CN111116714B - Wasp venom peptide reverse sequence analogue WVC-II and preparation method and application thereof - Google Patents

Abstract

The invention provides a wasp venom peptide reverse sequence analogue WVC-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 WVC-II is shown as SEQ ID NO: 1. The in vitro antibacterial activity test shows that the wasp venom peptide reverse sequence analogue WVC-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 WVC-II can be used as an active ingredient to replace the traditional antibiotics for treatment in clinic.

Description

A kind of vespidin reverse sequence analogue WVC-II and its preparation method and application

technical field

The invention relates to the technical field of polypeptide synthesis, in particular to a vespidin reverse sequence analogue WVC-II and its preparation method and application.

Background technique

Antimicrobial peptides can resist microbial invasion, and have specific or non-specific effects on Gram-negative bacteria, positive bacteria, fungi, viruses, tumors and eukaryotes. In addition to being antimicrobial, antimicrobial peptides have other functions. Antimicrobial peptides are mostly used in the form of peptide analogs or peptide derivatives, and these substances can be obtained by modifying the amino acids of natural antimicrobial peptides. Although a large number of natural antimicrobial peptides have been found to be expected to replace the problem of drug resistance caused by the abuse of traditional antibiotics, there are still many shortcomings of natural antimicrobial peptides, which limit their application as clinical drugs, especially in terms of toxicity and Aspects of enzymatic stability.

Contents of the invention

The object of the present invention is to provide a vespidin reverse analogue WVC-II and its preparation method and application. The vespidin reverse analogue WVC-II has better antibacterial activity.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a vespidin reverse analog WVC-II, the amino acid sequence of the vespidin reverse analog WVC-II is shown in SEQ ID NO:1.

The present invention also provides a preparation method of the vespidin reverse analogue WVC-II described in the above scheme, comprising the following steps:

1) sequentially coupling amino acids on the resin according to the amino acid sequence of the reverse sequence analog WVC-II of the vespatin to obtain a peptide resin;

2) Cleavage the peptide resin described in step 1), add a side chain protecting group scavenger, precipitate the target polypeptide, centrifuge, collect the precipitate to obtain the vespa venom reverse analogue WVC-II.

Preferably, the resin described in step 1) includes 4-(2′,4′-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamido-methylbenzhydrylamine resin .

Preferably, the coupling reagent used for coupling amino acids in step 1) is selected from dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-diaminopropyl-N-ethylcarbodiimide, 2-(1H-Benzotrisazo 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-oxytripyrrolidinylphosphonium hexafluorophosphate and one or more of 1-hydroxybenzotriazoles.

Preferably, the lysate used for cleavage in step 2) includes trifluoroacetic acid and/or hydrofluoric acid.

Preferably, the ratio of the mass of the peptide resin to the volume of the lysate in step 2) is 1g:5-50mL.

Preferably, the side chain protecting group scavenger in step 2) is selected from any two of thioanisole, thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol and m-cresol one or a combination of two or more.

Preferably, the reagent for precipitating the target polypeptide in step 2) includes ice anhydrous ether.

Preferably, after the step 2) obtains the reverse sequence analog WVC-II of vespatin, it also includes purifying the reverse sequence analog WVC-II of vespidin.

The present invention also provides the application of the vespidin reverse sequence analog WVC-II described in the above scheme in broad-spectrum antibacterial.

Beneficial effects of the present invention: the present invention provides a vespidin reverse analog WVC-II, the amino acid sequence of the vespidin reverse analog WVC-II is shown in SEQ ID NO:1. The N-terminal of the vespidin reverse analog WVC-II of the present invention has a positive charge and can combine with negatively charged cell membranes, and the hydrophobic amino acids at the C-terminal form an α-helical structure, which can perforate the surface of the cell membrane and cause bacterial death. The in vitro antibacterial activity of the vespidin reverse analogue WVC-II of the present invention shows that it has a significant bactericidal effect on Gram-positive bacteria, Gram-negative bacteria and fungi, and has better stability. Hemolytic toxicity is low, with the characteristics of broad spectrum, high efficiency and safety. The vespidin reverse analogue WVC-II of the present invention can be used as an active ingredient in clinical treatment instead of traditional antibiotics.

Description of drawings

Figure 1 is the antibacterial experiment results of vespidin reverse analogue WVC-II against Candida albicans (left), Staphylococcus aureus (middle) and Escherichia coli (right), in the figure: 1 represents the antibacterial activity of penicillin sodium; 2 is the negative control dd H ₂ O; 3 is the antibacterial activity of 1 mg·mL ^-1 vespidin reverse analogue.

