CN103288925A - Melittin antitone analogue and preparation method for same - Google Patents
Melittin antitone analogue and preparation method for same Download PDFInfo
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Abstract
The invention relates to a melittin antitone analogue and a preparation method for the same. The amino acid sequence of the melittin antitone analogue is RKRKRWSILAPLG. The preparation method for the melittin antitone analogue comprises the steps of taking materials, reacting, purifying, performing mass spectrometry analysis-identification, and the like. According to the melittin antitone analogue and the preparation method for the same disclosed by the invention, the bacteriostatic activity is improved.
Description
Technical field
The present invention relates to polypeptide synthetic field, specifically a kind of mellitin (Melittin) inverted sequence analogue and preparation method thereof.
Background technology
Antibacterial peptide is efficient with it, wide spectrum, fast, specific effect and antibiotic, antiviral, antitumor isoreactivity and to the characteristic of eukaryotic cell low toxicity, become the newtype drug with important potential value.Face today that the pathogenic bacteria resistance improves constantly at traditional microbiotic, the appearance of antibacterial peptide becomes valuable novel antibacterial medicine undoubtedly and is subject to people's attention.Bringing into play its active and reduction toxicity to greatest extent, is the matter of utmost importance of antibacterial peptide new drug development.Transform antibacterial peptide with the molecular designing means and become the key that addresses this problem.Be object with amphiphilic alpha-helix antibacterial peptide, to observing cationic and amphipathic principle and the active the physical-chemical parameters of influence in the molecular designing process, can take the method for sequence modification or brand-new design, to reach the purpose of directional transformation.
Antibacterial peptide shows diversity in sequence and structure, but its molecular designing has 2 common requirements, i.e. cationic and amphipathic.The former determines its selectivity, can attract each other with showing electronegative bacterial cell film outer surface specifically, does not interact and do not produce with the eukaryotic cell membrane outside surface that is neutrality.Here it is, and they do not have the major cause of toxic action to mammalian cell, and can the latter determine it effectively insert in the bacterial cell membrane, form hydrophobic channel.
Mellitin is the main active ingredient of apis mellifera Linnaeus (Apismellifera) venom, formed by 26 amino acid, primary structure is: GIGAVLKVLTTGLPALISWIKRKRQQ-COOH, mellitin can form amphipathic α-Luo Xuanjiegou, 4 C ends (KRKR) that concentrate on peptide chain in 5 basic aminoacidss are typical cationic antibacterial peptides.Mellitin has very strong restraining effect to gram-positive microorganism and negative bacterium, also has very strong hemolytic activity, has limited its clinical application.Mellitin has very high affinity interaction to calmodulin, can be in conjunction with the model of polypeptide combining site according to calmodulin, prediction also proves by experiment, the combining site of mellitin and calmodulin is 12～26 fragment Mel (12～26), and find that this fragment still keeps certain anti-microbial activity, but hemolytic activity disappears.
Summary of the invention
The object of the present invention is to provide a kind of mellitin inverted sequence analogue and preparation method thereof, it improves bacteriostatic activity.
The present invention solves above-mentioned technical problem by following technical proposals: a kind of mellitin inverted sequence analogue is characterized in that the aminoacid sequence of described mellitin inverted sequence analogue is RKRKRWSILAPLG.
Preferably, described mellitin inverted sequence analogue is for adopting the synthetic polypeptide products of solid state chemistry synthetic technology.
