Use of a polypeptide toxin mp3c derived from the spider comipsaertsi

Abstract

This invention discloses the application of a polypeptide toxin Mp3c derived from the spider *Gymnospermum tectorum*. Patch-clamp experiments verified that Mp3c effectively inhibits the voltage-gated potassium channel Kv1.3, with inhibitory effects approximately 3 times and 18 times greater than those of Kv1.4 and Kv1.5, respectively. This Kv1.3 polypeptide toxin inhibitor, as an immunosuppressant, specifically inhibits T cells involved in autoimmune diseases. EM Based on the strong inhibitory effect of Mp3c on Kv1.3, it is speculated that it may selectively inhibit T cells. EM The activation process plays a role, thus possessing therapeutic potential for autoimmune diseases. Furthermore, Mp3c has no significant inhibitory effect on other voltage-gated potassium channels, including Herg channels, which can, to some extent, avoid post-drug cardiotoxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of a polypeptide toxin Mp3c derived from the spider Macrothele proingens. BACKGROUND

[0002] Ion channels, as transmembrane proteins, regulate the movement of ions across biological membranes. Ion channels can regulate specific stages of cancer occurrence and progression, and their dysregulation can also lead to many diseases, such as cardiovascular and nervous system diseases.

[0003] Voltage-gated potassium ion channels are the most diverse class of all ion channels, and are involved in the regulation of many important processes. Dysfunction of potassium channels often leads to ion channel diseases such as epilepsy and arrhythmia. A large number of studies have reported dysregulation of potassium channel expression in human cancers. For example, overexpression of Kv10.1 was found in more than 70% of human cancer types, and high expression of Kv1.3 was detected in many human malignancies, including breast cancer, colon cancer, and prostate cancer. Blocking the activity or protein expression of potassium channels impairs the growth of certain tumors. Therefore, potassium channels are considered as new targets in oncology, but targeting ion channels in cancer is still an underutilized therapeutic strategy. Targeting ion channels on tumor cells is achieved by chemical blockers and antibodies that are found to inhibit several human prostate tumors, liver cancer, mesothelioma, colon cancer, breast cancer, glioma, and melanoma, etc. +

[0004] Kv1.3 is mainly expressed on immune system cells such as T lymphocytes, and in human T lymphocytes, Kv1.3 can provide driving force for sustained calcium ion influx by regulating cell resting membrane potential, and plays a key role in T cell activation, proliferation, and secretion of inflammatory factors. T EM Lymphocytes are associated with autoimmune diseases such as multiple sclerosis, type 1 diabetes, ankylosing spondylitis, rheumatoid arthritis, etc. After being activated by autoimmune antigens, T EM The expression of Kv1.3 on the cell membrane increases by about 300 times. Therefore, voltage-gated potassium channel Kv1.3 can be used as a starting point for autoimmune disease research, and by exploring potent inhibitors of Kv1.3, the exacerbation of autoimmune diseases mediated by T lymphocytes can be controlled. Currently, most treatments for autoimmune diseases use immunosuppressants that are not specific in their mode of action, which not only causes unnecessary side effects, but in more serious cases can even compromise the immune system. Due to the increasing incidence of autoimmune complications, the use of biologics has increased, and the cost of treating autoimmune diseases worldwide has also increased. Therefore, there is an urgent need to develop improved low-cost, safer, and specifically targeted treatments for these diseases. SUMMARY ​

[0005] The technical problem solved by the present application is to provide an application of a polypeptide toxin Mp3c derived from Macrothele palpator.

[0006] A polypeptide toxin κ-macrotoxin-Mp3c (Mp3c for short) derived from Macrothele palpator venom, the amino acid sequence of which is as follows:

[0007] Ala-Cys-Gly-Val-Phe-Asn-Asp-Arg-Cys-Pro-Leu-Phe-Lys-Cys-Cys-Pro-Gln-Tyr-Val-Cys-Lys-Gly-Trp-Arg-Thr-Lys-Arg-Cys-Leu-Asn-Pro. It consists of 31 amino acid residues, contains 6 cysteines, 3 pairs of disulfide bonds form a classic ICK motif, the molecular weight is 3607.32 Da, and the isoelectric point is 9.15.