Detailed ways

The present invention provides a vespidin reverse analog WVC-II, the amino acid sequence of the vespidin reverse analog WVC-II is shown in SEQ ID NO: 1, specifically: KHRPXGFSAAPVFLRPPD, wherein X is hydroxyl Proline Hrp, the amino group (NH ₂ ) is located at the K terminal, and the carboxyl group (COOH) is located at the D terminal.

The amino acid sequence of the reverse sequence of vespidin is shown in SEQ ID NO: 2, specifically: KGRPXGFSAAPVFLRPID, wherein X is hydroxyproline Hrp, the amino group (NH ₂ ) is located at the K terminal, and the carboxyl group (COOH) is located at the D terminal. In the present invention, the second position G of the above-mentioned vespidin reverse sequence is replaced by H, and the sixteenth position I is replaced by P, and the positive charge is further enhanced by adding a basic amino acid (histidine H). At the same time, the hemolytic activity of erythrocytes is reduced, and the antibacterial activity is increased. The N-terminal of the vespidin reverse analogue WVC-II of the present invention has a positive charge, which can combine with negatively charged cell membranes, and the hydrophobic amino acids at the C-terminal form an α-helical structure, which can perforate the surface of the cell membrane, thereby leading to bacterial death .

The present invention also provides a preparation method of the vespidin reverse analogue WVC-II described in the above scheme, comprising the following steps:

1) sequentially coupling amino acids on the resin according to the amino acid sequence of the reverse sequence analog WVC-II of the vespatin to obtain a peptide resin;

2) Cleavage the peptide resin described in step 1), add a side chain protecting group scavenger, precipitate the target polypeptide, centrifuge, collect the precipitate to obtain the vespa venom reverse analogue WVC-II.

The present invention preferably also includes the process of washing the resin, soaking the resin, removing the amino protecting group and washing the resin again before sequentially coupling amino acids on the resin according to the amino acid sequence of the vespidin reverse analogue WVC-II ; The resin preferably includes Rink Amide MBHA resin (4-(2′,4′-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamido-methylbenzhydrylamine Resin); The effect of described resin is as carrier.

In the present invention, the reagent used in the washing resin is preferably dimethylformamide (DMF); the number of washings is preferably 5 to 7 times, more preferably 6 times; the solvent used in the soaking resin is preferably two Methylformamide (DMF) or dichloromethane (DCM); The quality of the resin and the volume ratio of the solvent in the soaking process are preferably 0.5g:10mL; the soaking time is preferably 20 to 40min, more preferably 30min preferably also include feeding nitrogen in the soaking process, so that the resin can be turned up and down in the solution; the reagent for removing the amino protecting group is preferably a piperidine (PIP) solution; the piperidine (PIP) solution Concentration is preferably 10%～30%, more preferably 20%～25%; The time of described removal is preferably 5～30min, more preferably 10～20min; The consumption of described piperidine (PIP) solution is preferably 2 ~3 times the volume of the resin; the reagent used to wash the resin again is preferably dimethylformamide (DMF); the number of washings is preferably 2-10 times, more preferably 6-8 times. During the specific implementation of the present invention, after washing the resin again, the present invention also repeats the process of removing the amino protecting group and washing the resin again.

After the above-mentioned treatment, the present invention sequentially couples amino acids on the resin according to the amino acid sequence of the reverse sequence analogue WVC-II of the vespatin to obtain a peptide resin; the coupling reagent used for coupling amino acids is preferably selected from dicyclohexyl Carbodiimide (DCC), Diisopropylcarbodiimide (DIC), N-diaminopropyl-N-ethylcarbodiimide (EDC), 2-(1H-benzotrisazo L -1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluoro Phosphate (HBTU), benzotriazol-1-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP ) and 1-hydroxybenzotriazole (HOBt), more preferably a combination of DIC and HOBt, TBTU and HOBt or HBTU and HOBt, most preferably HBTU and HOBt; when coupling amino acids in sequence , based on the resin used as 0.5g, the amount of the corresponding amino acid added sequentially each time is preferably 0.45mmol, and the amount of the coupling agent is preferably 0.45mmol; the coupling temperature is preferably 16-30°C, More preferably 20-25°C; during the coupling process, the amino acid sequences of the polypeptides are coupled one by one from the C-terminus to the N-terminus; the present invention has no special limitation on the coupling time, and the coupling process uses indene The ketone reaction monitors the condensation reaction of the peptide to ensure successful coupling.

