CN111100190A - Wasp toxin peptide reverse order analogue WVD-II and preparation method and application thereof - Google Patents
Wasp toxin peptide reverse order analogue WVD-II and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a wasp venom peptide reverse order analogue WVD-II and 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 order analogue WVD-II is shown as SEQ ID NO. 1. the N end of the wasp venom peptide reverse order analogue WVD-II is positively charged and can be combined with a negatively charged cell membrane, and hydrophobic amino acid at the C end forms an α spiral structure and can be perforated on the surface of the cell membrane to cause bacterial death.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a wasp toxic peptide reverse order analogue WVD-II and a preparation method and application thereof.
Background
The antibacterial peptide can resist microbial invasion, and has specific or non-specific effects on gram-negative bacteria, positive bacteria, fungi, viruses, tumors, eukaryotes and the like. In addition to having antimicrobial effects, antimicrobial peptides have other functions. The antibacterial peptide is mostly applied in the form of polypeptide analogs 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 promising candidates for replacing the resistance problems associated with the abuse of conventional antibiotics, the natural antimicrobial peptides have a number of disadvantages that limit their use as clinical drugs, particularly in terms of toxicity and enzymatic stability.
Disclosure of Invention
The invention aims to provide a wasp toxic peptide reverse order analogue WVD-II and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a wasp toxic peptide reverse order analogue WVD-II, wherein the amino acid sequence of the wasp toxic peptide reverse order analogue WVD-II is shown as SEQ ID NO: 1 is shown.
The invention also provides a preparation method of the wasp venom peptide reverse order analogue WVD-II, which comprises the following steps:
1) sequentially coupling amino acids on resin according to the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II to obtain peptide resin;
2) and (2) cracking the peptide resin in the step 1), adding a side chain protecting group scavenger, precipitating target polypeptide, centrifuging, and collecting precipitate to obtain the wasp venom peptide reverse order analogue WVD-II.
Preferably, the resin in step 1) comprises 4- (2 ', 4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzhydrylamine resin.
Preferably, the coupling reagent used for coupling the amino acid in step 1) is one or more selected from the group consisting of dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-diaminopropyl-N-ethylcarbodiimide, 2- (1H-benzotriazol-L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate, O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate, benzotriazol-1-oxytris (dimethylamino) phosphonium hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate, and 1-hydroxybenzotriazole.
Preferably, the lysis solution used in the step 2) comprises trifluoroacetic acid and/or hydrofluoric acid.
Preferably, the ratio of the mass of the peptide resin to the volume of the lysate in the step 2) is 1g: 10-50 mL.
Preferably, the side chain protecting group scavenger in step 2) is selected from any two or a combination of two or more of thioanisole, 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-order analogue WVD-II of the wasp venom peptide is obtained in the step 2), the purification of the reverse-order analogue WVD-II of the wasp venom peptide is further included.
The invention also provides application of the wasp toxic peptide reverse order analogue WVD-II in broad-spectrum bacteriostasis.
The invention has the beneficial effects that the invention provides a wasp venom peptide reverse order analogue WVD-II, the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II is shown as SEQ ID NO. 1. the N end of the wasp venom peptide reverse order analogue WVD-II is provided with positive charges and can be combined with a negatively charged cell membrane, and hydrophobic amino acid at the C end forms an α spiral structure and can perforate on the surface of the cell membrane to cause bacterial death.
Drawings
FIG. 1 shows the results of the antibiotic experiments of the reverse-order Wasp venom peptide analogue WVD-II against Staphylococcus aureus (left), Candida albicans (middle) and Escherichia coli (right), in which: 1 represents the antibacterial activity of penicillin sodium; 2 is a negative control dd H2O; 3 is 1 mg. mL-1The antibacterial activity of the reverse-sequence peptide of (1).
Detailed Description
The invention provides a wasp toxic peptide reverse order analogue WVD-II, wherein the amino acid sequence of the wasp toxic peptide reverse order analogue WVD-II is shown as SEQ ID NO: 1, specifically: RIRLKRKIAALAKKLL amino (NH)2) At the R-terminus, a carboxyl group (COOH) at the L-terminus.