[0008] Experiments prove that Mp3c has a high affinity inhibitory effect on Kv1.3, and effectively inhibits Kv1.4 and Kv1.5, and has a voltage-dependent inhibitory effect on Kv1.1 and Kv1.2, which gradually weakens with the extension of depolarization stimulation time. This is different from the functions of other Kv1.3 inhibitors that have been found so far, so the discovery of this spider polypeptide toxin can be used as a molecular tool for analyzing the interaction mechanism of spider polypeptide toxin and Kv1.3, and as a reference for improving the specificity of the polypeptide sequence of the toxin and the channel. As for the enrichment of spider polypeptide toxin Mp3c, in addition to being obtained by separating the spider venom, the present application successfully synthesizes renatured Mp3c with the same activity by chemical synthesis, thereby providing certain insights and reference methods for obtaining Mp3c. BRIEF DESCRIPTION OF DRAWINGS

[0009] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0010] Figure 1 The figure shows the purification and verification of spider polypeptide toxin Mp3c, wherein: A is Macrothele palpator; B is the RP-HPLC chromatogram of the crude venom of Macrothele palpator, wherein the peak 15 containing Mp3c is marked; C is the second round of RP-HPLC subdivision results of peak 15, and the small graph in the upper left corner is the current trajectory graph of the arrow indicating the subdivided component acting on Kv1.3; D is the mass spectrum result of the chromatographic peak segment pointed by the arrow in C;

[0011] Figure 2Figure 1 shows the chemical synthesis and identification of Mp3c. Chromatogram (215 nm) The peak marked with an asterisk is the peak of interest. The loading amount is 100 μg / ml, acetonitrile gradient 15-50%, where A is the chromatogram of linear Mp3c after purification, the peak of interest appears at acetonitrile gradient 48%; B is the chromatogram of renatured Mp3c, the peak of interest appears at acetonitrile gradient 36%; C is the mass spectrum result of linear Mp3c; D is the mass spectrum result of renatured Mp3c;

[0012] Figure 3 Figure 2 shows the activity analysis of Mp3c on different subtypes of voltage-gated potassium ion channels. At +30 mV depolarization voltage, the channel current graphs before (Control) and after (1 μM and 5 μM Mp3c) treatment with the final concentration of 1 μM and 5 μM Mp3c are shown in sequence.

[0013] Figure 4 Figure 3 shows the concentration-effect relationship curve of Mp3c in inhibiting the channel current of Kv1.3, Kv1.4 and Kv1.5. The IC50of Mp3c in inhibiting the channel current of Kv1.3 is 15.57 nM ± 1.01; the IC50of Mp3c in inhibiting the channel current of Kv1.4 is 284.1 nM ± 0.89; and the IC50of Mp3c in inhibiting the channel current of Kv1.5 is 51.34 nM ± 0.90. The data is mean ± SEM, and the number of cells corresponding to each point is n = 5-6. 50 50 50

[0014] Figure 5 Figure 4 shows the activity analysis of Mp3c on different subtypes of voltage-gated sodium ion channels. At +10 mV depolarization voltage stimulation, the channel current graphs before (Control) and after (1 μM and 5 μM Mp3c) treatment with the final concentration of 1 μM and 5 μM Mp3c are shown in sequence. A-G represent Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.8 channels, none of which has obvious effect. The data is mean ± SEM, and the number of cells corresponding to each point is n = 3-5. DETAILED DESCRIPTION

[0015] In order to enable those skilled in the art to have a clearer and more intuitive understanding of the present application, the present application will be further described below in conjunction with the drawings.

