CN110627891B - Method for concealing hemolytic toxic and side effects of bee venom hemolytic peptide - Google Patents
Method for concealing hemolytic toxic and side effects of bee venom hemolytic peptide Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a method for concealing toxic and side effects of bee venom lysopeptide, which takes natural bee venom dry powder or bee venom lysopeptide crude peptide synthesized by adopting a biological polypeptide solid-phase synthesis method as a raw material, and prepares the bee venom lysopeptide with the mass content of more than 98% through gel filtration chromatographic column separation and reversed-phase high performance liquid chromatography purification, wherein the concealing agent is adopted for dissolution in the separation process of the gel filtration chromatographic column; then, the melittin without hemolytic activity is obtained after the melittin is treated by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane; the hemolytic toxic and side effects of the bee venom hemolytic peptide can be 'hidden' after the treatment by the method; if it is treated with less than 1mmol/L of the organic acid and its salt buffer, it has hemolytic activity, which is confirmed by treating it with pure water (water for injection).
Description
Technical Field
The invention belongs to the field of biological pharmacy, and in particular relates to a preparation method of high-purity and high-activity melittin, in particular to a method for concealing hemolytic toxic and side effects of melittin.
Background
As is well known, bee venom is the liquid that is expelled from a stinging needle when a worker bees stings against an enemy, and is called bee venom. The traditional Chinese medicine has long history of using bee venom therapy to treat diseases, and particularly has better curative effects on diseases such as rheumatism, rheumatoid arthritis and scapulohumeral periarthritis. In recent years, along with the continuous improvement of separation, purification and biosynthesis technologies, on the basis of researching the components and the structure of bee venom, the functions and the action mechanisms of the bee venom are discussed, the foundation is laid for further clinical application, and a better application prospect is shown.
Bee venom hemolytic peptideMelittin) The (or melittin) is one of the important candidate molecules for antibiotic resistance against drug-resistant pathogens, has a strong broad-spectrum antibacterial and anti-inflammatory activity, but has limited application due to its toxic side effects of hemolysis and difficulty in obtaining it from nature. Therefore, we eliminate their hemolytic activity on the basis of retaining the antibacterial activity. Melittin is capable of forming an amphipathic α -helical structure, with 4 of the 5 basic amino acids concentrated at the C-terminus of the peptide chain (krkrkr), and is a typical cationic antimicrobial peptide. The bee venom hemolysin has strong inhibition effect on gram-positive bacteria and gram-negative bacteria, and also has strong hemolytic activity, thus limiting the clinical application.
The bee venom dry powder contains main active ingredients of peptides, enzymes and non-peptides. Peptides mainly include melittin, melittin and mast cell degranulation polypeptides. Enzymes are mainly the major species responsible for allergic reactions after the phospholipase A2 is stinged by bees. Non-peptide substances include histamine, various biogenic amines, which are associated with pain following bee stings.
The bee venom hemolysin is main component and active component of bee venom, and accounts for about 50% of dry weight of bee venom; has antiinflammatory, antibacterial, antiviral, and antitumor effects. The melittin is white or white-like powder, has a molecular weight of 2840 and D, and consists of 26 amino acid residues, and has a primary structure with an amino acid residue sequence of:
NH 2 -GLY-ILE-GLY-ALA-VAL-LEU-LYS-VAL-LEU-THR-THR-GLY-LEU-PRO-ALA-LEU-ILE-SER-TRP-ILE-LYS-ARG-LYS-ARG-GLN-GLN-COOH
since the first separation of bee venom lysopeptides consisting of 26 amino acids from bee venom in 1967 by Habermann et al, the amino acid sequence was analyzed, and it was confirmed that there was no disulfide bond in the structure. Tertiary structure was observed by Anderson et al in 1980, and the results indicate that the spatial structure of melittin can be divided into four regions: namely, (1-10) N-terminal alpha helix structure, (11-13) C hinge structure connecting two helices at 120 DEG, (13-20) C alpha helix structure, and (2 l-26) C terminal is positive charge region. Such four monomers are linked by hydrophobic groups to form a stable structure of tetramer, and 4 of 5 basic amino acids are concentrated at the C-terminus of the peptide chain (krkrkr), which is a typical cationic antibacterial peptide. The bee venom hemolytic peptide can form an amphipathic alpha-helical structure, and has the characteristics of broad-spectrum antibacterial and antiviral functions, stronger hemolytic activity and the like. However, when the melittin is combined with cell membrane to act, it is arranged on the surface of cell membrane in the form of monomer, but it still has obvious cytoxic action (hemolytic activity). Melittin is a broad-spectrum antibacterial peptide, and the antibacterial and antiviral effects of melittin are that the melittin damages the bacterial cell membrane in a mode of being combined with the membrane so as to achieve the aim of sterilization.