The present invention also provides a kind of preparation method of mellitin inverted sequence analogue, it is characterized in that, this preparation method may further comprise the steps:
(1) the raw material reagent that will deposit in the refrigerator takes out, and places the moisture eliminator rewarming stand-by;
(2) behind the accurate weighing Fmoc-AA-Resin 0.1mmol, place synthesis reactor, with DMF flushing six times, the DMF that adds 2～3 times of amounts of about Fmoc-AA-Resin volume then soaked 30 minutes, feeds nitrogen Fmoc-AA-Resin can be stirred up and down in solution;
(3) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes feeds nitrogen reaction 10 minutes in synthesis reactor, then with twice of DMF flushing;
(4) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes again feeds nitrogen reaction 20 minutes in synthesis reactor, then with DMF flushing six times;
(5) Fmoc-AA of weighing 0.45mmol, HOBt and HBTU, earlier with 1ml DMF dissolving HOBT and HBTU, after solution is added among the Fmoc-AA, activate 15 minutes;
(6) will activate good Fmoc-AA solution and 1mL DIEPA adds among the Fmoc-AA-Resin simultaneously;
(7) flow of adjusting nitrogen can stir Resin up and down in solution, makes reaction more complete, and the reaction times was generally 2 hours;
(8) reaction is respectively washed Fmoc-AA-Resin twice successively with DMF, DCM, DMF after finishing;
(9) repeating step (3) is to step (8), till the peptide sequence coupling is intact;
(10) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes reacts, operate continuously twice, and the reaction times was respectively 10 minutes and 20 minutes, respectively wash Resin twice successively with DCM, DMF, DCM respectively, after washing for the last time, Resin is drained as far as possible, namely get peptide resin;
(11) preparation lysate: the corresponding 10ml lysate of 1g peptide resin;
(12) lysate is joined in the synthesis reactor, feeds nitrogen reaction 2 hours, during need to replenish lysate frequently in case evaporate to dryness places anhydrous diethyl ether-20 ℃ of refrigerator and cooled to freeze standby in addition;
(13) after reaction is finished, lysate is collected in the 50ml centrifuge tube of weighing in advance, cleaned synthesis reactor 2-3 time with TFA again, washing lotion is collected in the 50ml centrifuge tube equally;
(14) anhydrous diethyl ether in the refrigerator is taken out, select suitable containers that ether is poured into wherein, by anhydrous diethyl ether: the volume ratio of lysate=10: 1 splashes into lysate in the freezing anhydrous diethyl ether, control speed, filtrate is dropwise splashed into, constantly be stirred to simultaneously till the no longer generation precipitation;
(15) precipitation mixture was left standstill after several minutes centrifugal, abandon supernatant, use fresh freezing anhydrous diethyl ether washing precipitation at least three times then;
(16) the gained throw out is air-dry, seal with sealing film afterwards, prick close hole, with the Freeze Drying Equipment vacuum freezedrying and weigh;
(17) NH
4HCO
3The thick peptide of natural oxidation method oxidation: configuration 0.1M NH
4HCO
3Solution is in the ratio adding NH of polypeptide final concentration 0.3mg/ml
4HCO
3In the extremely thick peptide of solution, stirred overnight at room temperature; Seal with sealing film afterwards, prick close hole, use the Freeze Drying Equipment vacuum freezedrying;
(18) the thick peptide after the oxidation is carried out purifying and mass spectroscopy and identify, and obtain mellitin inverted sequence analogue;
(19) with purifying and through the mellitin inverted sequence analogue Freeze Drying Equipment vacuum freezedrying of mass spectroscopy accreditation, it is standby to weigh.
Positive progressive effect of the present invention is: the present invention has good stability in animal digestive tract, have simultaneously that immunogenicity is little, good water solubility, broad-spectrum sterilization even can fungicidal, protozoon and do not produce advantages such as endurance strain, can tolerate the degraded of proteolytic enzyme and peptase in the gi tract.
Description of drawings
Fig. 1 is the mass spectroscopy figure of synthetic mellitin (Melittin) the inverted sequence analogue of solid state chemistry, and the molecular weight that records the inverted sequence peptide by analysis is consistent with theoretical value;
Fig. 2 be mellitin (Melittin) inverted sequence analogue to the antibacterial experiment result of streptococcus aureus, among the figure: 1 the expression penbritin anti-microbial activity; 2 negative contrast ddH
2O; 3,4,5,6,7 is the anti-microbial activity of the inverted sequence peptide of 1mg/ml.
