A bungarus multicinctus venom protein, and preparation method and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a kind of Bungarus multicinctus venom protein and a preparation method and application thereof.The application provides a new sperm motility promoter, which is derived from Bungarus multicinctus venom and has the application potential to be developed into a new sperm motility promoter.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a class of banded krait venom proteins, their preparation methods, and applications. Background Technology

[0002] Asthenospermia (AZS) is one of the core causes of male infertility. Its diagnostic criteria are that the proportion of progressively motile sperm (PR) in a fresh semen sample is less than 32% (WHO 5th edition criteria), which is often accompanied by decreased sperm viability and other abnormal parameters.

[0003] In current clinical practice, intervention strategies for male infertility still primarily rely on assisted reproductive technology (ART) and microsurgery. However, the high cost of these treatments makes them unaffordable for many families, limiting their accessibility in primary healthcare settings. Current drug treatments also generally suffer from low specificity and safety profiles. For example, antioxidants (such as vitamin C, vitamin E, and coenzyme Q)... 10 While widely used to improve semen quality, its mechanism primarily involves scavenging excess reactive oxygen species (ROS) and reducing lipid peroxidation damage. However, this type of intervention is broad-spectrum defensive and cannot specifically repair primary sperm dysfunction. Another commonly used class of drugs are sex hormone regulators (including GnRH agonists, hCG / hMG, clomiphene, and letrozole), which can promote spermatogenesis by regulating the hypothalamic-pituitary-testicular axis homeostasis; however, long-term use may induce fluctuations in testosterone levels and even increase the risk of cardiovascular events. In addition, energy metabolism enhancers such as pentoxifylline, although they can increase intracellular cAMP concentration by inhibiting phosphodiesterase, thereby promoting sperm capacitation and acrosome reaction, recent studies suggest that they have potential teratogenic effects, so their clinical application must be extremely cautious.

[0004] In summary, although existing treatments have shown some efficacy in some patients, they are still limited by issues such as unclear mechanisms of action, large individual differences in response, and safety concerns. Therefore, the development of new sperm motility enhancers with high efficiency and targeting has become an urgent clinical need.

[0005] The banded krait (Bungarus multicinctus), belonging to the genus Bungarus in the family Elapinae, is a venomous snake with premolded fangs. Its venom genome contains numerous gene copies of the three-finger toxin (3FTX) family and the snake venom Kunitz-type serine protease inhibitor family (KUN) (Zhang ZY et al., 2022). Currently, there are no studies on the effects of banded krait venom on enhancing sperm motility. Summary of the Invention

[0006] The purpose of this invention is to provide a class of banded krait venom proteins, their preparation methods, and applications.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a krait venom extract. The preparation method of the krait venom extract includes: preparing krait venom into a solution, filtering, collecting the filtrate, loading it onto a high-performance liquid chromatography (HPLC) C8 column, using water containing 0.1% trifluoroacetic acid as mobile phase A, and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B to form an elution system for gradient elution. The gradient elution conditions include: from 0 to 77 min, the proportion of mobile phase A in the eluent decreases uniformly from 95% to 30%, and the proportion of mobile phase B increases uniformly from 5% to 70%; the flow rate of the eluent is 0.5 mL / min, and the peak eluted from 38 to 40 min is collected to obtain the krait venom extract; the flow rate of the eluent is 0.5 mL / min, and the peak eluted from 38 to 40 min is collected to obtain the krait venom extract.

[0008] Preferably, the venom of the banded krait is first prepared into a freeze-dried powder, and then the freeze-dried powder of the banded krait is dissolved.

[0009] Accordingly, the silver ring snake venom protein BM01, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] Correspondingly, the krait venom protein BM02, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0011] Accordingly, the application of the banded krait venom extract and / or the banded krait venom protein BMO1 and / or the banded krait venom protein BMO2 in the preparation of drugs to improve sperm motility.

[0012] Accordingly, a drug for improving sperm motility, the drug containing the krait venom extract and / or the krait venom protein BMO1 and / or the krait venom protein BMO2.