The biological broad-spectrum antibacterial peptide (antibacterial peptides) is widely used in insects, plants, animals and human bodies, has nonspecific antibacterial, antifungal and antiviral effects, and can be called peptide antibiotic (peptide antibiotics). The number of amino acid residues of the polypeptide is less than 40, and the polypeptide can form an alpha-helical structure and contains more basic amino acids (positively charged under physiological conditions) and more hydrophobic amino acids. The biological antibacterial peptide is combined on a bacterial membrane through electrostatic attraction of amphiphilic positive charges and phospholipid molecular negative charges of a microbial cell membrane, a hydrophobic end is inserted into the cell membrane, and finally the biological antibacterial peptide is gathered together through intermolecular displacement in the membrane to form an ion channel, so that the bacteria lose membrane potential and cannot maintain normal osmotic pressure to die. Due to the unique antibacterial mechanism, bacteria are not easy to generate drug resistance; the biological antibacterial peptide has strong bactericidal capability, broad antibacterial spectrum and less adverse reaction.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for concealing the toxic and side effects of bee venom hemolytic peptide, which takes natural bee venom dry powder or bee venom hemolytic peptide crude peptide synthesized by adopting a biological polypeptide solid-phase synthesis method as a raw material, and prepares the bee venom hemolytic peptide with the mass content of more than 98 percent through gel filtration chromatographic column separation and reversed-phase high performance liquid chromatography purification, wherein the concealing agent is adopted for dissolution in the separation process of the gel filtration chromatographic column; then the melittin without hemolytic activity is obtained after the melittin is treated by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane.
The natural bee venom dry powder contains 45-55% of bee venom hemolysis peptide.
The method is characterized in that the biological solid-phase synthesis method is adopted to synthesize the crude peptide of the melittin hemolysin, the FMOC protected amino acid solid-phase synthesis method is adopted, chloromethyl resin with the crosslinking degree of 2 percent is used as a carrier according to the preparation method of literature (refer to the method in Lou Carpino 1972 (FMOC)), polypeptide with the C end as amide is sequentially synthesized into the melittin composed of 26 amino acid residues according to the amino acid residue sequence of a primary structure, the melittin is reacted for 1 hour at the temperature of 0 ℃ in anhydrous hydrofluoric acid containing 10 percent anisole by mass, and the melittin is cut off from the chloromethyl resin after the reaction is completed, and meanwhile, the protecting group of a side chain is removed.
The masking agent is an organic acid solution with the concentration of 1 mmol/L-1000 mmol/L or an organic acid-organic acid salt buffer solution with the concentration of 1 mmol/L-1000 mmol/L; including, for example, but not limited to, acetic acid-ammonium acetate buffer solution, acetic acid solution.
The gel filtration chromatographic column separation specific operation is to balance the gel filtration chromatographic column by using a masking agent, dissolve natural bee venom dry powder or bee venom hemolysis peptide crude peptide synthesized by adopting a biological solid phase synthesis method by using the masking agent, loading the dissolved bee venom hemolysis peptide crude peptide into the gel filtration chromatographic column, eluting by using the masking agent as eluent, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and freeze-drying.
The molecular weight cut-off of the ultrafiltration membrane is less than 10000 Da, and the molecular weight cut-off of the nanofiltration membrane is 100-1000 Da; the semipermeable membrane has a molecular weight cut-off of less than 10000 Da.
