CN111153966B - Wasp venom peptide reverse sequence analogue WVF-II and preparation method and application thereof - Google Patents
Wasp venom peptide reverse sequence analogue WVF-II and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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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
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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 provides a wasp venom peptide reverse sequence analogue WVF-II, a preparation method and application thereof, belonging to the technical field of polypeptide synthesis, wherein the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II is shown as SEQ ID NO: 1. The in vitro antibacterial activity test shows that the wasp venom peptide reverse sequence analogue WVF-II has remarkable bactericidal effect on gram-positive bacteria, gram-negative bacteria and fungi, has better stability and low hemolytic toxicity, and has the characteristics of broad spectrum, high efficiency and safety. The wasp venom peptide reverse sequence analogue WVF-II can be used as an active ingredient to replace the traditional antibiotics for treatment clinically.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a wasp venom peptide reverse sequence analogue WVF-II, a preparation method and application thereof.
Background
The antibacterial peptide can resist invasion of microorganism, and has specific or non-specific effect on gram-negative bacteria, positive bacteria, fungi, viruses, tumors, eukaryotes and the like. In addition to having antimicrobial action, the antimicrobial peptides have other functions. The antibacterial peptide is usually applied in the form of polypeptide analogues or polypeptide derivatives, and the substances can be obtained by modifying natural antibacterial peptide amino acids. Although a large number of natural antimicrobial peptides have been found to be expected to replace the problem of drug resistance caused by abuse of conventional antibiotics, the natural antimicrobial peptides have a number of disadvantages which limit their use as clinical drugs, particularly in terms of toxicity and enzymatic stability.
Disclosure of Invention
The invention aims to provide a wasp venom peptide reverse sequence analogue WVF-II, a preparation method and application thereof, wherein the wasp venom peptide reverse sequence analogue WVF-II has good antibacterial activity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a wasp venom peptide anti-sequence analogue WVF-II, wherein the amino acid sequence of the wasp venom peptide anti-sequence analogue WVF-II is shown as SEQ ID NO: 1.
The invention also provides a preparation method of the wasp venom peptide reverse sequence analogue WVF-II, which comprises the following steps:
1) Sequentially coupling amino acids on the resin according to the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II to obtain peptide resin;
2) And (3) cracking the peptide resin in the step (1), adding a side chain protecting group scavenger, precipitating the target polypeptide, centrifuging, and collecting the precipitate to obtain the wasp venom peptide reverse sequence analogue WVF-II.
Preferably, the resin in step 1) comprises 4- (2 ',4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzene methylamine resin.
Preferably, the coupling reagent used for coupling the amino acids in step 1) is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-diaminopropyl-N-ethylcarbodiimide, 2- (1H-benzotrisazo L-1-yl) -1, 3-tetramethylurea tetrafluoroborate, O-benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-1-oxy tris (dimethylamino) phosphorus hexafluorophosphate, benzotriazol-1-yl-oxy-tripyrrolidinyl phosphorus hexafluorophosphate and 1-hydroxybenzotriazole.
Preferably, the cleavage liquid used for cleavage in step 2) comprises trifluoroacetic acid and/or hydrofluoric acid.
Preferably, the ratio of the mass of the peptide resin in step 2) to the volume of the lysate is 1 g:5-50 mL.
Preferably, the side chain protecting group scavenger in step 2) is selected from any two or more of anisole, phenylsulfide, triisopropylsilane, phenol, water, 1, 2-ethanedithiol and m-cresol.
Preferably, the reagent for precipitating the polypeptide of interest in step 2) comprises ice-anhydrous diethyl ether.
Preferably, after the reverse-sequence wasp venom peptide analog WVF-II is obtained in the step 2), purification of the reverse-sequence wasp venom peptide analog WVF-II is further included.
The invention also provides application of the wasp venom peptide reverse sequence analogue WVF-II in broad-spectrum bacteriostasis.