After obtaining the peptide resin, the present invention preferably also includes removing the amino-protecting group from the peptide resin and washing the peptide resin sequentially with DCM, DMF, and DCM; the reagent for removing the amino-protecting group is preferably a piperidine (PIP) solution; The volume concentration of the piperidine (PIP) solution is preferably 10% to 30%, more preferably 20% to 25%; the coefficient of removing the amino protecting group is preferably 2 times, the first time removing the amino protecting group The time is preferably 10 min, and the time for removing the amino protecting group for the second time is preferably 20 min.

After the peptide resin is cleaned, the present invention cracks the cleaned peptide resin, adds a side chain protecting group scavenger, precipitates the target polypeptide, centrifuges, collects and precipitates to obtain the reverse sequence analogue of Vespa venom peptide WVC-II; The lysate includes trifluoroacetic acid and/or hydrofluoric acid; the ratio of the mass of the peptide resin to the volume of the lysate is preferably 1g:5-50mL, more preferably 1g:10-20mL; Nitrogen; the cracking time is preferably 1 to 3h, more preferably 2h; the side chain protecting group scavenger is preferably selected from the group consisting of thioanisole, thioanisole, triisopropylsilane, phenol, water, 1 , the combination of any two or more of 2-ethanedithiol and m-cresol, more preferably phenol, water, thioanisole and 1,2-dimercaptoethanol; the phenol, water, thioanisole The volume ratio of ether and 1,2-dimercaptoethanol is preferably 0.5～2:0.5～3:0.5～1:0.25～1, more preferably 1～1.5:1～2:0.8:0.5～0.75; the precipitation The reagent for the target polypeptide preferably includes ice anhydrous ether; the rotational speed of the centrifugation is preferably 3000 rpm; the centrifugation time is preferably 5 min.

After obtaining the vespidin reverse analog WVC-II, the present invention preferably also includes purifying the vespidin reverse analog WVC-II; the purification method is preferably reversed-phase chromatography or ion-exchange chromatography .

The present invention also provides the application of the vespidin reverse sequence analog WVC-II described in the above scheme in broad-spectrum antibacterial; the application includes the preparation of anti-infective drugs, cosmetics and feed; the objects of the broad-spectrum antibacterial include Gram-positive bacteria, Gram-negative bacteria and fungi; the Gram-positive bacteria are preferably Staphylococcus aureus; the Gram-negative bacteria are preferably Escherichia coli; and the fungi are preferably Candida albicans.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1 Solid Phase Chemical Synthesis of Vespatin Reverse Analogue WVC-II Polypeptide

Amino acid protected by Fmoc (fluorenylmethoxycarbonyl) was used for solid-phase synthesis, and RinkAmide MBHA resin (4-(2′,4′-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxy Acetamido-methyl benzhydrylamine resin) as the carrier, 1-hydroxybenzotriazole and O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate as the coupling Reagents, cleavage of peptides from the resin using lysate trifluoroacetic acid and side chain protective group scavenger (phenol/water/sulfide anisole/1,2-dimercaptoethanol), react at room temperature for 4-6 hours, and rotate to evaporate After removing most of the trifluoroacetic acid, diethyl ether was added dropwise at 0°C to obtain a flocculent precipitate, and the crude peptide was obtained after centrifugation, which was initially separated with a Sephadex G-15 column, and the column was equilibrated with 5% acetic acid, and the flow rate was 1 mL min after sample loading. It was eluted with 5% acetic acid at ^-1 , detected at 280 nm, and the first peak was collected and freeze-dried. Purify with semi-preparative high-performance liquid chromatography and collect the most abundant peak. The purity of the peptides was determined by analytical HPLC and amino acid analysis. The specific operation steps of the preparation method of the vespidin reverse analogue WVC-II are as follows:

1) Take out the materials, take out the raw material reagents stored in the refrigerator, and place them in a desiccator for rewarming for later use;

2) Accurately weigh 0.5g of RinkAmide MBHA resin, put it in the synthesis reactor, wash it with dimethylformamide six times, then add 10mL of dimethylformamide to soak for 30min, and let the resin flow up and down in the solution flip;

3) Add 2 times the volume of 20% piperidine into the synthesis reactor, pass through nitrogen to react for 10 minutes, and then rinse twice with dimethylformamide;