[0013] The present invention has the following beneficial effects: The present invention is the first to discover that some components of the venom of the banded krait have the effect of improving sperm motility, and further screening and extraction have yielded two new snake venom proteins that can improve sperm motility: BMO1 and BMO2, which have the potential to be developed into new sperm motility promoters. Attached Figure Description

[0014] Figure 1 This is a fractional HPLC chromatogram of crude venom from the banded krait.

[0015] Figure 2 A schematic diagram illustrating the effects of different components of crude venom from the banded krait on sperm motility;

[0016] Figure 3 SDS-PAGE results of crude venom components F3 and F18 from the banded krait;

[0017] Figure 4 This is a schematic diagram of the first 5 components of the 9.5 kDa band mass spectrum of the F18 fraction;

[0018] Figure 5 This is a schematic diagram of the first five components of the 14.4 kDa band mass spectrum of the F18 fraction;

[0019] Figure 6 Image of GST-BM01 plasmid;

[0020] Figure 7 The results of SDS-PAGE analysis of GST-BM01 expressed in prokaryotes;

[0021] Figure 8 The results of Western Blot analysis of GST-BM01 expressed in prokaryotes;

[0022] Figure 9 SDS-PAGE results of AKTA purified samples;

[0023] Figure 10 The circular dichroism chromatogram of BM01;

[0024] Figure 11 This is a schematic diagram of the BM01 spatial structure;

[0025] Figure 12 A schematic diagram illustrating the effect of temperature treatment on bovine sperm motility;

[0026] Figure 13 This is a schematic diagram illustrating the effect of BM01 on sperm motility. Detailed Implementation

[0027] This invention is the first to screen and extract two snake venom proteins from the banded krait: BM01 and BM02.

[0028] The amino acid sequence of BM01 is: MSSGGLLLLLGLLTLWAELTPVSSRKRHRDCDKPPDKGNCGRVRRAFYYDTKLETCKPFPYRGCKGNGNHFKTETLCRCECLVYP (SEQ ID NO: 1);

[0029] The amino acid sequence of BM02 is: MNPAHLLVLAAVCVSLLGAANIPPHPLNLINFMEMIRYTIPCEKTWGEYTNYGCYCGAGGSGRPIDALDRCCYVHDNCYGDAANIRDCNPKTQSYKLTKRTIICYGAAGTCARVVCDCDRTAALCFGDSEYIEGHKNIDTARFCQ (SEQ ID NO: 2).

[0030] The snake venom protein described can significantly improve sperm motility and is expected to be used to treat asthenospermia.

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0032] Example 1: Isolation and purification of components of crude venom from the banded krait and its effect on sperm motility

[0033] 1. Separation and purification

[0034] Banded kraits were collected from the wild in Longquan City, Zhejiang Province. The snake's head was fixed, venom glands were squeezed, and venom was collected into cryovials and rapidly immersed in liquid nitrogen. 210 mg of venom was freeze-dried (vacuum freeze-dried at -80℃ for 12 h) to prepare a freeze-dried powder, which was stored at -80℃. The banded krait venom freeze-dried powder was dissolved in a 50 mM Tris-HCl 50 mM NaCl (pH=7.8) buffer solution, filtered through a 0.45 µm filter membrane, and the filtrate was collected and loaded onto an analytical or preparative high-performance liquid chromatography (HPLC) C8 column (Shimadzu HPLC system used in this example). Gradient elution was performed using a water (mobile phase A, containing 0.1% trifluoroacetic acid): acetonitrile (mobile phase B, containing 0.1% trifluoroacetic acid) elution system, as shown in Table 1. Within the range of 0-77 min, the proportions of mobile phases A and B changed uniformly from the initial value (0 min) to the final value (77 min).

[0035] Table 1 Elution gradient of C8 column

[0036]

[0037] The absorption peak was detected at 214 nm, and the peaks were divided into 27 components based on their shape. The results are as follows: Figure 1 As shown. Each component was collected according to peak and then freeze-dried (vacuum freeze-dried at -80℃ for 24h).

[0038] 2. Effects on sperm motility

[0039] The obtained 27 lyophilized powder components were dissolved in ultrapure water, and the protein concentration of each component was detected using NanoDrop. The samples were then diluted with ultrapure water to a protein concentration of 0.5 mg / mL to obtain the test samples.