The invention has the beneficial effects that: the invention provides a wasp venom peptide anti-sequence analogue WVF-II, wherein the amino acid sequence of the wasp venom peptide anti-sequence analogue WVF-II is shown as SEQ ID NO: 1. The N end of the wasp venom peptide reverse sequence analogue WVF-II has positive charges, can be combined with negatively charged cell membranes, and hydrophobic amino acid at the C end forms an alpha helix structure, can be perforated on the surface of the cell membranes, so that bacteria death is caused. The wasp venom peptide reverse sequence of the inventionThe in vitro antibacterial activity test of the analogue WVF-II shows that the analogue WVF-II has remarkable bactericidal effect on gram-positive bacteria, gram-negative bacteria and fungi, has better stability and low hemolytic toxicity, and has the characteristics of broad spectrum, high efficiency and safety. The wasp venom peptide reverse sequence analogue WVF-II can be used as an active ingredient to replace the traditional antibiotics for treatment clinically. The minimum inhibitory concentration (Minimal inhibition concentration, MIC) of the wasp venom peptide reverse sequence analogue WVF-II of the invention on staphylococcus aureus, escherichia coli and candida albicans is 0.5 mug.mL respectively -1 、1μg·mL -1 And 0.5. Mu.g.mL -1 Does not show hemolytic activity in the bacteriostatic concentration range. The wasp venom peptide reverse sequence analogue WVF-II has unique structural sequence and action mechanism, and is more perfect in antibacterial activity, stability and hemolytic activity reduction.
Drawings
FIG. 1 is a graph showing the results of antibacterial experiments against the reverse sequence of the wasp venom analogue WVF-II against Staphylococcus aureus (left), candida albicans (middle) and Escherichia coli (right): 1 represents the antibacterial activity of penicillin sodium; 2 as negative control dd H 2 O;3 is 1 mg.mL -1 Antibacterial activity of the reverse-order peptide of (a).
Detailed Description
The invention provides a wasp venom peptide anti-sequence analogue WVF-II, wherein the amino acid sequence of the wasp venom peptide anti-sequence analogue WVF-II is shown as SEQ ID NO:1, specifically: NH2-LLGSILKGIRKKR-COOH.
The amino acid sequence of the wasp venom peptide reverse sequence is shown as SEQ ID NO:2, specifically: NH (NH) 2 LLGSGLKGIRKKR-COOH. The invention replaces the fifth G of the reverse sequence of the wasp venom peptide with I, namely replaces hydrophilic neutral glycine G with a hydrophobic amino acid (isoleucine I), thereby further increasing the cell selectivity of the antibacterial peptide, improving the antibacterial activity and weakening the hemolytic activity of erythrocytes. The N end of the reverse-sequence peptide molecule has positive charge, can be combined with negatively charged cell membrane, and the hydrophobic amino acid at the C end forms an alpha helical structure, can be perforated on the surface of the cell membrane, thereby leading to bacterial deathAnd (3) death.
The invention also provides a preparation method of the wasp venom peptide reverse sequence analogue WVF-II, which comprises the following steps:
1) Sequentially coupling amino acids on the resin according to the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II to obtain peptide resin;
2) And (3) cracking the peptide resin in the step (1), adding a side chain protecting group scavenger, precipitating the target polypeptide, centrifuging, and collecting the precipitate to obtain the wasp venom peptide reverse sequence analogue WVF-II.
The invention preferably also comprises the processes of washing resin, soaking resin, removing amino protecting group and washing resin again before amino acid is coupled on the resin according to the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II; the resin preferably comprises Rink Amide MBHA resin (4- (2 ',4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methyl benzhydrylamine resin); the resin acts as a carrier.