The amino acid sequence of the reverse sequence of the wasp venom peptide is shown as SEQ ID NO: 2, specifically: RINLKRAIAALAKKLL amino (NH)2) At the R-terminus, a carboxyl group (COOH) at the L-terminus. The invention replaces the third position N of the reverse sequence of the wasp poison peptide with R, and replaces the seventh position A with K. The electropositivity is further enhanced by adding two basic amino acids (i.e., arginine R, lysine K), while at the same time, the hemolytic activity of erythrocytes is overcome and the antibacterial activity is improved.
The N end of the wasp venom peptide reverse-order analogue WVD-II is positively charged and can be combined with a negatively charged cell membrane, and the hydrophobic amino acid at the C end forms an α spiral structure and can perforate the surface of the cell membrane, so that bacteria die.
The invention also provides a preparation method of the wasp venom peptide reverse order analogue WVD-II, which comprises the following steps:
1) sequentially coupling amino acids on resin according to the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II to obtain peptide resin;
2) and (2) cracking the peptide resin in the step 1), adding a side chain protecting group scavenger, precipitating target polypeptide, centrifuging, and collecting precipitate to obtain the wasp venom peptide reverse order analogue WVD-II.
Before amino acids are sequentially coupled on resin according to the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II, the process of washing the resin, soaking the resin, removing an amino protecting group and washing the resin again is preferably further included; the resin preferably includes Rink Amide MBHA resin (4- (2 ', 4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzhydrylamine resin); the resin functions as a carrier.
In the present invention, the reagent used for washing the resin is preferably Dimethylformamide (DMF); the washing frequency is preferably 5-7 times, and more preferably 6 times; the solvent adopted by the resin soaking 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 0.5g to 10 mL; the soaking time is preferably 20-40 min, and more preferably 30 min; the soaking process preferably also comprises the step of introducing nitrogen so that the resin can be turned over up and down in the solution; the reagent for removing the amino protecting group is preferably piperidine (PIP) solution; the concentration of the piperidine (PIP) solution is preferably 10-30% (v/v), more preferably 20-25% (v/v); the removing time is preferably 5-30 min, and more preferably 10-20 min; the dosage of the piperidine (PIP) solution is preferably 2-3 times of the volume of the resin; the reagent used for washing the resin again is preferably Dimethylformamide (DMF); the number of washing is preferably 2 to 10, more preferably 6 to 8. In the specific implementation process of the invention, after the resin is washed again, the process of removing the amino protecting group and washing the resin again is repeated once.
After the treatment, amino acids are sequentially coupled on resin according to the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II to obtain peptide resin; the coupling reagent used for coupling the amino acids is preferably selected from the group consisting of Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), N-diaminopropyl-N-Ethylcarbodiimide (EDC), 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N, one or more of N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxytriazolidinylphosphine (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; the dosage of the corresponding amino acid added in sequence every time is preferably 0.45mmol and the dosage of the coupling agent is preferably 0.45mmol based on 0.5g of the adopted resin; the coupling temperature is preferably 16-30 ℃, and more preferably 20-25 ℃; in the coupling process, the polypeptide amino acid sequences are coupled one by one from the C end to the N end; the invention has no special limit on the coupling time, and the condensation reaction of the polypeptide is monitored by a ninhydrin reaction during the coupling process to ensure the coupling success.
After obtaining the peptide resin, the invention preferably also comprises the steps of removing amino protecting groups from the peptide resin and sequentially cleaning the peptide resin by adopting DCM, DMF and DCM; the reagent for removing the amino protecting group is preferably piperidine (PIP) solution; the concentration of the piperidine (PIP) solution is preferably 10-30% (v/v), more preferably 20-25% (v/v); the removal of the amino protecting group is preferably performed 2 times, the time for the first removal of the amino protecting group is preferably 10min, and the time for the second removal of the amino protecting group is preferably 20 min.
After the peptide resin is cleaned, the cleaned peptide resin is cracked, a side chain protecting group scavenging agent is added, target polypeptide is precipitated, centrifugation is carried out, and the precipitate is collected to obtain the wasp venom peptide reverse order analogue WVD-II; the cracking liquid used for cracking comprises trifluoroacetic acid and/or hydrofluoric acid; the ratio of the mass of the peptide resin to the volume of the lysate is preferably 1g: 10-50 mL, and more preferably 1g: 15-25 mL; introducing nitrogen in the cracking process; the cracking time is preferably 1-3 h, and more preferably 2 h; the side chain protecting group scavenger is preferably selected from any two or a combination of more than two of thioanisole, triisopropylsilane, phenol, water, 1, 2-ethanedithiol and m-cresol, and is more preferably selected from phenol, water, thioanisole and 1, 2-dimercaptoethanol; the volume ratio of the phenol to the water to the dimethyl sulfide to the 1, 2-dimercaptoethanol is preferably 0.5-2: 0.5-3: 0.5-1: 0.25-1, and more preferably 1-1.5: 1-2: 0.8: 0.5-0.75; the reagent for precipitating the target polypeptide preferably comprises ice anhydrous diethyl ether; the volume ratio of the ice anhydrous ether to the reaction liquid is preferably 10: 1; the invention has no special limit on the precipitation time until no precipitation is generated; the rotation speed of the centrifugation is preferably 3000 rpm; the time for the centrifugation is preferably 5 min.