Embodiment
One, the solid state chemistry polypeptide building-up reactions of mellitin (Melittin) inverted sequence analogue
We have designed and synthesized the serial inverted sequence peptide analogs based on Mel (12～26) with different alkaline amino acid residue numbers and different hydrophobicity fragment chain lengths, the result shows, the positive charge of inverted sequence peptide and hydrophobicity are all very important for bacteriostatic activity, the analogue that the chain length that N end keeps the hydrophobicity fragment that 3 basic aminoacidss (positive charge〉4) and C hold at least is at least 8 amino-acid residues has higher bacteriostatic activity, and the hemolytic activity of these inverted sequence peptides is all very little.
Core Feature sequence according to mellitin (Melittin), reverse sequence with Melittin (12～24): the 5th I among the RKRKIWSILAPLG-COOH replaces with R, thereby increase a basic aminoacids and further strengthen positive polarity, keep 8 hydrophobic amino acids of C end constant simultaneously, its aminoacid sequence is: RKRKRWSILAPLG-COOH, this inverted sequence peptide molecule N end has positive charge, can be combined with electronegative cytolemma, the hydrophobic amino acid of C end forms α-Luo Xuanjiegou, can bore a hole at surface of cell membrane, thereby cause bacterium death.This inverted sequence peptide has been abandoned the hemolytic activity of mellitin, and its anti-microbial activity and stability all are better than single mellitin molecule.This inverted sequence peptide detects through fungicidal activity, to Gram
+Bacterium, Gram
-Bacterium and fungi etc. all have the obvious suppression effect, and hemolytic activity is very little.
The aminoacid sequence of mellitin (Melittin) inverted sequence analogue is RKRKRWSILAPLG, and mellitin inverted sequence analogue is for adopting the synthetic polypeptide products of solid state chemistry synthetic technology.Agar hole diffusion process records mellitin (Melittin) inverted sequence analogue to streptococcus aureus, intestinal bacteria K
12D
31, Pseudomonas aeruginosa, four kinds of bacteriums of moscow' typhisuis minimal inhibitory concentration (Minimal inhibition concentration MIC) is respectively 0.5,1,1 and 1 μ gmL
-1, in the Mlc scope, do not show hemolytic activity.Obtain a kind of novel antibacterial peptide with higher anti-microbial activity and safety non-toxic, provide new thinking for researching and developing desirable novel antibacterial peptide molecule.The present invention can be widely used in fields such as anti-infective medicine, makeup and livestock industry healthy aquaculture for research and development and the application of antibacterial peptide product provide new thinking.
Adopt the amino acid solid-phase synthesis of Fmoc protection; make carrier with Rink Amide MBH resin; HBTU/HOBt makes condensing agent; peptide is trifluoroacetic acid/phenol/water/thioanisole/1 from the cutting reagent on the resin; 2-3-mercaptoethanol (volume ratio 8: 0. 5: 0. 5: 0. 5: 0. 25); in room temperature reaction 4 ~ 6 h; after rotary evaporation is removed most of trifluoroacetic acid, after getting flocks, be centrifugal, 0 ℃ of dropping ether gets thick peptide; with the initial gross separation of Sephadex G-15 post; post is with 5% acetic acid balance, behind the last sample flow velocity be under 1 mL/min with 5% acetic acid wash-out, 280 nm detect; collect first peak, lyophilize.Carry out purifying with half preparative high performance liquid chromatography instrument, collect the abundance maximum peak.The purity of polypeptide is determined by analysis mode high performance liquid chromatography (C18) and amino acid analysis.The preparation method's of mellitin (Melittin) inverted sequence analogue concrete operations step is as follows:
(1) gets material, the raw material reagent of depositing in the refrigerator (as: reagent such as amino acid, ether, phenol) is taken out, place the moisture eliminator rewarming stand-by;
(2) behind the accurate weighing Fmoc-AA-Resin 0.1mmol, place synthesis reactor, use DMF(Dimethyl Fumarate, dimethyl fumarate) flushing is six times, the DMF that adds 2～3 times of amounts of about Fmoc-AA-Resin volume then soaked 30 minutes, feeds nitrogen Fmoc-AA-Resin can be stirred up and down in solution;
(3) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes feeds nitrogen reaction 10 minutes in synthesis reactor, then with twice of DMF flushing;
(4) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes again feeds nitrogen reaction 20 minutes in synthesis reactor, then with DMF flushing six times;
(5) Fmoc-AA of weighing 0.45mmol, HOBt and HBTU, earlier with 1ml DMF dissolving HOBT and HBTU, after solution is added among the Fmoc-AA, activate 15 minutes;
(6) will activate good Fmoc-AA solution and 1mL DIEPA adds among the Fmoc-AA-Resin simultaneously;
(7) flow of adjusting nitrogen can stir Resin up and down in solution, makes reaction more complete, and the reaction times was generally 2 hours;
(8) reaction is respectively washed Fmoc-AA-Resin twice successively with DMF, DCM, DMF after finishing;
(9) repeating step (3) is to step (8), till the peptide sequence coupling is intact;
(10) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes reacts, twice of operate continuously, reaction times was respectively 10 minutes and 20 minutes, respectively wash Resin twice successively with DCM, DMF, DCM respectively, after washing for the last time, Resin is drained as far as possible, namely get the Peptide-Resin(peptide resin);
(11) preparation lysate: the corresponding 10ml lysate of 1g peptide resin;
(12) lysate is joined in the synthesis reactor, feeds nitrogen reaction 2 hours, during need to replenish lysate frequently in case evaporate to dryness places anhydrous diethyl ether-20 ℃ of refrigerator and cooled to freeze standby in addition;
(13) after reaction is finished, lysate is collected in the 50ml centrifuge tube of weighing in advance, cleaned synthesis reactor 2-3 time with TFA again, washing lotion is collected in the 50ml centrifuge tube equally;
(14) anhydrous diethyl ether in the refrigerator is taken out, select suitable containers that ether is poured into wherein, in anhydrous diethyl ether: lysate=10: the ratio 1(volume ratio) splashes into lysate in the freezing anhydrous diethyl ether, control speed, filtrate is dropwise splashed into, constantly be stirred to simultaneously till the no longer generation precipitation;
(15) precipitation mixture was left standstill after several minutes centrifugal, abandon supernatant, use fresh freezing anhydrous diethyl ether washing precipitation at least three times then;
(16) the gained throw out is air-dry, seal with sealing film afterwards, prick close hole, with the Freeze Drying Equipment vacuum freezedrying and weigh;
(17) NH
4HCO
3The thick peptide of natural oxidation method oxidation: configuration 0.1M NH
4HCO
3(pH8.2) solution is in the ratio adding NH of polypeptide final concentration 0.3mg/ml
4HCO
3In the extremely thick peptide of solution, stirred overnight at room temperature.Seal with sealing film afterwards, prick close hole, use the Freeze Drying Equipment vacuum freezedrying;
(18) the thick peptide after the oxidation is carried out purifying and mass spectroscopy and identify (as described in reverse high-efficient liquid phase chromatogram purification and the mass spectrum evaluation of mellitin (Melittin) inverted sequence analogue " two, " of concrete as back), and obtain mellitin (Melittin) inverted sequence analogue;
(19) with purifying and through mellitin (Melittin) the inverted sequence analogue Freeze Drying Equipment vacuum freezedrying of mass spectroscopy accreditation, it is standby to weigh.
