand, foot and mouth disease (HFMD) is one of the key infectious diseases that need to be strengthened in prevention and control. It is an acute intestinal infectious disease caused by a variety of enteroviruses. Some cases can quickly induce complications such as the central nervous system and respiratory system, and the mortality and disability rates of critical cases are high. Enterovirus 71 (EV71) belongs to the Picornaviridae family and the Enterovirus genus. It is one of the main pathogenic microorganisms of HFMD and the main pathogen causing severe cases. At present, interferon, ribavirin and methylprednisolone are often used in clinical treatment of HFMD. The efficacy of severe HFMD caused by EV71 is poor, and it is particularly important to seek new and specific anti-EV71 drugs for the prevention and treatment of HFMD.

[0003] The whole scorpion is the dried body of the East Asian scorpion Butkus martensii Karsch, a member of the family Scorpionidae. According to the pharmacopoeia, the whole scorpion has the effects of "calming down wind and relieving spasms, attacking toxins and dispersing knots, unblocking meridians and relieving pain". It is used for infantile convulsions, convulsions and spasms, crooked mouth due to stroke, hemiplegia, tetanus, rheumatism, hemiplegia, sores, scrofula, etc. Modern research shows that scorpion toxin polypeptides derived from various scorpions of the order Scorpionales have inhibitory effects on a variety of viruses such as influenza, dengue fever, hepatitis C, herpes simplex and HIV. Zhiqiang Xia et al. (Zhiqiang Xia, Huijuan Wang, Weilie Chen, et al. 2024. Scorpion Venom Antimicrobial Peptide Derivative BmKn2-T5 Inhibits Enterovirus71 in the Early Stages of the Viral Life Cycle In Vitro[J]. Biomolecules, 14,545) studied the antiviral activity of the scorpion venom antimicrobial peptide BmKn2 and its five derivatives from the scorpion M. martensii, and found that BmKn2 and its derivative BmKn2-T5 had a significant inhibitory effect on EV71; they also studied the inhibitory activity of antimicrobial peptides from other scorpions on EV71 infection by the CPE reduction method. Compared with BmKn2 and its derivative BmKn2-T5, no other scorpion-derived antimicrobial peptides were found to exhibit significant antiviral ability against EV71 infection, indicating that scorpion-derived antimicrobial peptides with different structures or properties exhibit different pharmacological activities in EV71 infection. There is no report in the prior art on the anti-EV71 virus activity of polypeptide components prepared from Buthus scorpion. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an East Asian scorpion toxin polypeptide with anti-EV71 virus activity, a preparation method and an application thereof. The prepared East Asian scorpion toxin polypeptide has good anti-EV71 virus effect, providing a theoretical basis for its use in biomedical research and clinical application.

[0005] The present invention is achieved through the following technical solutions: A method for preparing a scorpion toxin polypeptide with anti-EV71 virus activity, comprising the following steps: (1) The crude venom of the East Asian scorpion was obtained by electric stimulation of the scorpion tail segment, and the freeze-dried powder of the East Asian scorpion toxin was obtained by centrifugation and freeze-drying; (2) The lyophilized powder of the East Asian scorpion toxin in step (1) is dissolved, filtered, the sediment is removed, and ultrafiltration centrifuged to separate and obtain the East Asian scorpion toxin polypeptide with anti-EV71 virus activity.

[0006] Furthermore, in step (2), ultrafiltration is performed using an ultrafiltration centrifuge tube with a cutoff relative molecular mass of 10 kDa to obtain a East Asian scorpion toxin polypeptide with a relative molecular mass of <10 kDa.

[0007] Furthermore, in step (2), the East Asian scorpion toxin polypeptide with a relative molecular mass <10 kDa is ultrafiltered using an ultrafiltration centrifuge tube with a cutoff relative molecular mass of 3 kDa to obtain the East Asian scorpion toxin polypeptide with a relative molecular mass <3 kDa and the East Asian scorpion toxin polypeptide with a relative molecular mass between 3 and 10 kDa (excluding 10 kDa).

[0008] Furthermore, the conditions of ultrafiltration centrifugation in step (2) are: 8000-10000 rpm, 4°C, 15-30 min.

[0009] Furthermore, the centrifugation conditions in step (1) are 4°C, 2500-4000 rpm, and centrifugation for 30-40 min.

[0010] Furthermore, in step (2), the lyophilized powder of the East Asian scorpion toxin is dissolved in PBS buffer and filtered through a 0.22 μm filter membrane to remove the sediment.

[0011] In the present invention, the preparation method described herein prepares an East Asian scorpion toxin polypeptide having anti-EV71 virus activity.