[0040] 200 µL of fresh boar semen was centrifuged at 1200 rpm for 6 min at 17 °C. After discarding the supernatant (seminal plasma), 190 µL of LBTS buffer (37 g glucose, 3 g sodium citrate, 1.25 g Na2-EDTA, 1.25 g sodium bicarbonate, and 0.75 g potassium chloride, diluted to 1 L with sterile water, and the pH was adjusted to 7.2–7.4) was added to resuspend the precipitate, obtaining a sperm suspension. 10 µL of the test sample and sperm suspension were added and incubated together at 37 °C for 10 min. An equal volume of ultrapure water was used as the blank control group instead of the test sample, with all other conditions remaining the same. After incubation, various parameters of sperm motility were detected using a CASA system. The counting chamber was preheated on a 37 °C stage. 3 µL of incubated sperm was slowly injected into the counting chamber, ensuring even distribution throughout the counting chamber. At least three fields of view were observed for each sample, and at least 100 sperm were counted in each field of view. A 20% increase in sperm VCL was used as the selection threshold for active ingredients.

[0041] The results are as follows Figure 2 As shown in the figure. The results showed that components F3 and F18 had the most significant effect on improving sperm motility, and could significantly increase the velocity-clinch (VCL) of normal sperm without obvious asthenospermia. SDS-PAGE electrophoresis analysis of F3 and F18 was performed, and the electrophoresis pattern is shown in the figure. Figure 3 As shown, F3 had no obvious band due to its low protein concentration. F18 had two obvious bands at 9.5 kDa and 14.4 kDa, so F18 was selected for further protein spectroscopy identification.

[0042] Separation and purification were performed according to the method described in this embodiment to obtain the following... Figure 1 The peak shape shown is the peak that elutes after 38–40 minutes, which is F18.

[0043] Example 2: Identification, analysis, expression, and purification of the protein corresponding to F18

[0044] 1. Identification and Analysis

[0045] The two bands of component F18 obtained in Example 1 were gel-extracted and analyzed by mass spectrometry. The mass spectrometry data were then used to search a library of protein sequences annotated from the *Bungarus fasciatus* genome. The top 5 components identified, sorted by iBAQ size, were compared to the following percentages: Figure 4 (Corresponding to 9.5kDa strip) Figure 5 (As shown in the corresponding 14.4 kDa band). The results show that the most abundant proteins in the two bands of F18 are B.multicinctus_HiC_scaffold421_G00001.t1 (abbreviated as Bm01) and B.multicinctus_HiC_scaffold12_G00235_236_1.t1 (abbreviated as Bm02).

[0046] Mass spectrometry identification of BM01 and BM02: Protein identification was performed by Shanghai Zhongke New Life Biotechnology Co., Ltd. The proteins were subjected to reduction and alkylation treatments (reduction at 37°C for 1 h with 10 mM DTT at 800 rpm in a constant temperature mixer, followed by incubation in the dark with 30 mM IAA for 40 min). Then, protease (protease to BM01 or BM02 mass ratio of 1:50) was added, and enzymatic hydrolysis was carried out at 37°C for approximately 20 hours. The hydrolysate was desalted, lyophilized, reconstituted in 0.1% formic acid (FA) solution, and stored at -20°C for later use.

[0047] Each sample was separated using a nanoElute HPLC system with a flow rate of nanoliters. Buffer solution A was a 0.1% formic acid aqueous solution, and solution B was a 0.1% formic acid-acetonitrile solution (acetonitrile concentration 99.9%). The column was equilibrated with 95% of solution A. Samples were loaded onto the analytical column (Thermo Scientific EASY column, 25 cm, ID75 μm, 1.9 μm, C18) via an autosampler at a flow rate of 300 nL / min. The HPLC gradient was as follows over 30 min: 0.00–18.00 min, linear gradient of solution B from 5% to 35%; 18.00–20.00 min, linear gradient of solution B from 35% to 80%; 20.00–30.00 min, solution B maintained at 80%. After chromatographic separation, samples were analyzed by mass spectrometry using a timsTOF Pro mass spectrometer. The detection method was positive ions, with the ion source voltage set to 1.5 kV. Both MS and MSMS were performed using TOF for detection and analysis. The mass spectrometry scan range was set to 100–1700 m / z. The data acquisition mode was Parallel Accumulation Serial Fragmentation (PASEF). After a single primary mass spectrometry acquisition, eight PASEF scans were performed to acquire the precursor ion, with a cycle window of 0.95 s. Secondary spectra were obtained for charge numbers in the range of 0–5. The dynamic exclusion time for tandem mass spectrometry was set to 24 s to avoid duplicate precursor ions.