The melittin without hemolytic activity prepared by the method is a biological broad-spectrum antibacterial peptide; the biological antibacterial peptide is melittin (26P) with the melittin hiding hemolytic activity, has broad-spectrum antibacterial, antifungal and antiviral activities, and has no hemolytic activity.
The melittin without hemolytic activity can be prepared into injection medicines, such as melittin for injection and melittin injection; can also be prepared into external preparations such as spray, gel and liniment for treating mosquito bite, detumescence, relieving pain and itching; can also be used for treating infantile eczema.
Principle of bee venom hemolytic peptide for concealing side effects of hemolytic toxin:
under the action of 1-1000 mmol/L of organic acid and salt thereof, the bee venom hemolysin restores the 120 DEG corner of 11-13 peptide chains of the primary structure of the bee venom hemolysin through molecular sieve filtration (ultrafiltration membrane ultrafiltration process, nanofiltration membrane nanofiltration process and/or biological semipermeable membrane action process), has no electrostatic adsorption effect with erythrocyte membranes, loses the perforation effect, has no hemolysis effect, solves the toxic and side effects of the bee venom hemolysin, has no influence on the perforation effect of procaryotic cells, and ensures that the sterilization effect is unchanged (the perforation effect of 13-26 peptides of the bee venom peptide).
The preparation method has simple process and easy operation, the bee venom hemolytic peptide is dissolved by an organic acid and a salt buffer thereof, is separated by a gel filtration chromatographic column, is purified by reverse phase high performance liquid chromatography, and can be used for 'concealing' the hemolytic toxic and side effects of the bee venom hemolytic peptide after being treated by ultrafiltration, nanofiltration, semipermeable membrane and other molecular sieves; experiments show that if it is treated with less than 1mmol/L of organic acid and its salt buffer, it will have hemolytic activity again, confirmed by treatment with pure water (water for injection).
Drawings
FIG. 1 shows the result of detecting the purity of melittin by analytical high performance liquid chromatography;
FIG. 2 is a schematic diagram showing the results of detection of the antibacterial activity of melittin without hemolytic activity.
Detailed Description
The present invention will be described in further detail by way of examples, but the scope of the present invention is not limited to the above.
Example 1: the method for concealing the toxic and side effects of the bee venom lysopeptide and the hemolytic detection content are as follows:
1. dissolving 1.0g of bee venom dry powder containing 50% bee venom hemolysin in 10mL of acetic acid-ammonium acetate buffer solution (100 mmol/L), centrifuging at 2000 rpm for 10min, collecting supernatant, performing preliminary separation by using SephadexG-50 gel filtration chromatographic column (50 mm multiplied by 600 mm), balancing the chromatographic column with 100mmol/L acetic acid-ammonium acetate buffer solution, eluting with 100mmol/L acetic acid-ammonium acetate buffer solution at a flow rate of 1.0mL/min after loading, detecting with ultraviolet detector 280nm, collecting the first peak, and freeze drying; purifying with semi-preparative high performance liquid chromatograph, and collecting peak with maximum abundance;
wherein the semi-preparative high performance liquid chromatograph performs purification using a C18 reverse phase column (300A, 10 μm,19 mm. Times.300 mm); dissolving the product obtained by gel filtration chromatographic column separation in 2mL of ultrapure water, centrifuging 10000g for 5min, and loading onto a reversed-phase column which is well balanced by BufferA (0.1% trifluoroacetic acid); gradient elution with BufferB (acetonitrile containing 0.1% trifluoroacetic acid); detecting at a flow rate of 10mL/min and 215nm, and collecting the maximum abundance peak;
2. and (3) sequentially carrying out ultrafiltration on the collected liquid by an ultrafiltration membrane with a molecular weight cut-off of 5000Da, nanofiltration by a nanofiltration membrane with a molecular weight cut-off of 200Da and dialysis by a dialysis membrane with a molecular weight cut-off of 3000Da to obtain the melittin (26P) without hemolytic activity.
The purity of melittin reaches 99.2% by analytical high performance liquid chromatography (C18) HPLC; the results are shown in FIG. 1; the melittin obtained by amino acid analysis and determination by MALDI-TOF mass spectrometer is the target product melittin.