[0016] MATERIALS AND METHODS

[0017] 1. Venom collection and toxin purification

[0018] Electrical stimulation method for collecting the spider Macrothele proingens (Chen et al., 2005) Figure 1 ​​​Spider venom (as shown in -A) was freeze-dried and stored at -20℃ for subsequent experiments. The separation and purification of Mp3c involved two steps: (1) using an XB-C18 column (…). (4.6mm × 250mm, Welch Materials Inc., Shanghai, China) was separated and purified using a Shimadzu high-performance liquid chromatography system, with approximately 1 mg of crude toxin loaded each time. Solution A was acetonitrile (with 0.1% trifluoroacetic acid added), and solvent B was water (with 0.1% trifluoroacetic acid added). A linear gradient elution was performed at a flow rate of 1 ml / min: washing with 10% A for 5 min, followed by a gradient run of A from 10% to 65% for 55 min. The absorbance was monitored at 215 nm, and chromatographic peaks (e.g., [missing information]) were collected. Figure 1 -B As shown, the rectangular boxes are marked as chromatographic peaks 14 and 15 containing the target polypeptide. After freezing at -80℃, they were freeze-dried. (2) The target components 14 and 15 were processed using an XB-C18 column ( The second round of high-performance liquid chromatography (HPLC) analysis was performed using a Watersalliance 2695 HPLC system (4.6 mm × 250 mm, Welch Materials Inc., Shanghai, China). The acetonitrile gradient was from 15% to 30% (acetonitrile increased by 0.5% per minute, flow rate 1 ml / min). Arrows marked the target peak segments collected (e.g., ...). Figure 1 -C is shown).

[0019] 2. Mass spectrometry identification

[0020] CCA powder was dissolved in 50% acetonitrile (containing 0.1% TFA) to form a saturated solution. 0.5 μL of the sample solution and 0.5 μL of CCA solution were mixed on a spotting plate. After the solution crystallized naturally, the molecular weight of the peptide was determined by MALDI-TOF-TOFMS mass spectrometry (ABSCIEXTOF / TOFTM 5800 system, Applied Biosystems, USA).

[0021] 3. Cell culture and transfection

[0022] CHO-K1 cells were cultured in DMEM F / 12 medium, ND7 / 23 and HEK293T cells were cultured in high glucose DMEM liquid medium, in a 37 °C incubator with 5% CO2, containing 10% FBS and 1% PS. Cells were trypsinized, diluted with culture medium, and cultured in 35 mm dishes. When the cell density reached 90%, transfection was performed according to the operation steps in the product manual of X-treme GENE HP DNA Transfection Reagent (Roche, Basel, Switzerland), and the channel plasmid and eGFP were co-transfected. Nav1.6 and Nav1.8 transfected ND7 / 23 cells, other sodium channels transfected Hek293T cells, and potassium channels transfected CHO-K1 cells. After 4-6 hours of transfection, the cells were seeded in new dishes and cultured at 37 °C in 5% CO2 for 24 hours before whole-cell patch clamp recording. Green fluorescence was used to identify positive transfection cells.

[0023] 4. Electrophysiological recording

[0024] The data acquisition amplifier used was EPC 10USB Amplifier (HEKA, Germany). Borosilicate glass capillary electrodes (electrode liquid entry resistance controlled at 2.0-2.5 MΩ) were made by P-97 electrode puller (PC-10, Narishige) with 1.5 mm capillary glass. After thermal polishing of the glass electrode, the electrode tip diameter was 1.5-3.0 μm, and after completion of the drawing, the glass electrode was filled with intracellular solution. 80% series resistance compensation was used to minimize voltage error. In the EPC-10USB amplifier, the liquid junction potential was corrected during whole-cell recording. After establishing the whole-cell mode, patchmaster was used to record the voltage-dependent current, sampling at 30 kHz and filtering at 2.9 kHz.

[0025] 5. Data processing

[0026] Patch Master v2x73, Igor Pro6, Office Excel 2010 and Graph Pad Prism 8 were used to analyze the data. All data points were shown as the mean ± standard error of independent experimental units. Hill equation was used to fit the concentration-dependent activation curve. One-way ANOVA was used to evaluate the differences between multiple groups.