[0048] A theoretical secondary spectrum database was constructed based on protein sequences from the provided database. Then, the secondary spectra generated by mass spectrometry were searched and compared with the theoretical secondary spectra. After algorithmic scoring and filtering, correctly matching theoretical peptide sequences were obtained. Protein-specific peptides were identified to determine the contained protein information. Further data filtering was performed on the database search results. The accuracy (FDR) for identification at the spectrum, peptide, and protein levels was set to 1%, and each identified protein must contain at least one unique peptide.

[0049] BM01 belongs to the Kunitz-type serine protease inhibitor family, has 85 amino acids, a protein molecular weight of 9.6832 kDa, and an isoelectric point of 9.03. BM02 belongs to the phospholipase A2 (PLA2) family, has 147 amino acids, a protein molecular weight of 16.152 kDa, and an isoelectric point of 6.38.

[0050] The amino acid sequence of BM01 is: MSSGGLLLLLGLLTLWAELTPVSSRKRHRDCDKPPDKGNCGRVRRAFYYDTKLETCKPFPYRGCKGNGNHFKTETLCRCECLVYP (SEQ ID NO: 1);

[0051] The amino acid sequence of BM02 is: MNPAHLLVLAAVCVSLLGAANIPPHPLNLINFMEMIRYTIPCEKTWGEYTNYGCYCGAGGSGRPIDALDRCCYVHDNCYGDAANIRDCNPKTQSYKLTKRTIICYGAAGTCARVVCDCDRTAALCFGDSEYIEGHKNIDTARFCQ (SEQ ID NO: 2).

[0052] 2. Expression purification

[0053] Two proteins, BM01 and BM02, were recombinantly expressed in vitro using an E. coli expression system. During the experiment, it was found that BM02 exhibited strong toxicity to E. coli, making efficient expression difficult. Therefore, this example primarily showcases BM01.

[0054] (1) Plasmid transformation and culture

[0055] Construct a recombinant plasmid with a GST tag at the N-terminus, a His tag at the C-terminus, and restriction enzyme sites, as shown in the figure. Figure 6 As shown. Recombinant expression of GST-BM01 protein was performed using the colic sensory system, specifically as follows: BL21(DE3) competent cells were thawed, 5 µL of recombinant plasmid was added, and the mixture was pipetted and incubated on ice for 20 min. Subsequently, the cells were heat-shocked in a 42℃ water bath for 90 s, followed by ice incubation for 3 min. 1 mL of antibiotic-free LB medium was added, and the cells were incubated on a shaker at 37℃ and 220 rpm for 1 h to complete the transformation of the recombinant plasmid.

[0056] (2) Protein expression and purification

[0057] Centrifuge the bacterial culture at 4800 rpm for 4 min, discard part of the supernatant, and resuspend the remaining bacterial culture (100 µL) evenly. Spread the mixture onto ampicillin (Amp) resistant LB agar plates (subsequent plates containing the same antibiotic as described here) and incubate overnight at 37°C inverted. After overnight incubation, select single clones and incubate them in 10 mL of liquid LB medium (containing the antibiotic) at 37°C for 5–6 hours. Then transfer them to 100 mL of LB medium (containing the antibiotic) and continue incubating for 3–4 hours. Finally, expand the culture to 1 L of LB medium (containing the antibiotic) until OD reaches 100%. 600=0.6~1.0, add IPTG to a final concentration of 0.2mM, and induce overnight at 16℃ and 180rpm. Centrifuge at 3800rpm for 10min to collect bacterial cells, resuspend in 50mL of 50mM HEPES buffer, centrifuge at 8000rpm for 10min, remove the supernatant, resuspend again in 50mM HEPES buffer, add PMSF and homogenize the bacterial cells using a homogenizer with parameters set to 800bar pressure and 50mL / min, repeat at least 3 times until the bacterial solution is clear.