3. Hemolytic activity test of melittin without hemolytic Activity
The hemolytic activity was determined according to the activity determination method of the national pharmaceutical standard WS1-XG-016-2001 bee venom quality standard, as follows:
adding sodium chloride solution with the mass concentration of 0.9% into the melittin prepared in the step 2 to prepare 8 mug/mL melittin solution serving as a test sample;
preparation of anticoagulated rabbit blood: adding 20mL of freshly collected rabbit blood into 2mL of 2% potassium oxalate solution, and slowly shaking to prepare anticoagulated rabbit blood; taking 8mL of anticoagulated rabbit blood, adding 10mL of 0.9% sodium chloride solution, and shaking uniformly to obtain diluted rabbit blood;
taking 9 test tubes, 3 test tubesRespectively adding 10mL of test sample; 3 branches of the negative control solution are respectively added with 10mL of 0.9% sodium chloride solution; adding 10mL of water into 3 branches respectively to serve as positive control; placing each tube in a water bath at 37 ℃ for 20min, respectively adding 0.2mL of diluted rabbit blood, slightly shaking, continuously keeping the temperature in the water bath at 37 ℃ for 30min at a certain time, taking out, placing in an ice water bath for 5min, centrifuging at 2000 rpm for 5min, respectively sucking the supernatant, and measuring the absorbance A at a wavelength of 545 nm; average of negative controlA nc Not more than 0.03, mean value of positive control tubeA pc Should be 0.8.+ -. 0.03, if not within this range, should be tested separately; the hemolytic activity was calculated as follows:
A i -A nc ()×100%
A
pc
-A
nc
in the middle ofA i -the sample absorbance;
A nc -negative control absorbance;
A pc positive control absorbance;
the results are shown in Table 1, and the hemolytic activity of the melittin without hemolytic activity obtained in step 2 is 0;
TABLE 1
Example 2: the method for concealing the toxic and side effects of the bee venom lysopeptide and the hemolytic property detection are as follows:
1. adopting an FMOC protected amino acid solid-phase synthesis method, preparing chloromethyl resin with 2% of crosslinking degree as a carrier according to a literature method, sequentially synthesizing a bee venom hemolysis peptide (26P) consisting of 26 amino acid residues according to an amino acid residue sequence of a primary structure of polypeptide with C-terminal as an amide, reacting in anhydrous hydrofluoric acid containing 10% of anisole at 0 ℃ for 1h, cutting the bee venom hemolysis peptide from the resin, and removing a protecting group of a side chain to obtain a crude bee venom hemolysis peptide; wherein the reagent for cutting the bee venom lysopeptide from the resin is trifluoroacetic acid/phenol/water/phenyl sulfide/1, 2-dimercaptoethanol (the volume ratio of trifluoroacetic acid, phenol, water, phenyl sulfide, 1, 2-dimercaptoethanol is 8:0.5:0.5:0.25), the reaction is carried out for 5 hours at room temperature, after removing most of the trifluoroacetic acid by rotary evaporation, diethyl ether is dripped at 0 ℃ to obtain flocculent precipitate, and the bee venom lysopeptide crude peptide is obtained after centrifugation;
2. performing preliminary separation by using a SephadexG-25 gel filtration chromatographic column, balancing the column by using acetic acid solution with the mass concentration of 30%, loading the sample, eluting by using acetic acid with the mass concentration of 30% at the flow rate of 1mL/min, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and freeze-drying; purifying with semi-preparative high performance liquid chromatograph, and collecting peak with maximum abundance;
3. ultrafiltering the collected solution with ultrafilter membrane with molecular weight cut-off of 5000Da, nanofiltration with nanofiltration membrane with molecular weight cut-off of 200Da and dialysis with dialysis membrane with molecular weight cut-off of 3000Da to obtain melittin (26P) without hemolytic activity;
the purity of melittin reaches 99.5% by analytical high performance liquid chromatography (C18) HPLC; determining that the prepared melittin is a target product melittin by amino acid analysis and analysis with MALDI-TOF mass spectrometer;
4. hemolytic activity test of melittin without hemolytic activity the method was the same as in step 3 of example 1, and the hemolytic activity of melittin without hemolytic activity obtained by detecting step 3 of this example was 0 (see Table 2);
TABLE 2
Example 3: the method for concealing the toxic and side effects of the bee venom lysopeptide and the hemolytic property detection are as follows:
1. dissolving 1.0g of bee venom dry powder containing 52% bee venom hemolysin in 10mL of acetic acid-ammonium acetate buffer solution (200 mmol/L), centrifuging at 2000 rpm for 10min, collecting supernatant, performing preliminary separation by using SephadexG-50 gel filtration chromatographic column (50 mm multiplied by 600 mm), balancing the chromatographic column with 200mmol/L acetic acid-ammonium acetate buffer solution, eluting with 200mmol/L acetic acid-ammonium acetate buffer solution at a flow rate of 1.0mL/min after loading, detecting with ultraviolet detector 280nm, collecting the first peak, and freeze drying; purifying with semi-preparative high performance liquid chromatograph, collecting peak with maximum abundance, and drying the collected liquid;
2. treating the dried product obtained in the step 1 by using a dialysis membrane
The molecular weight cutoff is 3500 (7000) Da by adopting an inlet MD34-3.5 (7) dialysis bag; shearing a dialysis bag with the size of 1-3m according to the feeding amount of each time;
new dialysis bag treatment: boiling the dialysis bag with water solution containing 2% sodium bicarbonate and 1mol/L disodium edetate for 10min, cooling, and washing with injectable water for 3-5 times; boiling with 1mol/L disodium edetate solution for 10min, cooling, washing with injectable water for 3-5 times, soaking in injectable water at 4deg.C, and preserving; the treated dialysis bag should be put on gloves.
Preparing a dialysis solution: weighing 7.7 g of ammonia acetate, adding 0.8L of water for injection for dissolution, and adding 10-20mL of acetic acid to adjust the pH to 4.5-5.5; water for injection was added to 1L.
Dissolving bee venom powder: weighing the dried product in the step 1, and dissolving the dried product in the dialyzate according to the proportion of 6 percent to fully dissolve the dried product; taking out the treated dialysis bag, firstly, pricking one end with a rubber ring, pouring 2/3 of water for injection, reversing the dialysis bag for several times, discarding the water in the dialysis bag, and cleaning for 2-3 times; then putting the bee venom dissolved with the dried product into a dialysis bag (about 2/3), rolling up the other end by a rubber ring, and putting into the dialysis liquid with the volume of 5 times of that of the bee venom for dialysis; the dialysis extraction is carried out for more than 30 hours, the dialysis bag is taken out, and the solution in the container is the bee venom hemolysis peptide dialysate I liquid;
and (3) secondary dialysis: transferring the dialysis bag into water for injection with 6 times of the volume of the dialysis bag, and continuing dialysis for 30 hours; taking out the dialysis bag, wherein the solution in the container is the bee venom hemolysis peptide dialysate II; and then carrying out dialysis for the 3 rd to the 4 th times; obtaining a bee venom hemolysis peptide dialysate III and IV; namely, melittin stock solution, ultraviolet content detection and hemolytic activity measurement. The bee venom hemolysis peptide dialysate I and II have no hemolysis activity through measurement; the bee venom hemolysis peptide dialyzates III and IV have hemolytic activity, and the hemolytic activity of the solution is more than 60% in 8 mug/mL (see table 3); the result shows that the bee venom hemolysin has hemolytic activity after being treated by water for injection;
TABLE 3 Table 3
Example 4: antibacterial activity detection of melittin without hemolytic activity
The antibacterial activity of the non-hemolytic melittin obtained in the above example was measured by heating and melting beef extract peptone agar medium in water bath, cooling to about 50deg.C, and sucking 60 μl of the test bacterial liquid (OD) 600 =0.3) adding the mixture into 20mL of beef extract peptone agar medium, quickly and uniformly mixing, pouring the mixture into a sterile flat dish with the diameter of 9cm, and placing the mixture horizontally at room temperature to be coagulated, wherein the thickness is about 1.5 mm; punching round holes with the diameter of 2.7mm on agar, respectively adding 10 μl of melittin without hemolytic activity into the holes, using sterile water as negative control, and using Amp as positive control; after the sample was added, the plate was placed in a refrigerator at 4℃and after the sample had diffused sufficiently to the agar, the plate was inverted and incubated overnight at 37℃and the results were observed the next day. The test bacteria in the test are staphylococcus aureus, and the experimental result is shown in figure 2; the results show that melittin without hemolytic activity has antibacterial activity against staphylococcus aureus.