Two, the reverse high-efficient liquid phase chromatogram purification of mellitin (Melittin) inverted sequence analogue and mass spectrum are identified
Carry out purifying at high performance liquid chromatograph, use Hypersil BDS C18 reversed-phase column (300,5 μ m, 4.6 mm * 300 mm).Thick peptide sample after the oxidation is dissolved in the 2mL ultrapure water, and the centrifugal 5min of 10000g is loaded into and uses Buffer A(0.1%TFA) on the good reversed-phase column of balance.The acetonitrile that contains 0.1% TFA with Buffer B() gradient elution (table 1).Flow velocity is 1 ml/min, and 215 nm and 254nm two channels detect, and collects elution peak, uses the MOLDI-TOF mass spectroscopy, and qualification result is seen Fig. 1, and the molecular weight of the mutant of mass spectroscopy is consistent with theoretical value.With the sample of purifying Freeze Drying Equipment vacuum freezedrying, it is standby to weigh.The purity of thick peptide is determined by analysis mode high performance liquid chromatography (C18) and amino acid analysis.
Table 1 RPLC gradient
A:0.1%TFA B: acetonitrile (containing 0.1% TFA)
| Time (min) | Flow velocity (ml/min) | A﹪ | B﹪ | Curve |
| ﹡ | 1 | 95 | 5 | ﹡ |
| 28 | 1 | 5 | 95 | 6 |
| 29 | 1 | 5 | 95 | 6 |
Three, the anti-microbial activity of mellitin (Melittin) inverted sequence analogue detects
3.1 the anti-microbial activity of mellitin (Melittin) inverted sequence analogue is measured:
Nutrient agar is heat fused in water-bath, is cooled to about 50 ℃, draws giving birth to of 60 μ L with aseptic method and surveys bacterium (OD
600=0.3), add in the 20 mL nutrient agars, rapid mixing, pouring into diameter is that thickness is about 1.5 mm in the aseptic plane ware of 9 cm, horizontal positioned is waited to solidify.Beat the circular hole that diameter is 2.7 mm at agar, in the hole, add each 10 μ L of the mellitin of purifying (Melittin) inverted sequence analogue sample respectively, do negative control with sterilized water, do positive control with Amp.Behind the application of sample plate is put into 4 ℃ of refrigerators, treat that sample fully is diffused into agar after, plate is inverted in 37 ℃ of overnight incubation, next day observations as shown in Figure 2.Supplying the examination bacterium in this test is streptococcus aureus (Staphylococcus aureus).
3.2 the mensuration of mellitin (Melittin) inverted sequence analogue minimal inhibitory concentration (MIC)
MIC: test strain is cultured to the bacterium liquid dilution of logarithmic phase for about 5 * 10
6CFU/mL adds in 96 well culture plates, and the every hole of test hole adds bacterium liquid 90uL, adds the inverted sequence peptide solution of the different concns of doubling dilution then, the 10uL/ hole.Positive control is the bacterium liquid in 100uL/ hole, and negative control is corresponding 100uL substratum.Slowly shake in 37 ℃ then and cultivate about 16h, survey OD with microplate reader
630The minimum concentration of bacteria growing inhibiting is MIC, the results are shown in Table 2.
The MIC of table 2 mellitin (Melittin) inverted sequence analogue
| Bacterial strain | MIC (ug/mL) |
| Streptococcus aureus | 0.5 |
| Intestinal bacteria K 12D 31 | 1 |
| Pseudomonas aeruginosa | 1 |
| Moscow' typhisuis | 1 |
| Staphylococcus haemolyticus | 2 |
| Escherichia coli | 4 |
| Staphylococcus saprophyticus | 4 |
| Staphylococcus warneri | 5 |
| Urine enterococcus (resistance) | 5 |
| Morganella morganii | 40 |
| Vibrio vulnificus | 150 |
3.3 hemolytic activity experiment
(the 35mmol/L phosphate buffered saline buffer contains 150 mmol/L NaCl, is suspended in the PBS damping fluid after pH=7.4) washing 4 times, gets erythrocyte suspension (volume fraction 2.5%) with the PBS damping fluid with human red cell.The inverted sequence peptide is dissolved in the PBS damping fluid, be made into storing solution, get the polypeptide storing solution of different volumes in 0.5 mL, 2.5% erythrocyte suspension, add the PBS damping fluid to final volume 1 mL, shake up, behind 37 ℃ of insulation 60min, with centrifugal 10 min of 4000r/min, get supernatant colorimetric under 414 nm, being suspended in the PBS damping fluid with erythrocyte is blank, and being suspended among the TritonX 100 with erythrocyte is 100% haemolysis.After testing, the inverted sequence peptide of preparation does not have obvious hemolytic activity.