[0027] Example 1

[0028] The amino acid sequence of the acinonyx polypeptide toxin Mp3c is composed of ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP, and the nucleotide sequence of the cDNA gene encoding the acinonyx polypeptide toxin Mp3c is as follows:

[0029] ATGAATTTTTCAATCCTTTTCATTACATTCTCTATGATGCTGACGCTTGGTGTTCTGGTC

[0030] ATTGCTGAAGAAGAGAATGAAGAAAACTCTTTGGAAGAAATGCCTCTGCTTTCCTTGA

[0031] AGGAATTCATTCAATCGCTAGCGAGAGCTGAAGAAAGGGCTTGTGGTGTTTTTAATGA

[0032] TCGCTGCCCTCTATTTAAATGCTGCCCACAGTACGTATGCAAAGGTTGGAGGACGAAG

[0033] AGATGCTTGAATCCT。

[0034] The venom secreted by the chelicera of the spider is collected and separated and purified by RP-HPLC to obtain a natural animal polypeptide toxin, generally, the crude venom of the spider is only separated and purified once, and most of the elution peak components are difficult to completely separate, the spider polypeptide toxin of the present application is still difficult to obtain a single toxin component with high purity through a second round of fine separation means, and low content and impure components lead to the fact that the polypeptide toxin amino acid sequence cannot be obtained through Edman degradation, therefore, the present application selects to construct a spider venom gland cDNA library to obtain the amino acid sequence of Mp3c. Figure 1 As shown in -B, the peak No. 15 of the first round of RP-HPLC is collected, freeze-dried into a powder, dissolved in 50-100ul of ultrapure water for a second round of fine separation, and the main active component of peak No. 15 is collected (as shown in Figure 1 The peak segment indicated by the arrow of -C), and the molecular weight thereof is identified by mass spectrometry as Figure 1 As shown in -D, the mass spectrometry result of the fine separation of peak No. 15 is 3601Da. The activity is verified by patch clamp, and the latter half of the main peak of the fine separation of peak No. 15 has strong inhibitory activity on Kv1.3 (as shown in Figure 1Mp3c is the active ingredient of the venom of the spider Macrothele proingens, and its molecular weight is 3601 Da (as shown in the upper left corner of the current trajectory diagram), and the molecular weight of the active ingredient is determined according to the mass spectrum result, and finally the encoding gene and amino acid sequence of the polypeptide toxin are successfully found in the Macrothele proingens cDNA library according to the relative molecular mass of the polypeptide toxin. By comparing the sub-mass spectrum result with the polypeptide sequence molecular weight of the cDNA library, the full length of Mp3c sequence is obtained, and it is proved that Mp3c is the active ingredient by subsequent patch clamp experiment.

[0035] 1. Isolation of the venom gland of Macrothele proingens

[0036] The prepared ophthalmic scissors, Venus scissors and forceps were high-temperature sterilized and dried, and then ultraviolet sterilized for 30 min and cooled to room temperature. Four Macrothele proingens were taken as materials for extracting total RNA of venom glands, the head and thorax of the spider was fixed using forceps, the surface of the spider was disinfected by spraying 75% alcohol, four pairs of legs and abdomen were cut off to leave only the head and thorax, the exoskeleton of the spider was cut open along the edge towards the chelicera direction to expose the muscle tissue. The water droplet-shaped venom gland near the venom tooth was found, and the venom gland and the two venom ducts for transporting venom were gently pulled out with forceps and placed in a pre-cooled RNAase-free centrifuge tube. The venom glands of 10 spiders were extracted, and liquid nitrogen was continuously poured into the centrifuge tube containing the venom glands to maintain low temperature during the process.

[0037] 2. Extraction of total RNA of Macrothele proingens venom gland: (1) The mortar was pre-cooled in liquid nitrogen, and the venom glands were ground into powder. Liquid nitrogen was continuously added during the grinding process to protect the RNA of the venom glands from being damaged. (2) The ground and fully ground Macrothele proingens venom gland powder was collected in a centrifuge tube that had been high-temperature inactivated RNAase, 750 μL Trizol reagent was added, and the reaction was performed for 5 min. 150 μL chloroform was added, mixed uniformly by shaking, and stood for 5 min. 4°C, 12000 rpm, centrifugation for 10 min. (3) Transfer the supernatant to a new centrifuge tube (RNase-free), add 750 μL 70% ethanol (DEPC water preparation) and mix well. 4°C, 8000 rpm, centrifugation for 5 min, discard the supernatant, dry the precipitate in a clean bench, resuspend with DEPC water, and store in an ultra-low temperature freezer (-80°C).