In the present invention, the reagent used for washing the resin is preferably Dimethylformamide (DMF); the number of times of the washing is preferably 5 to 7 times, more preferably 6 times; the solvent used for soaking the resin is preferably Dimethylformamide (DMF) or Dichloromethane (DCM); the volume ratio of the mass of the resin to the solvent in the soaking process is preferably 0.5 g/10 mL; the soaking time is preferably 20-40 min, more preferably 30min; the soaking process preferably further comprises the step of introducing nitrogen gas to enable the resin to turn up and down in the solution; the reagent for removing amino protecting group is preferably piperidine (PIP) solution; the concentration of the piperidine (PIP) solution is preferably 10% to 30%, more preferably 20% to 25%; the time for the removal is preferably 5 to 30min, more preferably 10 to 20min; the piperidine (PIP) solution is preferably used in an amount of 2 to 3 times the volume of the resin; the reagent used for the re-washing of the resin is preferably Dimethylformamide (DMF); the number of times of washing is preferably 2 to 10 times, more preferably 6 to 8 times. In the implementation process of the invention, after the resin is washed again, the invention also repeats the processes of removing the amino protecting group once and washing the resin again.
Through the treatment, amino acid is sequentially coupled on the resin according to the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II to obtain peptide resin; the coupling reagent used for coupling the amino acid is preferably one or more selected from Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-diaminopropyl-N-Ethylcarbodiimide (EDC), 2- (1H-benzotrisazo L-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N, N' -tetramethyluronium Hexafluorophosphate (HBTU), benzotriazol-1-oxybis (dimethylamino) phosphorus (BOP), benzotriazol-1-yl-oxybis (PyBOP) and 1-hydroxybenzotriazole (HOBt), more preferably DIC and HOBt, TBTU and HOBt or a combination of HBTU and HOBt, most preferably HBTU and HOBt; when amino acids are coupled sequentially, the dosage of the corresponding amino acid is preferably 0.45mmol and the dosage of the coupling agent is preferably 0.45mmol, calculated by adopting 0.5g of resin; the temperature of the coupling is preferably 16 to 30 ℃, more preferably 20 to 25 ℃; in the coupling process, the polypeptide amino acid sequences are coupled one by one from the C end to the N end; the time of the coupling is not particularly limited in the invention, and the condensation reaction of the polypeptide is monitored by ninhydrin reaction during the coupling process to ensure the coupling success.
After the peptide resin is obtained, the invention preferably further comprises removing amino protecting groups from the peptide resin and sequentially cleaning the peptide resin by adopting DCM, DMF, DCM; the reagent for removing amino protecting group is preferably piperidine (PIP) solution; the volume concentration of the piperidine (PIP) solution is preferably 10-30%, more preferably 20-25%; the coefficient of removing the amino protecting group is preferably 2 times, the time for removing the amino protecting group for the first time is preferably 10min, and the time for removing the amino protecting group for the second time is preferably 20min.
After the peptide resin is cleaned, the invention carries out cracking on the cleaned peptide resin, adds a side chain protecting group scavenger, precipitates target polypeptide, and centrifugally collects precipitate to obtain a wasp venom peptide reverse sequence analogue WVF-II; the cracking liquid used in the cracking of the invention comprises trifluoroacetic acid and/or hydrofluoric acid; the ratio of the mass of the peptide resin to the volume of the lysate is preferably 1 g:5-50 mL, more preferably 1 g:10-20 mL; nitrogen is introduced in the cracking process; the time of the cleavage is preferably 3 to 5 hours, more preferably 4 hours; the side chain protecting group scavenger is preferably selected from any two or more of anisole sulfide, phenyl sulfide, triisopropyl silane, phenol, water, 1, 2-ethylene dithiol and m-cresol, more preferably phenol, water, phenyl sulfide and 1, 2-dimercaptoethanol; the volume ratio of the phenol, the water, the phenyl sulfide and the 1, 2-dimercaptoethanol is preferably 0.5-2:0.5-3:0.5-1:0.25-1, more preferably 1-1.5:1.5-2:0.8:0.55-0.75; the reagent for precipitating the polypeptide of interest preferably comprises ice-anhydrous diethyl ether; the rotational speed of the centrifugation is preferably 3000rpm; the time of the centrifugation is preferably 5min.