[0012] Furthermore, the relative molecular mass of the East Asian scorpion toxin polypeptide is <10 kDa.

[0013] Furthermore, the East Asian scorpion toxin polypeptide is an East Asian scorpion toxin polypeptide with a relative molecular mass of <3 kDa or an East Asian scorpion toxin polypeptide with a relative molecular mass of 3-10 kDa (excluding 10 kDa).

[0014] In the present invention, the East Asian scorpion toxin polypeptide with anti-EV71 virus activity is used in the preparation of anti-EV71 virus drugs.

[0015] Beneficial Effects The East Asian scorpion toxin polypeptides S-Ⅰ (<3 kDa) and S-Ⅱ (3 ~10 kDa) obtained from the East Asian scorpion in the present invention have good anti-EV71 virus effect; at the same concentration, the East Asian scorpion toxin polypeptide with a relative molecular mass of <3 kDa has better anti-EV71 virus activity and has no toxic effect on cells, and has the potential to be used as an anti-EV71 virus drug, providing a theoretical basis for its biomedical research and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of the toxicity experiment of the East Asian scorpion toxin polypeptide S-Ⅰ (<3 kDa) on Vero cells; Figure 2 This is the result of the toxicity experiment of the East Asian scorpion toxin polypeptide S-Ⅱ (3 ~10 kDa) on Vero cells; Figure 3 This is a light microscopic observation of the inhibitory effect of the East Asian scorpion toxin polypeptide S-Ⅰ (<3 kDa) on EV71 virus; Figure 4 This is a light microscopic observation of the inhibitory effect of the East Asian scorpion toxin polypeptide S-Ⅱ (3 ~10 kDa) on EV71 virus; Figure 5 This is the result graph showing the inhibition rate of the East Asian scorpion toxin polypeptide S-Ⅰ (<3 kDa) on EV71 virus; Figure 6 This is the result graph of the inhibition rate of East Asian scorpion toxin polypeptide S-Ⅱ (3 ~10 kDa) on EV71 virus. DETAILED DESCRIPTION

[0017] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0018] Example 1 Preparation of East Asian scorpion toxin peptide with anti-EV71 virus activity: (1) Collection of crude venom of East Asian scorpion and preparation of freeze-dried powder: The scorpion tail segment was obtained by electrical stimulation (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: Take 6-7 year old scorpions and place them in a basin at room temperature above 25°C; operating steps of the electric pulse venom extraction instrument: turn on the venom extraction instrument switch, adjust the voltage to 8-12 volts, adjust the frequency to 128 Hz, insert the electrode soft pen wire and the wire of the harvesting rack into the corresponding jacks, and turn on the power; soak the tip of the electrode soft pen of the harvesting rack with physiological saline, grasp the last two sections of the scorpion's tail, and use the electrode to continuously touch the scorpion's front pincers to stimulate the scorpion to expel venom, which is collected in a 50 ml centrifuge tube, and the 50 ml centrifuge tube is placed on ice; the collected venom is centrifuged at 4°C and 3000 rpm for 40 minutes to separate impurities such as dust and calcium to obtain pure scorpion venom; vacuum freeze-drying is performed to obtain East Asian scorpion toxin freeze-dried powder, which is stored at low temperature for later use.

[0019] (2) Isolation of East Asian scorpion toxin peptides with anti-EV71 activity: 1) Take 100 mg of the freeze-dried powder of the East Asian scorpion toxin in step (1), add 10 ml of PBS to prepare a 10 mg / mL solution, pre-filter with a 0.22 μm filter membrane, remove the sediment and set aside to obtain the East Asian scorpion toxin solution; 2) Add ultrapure water to 10 KDa ultrafiltration tube (retention molecular weight is 10 kDa) and 3 KDa ultrafiltration tube (retention molecular weight is 3 kDa), and allow the water to completely pass through the membrane. Precool in an ice bath for 5 minutes. 3) Pour out the water in the 10 KDa ultrafiltration tube and add the East Asian scorpion toxin solution prepared in step 1) to the ultrafiltration tube, not exceeding the white line on the top of the tube. The operation should be gentle. Before adding the East Asian scorpion toxin solution, the ultrafiltration tube must be inserted into an ice bath for precooling. After adding the East Asian scorpion toxin solution to the ultrafiltration tube, centrifuge at 9000 rpm and 4 ℃ for 15 min; carefully aspirate the liquid in the 10 kDa ultrafiltration centrifuge tube to obtain the relative molecular mass > 10 KDa component, and the liquid at the bottom of the centrifuge tube is the East Asian scorpion toxin polypeptide with a relative molecular mass < 10kDa; 4) Take the liquid at the bottom of the 10 kDa ultrafiltration centrifuge tube (relative molecular mass <10 kDa) and put it into the inner tube of a 3 kDa ultrafiltration tube. Centrifuge at 9000 rpm and 4 °C for 30 min. The liquid in the inner tube of the 3 kDa ultrafiltration tube is the East Asian scorpion toxin polypeptide (S-Ⅱ) of 3~10 kDa (excluding 10 kDa), and the liquid outside the centrifuge tube is the East Asian scorpion toxin polypeptide (S-Ⅰ) of relative molecular mass <3 kDa.