Determination of Minimum Inhibitory Concentration (MIC) of melittin without hemolytic Activity
Diluting the bacterial liquid of the test strain cultured to the logarithmic phase to 5×10 6 CFU/mL is added into a 96-well culture plate, 90 mu l of bacterial liquid is added into each well of a test well, and then 10 mu l/well of melittin solution with different concentrations diluted in a double ratio is added; the positive control is 100 mu L/hole bacterial liquid, and the negative control is 100 mu L of corresponding culture medium; then slowly shaking and culturing at 37 ℃ for about 16 hours, and measuring OD by using an enzyme labeling instrument 630 The method comprises the steps of carrying out a first treatment on the surface of the The minimum concentration for inhibiting bacterial growth is MIC, and the results are shown in table 4;
table 4: melittin MIC (minimum concentration for inhibiting bacterial growth)
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention should be made by those skilled in the art without departing from the design spirit of the present invention, and the scope of the present invention is defined by the claims.
Claims (4)
1. A method for concealing hemolytic toxic side effects of bee venom hemolytic peptide is characterized in that: taking natural bee venom dry powder or bee venom hemolysis peptide crude peptide synthesized by adopting a biological polypeptide solid-phase synthesis method as a raw material, and separating by a gel filtration chromatographic column and purifying by reverse-phase high performance liquid chromatography to obtain bee venom hemolysis peptide with the mass content of more than 98%, wherein a masking agent is adopted for dissolution in the separation process of the gel filtration chromatographic column; then, the melittin without hemolytic activity is obtained after the melittin is treated by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane;
the masking agent is an organic acid solution with the concentration of 1 mmol/L-1000 mmol/L or an organic acid-organic acid salt buffer solution with the concentration of 1 mmol/L-1000 mmol/L, and comprises an acetic acid-ammonium acetate buffer solution and an acetic acid solution;
the molecular weight cut-off of the ultrafiltration membrane is less than 10000 Da, and the molecular weight cut-off of the nanofiltration membrane is 100-1000 Da; the semipermeable membrane is a dialysis membrane with molecular weight cut-off less than 10000 Da.
2. The method for concealing a hemolytic toxic side effect by a melittic hemolytic peptide according to claim 1, wherein: the natural bee venom dry powder contains 45-55% bee venom hemolysin.
3. The method for concealing a hemolytic toxic side effect by a melittic hemolytic peptide according to claim 1, wherein: the gel filtration chromatographic column separation specific operation is to balance the gel filtration chromatographic column by using a masking agent, dissolve natural bee venom dry powder or bee venom hemolysis peptide crude peptide synthesized by adopting a biological solid phase synthesis method by using the masking agent, loading the dissolved bee venom hemolysis peptide crude peptide into the gel filtration chromatographic column, eluting by using the masking agent as eluent, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and freeze-drying.
4. The method for concealing a hemolytic toxic side effect by a melittic hemolytic peptide according to claim 1, wherein: the method for synthesizing the crude peptide of the melittin hemolysin by adopting the biological polypeptide solid-phase synthesis method is characterized in that the FMOC protected amino acid solid-phase synthesis method is adopted, chloromethyl resin with the crosslinking degree of 2% is used as a carrier, polypeptide with the C end as an amide is sequentially synthesized into the melittin hemolysin consisting of 26 amino acid residues according to the amino acid residue sequence of a primary structure, the melittin hemolysin is reacted in anhydrous hydrofluoric acid containing anisole with the mass percentage of 10% at the temperature of 0 ℃ for 1h, and the melittin hemolysin is cut off from the chloromethyl resin after the reaction is finished, and meanwhile, the protecting group of a side chain is removed.
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