[0038] 3. Construction of Macrothele proingens cDNA library

[0039] The CLONTECH company Creator TM SMARTTM cDNA Library Construction Kit plasmid cDNA library construction kit was used.

[0040] A. Synthesis of cDNA first strand:

[0041] The following reagents were added to the PCR tube, the reagents in the centrifuge tube were mixed and centrifuged briefly, and 72°C was incubated for 2 min. Immediately incubate the centrifuge tube on ice for 2 min.

[0042] Table 1-1 Reaction System

[0043] Reaction Component Per rxn Total RNA (1 μg / μL) 1 μL SMART IV Oligonucleotide 1 μL CDS III / 3' PCR Primer 1 μL ddH2O 2 μL

[0044] Add the following reagents to the centrifuge tube, mix the reagents in the centrifuge tube and centrifuge briefly, incubate at 42°C for 1 hour. Stop the first strand synthesis by placing the centrifuge tube on ice. Take 2 μL of the synthesized first strand cDNA from the centrifuge tube for later use.

[0045] Table 1-2 Reaction System

[0046]

[0047]

[0048] B. Long Distance PCR to Synthesize Double-Stranded cDNA

[0049] Add the following reagents to the PCR tube:

[0050] Table 1-3 PCR Reaction System

[0051] Reaction Component Per rxn cDNA first strand product 2 μL ddH2O 80 μL 10X Advantage 2 PCR Buffer 10 μL CDS III / 3' PCR Primer 2 μL 50X dNTP Mix 2 μL 5' PCR Primer 2 μL 50X Advantage 2 Polymerase Mix 2 μL

[0052] Mix, centrifuge briefly, and place in the PCR machine for reaction:

[0053] Table 1-4 PCR Reaction Procedure

[0054] Reaction Program Reaction Temperature Reaction Time Step 1 95℃ 1 min Step 2 95℃ 15s Step 3 66℃ 20s Step 4 72℃ 4 min

[0055] Note: Perform 18 cycles for the 2nd, 3rd, and 4th steps. After the cycles are completed, extract the synthesized double-stranded cDNA in the centrifuge tube.

[0056] C. Extract and recover the PCR product using the SV Gel and PCR Clean-Up System kit from PROMEGA, following the steps below:

[0057] 1. Add the double-stranded cDNA to an equal volume of membrane binding buffer, mix well, and then transfer the mixture to the centrifuge purification column. Allow the DNA to bind to the silica gel membrane at room temperature for 5 minutes. Centrifuge at 16,000 g for 1 minute, and discard the waste liquid in the collection tube.

[0058] 2. Add 700 μL of elution buffer (containing ethanol) to the centrifuge purification column, centrifuge at 16,000 g for 1 minute, and discard the waste liquid in the collection tube.

[0059] 3. Repeat step 2.

[0060] 4. Centrifuge at 16,000 g for 5 minutes.

[0061] ​5. Place the spin column in a new centrifuge tube.

[0062] 6. Add 30 μL ultrapure water and let stand at room temperature for 5 minutes.

[0063] 7. Centrifuge at 16,000 g for 1 minute. The solution at the bottom of the tube is the purified cDNA duplex.

[0064] 4. Enzymatic digestion, ligation, and transformation of the ligation product:

[0065] 1. Add 1 μL Takara pMD19-T vector, 4 μL of the solution of the cDNA duplex of the venom gland of A. chrysippus, and 5 μL of water to a microcentrifuge tube.

[0066] 2. Add 5 μL (equal volume) of the ligation enzyme buffer mixture.

[0067] 3. Incubate at 16°C for 2 hours.

[0068] 4. Add the entire volume (10 μL) to 100 μL of DH5α competent cells and place on ice for 30 minutes.

[0069] 5. Heat at 42°C for 90 seconds and then place on ice for 1 minute.

[0070] 6. Add 890 μL of LB medium warmed at 37°C and incubate at 37°C with slow shaking for 60 minutes.

[0071] 7. Take 200 μL and spread on LB medium containing X-Gal, IPTG, and Amp and incubate at 37°C for 16 hours to form single colonies.