[0058] Centrifuge at 12000 rpm for 50 min to separate and collect the supernatant and precipitate. Incorporate the GST affinity chromatography packing material with the supernatant at 4℃ for 1 h, allow it to flow through naturally, and wash with 150 mL or more of 50 mM HEPES (pH=7.8) buffer until the eluent is free of protein (no color change on G250 detection). Elute with 10 mM reduced glutathione, collect the eluent, and measure for protein concentration using a micro spectrophotometer. Perform SDS-PAGE electrophoresis on the flow-through, washings, and eluent, and verify the results using Western blotting. Add the eluted protein solution to a dialysis bag and dialyze in dialysis buffer (1×PBS, pH=7.4) at 4℃ for 12 h.

[0059] (3) GST tag digestion

[0060] The GST tag of the fusion protein was isolated using Prescission Protease (PSP, Biomed). The reaction mixture was prepared according to the manufacturer's instructions and reacted at 4°C for 16 hours to cleave the GST tag. SDS-PAGE was performed on the cleaved solution to confirm successful digestion.

[0061] (4) Protein purification

[0062] Purification was performed using an AKTA protein purification system. The enzymatically digested mixture was filtered through a 0.22 µm filter and loaded onto a Sephadex G-75 column equilibrated with the same buffer. Elution was performed with 1×PBS at a flow rate of 0.4 mL / min, with 2.5 mL collected per tube. The collected samples were then analyzed by SDS-PAGE electrophoresis.

[0063] (5) Detection of target protein concentration

[0064] The target protein concentration was quantified according to the operating procedure of the BCA protein quantification kit, a standard curve was generated, and the concentration of the target protein was calculated.

[0065] (6) Western Blot

[0066] Add the protein solution to SDS loading buffer and heat at 95°C for 5 minutes. Using a precast protein gel, load the protein and perform electrophoresis at 80V for a period of time. Once the marker bands are clearly separated, increase the voltage to 150V. The termination time for electrophoresis depends on the size of the target protein. Next, the target protein needs to be transferred to a PVDF membrane. The transfer conditions are: 200mA for 80 minutes.

[0067] After transfer, check for marker marks on the membrane. Wash the membrane with PBST for 5 min, then block with 5% skim milk for 2 h to avoid nonspecific bands. After milk blocking, wash the membrane three times with PBST, 5 min each time. Then, place the membrane in GST primary antibody and gently shake at room temperature for 2 h, followed by overnight incubation at 4°C. The next day, the primary antibody should be recovered for future use. Wash the membrane three times with PBST, 5 min each time, then incubate with the corresponding species' secondary antibody for 1.5 h. Finally, wash the membrane three times with PBST, 5 min each time, and develop using a multi-functional imaging system.

[0068] The SDS-PAGE and Western Blot results of GST-BM01 prokaryotic expression are as follows: Figure 7 , 8 As shown.

[0069] To further eliminate the interference of the GST tag on protein activity, GST-BM01 was digested with Prescission Protease, and the digested sample was purified using an AKTA purification system. SDS-PAGE results are shown below. Figure 9 As shown in the figure. The results showed that 16–18.5 mL of the effluent was pure BM01 protein. The concentration of purified BM01 was determined using a BCA kit, and the protein concentration of purified BM01 was 0.45 mg / mL.

[0070] 3. Structural Analysis of BM01

[0071] (1) Secondary structure

[0072] The secondary structure of the prokaryotically expressed active protein BM01 was determined by circular dichroism (CD) chromatography. The specific method included: replacing the BM01 protein buffer with 10 mM phosphate buffer (200 ml, NaH2PO4•2H2O 0.2 g, Na2HPO4•12H2O 0.27 g, pH=7.0) by dialysis, removing particles through a 0.22 μm filter membrane, and then determining its concentration using NanoDrop.

