A high-purity cobra venom active peptide and its preparation method and application

Abstract

The invention discloses a high-purity cobra venom active peptide and a preparation method and application thereof, and belongs to the field of biomedical technology. The cobra venom active peptide is a brand-new cobra venom active peptide, which is not disclosed in the prior art, and after purification, the HPLC purity reaches 100%. The cobra venom active peptide has a good analgesic effect, and the onset time of the analgesic effect is significantly shorter than that of cobot peptide, and is very suitable for use as an analgesic drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a high-purity cobra venom active peptide and a preparation method and application thereof. Background Art

[0002] Biotoxins are a class of biologically significant natural chemical products with a wide range of practical applications. As an important source of traditional toxins, some biotoxins have been used as effective clinical drugs and actively promoted drug innovation; on the other hand, many biotoxins can be used as lead compounds for innovative drugs, playing an irreplaceable role in drug screening, molecular design and the discovery of new drug targets.

[0003] As an important component of biological toxins, the composition of snake venom is very complex, mainly including neurotoxins (NT), cytotoxins (CTXs), phospholipase A2 (PLA2), bioactive peptides and other small molecules. Snake venom components have multiple biological activities, including anti-inflammatory, anti-tumor, hemostasis / anticoagulant, thrombolysis, analgesia, etc.

[0004] Neurotoxins are important active components in snake venoms, mainly distributed in the venom of rattlesnakes in the cobra family, sea snake family and viper family. In ancient my country, there was a folk remedy of using cobra to soak wine and drink to treat low back and leg pain. Cobra venom neurotoxins have a central analgesic effect similar to morphine, and are used for analgesic treatment of intractable pain, malignant tumor pain and joint pain. The analgesic effect of neurotoxins is different from that of opioids, and is characterized by high potency, long duration, no tolerance and addiction. It is a new type of analgesic with great potential. Cobratide is a neurotoxin extracted from the venom of the Chinese cobra snake. It is mainly used in the treatment of chronic pain such as advanced cancer pain, chronic joint pain, sciatica, trigeminal neuralgia, leprosy reaction neuralgia, etc. The National Drug Standard for Cobratide (WS1-XG-009-2000) stipulates that the neurotoxin protein in cobratide shall not be less than 35.0% calculated on a dry basis, and the relative percentage of the main peak in its HPLC purity test shall not be less than 90.0%. Therefore, the commercially available cobotin may be a mixture of two or more neurotoxins. A patent has been filed to separate and identify two other neurotoxin impurities from cobotin raw materials (CN 202210094328.9). The content and purity of pharmaceutical raw materials play a decisive role in ensuring the quality, efficacy, safety, and dosage form of drugs. The presence of impurities may cause serious safety problems. The cobra neurotoxins and cobotin obtained by the existing purification methods may have problems such as relatively low content and purity between batches of product raw materials, unstable clinical efficacy, and slow onset time. Importantly, the use of organic solvents and high-concentration salt solutions in the purification process may cause them to denature, which is particularly unfavorable for maintaining their biological activity.

[0005] As a biochemical drug of natural origin, cobotide has the characteristics of good analgesic effect, long-lasting analgesic effect, small side effects, no drug tolerance and no dependence. However, the purity of commercially available cobotide is not high, and the onset time is slow. It usually needs to be used in combination with fast-acting analgesics.

[0006] Therefore, it is necessary to develop an active peptide from cobra venom with fast onset of action, strong analgesic effect and high purity.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] The purpose of the present invention is to solve the problems that the commercially available cobot peptide has low purity, slow onset time, and generally needs to be used as a compound drug with fast-acting analgesics, and to provide a cobra venom active peptide with fast onset time, strong analgesic effect and high purity.

[0009] The first aspect of the present invention provides a high-purity active peptide from cobra venom, the amino acid sequence of the active peptide from cobra venom being: LECHNQQSSETPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKKGIEINCCTTDRCNN.

[0010] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned cobra venom active peptide.