Those skilled in the art can carry out various remodeling and change to the present invention.Therefore, the present invention has covered various remodeling and the change in the scope that falls into appending claims and equivalent thereof.
＜110〉Guangzhou Ge Lamu bio tech ltd
＜120〉a kind of mellitin inverted sequence analogue and preparation method thereof
< 160> 1
< 210> 1
< 211> 25
< 212> PRT
＜213〉artificial sequence
< 220>
< 222>（1）…（13）
< 400> 1
Arg Lys Arg Lys Arg Trp Ser Ile Leu Ala Pro Leu Gly
1 5 10
Claims (3)
1. a mellitin inverted sequence analogue is characterized in that, the aminoacid sequence of described mellitin inverted sequence analogue is RKRKRWSILAPLG.
2. according to the described mellitin inverted sequence of claim 1 analogue, it is characterized in that described mellitin inverted sequence analogue is for adopting the synthetic polypeptide products of solid state chemistry synthetic technology.
3. the preparation method of a mellitin inverted sequence analogue is characterized in that, this preparation method may further comprise the steps:
(1) the raw material reagent that will deposit in the refrigerator takes out, and places the moisture eliminator rewarming stand-by;
(2) behind the accurate weighing Fmoc-AA-Resin 0.1mmol, place synthesis reactor, with DMF flushing six times, the DMF that adds 2～3 times of amounts of about Fmoc-AA-Resin volume then soaked 30 minutes, feeds nitrogen Fmoc-AA-Resin can be stirred up and down in solution;
(3) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes feeds nitrogen reaction 10 minutes in synthesis reactor, then with twice of DMF flushing;
(4) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes again feeds nitrogen reaction 20 minutes in synthesis reactor, then with DMF flushing six times;
(5) Fmoc-AA of weighing 0.45mmol, HOBt and HBTU, earlier with 1ml DMF dissolving HOBT and HBTU, after solution is added among the Fmoc-AA, activate 15 minutes;
(6) will activate good Fmoc-AA solution and 1mL DIEPA adds among the Fmoc-AA-Resin simultaneously;
(7) flow of adjusting nitrogen can stir Resin up and down in solution, makes reaction more complete, and the reaction times was generally 2 hours;
(8) reaction is respectively washed Fmoc-AA-Resin twice successively with DMF, DCM, DMF after finishing;
(9) repeating step (3) is to step (8), till the peptide sequence coupling is intact;
(10) 20% piperidines that adds 2 times of Fmoc-AA-Resin volumes reacts, operate continuously twice, and the reaction times was respectively 10 minutes and 20 minutes, respectively wash Resin twice successively with DCM, DMF, DCM respectively, after washing for the last time, Resin is drained as far as possible, namely get peptide resin;
(11) preparation lysate: the corresponding 10ml lysate of 1g peptide resin;
(12) lysate is joined in the synthesis reactor, feeds nitrogen reaction 2 hours, during need to replenish lysate frequently in case evaporate to dryness places anhydrous diethyl ether-20 ℃ of refrigerator and cooled to freeze standby in addition;
(13) after reaction is finished, lysate is collected in the 50ml centrifuge tube of weighing in advance, cleaned synthesis reactor 2-3 time with TFA again, washing lotion is collected in the 50ml centrifuge tube equally;
(14) anhydrous diethyl ether in the refrigerator is taken out, select suitable containers that ether is poured into wherein, by anhydrous diethyl ether: the volume ratio of lysate=10: 1 splashes into lysate in the freezing anhydrous diethyl ether, control speed, filtrate is dropwise splashed into, constantly be stirred to simultaneously till the no longer generation precipitation;