[0073] Before turning on the circular dichroism spectrometer, allow nitrogen to flow into the spectrometer for at least 30 minutes. Add 200–300 μL of the BM01 protein buffer solution (10 mM phosphate buffer) to a 1 mm diameter quartz cuvette, and record the background blank spectrum between 250 and 195 nm. Subtract the buffer background spectrum for spectral correction. Add 200 μL of 0.3 mg / mL BM01 protein buffer solution to a 1 mm diameter quartz cuvette and seal the anaerobic chamber with a tight lid. Transfer the cuvette to the CD spectrometer for scanning. Experimental conditions: Wavelength range: 185–260 nm; Test temperature: 25 °C; Bandwidth: 1 nm; Step size: 0.5–1.0 nm; Scan speed: 20–50 nm / min; Response time: 0.5–1 s; Number of sample scans: 3.

[0074] The average value of three consecutive spectral scans was taken, and then the CD signal of the solvent was subtracted. The secondary structure of the peptide was determined based on the results. The results are as follows: Figure 10 As shown in the figure. The results show that after BM01 was dissolved in 10mM phosphate buffer, the CD spectrum showed a strong positive peak at 195nm, which indicates that the secondary structure of BM01 is mainly β-sheet.

[0075] The component content of the secondary structure of BM01 was calculated, as shown in Table 2.

[0076] Table 2. Component content of BM01 secondary structure

[0077]

[0078] The results showed that BM01 dissolved in 10 mM phosphate buffer had an α-helix content of 12.38% and a β-sheet content of 40.44%. This structural feature is highly consistent with the structural features of classic Kunitz-type serine protease inhibitors, confirming the correctness of protein refolding from a structural biology perspective.

[0079] (3) Spatial structure

[0080] The spatial structure of BM01 was predicted using Alphafold3, and the results are as follows: Figure 11 As shown.

[0081] Example 3: Effect of BM01 protein on sperm motility

[0082] Fresh boar semen was collected and incubated in a 39°C water bath for 1 hour. Total sperm motility was then measured using the CASA system. Results showed that after high-temperature treatment, sperm motility... Figure 12 As shown, the overall sperm motility, progressive motility, VCL, VAP and other parameters were all significantly reduced (P<0.05), proving that low motility and asthenospermia sperm were successfully constructed.

[0083] Porcine sperm were treated with purified BM01 protein (0.54 mg / mL, 10 µL) via prokaryotic expression, following the same treatment method as in Example 1. A blank control group and a positive control group were also included. The positive control group used 0.54 mg / mL, 10 µL of pentoxifylline (PTX) instead of BM01. The results were then analyzed using a CASA system. The results are as follows: Figure 13 As shown in the figure. The results showed that BM01 protein could significantly improve the overall motility, progressive motility and VCL of weak porcine sperm (P < 0.05), indicating that prokaryotic expression of BM01 has the function of promoting sperm activity.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A venom extract from the banded krait, characterized in that: The preparation method of the banded krait venom extract includes: preparing banded krait venom into a solution, filtering, collecting the filtrate and loading it onto a high-performance liquid chromatography (HPLC) C8 column. A gradient elution system is formed using water containing 0.1% trifluoroacetic acid as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B. The gradient elution conditions include: from 0 to 77 min, the proportion of mobile phase A in the eluent decreases uniformly from 95% to 30%, and the proportion of mobile phase B increases uniformly from 5% to 70%. The flow rate of the eluent is 0.5 mL / min. The peak eluted from 38 to 40 min is collected to obtain the banded krait venom extract.

2. The silver-ringed snake venom extract according to claim 1, characterized in that: First, prepare the venom of the banded krait into a freeze-dried powder, and then dissolve the freeze-dried venom powder.

3. The venom protein BM01 of the banded krait, characterized by: The amino acid sequence of the protein BM01 is shown in SEQ ID NO:

1.

4. BMO2, a protein from the venom of the banded krait, is characterized by: The amino acid sequence of the protein BM01 is shown in SEQ ID NO:

2.

5. The use of the silver ring snake venom extract of claim 1 and / or the silver ring snake venom protein BMO1 of claim 2 and / or the silver ring snake venom protein BMO2 of claim 3 in the preparation of a drug for improving sperm motility.

6. A drug for improving sperm motility, characterized in that: The drug contains the venom extract of the banded krait as described in claim 1 and / or the venom protein BMO1 of the banded krait as described in claim 2 and / or the venom protein BMO2 of the banded krait as described in claim 3.

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