[0011] The third aspect of the present invention provides an expression cassette, a recombinant vector or a transgenic cell comprising the above nucleic acid molecule, wherein the transgenic cell does not contain any propagation material.

[0012] A fourth aspect of the present invention provides a method for purifying active peptides from cobra venom, comprising the following steps:

[0013] 1) Take the lyophilized powder of cobra venom, dissolve it in 0.2M Tris-HCl buffer (pH 7.5), centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use;

[0014] 2) After equilibration of the CM-sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P11 fraction (770-820 mL) with analgesic activity was collected;

[0015] 3) The collected fractions were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain a crude product of cobra venom active peptide P11;

[0016] 4) Take the crude active peptide P11 from cobra venom, dissolve it in 0.1M NH4OAc aqueous solution, centrifuge it at 10000rpm for 10min at 4℃, and take the supernatant for later use;

[0017] 5) Continue to take the supernatant of the cobra venom active peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with a 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 (56-58mL) containing the cobra venom active peptide P11;

[0018] 6) The collected fractions in step 5) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain the pure product of cobra venom active peptide P11.

[0019] The fifth aspect of the present invention provides the use of the above-mentioned cobra venom active peptide in the preparation of analgesic drugs.

[0020] The sixth aspect of the present invention provides the use of the above nucleic acid molecules in the preparation of analgesic drugs.

[0021] A seventh aspect of the present invention provides the use of the above-mentioned expression cassette, recombinant vector or transgenic cell in the preparation of analgesic drugs.

[0022] In one embodiment of the present invention, the analgesic drug is used to relieve neuropathic pain or bone metastasis cancer pain.

[0023] The eighth aspect of the present invention provides a medicine comprising at least one of (a) to (c):

[0024] (a) the active peptide from cobra venom according to claim 1;

[0025] (b) the nucleic acid molecule according to claim 2;

[0026] (c) The expression cassette, recombinant vector or transgenic cell according to claim 3.

[0027] A ninth aspect of the present invention provides a preparation characterized by comprising at least one of (a) to (c):

[0028] (a) the active peptide from cobra venom according to claim 1;

[0029] (b) the nucleic acid molecule according to claim 2;

[0030] (c) The expression cassette, recombinant vector or transgenic cell according to claim 3.

[0031] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0032] (1) The cobra venom active peptide of the present invention is a new cobra venom active peptide, which has not been disclosed in the prior art, and has been purified with a HPLC purity of 100%;

[0033] (2) The active peptide from cobra venom of the present invention has a good analgesic effect, and the onset time of the analgesic effect is significantly shorter than that of cobot peptide, and is very suitable for use as an analgesic drug;

[0034] (3) The purification method of the cobra venom active peptide of the present invention has simple steps, and is purified by only two column chromatography steps of CM-Sepharose cation exchange column and size exclusion chromatography column. No organic solvent is used in the purification process, and no high concentration salt is used. All operations are carried out at room temperature or below, which can maximize the biological activity of the cobra venom active peptide. In addition, the sample loading amount is large, which is conducive to the control of the purification process and the improvement of the degree of automation, and is conducive to the realization of large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a cation exchange chromatography spectrum according to Example 1 of the present invention;

[0036] Figure 2 is the size exclusion chromatogram of P11 in Example 1 of the present invention;

[0037] Figure 3 This is the HPLC spectrum of the cobra venom active peptide P11 obtained according to Example 1 of the present invention;

[0038] Figure 4 is a high-resolution mass spectrum of the cobra venom active peptide P11 obtained according to Example 1 of the present invention;

[0039] Figure 5 This is a diagram showing the N-terminal sequence analysis result of the cobra venom active peptide P11 obtained according to Example 1 of the present invention;

[0040] Figure 6 This is a diagram showing the results of De novo full sequence analysis of the cobra venom active peptide P11 obtained according to Example 1 of the present invention. DETAILED DESCRIPTION

[0041] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0042] The technical solution of the present invention is described below by specific embodiments. It should be understood that one or more steps mentioned in the present invention do not exclude the existence of other methods and steps before and after the combination step, or other methods and steps can be inserted between these explicitly mentioned steps. It should also be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the numbering of each method step is only for the purpose of identifying each method step, and does not limit the order of arrangement of each method or limit the scope of implementation of the present invention. The change or adjustment of the relative relationship thereof can also be regarded as the scope of implementation of the present invention without substantial changes in the technical content.