(15) precipitation mixture was left standstill after several minutes centrifugal, abandon supernatant, use fresh freezing anhydrous diethyl ether washing precipitation at least three times then;
(16) the gained throw out is air-dry, seal with sealing film afterwards, prick close hole, with the Freeze Drying Equipment vacuum freezedrying and weigh;
(17) NH
4HCO
3The thick peptide of natural oxidation method oxidation: configuration 0.1M NH
4HCO
3Solution is in the ratio adding NH of polypeptide final concentration 0.3mg/ml
4HCO
3In the extremely thick peptide of solution, stirred overnight at room temperature; Seal with sealing film afterwards, prick close hole, use the Freeze Drying Equipment vacuum freezedrying;
(18) the thick peptide after the oxidation is carried out purifying and mass spectroscopy and identify, and obtain mellitin inverted sequence analogue;
(19) with purifying and through the mellitin inverted sequence analogue Freeze Drying Equipment vacuum freezedrying of mass spectroscopy accreditation, it is standby to weigh.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551191A (en) * | 2019-09-10 | 2019-12-10 | 中国医学科学院基础医学研究所 | Improved melittin with low hemolytic activity and application thereof |
| CN110951820A (en) * | 2019-12-26 | 2020-04-03 | 杨威 | Bee venom activity detection method and detection device |
| CN111100190A (en) * | 2020-01-10 | 2020-05-05 | 大理大学 | Wasp toxin peptide reverse order analogue WVD-II and preparation method and application thereof |
| CN111116714A (en) * | 2020-01-10 | 2020-05-08 | 大理大学 | Wasp toxic peptide reverse order analogue WVC-II and preparation method and application thereof |
| CN111153966A (en) * | 2020-01-10 | 2020-05-15 | 大理大学 | Wasp toxic peptide reverse order analogue WVF-II and preparation method and application thereof |
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| CN1704431A (en) * | 2004-06-04 | 2005-12-07 | 中国人民解放军第三军医大学第一附属医院 | Melittin and use thereof |
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| CN1704431A (en) * | 2004-06-04 | 2005-12-07 | 中国人民解放军第三军医大学第一附属医院 | Melittin and use thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551191A (en) * | 2019-09-10 | 2019-12-10 | 中国医学科学院基础医学研究所 | Improved melittin with low hemolytic activity and application thereof |
| CN110951820A (en) * | 2019-12-26 | 2020-04-03 | 杨威 | Bee venom activity detection method and detection device |
| CN111100190A (en) * | 2020-01-10 | 2020-05-05 | 大理大学 | Wasp toxin peptide reverse order analogue WVD-II and preparation method and application thereof |
| CN111116714A (en) * | 2020-01-10 | 2020-05-08 | 大理大学 | Wasp toxic peptide reverse order analogue WVC-II and preparation method and application thereof |
| CN111153966A (en) * | 2020-01-10 | 2020-05-15 | 大理大学 | Wasp toxic peptide reverse order analogue WVF-II and preparation method and application thereof |
| CN111116714B (en) * | 2020-01-10 | 2023-04-25 | 大理大学 | Wasp venom peptide reverse sequence analogue WVC-II and preparation method and application thereof |
| CN111100190B (en) * | 2020-01-10 | 2023-04-28 | 大理大学 | Wasp venom peptide reverse sequence analogue WVD-II and preparation method and application thereof |
| CN111153966B (en) * | 2020-01-10 | 2023-05-16 | 大理大学 | Wasp venom peptide reverse sequence analogue WVF-II and preparation method and application thereof |
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Application publication date: 20130911 |