After obtaining the reverse-sequence analogue WVF-II of the wasp venom peptide, the invention preferably further comprises purifying the reverse-sequence analogue WVF-II of the wasp venom peptide; the purification method is preferably reverse phase chromatography or ion exchange chromatography.
The invention also provides application of the wasp venom peptide reverse sequence analogue WVF-II in broad-spectrum bacteriostasis; such applications include the preparation of anti-infective drugs, cosmetics and feeds; the broad-spectrum antibacterial objects comprise gram-positive bacteria, gram-negative bacteria and fungi; the gram positive bacterium is preferably staphylococcus aureus; the gram-negative bacteria are preferably E.coli; the fungus is preferably candida albicans.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 solid phase chemical Synthesis of the Wasp peptide anti-sequence analog WVF-II polypeptide
Fmoc (fluorenylmethoxycarbonyl) -protected amino acid was used for solid phase synthesis using rinkle amide MBHA resin (4- (2 ',4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methyl benzhydrylamine resin) as the carrier, 1-hydroxybenzotriazole and O-benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate as the coupling reagent, cleavage of the peptide from the resin using the cleavage solution trifluoroacetic acid and side chain protecting group scavenger (phenol/water/benzothiophene)1, 2-dimercaptoethanol), reacting for 4-6 h at room temperature, rotary evaporating to remove most trifluoroacetic acid, dripping diethyl ether at 0deg.C to obtain flocculent precipitate, centrifuging to obtain crude peptide, initially separating with Sephadex G-15 column, balancing with 5% acetic acid, loading sample at flow rate of 1mL min -1 The first peak was collected by eluting with 5% acetic acid, 280nm, and freeze-dried. Purifying with semi-preparative high performance liquid chromatograph, and collecting peak with maximum abundance. The purity of the polypeptide was determined by analytical high performance liquid chromatography and amino acid analysis. The preparation method of the wasp venom peptide reverse sequence analogue WVF-II comprises the following specific operation steps:
1) Taking out the raw material reagent stored in the refrigerator, and placing the raw material reagent in a dryer for Wen Daiyong;
2) Accurately weighing 0.5g of RinkAmide MBHA resin, placing the resin in a synthesis reactor, flushing with dimethylformamide for six times, adding 10mL of dimethylformamide for soaking for 30min, and introducing nitrogen to enable the resin to turn up and down in the solution;
3) Adding 20% piperidine with the volume being 2 times that of the piperidine into a synthesis reactor, introducing nitrogen to react for 10min, and then flushing with dimethylformamide twice;
4) Adding 20% piperidine with the volume being 2 times of that of the synthetic reactor again, introducing nitrogen to react for 20min, and then washing with DMF for six times;
5) 0.45mmol of Fmoc-protected amino acid, equimolar coupling agent 1-hydroxybenzotriazole and O-benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate were weighed for room temperature reaction, monitored by ninhydrin reaction to ensure successful coupling, and the resin was washed 7 times with DMF. After the first amino acid is coupled to the resin, the coupling reaction of the next amino acid can be continued according to the method, and the cycle is performed until all the amino acids are coupled;
6) Adding 20% piperidine with volume of 2 times for reaction, and continuously operating for two times for 10min and 20min respectively, sequentially cleaning resin (resin) with DCM, DMF, DCM respectively, and pumping to the greatest extent to obtain NH2-KHRPXGFSAPVFLRPPD-resin (peptide resin), wherein X is hydroxyproline Hrp;
7) 5mL of trifluoroacetic acid lysate and a side chain protecting group scavenger (phenol/water/phenylsulfide/1, 2-dimercaptoethanol: 15:1.5:1:0.8:0.55) is added into a synthesis reactor, nitrogen is introduced for reaction for 3 hours, and anhydrous diethyl ether is placed in a refrigerator at the temperature of minus 20 ℃ for later use;
8) After the reaction is finished, collecting the reaction liquid into a pre-weighed 50mL centrifuge tube, cleaning the synthesis reactor for 2 times by using trifluoroacetic acid, and collecting the washing liquid into the 50mL centrifuge tube;
9) Taking out ice anhydrous diethyl ether in a refrigerator, selecting a proper container, pouring diethyl ether into the container, and performing absolute diethyl ether: dropping the lysate into frozen anhydrous diethyl ether according to the ratio of reaction liquid=10:1 (volume ratio), controlling the speed to drop the filtrate dropwise as much as possible, and continuously stirring until no precipitate is generated;
10 Standing the precipitate mixture for several minutes, centrifuging, discarding, and washing the precipitate with fresh frozen anhydrous diethyl ether for at least three times;
11 Freeze-drying the obtained precipitate in vacuum by a freeze dryer to obtain crude peptide and weighing;
12 Purifying and mass spectrometry identification of the crude peptide, and obtaining WVF-II;
13 Purifying and identifying qualified WVF-II reverse-sequence analogues through mass spectrometry, freeze-drying the analogues in vacuum by a freeze dryer, and weighing the analogues for later use.