[0043] The sources of the raw materials and instruments used in the examples are not particularly limited and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0044] Example 1

[0045] The method for purifying active peptides from cobra venom comprises the following steps:

[0046] 1) Weigh 2 g of lyophilized cobra venom powder and dissolve it in 10 mL of 0.2 M Tris-HCl buffer (pH 7.5). Centrifuge at 10,000 rpm for 10 min at 4°C and take the supernatant for later use.

[0047] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P11 fraction (770-820 mL) with analgesic activity was collected.

[0048] 3) The collected fractions were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain a crude product of cobra venom active peptide P11.

[0049] 4) Weigh 30 mg of crude cobra venom active peptide P11, dissolve it in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0050] 5) Continue to load the supernatant of the cobra venom active peptide P11 obtained in step 4) onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 (56-58mL) containing the cobra venom active peptide P11.

[0051] 6) The collected fractions in step 5) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain the pure product of cobra venom active peptide P11.

[0052] HPLC analysis: Use mobile phase to prepare 1 mg / mL cobra venom active peptide P11 solution, inject 10 μL, select C18 column for HPLC analysis, HPLC spectrum as shown Figure 3 As shown in the figure, the purity of the active peptide P11 from cobra venom was 100%.

[0053] Mass spectrometry analysis: A 1 mg / mL solution of cobra venom active peptide P11 was prepared with the mobile phase, and after dilution, it was analyzed using ESI high-resolution mass spectrometry. The high-resolution mass spectrum is shown in Figure 4 As shown in Figure 2, the molecular weight of the active peptide P11 from cobra venom was 6959.0075 Da.

[0054] N-terminal sequence analysis: The cobra venom active peptide P11 was reduced with DTT and derivatized with IAM, and then the N-terminal sequence of the cobra venom active peptide P11 derivative was analyzed by Edman degradation. The N-terminal sequence analysis results are shown in the figure below. Figure 5After testing, the N-terminal sequence of the cobra venom active peptide P11 is LECHNQQSSETPTTTGCSGGETNCY.

[0055] De novo full sequence analysis results: After the cobra venom active peptide P11 was digested with Pepsin, Trypsin and Lyc-C, De novo full sequence analysis was performed by LC-MS / MS. The De novo full sequence analysis results are shown in the figure below. Figure 6 After testing, the full sequence of the active peptide P11 from cobra venom is LECHNQQSSETPTTTGCSGGETNCYKKRWR DHRGYRTERGCGCPSVKKGIEINCCTTDRCNN.

[0056] Experimental example

[0057] Acetic acid writhing analgesia test in mice:

[0058] Thirty-six SPF male mice, weighing 18-22 g, were randomly divided into two treatment groups and a saline control group according to body weight, with 12 mice in each group. Each group of animals was intramuscularly injected with the corresponding 0.9% sodium chloride injection (control group), 20 μg / kg cobra venom active peptide P11 and 20 μg / kg cobotin (purchased from China Food and Drug Inspection Institute), with a dosage volume of 2 mL / kg. One hour after administration, each mouse was intraperitoneally injected with 0.8% glacial acetic acid solution, with an injection volume of 10 mL / kg, and the writhing reactions (times / 15 min) at 0-15 min and 16-30 min and the writhing reactions (times / 30 min) at 0-30 min and 31-60 min were recorded respectively.

[0059] The writhing inhibition rate of each dosage group was calculated according to the following formula. The results are shown in Table 1.