[0020] 5) Store the East Asian scorpion toxin polypeptides (S-Ⅰ and S-Ⅱ) of different relative molecular weights at -80℃.

[0021] (3) Freeze-drying The East Asian scorpion toxin polypeptides (S-Ⅰ and S-Ⅱ) obtained in step (2) are concentrated and freeze-dried to obtain East Asian scorpion toxin polypeptide freeze-dried powder.

[0022] Example 2 Detection of the anti-EV71 virus activity of East Asian scorpion toxin peptides: (1) The lyophilized powders of the scorpion toxin polypeptides S-Ⅰ and S-Ⅱ in Example 1 were added with phosphate buffer to prepare a uniform solution, and the concentration was determined by BCA method. The solution was sterilized by filtration with a 0.22 μm microporous filter membrane, divided into aliquots, and stored at -80°C; (2) CCK8 method to detect the cytotoxicity of East Asian scorpion toxin peptides: Vero cells were cultured in DMEM containing 10% FBS in a 5% CO 2 , 37 ℃; Vero cells in the logarithmic growth phase were digested with trypsin and inoculated into 96-well plates at 1× 10 4 After the cells have grown into a monolayer, the culture medium was discarded, S-Ⅰ and S-Ⅱ were added to a 96-well plate, and 3 wells of each dilution concentration were inoculated as the experimental group. At the same time, a normal cell control group and a blank control group were set up, with a final volume of 100 μL culture medium in each well. The plate was placed at 37 °C and 5% CO. 2 The cells were cultured in an incubator, and the OD value was determined by CCK8 method after 72 h to calculate the cell survival rate.

[0023] The cell survival rate results of the East Asian scorpion toxin polypeptides (S-Ⅰ and S-Ⅱ) are shown in the figure below. Figure 1 and Figure 2 As shown, the maximum non-toxic concentration of East Asian scorpion toxin polypeptide S-Ⅰ (<3 kDa) is 50 μg / mL, and the maximum non-toxic concentration of East Asian scorpion toxin polypeptide S-Ⅱ (3~10 kDa) is 100 μg / mL, which are used as the maximum concentrations for subsequent experiments.

[0024] (3) The improvement effect of East Asian scorpion toxin peptides S-Ⅰ and S-Ⅱ on EV71 virus-induced cytopathic effects Vero cells in the logarithmic growth phase were digested with trypsin and inoculated into 96-well plates at 1×10 4 / well, when the cells have grown a full monolayer, the culture medium is discarded, and the cells are washed three times with PBS. After Vero cells are infected with 100 TCID50 EV71 virus for 1 h, 2% FBS DMEM culture medium containing different concentration gradients of East Asian scorpion toxin peptides S-Ⅰ and S-Ⅱ are added respectively, and the culture is continued for about 72 h. At the same time, a normal cell control group, an EV71 virus control group and a blank control group are set up; when about 90% CPE (cytopathic effect) lesions appear in the virus control wells, the cytopathic condition is observed under a microscope.

[0025] The lesion rate of 0% CPE is “-”.

[0026] CPE between 1% and 25% is “+”; CPE between 25% and 50% is judged as “++”; CPE between 50% and 75% was judged as “+++”; CPE greater than 75% is “+ + + +”.

[0027] The inhibitory effects of the East Asian scorpion toxin polypeptide S-Ⅰ (<3 kDa) and the East Asian scorpion toxin polypeptide S-Ⅱ (3 ~10 kDa) on EV71 virus were observed under a light microscope. Figure 3 (A: normal cell control group, B: EV71 virus control group, C: S-Ⅰ 3.125μg / mL, D: S-Ⅰ 6.25μg / mL, E: S-Ⅰ 12.5μg / mL, F: S-Ⅰ 25μg / mL, G: S-Ⅰ 50μg / mL) and Figure 4 As shown (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); it can be seen from the figure that the cells in the normal cell control group have normal morphology and good growth status; the cells in the EV71 virus group are wrinkled and rounded, the cell gaps become larger, and some cells fall off and necrotize; compared with the EV71 virus group, scorpion toxin polypeptides S-Ⅰ 50μg / mL and S-Ⅱ 100μg / mL can effectively inhibit the cell pathological changes caused by EV71.