Example 2 WVF-II reverse HPLC purification and Mass Spectrometry characterization
Purification was performed by high performance liquid chromatography using Sepax Bio-C18 reverse phase column (10.0X105 mm,10 μm,
). The oxidized crude peptide sample was dissolved in 1mL of ultrapure water, centrifuged at 10000rpm for 5min, and detected by a W2998 detector at 215, 254, 280nm. The mobile phase is a four-phase elution system, and the flow rate is 3 mL.min -1 . The eluent is aqueous solution (A) of 0.1% TFA and acetonitrile solution (B) of 0.1% TFA respectively, the column temperature is 25 ℃, the gradient of reversed-phase high performance liquid chromatography elution is shown in Table 1, the elution peak is collected, the analysis by Q-TOF mass spectrum is carried out, the identification result is shown in Table 1, the molecular weight of the mutant analyzed by mass spectrum accords with the theoretical value, which indicates that WV is successfully synthesizedF-II. The purified samples were lyophilized and stored for later use. The purity of the crude peptide was determined by high performance liquid chromatography and amino acid analysis.
TABLE 1 reversed phase high performance liquid chromatography elution gradient
TABLE 2 relative molecular masses of WVF-II Single Components
Example 3 detection of antibacterial Activity of the reverse sequence analog WVF-II of wasp venom peptide
(1) Determination of the antibacterial Activity of WVF-II
The agar medium is heated and melted in a water bath, cooled to 50 ℃, 60 mu L of bacterial suspension is sucked by a sterile operation method, added into 20mL of agar medium, quickly and evenly mixed, poured into a sterile flat dish with the diameter of 9cm, the thickness is about 1.5mm, and horizontally placed for solidification. Round holes with the diameter of 2.7mm are made on agar, 10 mu L of purified wasp venom peptide reverse sequence analogue WVF-II samples are respectively added into the holes, sterile water is used as a negative control, and penicillin sodium is used as a positive control. After loading, 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℃with the observations shown in FIG. 1. (FIG. 1 shows the results of antibacterial experiments on Staphylococcus aureus (left), candida albicans (middle) and Escherichia coli (right) with the reverse sequence analogue WVF-II of wasp venom, wherein 1 represents the antibacterial activity of penicillin sodium and 2 is the negative control dd H 2 O;3 is 1 mg.mL -1 An antibacterial activity of the wasp venom peptide reverse sequence analogue. As can be seen from FIG. 1, the WVF-II sample (No. 3 zone) has a good antibacterial effect.