[0060] Torsion inhibition rate = (number of torsion in the control group - number of torsion in the drug-treated group) / number of torsion in the control group × 100%

[0061] Table 1 Comparison of the writhing activity of cobra venom active peptide P11 and cobotide

[0062]

[0063] As can be seen from Table 1, the cobra venom active peptide P11 prepared in Example 1 has a significant inhibitory effect on mouse writhing, which is significantly different from the control group and has a significant analgesic effect. The writhing inhibition rate of P11 at 0-15min, 16-30min, and 0-30min is significantly higher than that of cobotin, and the writhing inhibition rate at 31-60min is comparable to that of cobotin.

[0064] Analgesia test in the rat chronic sciatic nerve compression injury model:

[0065] Fifty qualified SPF male SD rats were selected and randomly divided into a sham operation group (10 rats) and a modeling group (40 rats) according to their body weight. The animals in the modeling group were anesthetized with 10% chloral hydrate (3 mL / kg) and the sciatic nerve of the right hind limb was surgically exposed and four loose ligatures were made with an interval of 1-2 mm. The nerves of the animals in the sham operation group were exposed but not ligated. On the 8th day after surgery, the mechanical withdrawal thresholds of the left and right hind limbs of the animals in each group were tested. The animals after successful modeling were randomly divided into a modeling group, a P11 group, and a cobotide group according to the mechanical withdrawal threshold of the right foot combined with body weight, with 10 rats in each group and 10 rats in the sham operation group, for a total of 40 animals. The animals in each group were given 0.9% sodium chloride injection, P11 (20 μg / kg), and cobotide (purchased from China Food and Drug Inspection Institute, 20 μg / kg) by intramuscular injection of the hind limbs, respectively, with a dosing volume of 500 μL / kg / time. The mechanical pain threshold of the right foot on the operated side and the left foot on the control side was measured using a plantar pain meter 1 hour and 3 hours after administration.

[0066] Quantitative data were expressed as mean and standard deviation, and SPSS 19.0 analysis software was used for data analysis.

[0067] On the day of grouping, compared with the sham operation group, the mechanical withdrawal threshold of the right foot of the animals in the modeling group, P11 group and cobotide group was extremely significantly lower (p<0.001), and was extremely significantly lower than that of the left foot in the same group (p<0.001), indicating that the chronic sciatic nerve compression injury model in rats was successfully established (see Table 2).

[0068] Table 2 Mechanical withdrawal threshold of bilateral hind paws of animals in each group after secondary grouping (g, Mean±SD)

[0069]

[0070] Note: Compared with the sham operation group, ***p<0.001; compared with the left foot mechanical withdrawal threshold in the same group, &&& p<0.001

[0071] The results of the mechanical withdrawal threshold test of the bilateral hind paws of the animals 1 hour and 3 hours after administration are shown in Table 3. As shown in Table 3, 1 hour and 3 hours after administration, the mechanical withdrawal threshold of the right paw of the animals in the model group was significantly lower than that in the sham operation group (p<0.001). 1 hour and 3 hours after administration of P11, the mechanical withdrawal threshold of the right paw of the animals in this group was significantly or extremely significantly higher than that in the model group (p<0.01 or p<0.001); 1 hour and 3 hours after administration of cobotide, the mechanical withdrawal threshold of the right paw of the animals in this group was significantly or extremely significantly lower than that in the model group (p<0.05 or p<0.01 or p<0.001); 1 hour after administration, the mechanical withdrawal threshold of the right paw of the animals in the cobotide group was significantly lower than that in the P11 group (p<0.05).

[0072] Table 3 Mechanical withdrawal threshold of bilateral hind paws of animals 3 hours after drug administration (g, Mean±SD)

[0073]

[0074] Note: Compared with the sham operation group, *p<0.05, **p<0.01, ***p<0.001; compared with the modeling group, #p<0.05, ##p<0.01, ###p<0.001; compared with the P11 group, &p<0.05

[0075] The results showed that a single intramuscular injection of P11 and cobotin had a significant analgesic effect on the neuropathic pain model caused by chronic sciatic nerve compression injury in rats; the analgesic effect of P11 was stronger than that of cobotin 1 hour after administration, and the analgesic effect of P11 was equivalent to that of cobotin 3 hours after administration.