[0028] (4) Inhibitory effects of East Asian scorpion toxin peptides S-Ⅰ and S-Ⅱ on EV71 virus Vero cells in the logarithmic growth phase were digested with trypsin and then inoculated into 96-well plates at 1×10 4 100 μL of virus suspension containing 100 TCID50EV71 was added and cultured for about 72 h. At the same time, a normal cell control group, a virus control group, and a blank control group were set up. The 96-well plate was placed at 37 °C and 5% CO. 2 The cells were cultured in an incubator and the CPE of the cells was continuously observed under a microscope. The cells were stained with CCK-8 and the OD value was detected by an enzyme marker, and the virus inhibition rate was calculated.

[0029] The results of the inhibitory effects of the East Asian scorpion toxin peptides S-Ⅰ and S-Ⅱ on EV71 virus are shown in the figure below. Figure 5 and Figure 6 As shown in the figure, it can be seen that the East Asian scorpion toxin polypeptides S-Ⅰ and S-Ⅱ both have inhibitory effects on EV71 virus. The inhibition rate of S-Ⅰ on EV71 virus is better than that of S-Ⅱ, showing better anti-EV71 virus activity.

Claims

1. A method for preparing a scorpion toxin polypeptide with anti-EV71 virus activity, characterized in that: The following steps are involved: (1) The crude venom of the East Asian scorpion was obtained by electric stimulation of the scorpion tail segment, and the freeze-dried powder of the East Asian scorpion toxin was obtained by centrifugation and freeze-drying; (2) The freeze-dried powder of the East Asian scorpion toxin in step (1) is dissolved, the sediment is removed by filtration, and the East Asian scorpion toxin polypeptide with anti-EV71 virus activity is isolated by ultrafiltration and centrifugation.

2. The method for preparing the East Asian scorpion toxin polypeptide with anti-EV71 virus activity according to claim 1, characterized in that: In step (2), ultrafiltration is performed using an ultrafiltration centrifuge tube with a cutoff relative molecular mass of 10 kDa to obtain a East Asian scorpion toxin polypeptide with a relative molecular mass of <10 kDa.

3. The method for preparing the East Asian scorpion toxin polypeptide with anti-EV71 virus activity according to claim 2, characterized in that: In step (2), the East Asian scorpion toxin polypeptide with a relative molecular mass of <10 kDa is ultrafiltered using an ultrafiltration centrifuge tube with a cutoff relative molecular mass of 3 kDa to obtain the East Asian scorpion toxin polypeptide with a relative molecular mass of <3 kDa and the East Asian scorpion toxin polypeptide with a relative molecular mass of 3-10 kDa.

4. The method for preparing the Buthus scorpion toxin polypeptide with anti-EV71 virus activity according to claim 1, characterized in that: The ultrafiltration centrifugation conditions in step (2) are: 8000-10000 rpm, 4°C, 15-30 min.

5. The method for preparing the Buthus scorpion toxin polypeptide with anti-EV71 virus activity according to claim 1, characterized in that: The centrifugal conditions in step (1) are 4°C, 2500-4000 rpm, and centrifugation for 30-40 min.

6. The method for preparing the Buthus scorpion toxin polypeptide with anti-EV71 virus activity according to claim 1, characterized in that: Step (2) The freeze-dried powder of the East Asian scorpion toxin is dissolved in PBS buffer and filtered through a 0.22 μm filter membrane to remove the sediment.

7. A Buthus scorpion toxin polypeptide with anti-EV71 virus activity prepared by the preparation method according to any one of claims 1 to 6.

8. The Buthus scorpion toxin polypeptide with anti-EV71 virus activity according to claim 7, characterized in that: The relative molecular mass of the East Asian scorpion toxin polypeptide is <10 kDa.

9. The Buthus scorpion toxin polypeptide with anti-EV71 virus activity according to claim 8, characterized in that: The East Asian scorpion toxin polypeptide is an East Asian scorpion toxin polypeptide with a relative molecular mass of <3 kDa or an East Asian scorpion toxin polypeptide with a relative molecular mass of 3-10 kDa.

10. Use of the Buthus crenata venom polypeptide with anti-EV71 virus activity according to any one of claims 7 to 9 in the preparation of anti-EV71 virus drugs.

Images