[0072] 8. Wash the colonies with 5 mL of LB liquid medium per LB plate and store in 30% glycerol. The constructed cDNA library contains approximately 1 x 10 6 individual clones.

[0073] 5. Sequencing of single colonies of the cDNA library

[0074] From the plates inoculated with a 10 6 fold dilution of the primary cDNA library, randomly pick single colonies and inoculate into LB liquid medium containing ampicillin and incubate for 4-6 hours (the bacterial solution becomes turbid) for sequencing. The sequencing method is Sanger sequencing, and the forward sequencing primer is M13F. The coding gene of Mp3c obtained from the 726th sequencing result is as follows:

[0075] ATGAATTTTTCAATCCTTTTCATTACATTCTCTATGATGCTGACGCTTGGTGTTCTGGTC

[0076] ATTGCTGAAGAAGAGAATGAAGAAAACTCTTTGGAAGAAATGCCTCTGCTTTCCTTGA

[0077] AGGAATTCATTCAATCGCTAGCGAGAGCTGAAGAAAGGGCTTGTGGTGTTTTTAATGA

[0078] TCGCTGCCCTCTATTTAAATGCTGCCCACAGTACGTATGCAAAGGTTGGAGGACGAAGAGATGCTTGAATCCT.

[0079] The open reading frame in the 5'→3' direction was predicted by the online website Translatetool (https: / / web.expasy.org / translate / ), and the corresponding protein was translated. The longest open reading frame was selected as the correct prediction: MNFSILFITFSMMLTLGVLVIAEEENEENSLEEMPLLSLKEFIQSLARAEERACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP; the signal peptide sequence of the toxin precursor was predicted by the online website SignalP-5.0 (https: / / www.novopro.cn / tools / signalp.html). The cleavage site between the prepeptide and the predicted mature peptide was determined by the Processing Quadruplet Motif (PQM) mode, and the middle peptide of this sequence was AEER. The final mature peptide amino acid sequence of Mp3c was: ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP, with a molecular weight of 3607.32 Da, and a molecular weight of 3601.32 Da after subtracting three pairs of disulfide bonds.

[0080] Example 2

[0081] The polypeptide was chemically synthesized by Fmoc solid-phase synthesis and successfully refolded, as shown in Figure 2 The polypeptide was synthesized in the order from the C-terminal to the N-terminal.

[0082] 1. Chemical synthesis of polypeptide toxin:

[0083] 1. Swelling resin: weigh 0.1 mM Rink resin into the synthesis tube, and add about 3 mL of DMF solution, soak for 50 min, so that the resin is fully swollen, and then use a vacuum pump to filter out the liquid.

[0084] 2. Deprotection: Add 3 mL of 20% piperidine to the swelled resin in the synthesis tube and shake on a rotator for 7 min. This is to remove the protecting group from the amino group. Remove the piperidine by suction filtration and add another 3 mL of 20% piperidine to the tube and shake on a rotator for 7 min. This is the second deprotection. Remove the liquid by suction and wash the tube with 2 mL of DMF for 8 times to ensure that the piperidine is removed.

[0085] 3. Activation of amino acid: Start the activation of amino acid at the same time when the first protection is performed. That is, take 0.1 mM of HATU and HOBT in an EP tube and add 750 μL of N-methyl morpholine to each of them, which are stored in the dark. Mix them and add to the weighed amino acid. Mix well and place on a rotator for 15-20 min for activation.

[0086] 4. Coupling of amino acid: Add the activated amino acid to the deprotected resin in the synthesis tube and mix well. Place on a rotator for 60 min at 25 °C for coupling at 180 rpm. Remove the liquid by suction and wash the tube with 2 mL of DMF for 8 times. Wash the top and bottom caps. Repeat the activation and coupling of amino acid until the last amino acid is coupled.