After the wasp venom peptide reverse-order analogue WVD-II is obtained, the method preferably further comprises the steps of washing, drying and purifying the wasp venom peptide reverse-order analogue WVD-II; the washing solvent is preferably anhydrous frozen diethyl ether; the number of washing is preferably 3; the drying mode is preferably vacuum freeze drying; the purification method is preferably reverse phase chromatography or ion exchange chromatography.
The invention also provides application of the wasp toxic peptide reverse order analogue WVD-II in broad-spectrum bacteriostasis; the application comprises preparing anti-infective drugs, cosmetics and feeds; the broad-spectrum bacteriostatic objects comprise gram-positive bacteria, gram-negative bacteria and fungi; the gram-positive bacterium is preferably staphylococcus aureus; the gram-negative bacterium is preferably escherichia 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
The Fmoc protected amino acid is adopted for solid phase synthesis, Rinkamide MBHA resin is used as a carrier, and 1-hydroxybenzotriazole and O-benzotriazole-N, N, N ', N' -tetramethyl are adoptedUsing urea hexafluorophosphate as coupling reagent, using lysate trifluoroacetic acid and side chain protecting group scavenger (phenol/water/thioanisole/1, 2-dimercaptoethanol) for cutting peptide from resin, reacting at room temperature for 4-6 h, removing most of trifluoroacetic acid by rotary evaporation, dripping ether at 0 deg.C to obtain flocculent precipitate, centrifuging to obtain crude peptide, primarily separating with Sephadex G-15 column, balancing with 5% acetic acid, loading sample at flow rate of 1 mL/min-1Eluting with 5% acetic acid, detecting at 280nm, collecting the first peak, and freeze drying. 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 WVD-II comprises the following specific operation steps:
1) taking materials, namely taking out the raw material reagent stored in the refrigerator, and putting the raw material reagent in a dryer for rewarming for later use;
2) accurately weighing 0.5g of MBHA resin, placing the MBHA resin in a synthesis reactor, flushing the MBHA resin with dimethylformamide for six times, adding 10mL of dimethylformamide to soak the MBHA resin for 30min, and introducing nitrogen to ensure that the resin can be turned over up and down in the solution;
3) adding 20% piperidine with the volume 2 times that of Fmoc-AA-resin into a synthesis reactor, introducing nitrogen to react for 10min, and then flushing twice with dimethylformamide;
4) adding 20% piperidine with 2 times of Fmoc-AA-resin volume into the synthesis reactor again, introducing nitrogen for reaction for 20min, and then washing with DMF for six times;
5) 0.45mmol of Fmoc protected amino acid, equimolar amounts of coupling reagents 1-hydroxybenzotriazole and O-benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate were weighed for reaction at room temperature, 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, continuing the coupling reaction of the next amino acid according to the method, and circulating the steps until all amino acids are coupled;
6) adding 2 times volume of 20% piperidine for reaction, continuously operating twice, respectively reacting for 10min and 20min, sequentially cleaning resin with DCM, DMF and DCM respectively, and draining to obtain NH 2-RIRLKRKIAALAKKLL-resin;
7) 5mL of trifluoroacetic lysate and side chain protecting group scavenger (phenol/water/thioanisole/1, 2-dimercaptoethanol: 0.5:0.5:0.5:0.25) into a synthesis reactor, introducing nitrogen gas for reaction for 2 hours, and additionally placing anhydrous ether in a refrigerator at the temperature of minus 20 ℃ for freezing for later use;
8) after the reaction is finished, collecting the reaction solution into a pre-weighed 50mL centrifuge tube, cleaning the synthesis reactor for 2 times by using trifluoroacetic acid, and collecting the cleaning solution into the 50mL centrifuge tube;
9) taking out the ice anhydrous ether in a refrigerator, selecting a proper container, pouring the ether into the container, dropping the lysate into the frozen anhydrous ether according to the ratio of the anhydrous ether to the reaction solution of 10:1 (volume ratio), controlling the speed, dropping 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 using a freeze dryer to obtain crude peptide and weighing the crude peptide;
12) purifying the crude peptide, analyzing and identifying by mass spectrometry, and obtaining a wasp toxic peptide reverse sequence analogue WVD-II;
13) and (3) freezing and vacuum-drying the purified and qualified wasp toxic peptide reverse-sequence analogue WVD-II by mass spectrometry with a freeze dryer, and weighing for later use.