(2) Determination of Minimum Inhibitory Concentration (MIC) of WVF-II
MIC: diluting the bacterial liquid of the test strain cultured to the logarithmic phase to about 5×10 6 CFU·mL -1 Added into a 96-well plate culture plate,the test wells were filled with 90. Mu.L of bacterial solution per well, followed by 10. Mu.L/well of the diluted reverse-order peptide solution at different concentrations. The positive control was 100. Mu.L/well of bacterial solution and the negative control was the corresponding 100. Mu.L of medium. Then slowly shaking and culturing at 37 ℃ for about 16 hours, and measuring OD by using an enzyme labeling instrument 630 . The minimum concentration that inhibited bacterial growth was MIC, and the results are shown in table 3.
Table 3 MIC of the reverse-sequence analog of WVF-II
(3) Hemolytic Activity assay
Human blood erythrocytes were washed 4 times with PBS buffer (35 mM phosphate buffer, 150mM nacl, ph=7.4) and suspended in PBS buffer to give a red blood cell suspension (volume fraction 2.5%). Dissolving the reverse-order peptide in PBS buffer solution to prepare stock solution, taking polypeptide stock solutions with different volumes into 0.5mL of 2.5% red blood cell suspension, adding PBS buffer solution to a final volume of 1mL, shaking uniformly, incubating for 1h at 414nm and then a constant temperature incubator at 37 ℃, centrifuging at 1200rpm for 15min, taking supernatant to color at 414nm, taking red blood cells suspended in PBS buffer solution as blank, and taking red blood cells suspended in TritonX100 as 100% hemolysis. The test shows that the prepared reverse peptide has no obvious hemolytic activity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of university
<120> a wasp venom peptide reverse sequence analogue WVF-II, its preparation method and application
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Leu Leu Gly Ser Gly Leu Lys Gly Ile Arg Lys Lys Arg
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Claims (10)
1. A wasp venom peptide anti-sequence analogue WVF-ii, wherein the amino acid sequence of the wasp venom peptide anti-sequence analogue WVF-ii is shown in SEQ ID NO: 1.
2. The method for preparing the wasp venom peptide anti-sequence analogue WVF-II according to claim 1, which comprises the following steps:
1) Sequentially coupling amino acids on the resin according to the amino acid sequence of the wasp venom peptide reverse sequence analogue WVF-II to obtain peptide resin;
2) And (3) cracking the peptide resin in the step (1), adding a side chain protecting group scavenger, precipitating the target polypeptide, centrifuging, and collecting the precipitate to obtain the wasp venom peptide reverse sequence analogue WVF-II.
3. The method according to claim 2, wherein the resin in step 1) comprises 4- (2 ',4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzene methylamine resin.
4. A process according to claim 2 or 3, wherein the coupling reagent used for coupling the amino acid in step 1) is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-diaminopropyl-N-ethylcarbodiimide, 2- (1H-benzotrisazo L-1-yl) -1, 3-tetramethylurea tetrafluoroborate, O-benzotriazole-N, N' -tetramethylurea hexafluorophosphate, benzotriazole-1-oxytris (dimethylamino) phosphorus hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinylphosphos and 1-hydroxybenzotriazole.
5. The method according to claim 2, wherein the cleavage liquid used for cleavage in step 2) comprises trifluoroacetic acid and/or hydrofluoric acid.
6. The method according to claim 5, wherein the ratio of the mass of the peptide resin to the volume of the lysate in step 2) is 1 g:5-50 mL.
7. The method according to claim 2, wherein the side chain protecting group scavenger in step 2) is selected from any two or more of anisole, phenylsulfide, triisopropylsilane, phenol, water, 1, 2-ethanedithiol and m-cresol.
8. The method according to claim 2, wherein the reagent for precipitating the polypeptide of interest in step 2) comprises ice-anhydrous diethyl ether.
9. The method of claim 2, further comprising purifying the reverse wasp venom peptide analog WVF-ii after obtaining the reverse wasp venom peptide analog WVF-ii in step 2).
10. Use of the reverse wasp venom peptide analog WVF-ii according to claim 1 for the preparation of a medicament for inhibiting staphylococcus aureus, escherichia coli and candida albicans.
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