[0087] 5. Cleavage of polypeptide chain: After the last amino acid is coupled, add 20% piperidine to the synthesis tube and shake on a rotator for 7 min for deprotection. Wash with DMF and methanol in sequence and remove the liquid by suction. Add 6 mL of cleavage solution (TFA: dimercaptoethane: anisole: benzyl mercaptide = 90:5:2:3) which is prepared in advance and mix well. Place on a rotator for 3.5 h for cleavage. After the reaction is completed, transfer the cleavage solution from the synthesis tube to a 50 mL centrifuge tube, taking care not to mix the resin in the synthesis tube. Add 30 mL of ice ethyl ether to the cleavage solution, mix well, stand for 2 min and centrifuge at 4000 r / min for 3 min. Discard the supernatant and add an appropriate amount of ethyl ether again to repeat the centrifugation. Discard the supernatant and obtain the lower precipitate, which is the crude polypeptide. Dry in air and perform separation and purification of the linear polypeptide.

[0088] 2. Separation and purification of linear polypeptide

[0089] Dissolve the linear polypeptide in an appropriate amount of ddH2O and separate by preparative high performance liquid chromatography (Shimadzu), C18 chromatographic column (10 x 250 mm, 5 μm) with acetonitrile elution gradient from 15% to 50%. Collect the chromatographic peaks for mass spectrometry identification. Collect the linear polypeptide target peak, freeze and dry, and store in a -20 °C refrigerator for later use. Dissolve 100 μg of the purified linear polypeptide in 1 mL of ultrapure water and load 1 mL for high performance liquid chromatography verification (as shown in Figure 2 - A), with a peak time of 38 min and an acetonitrile gradient of 48%. The mass spectrometry result is shown in Figure 2- C) 3607 Da, which is in agreement with the results described above.

[0090] 3. Polypeptide renaturation

[0091] At room temperature, 50 mL of renaturation solution was prepared in the dark: 0.1 M NaCl, 0.1 M Tris-HCl, 5 mM GSH and 0.5 mM GSSG, 400 mM guanidine hydrochloride, 500 mM ammonium acetate, pH 7.0. The prepared renaturation solution was wrapped with tin foil paper, and the dissolved sample was slowly dripped into the divided 1 mL renaturation solution at a final concentration of 100 μg / mL. The centrifuge tube was covered, and the sample was placed at 4°C for 24 h. Then, 10% 50% TFA was added to terminate the reaction. After termination of the reaction, the renatured polypeptide was further separated and purified by reverse-phase high-pressure liquid chromatography on a C18 reverse-phase chromatographic column (10 x 250 mm, 5 μm) with an acetonitrile elution gradient from 15% to 50%, and the target peak was at 26 min (acetonitrile gradient 36%), as shown in Figure 2. Figure 2 - B, mass spectrometry determined that the molecular weight was 3601 Da Figure 2 - D).