Example 2 reverse-phase HPLC purification and Mass Spectrometry identification of Wasp venom peptide reverse-order analog WVD-II
Purification was performed by HPLC using a Sepax Bio-C18 reverse phase column (10.0X 250mm,10 μm,
). Dissolving the oxidized crude peptide sample in 1mL of ultrapure water, centrifuging at 10000rpm for 5min, and detecting by a W2998 detector at the detection wavelengths of 215 nm, 254 nm and 280 nm. The mobile phase is a four-phase elution system with the flow rate of 3 mL/min-1. The eluates were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B), respectively, at 25 ℃ and reversed phaseThe elution gradient of the high performance liquid chromatography is shown in table 1, the elution peak is collected, Q-TOF mass spectrometry is used, the identification result is shown in table 2, the molecular weight of the mutant subjected to mass spectrometry is consistent with the theoretical value, and the result shows that the wasp venom peptide reverse-order analogue WVD-II is successfully synthesized. The purified samples were lyophilized and stored for future use. The purity of the crude peptide was determined by high performance liquid chromatography and amino acid analysis.
TABLE 1 reversed-phase HPLC elution gradient
TABLE 2 relative molecular masses of individual components of the reverse order analogue WVD-II of wasp venom peptides
Example 3 detection of antibacterial Activity of reverse-order analogs of Wasp venom peptides
(1) Determination of antibacterial activity of wasp venom peptide reverse order analogue WVD-II
Heating agar culture medium in water bath for melting, cooling to about 50 deg.C, sucking 60 μ L bacterial suspension by aseptic technique, adding into 20mL agar culture medium, rapidly mixing, pouring into sterile flat dish with diameter of 9cm and thickness of about 1.5mm, and standing horizontally for solidification. Round holes with the diameter of 2.7mm are punched on agar, 10 mu L of each purified wasp toxic peptide reverse order analogue WVD-II sample is respectively added into the holes, sterile water is used as a negative control, and penicillin sodium is used as a positive control. Placing the plate into a 4 ℃ refrigerator after sample application, placing the plate upside down at 37 ℃ for overnight culture after the sample is fully diffused into agar, and observing results are shown in figure 1 (figure 1 is the result of the antibacterial experiment of the wasp toxic peptide reverse order analogue WVD-II on staphylococcus aureus (left), candida albicans (middle) and escherichia coli (right), wherein 1 shows the antibacterial activity of penicillin sodium, and 2 is a negative control dd H2O; 3 is 1 mg. mL-1The antibacterial activity of the reverse-order peptide of (1). The test bacterium in this test was Staphylococcus aureus (Staphylococcus aureus). As can be seen from FIG. 1, the wasp venom peptide reverse-order analogue WVD-The Minimum Inhibitory Concentrations (MICs) of II on Staphylococcus aureus, Escherichia coli and Candida albicans are 0.5, 1 and 0.5 μ g/mL respectively-1And hemolytic activity is not shown in the bacteriostatic concentration range, and the WVD-II sample (No. 3 bacteriostatic zone) has better bacteriostatic effect.
(2) Determination of Minimum Inhibitory Concentration (MIC) of Wasp toxin peptide reverse-order analogue WVD-II
MIC: the dilution of the broth from the test strain culture to logarithmic growth phase was about 5X 106CFU·L-1And m, adding the mixture into a 96-well plate culture plate, adding 90 mu L of bacterial liquid into each test hole, and then adding 10 mu L/hole of reverse-order peptide solution with different concentrations diluted in multiple proportion. The positive control was 100. mu.L/well of the broth, and the negative control was the corresponding 100. mu.L of medium. Then, the cells were incubated at 37 ℃ for about 16 hours with slow shaking, and OD630 was measured with a microplate reader. The minimum concentration at which bacterial growth is inhibited is the MIC, and the results are shown in Table 3. As can be seen from Table 3, WVD-II has better MIC values for Staphylococcus aureus, Escherichia coli and Candida albicans.