[0076] Mouse bone metastasis pain analgesia test:

[0077] Human breast cancer cells MDA-MB-231 in the logarithmic growth phase were digested and collected by trypsin, and then washed and resuspended in PBS (the final cell concentration reached 2.5×107 cells / mL, and inoculated into the left posterior tibia of BALB / c Nude mice anesthetized by isoflurane breathing method at 4 μL / mouse. Seven days after tumor cell injection, the animal's paw withdrawal pain threshold was measured by Von Frey method, and the animals were randomly divided into model group, P11 group and cobotide (purchased from China Food and Drug Inspection Institute) group according to the pain threshold and animal weight, with 8-10 animals in each group. A single intramuscular injection was given at a dose of 20 μg / kg. During the measurement, the animal was placed in the test container, and the animal's left hind paw withdrawal pressure was measured after the animal was quiet (when the pressure reached the maximum value of 15 g, the incubation period lasted for 20 s, and the animal still did not show a paw withdrawal reaction, to avoid tissue damage, it was manually ended, and the paw withdrawal pressure value was recorded as 15 g. ), when the animal quickly retracts its paw or licks its paw, manually record the pain value displayed on the instrument interface. If the measurement process is stopped due to spontaneous activity of the animal, wait until the animal is quiet and then re-measure. The paw retraction threshold was measured once before administration, 1 h, 3 h, and 6 h after administration (see Table 4).

[0078] Quantitative data were expressed as mean and standard deviation, and SPSS 19.0 analysis software was used for data analysis.

[0079] Table 4 Mechanical withdrawal threshold of the left hind paw of animals after drug administration (g, Mean±SD)

[0080]

[0081] Note: Compared with the normal control group, * p<0.05, **p<0.01, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001.

[0082] 1 to 6 hours after administration, the mechanical withdrawal threshold of the left paw of the animals in the model group was significantly or extremely significantly lower than that of the normal control group (p<0.01 or p<0.001), while 1 to 6 hours after administration of P11 or cobotide, the mechanical withdrawal threshold of the left paw of the animals was significantly higher than that of the animals in the model group, among which the highest increase was 1 hour after administration (p<0.001) (see Table 4), indicating that a single intramuscular injection of P11 and cobotide had a significant analgesic effect on bone metastasis pain in mice; at the same dose, the analgesic effect of P11 was slightly better than that of cobotide.

[0083] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A high-purity active peptide from cobra venom, characterized in that: The amino acid sequence of the cobra venom active peptide is: LECHNQQSSETPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKKGIEINCCTTDRCNN.

2. A nucleic acid molecule encoding the cobra venom active peptide according to claim 1.

3. An expression cassette, a recombinant vector or a transgenic cell comprising a nucleic acid molecule according to claim 2, said transgenic cell not comprising propagation material.

4. Use of the active peptide from cobra venom according to claim 1 in the preparation of analgesic drugs.

5. Use of the nucleic acid molecule according to claim 2 in the preparation of analgesic drugs.

6. Use of the expression cassette, recombinant vector or transgenic cell according to claim 3 in the preparation of analgesic drugs.

7. The use according to any one of claims 4 to 6, wherein the analgesic drug is used to relieve neuropathic pain or bone metastasis cancer pain.

8. A medicine, characterized in that Contains at least one of (a) to (c): (a) the active peptide from cobra venom according to claim 1; (b) the nucleic acid molecule according to claim 2; (c) The expression cassette, recombinant vector or transgenic cell according to claim 3.

9. A preparation characterized in that Contains at least one of (a) to (c): (a) the active peptide from cobra venom according to claim 1; (b) the nucleic acid molecule according to claim 2; (c) The expression cassette, recombinant vector or transgenic cell according to claim 3.

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