[0092] Example 3

[0093] The effect of the spider polypeptide toxin Mp3c on potassium channel subtypes was verified, and the results are shown in Figure 6. Figure 3 Before the experiment, the extracellular solution must be equilibrated at room temperature, and then the culture solution in the culture dish is replaced to prevent a sharp change in solution temperature. When the solution is replaced, the cells must be prevented from falling off the bottom of the culture dish. The cells with smooth membranes and uniform cytoplasm are selected under an inverted microscope, and the patch clamp experiment is performed at room temperature at 20-25°C. After the whole-cell recording mode is formed, the cell is clamped to -90 mV, and the potassium channel is given a +30 mV voltage for single stimulation to record the current. The effects of Mp3c at final concentrations of 5 μM and 1 μM on sodium channels and potassium channel subtypes are recorded, and the Kv1.1 and Kv1.2 channel currents are partially inhibited and gradually weakened with increasing stimulation time, as shown in Figures 7A and 7B. Figure 3 - A and Figure 3 - B. Mp3c at a final concentration of 100 nM can almost completely inhibit the Kv1.3 channel current, as shown in Figure 7C. Mp3c at final concentrations of 1 μM and 5 μM has different degrees of inhibition on the Kv1.4 current, as shown in Figure 7D. Mp3c at final concentrations of 1 μM and 5 μM almost completely inhibits the Kv1.5 channel current, as shown in Figure 7E. Figure 3 - C. Mp3c at final concentrations of 1 μM and 5 μM has different degrees of inhibition on the Kv1.4 current, as shown in Figure 7D. Mp3c at final concentrations of 1 μM and 5 μM almost completely inhibits the Kv1.5 channel current, as shown in Figure 7E. Figure 3 - C. Mp3c at final concentrations of 1 μM and 5 μM has different degrees of inhibition on the Kv1.4 current, as shown in Figure 7D. Mp3c at final concentrations of 1 μM and 5 μM almost completely inhibits the Kv1.5 channel current, as shown in Figure 7E. Figure 3 - C. Mp3c at final concentrations of 1 μM and 5 μM has different degrees of inhibition on the Kv1.4 current, as shown in Figure 7D. Mp3c at final concentrations of 1 μM and 5 μM almost completely inhibits the Kv1.5 channel current, as shown in Figure 7E. Figure 3As shown in F-M, the current size of Kv2.1, Kv3.2, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv4.3 and Herg channels did not change significantly, indicating that Mp3c at final concentrations of 1 μM and 5 μM had no effect on these potassium channel subtypes. Mp3c data are mean ± SEM, and the number of cells corresponding to each point n = 3-5. It was found that Mp3c showed high inhibition on Kv1.3, with an IC50 value of 15.57 nM ± 1.01, and different degrees of inhibition on Kv1.4 and Kv1.5, with IC50 values of 284.1 nM ± 0.89 and 51.34 nM ± 0.90, respectively (as shown in Figs. 1-3). Figure 4 ), the inhibition of Kv1.3 by Mp3c was about 3 times and 18 times that of Kv1.4 and Kv1.5, respectively, and the inhibition of Kv1.1 and Kv1.2 decreased with the extension of depolarization stimulation time, while there was no effect on other potassium channels.

[0094] Example 4

[0095] The effect of the spider polypeptide toxin Mp3c on sodium ion channel subtypes was verified, and the cell was clamped to -100 mV or -120 mV during sodium channel activity identification. After establishing the whole cell mode, a single stimulus was given at +10 mV voltage to start recording the current after 5 minutes. The effect of Mp3c at final concentrations of 5 μM and 1 μM on sodium channel subtypes was recorded, as shown in Figs. 4-6. Figure 4 As shown in Figs. 4-6, the current size of Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.8 channels did not change significantly under the action of Mp3c at final concentrations of 1 μM and 5 μM, so Mp3c had no significant inhibitory effect on most voltage-gated sodium channels (as shown in Figs. 4-6). Figure 5

[0096] In summary, the present application relates to the application of the spider polypeptide toxin Mp3c, belonging to the field of biology. Experiments have shown that Mp3c has strong inhibitory effect on Kv1.3, can also produce inhibitory effect on Kv1.4 and Kv1.5, has weak or no effect on other potassium channels, and has almost no effect on sodium channels, so it is a selective voltage-gated potassium ion channel inhibitor, which can to some extent avoid the cardiotoxicity after drug development. The present application also provides certain insights and reference methods for obtaining the above-mentioned polypeptide toxin by chemical synthesis.

[0097] ​The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as of their onset and might not represent the only or even the dominant form of the application. It is intended that the scope of the application embrace all techniques capable of attaining the same results caused by the embodiments described herein, including all equivalents thereof within the scope of the appended claims.

Claims

1. A voltage-gated potassium ion channel Kv1.3 inhibitor, characterized in that, A polypeptide toxin comprising Mp3c from Macrothele proingens, the amino acid sequence of the polypeptide toxin Mp3c consists of: ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP.

2. A voltage-gated potassium ion channel Kv1.4 inhibitor, characterized in that, A polypeptide toxin comprising Mp3c from Macrothele proingens, the amino acid sequence of the polypeptide toxin Mp3c consists of: ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP.

3. A voltage-gated potassium ion channel Kv1.5 inhibitor, characterized in that, A polypeptide toxin comprising Mp3c from Macrothele proingens, the amino acid sequence of the polypeptide toxin Mp3c consists of: ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP. A polypeptide toxin comprising Mp3c from Macrothele proingens, the amino acid sequence of the polypeptide toxin Mp3c consists of: ACGVFNDRCPLFKCCPQYVCKGWRTKRCLNP.

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