TABLE 3 MIC of reverse-order analogs of Wasp venom peptide reverse-order analogs WVD-II
(3) Hemolytic Activity test
Human blood red blood cells were washed 4 times with PBS buffer (35mM phosphate buffer, containing 150mM NaCl, pH 7.4) and then suspended in PBS buffer to obtain a red blood cell suspension (volume fraction: 2.5%). Dissolving the reverse sequence peptide in PBS buffer solution to prepare stock solution, taking polypeptide stock solution with different volumes to 0.5mL of 2.5% red blood cell suspension, adding PBS buffer solution to the final volume of 1mL, shaking up, carrying out color comparison at 414nm, then incubating for 1h in a constant-temperature incubator at 37 ℃, then using 1200rpm, centrifuging for 15min, taking supernatant to carry out color comparison at 414nm, using the suspension of the red blood cells in the PBS buffer solution as a blank, and using the suspension of the red blood cells in TritonX100 as 100% hemolysis. The prepared reverse-sequence peptide has no obvious hemolytic activity through test detection.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of university
<120> wasp venom peptide reverse sequence analogue WVD-II, preparation method and application thereof
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<213> Artificial Sequence (Artificial Sequence)
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Arg Ile Arg Leu Lys Arg Lys Ile Ala Ala Leu Ala Lys Lys Leu Leu
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<213> Artificial Sequence (Artificial Sequence)
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Arg Ile Asn Leu Lys Arg Ala Ile Ala Ala Leu Ala Lys Lys Leu Leu
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Claims (10)
1. A wasp venom peptide reverse sequence analogue WVD-II, wherein the amino acid sequence of the wasp venom peptide reverse sequence analogue WVD-II is shown as SEQ ID NO: 1 is shown.
2. A method for preparing the wasp venom peptide reverse-order analog WVD-ii according to claim 1, comprising the steps of:
1) sequentially coupling amino acids on resin according to the amino acid sequence of the wasp venom peptide reverse order analogue WVD-II to obtain peptide resin;
2) and (2) cracking the peptide resin in the step 1), adding a side chain protecting group scavenger, precipitating target polypeptide, centrifuging, and collecting precipitate to obtain the wasp venom peptide reverse order analogue WVD-II.
3. The method of claim 2, wherein the resin in step 1) comprises 4- (2 ', 4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzhydrylamine resin.
4. The process according to claim 2 or 3, wherein the coupling reagent used for coupling the amino acid in step 1) is one or more selected from the group consisting of dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-diaminopropyl-N-ethylcarbodiimide, 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate, O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate, benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate, benzotriazole-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate and 1-hydroxybenzotriazole.
5. The method according to claim 2, wherein the lysis solution used in the 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 1g:10 to 50 mL.
7. The method according to claim 2, wherein the side chain protecting group scavenger in step 2) is selected from the group consisting of thioanisole, triisopropylsilane, phenol, water, 1, 2-ethanedithiol, and m-cresol, in any combination of two or more.
8. The method according to claim 2, wherein the reagent for precipitating the polypeptide of interest in step 2) comprises ice-dry diethyl ether.
9. The method of claim 2, further comprising purifying the reverse-sequence wasp venom peptide analogue WVD-ii after obtaining the reverse-sequence wasp venom peptide analogue WVD-ii in step 2).
10. The use of the wasp venom peptide reverse-order analog WVD-II of claim 1 in broad-spectrum bacteriostasis.
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| CN104292324A (en) * | 2011-05-06 | 2015-01-21 | 上海医药工业研究院 | HRP5 analogues and preparation method thereof |
| CN103288925A (en) * | 2012-10-24 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Melittin antitone analogue and preparation method for same |
| WO2017107241A1 (en) * | 2015-12-22 | 2017-06-29 | 何润泽 | Method for preparing polypeptide of kpv peptide and analogue thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112194707A (en) * | 2020-10-15 | 2021-01-08 | 中国科学院西双版纳热带植物园 | Novel melittin and application thereof |
| CN112194707B (en) * | 2020-10-15 | 2023-08-29 | 中国科学院西双版纳热带植物园 | Novel melittin and application thereof |
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| CN111100190B (en) | 2023-04-28 |
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