4) Add 2 times the volume of 20% piperidine to the synthesis reactor again, pass nitrogen gas to react for 20 minutes, and then rinse with DMF six times;

5) Weigh 0.45 mmol of Fmoc-protected amino acid, equimolar coupling agent 1-hydroxybenzotriazole and O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate The salt was reacted at room temperature, monitored by ninhydrin reaction to ensure the coupling was successful, 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 above method, and this cycle is repeated until all amino acid couplings are completed;

6) Add 2 times the volume of 20% piperidine to react, operate twice continuously, the reaction time is 10min and 20min respectively, wash the resin (resin) successively with DCM, DMF, DCM respectively, and drain as much as possible to obtain NH2-KHRPXGFSAPVFLRPPD- resin (peptide resin), wherein X is hydroxyproline Hrp;

7) Add 5 mL of trifluoroacetic acid lysate and side chain protecting group scavenger (phenol/water/sulfide anisole/1,2-dimercaptoethanol: 0.5:0.5:0.5:0.25) into the synthesis reactor, and pass Nitrogen gas was added to react for 2 hours, and anhydrous diethyl ether was placed in a -20°C refrigerator to freeze for later use;

8) After the reaction is completed, collect the reaction solution into a pre-weighed 50mL centrifuge tube, then wash the synthesis reactor twice with trifluoroacetic acid, and collect the washing solution into a 50mL centrifuge tube;

9) Take out the ice anhydrous ether in the refrigerator, choose a suitable container and pour the ether into it, and drop the lysate into the frozen anhydrous ether according to the ratio of anhydrous ether: reaction liquid = 10:1 (volume ratio) , control the speed, try to make the filtrate drop by drop, while stirring continuously until no precipitation occurs;

10) Centrifuge the precipitation mixture after standing for several minutes, discard it, and then wash the precipitation at least three times with fresh frozen anhydrous ether;

11) Freeze and vacuum-dry the obtained precipitate with a lyophilizer to obtain a crude peptide and weigh it;

12) Purify the crude peptide and identify it by mass spectrometry, and obtain WVC-II;

13) Freeze and vacuum-dry the purified WVC-II reverse-sequence analogue qualified by mass spectrometry analysis, and weigh it for future use.

Reverse HPLC purification and mass spectrometry identification of embodiment 2WVC-Ⅱ

)。将氧化后的粗肽样品溶于1mL的超纯水中，10000rpm/min离心5min，W2998检测器检测，检测波长为215、254、280nm。流动相为四相洗脱系统，流速为3mL·min^-1。洗脱液分别为0.1％TFA水溶液(A)和0.1％TFA乙腈溶液(B)，柱温为25℃，反相高效液相色谱洗脱梯度见表1，收集洗脱峰，用Q-TOF质谱分析，鉴定结果见表2，质谱分析的突变体的分子量与理论值相符合，说明成功合成了WVC-Ⅱ。将纯化的样品冻干后储存备用。粗肽的纯度通过高效液相色谱和氨基酸分析确定。Purify in high performance liquid chromatography, using Sepax Bio-C18 reverse phase column (10.0 × 250mm, 10μm,

). The oxidized crude peptide sample was dissolved in 1 mL of ultrapure water, centrifuged at 10,000 rpm/min for 5 min, and detected by a W2998 detector with detection wavelengths of 215, 254, and 280 nm. The mobile phase was a four-phase elution system with a flow rate of 3mL·min ^-1 . The eluents were 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile solution (B) respectively, the column temperature was 25°C, the elution gradient of reversed-phase high-performance liquid chromatography was shown in Table 1, and the elution peaks were collected and analyzed by Q-TOF Mass spectrometry analysis, the identification results are shown in Table 2, the molecular weight of the mutants analyzed by mass spectrometry is consistent with the theoretical value, indicating that WVC-II was successfully synthesized. The purified samples were freeze-dried and stored for later use. The purity of the crude peptide was determined by HPLC and amino acid analysis.

Table 1 Reversed phase high performance liquid chromatography elution gradient

Table 2 The relative molecular mass of the single component of WVC-Ⅱ

Antibacterial activity detection of the reverse sequence analogue WVC-II of embodiment 3 vespidin

(1) Determination of antibacterial activity of WVC-Ⅱ

The agar medium was heated and melted in a water bath, cooled to about 50°C, and 60 μL of bioassay bacteria was absorbed by aseptic operation, added to 20 mL of agar medium, mixed quickly, and poured into a sterile flat dish with a diameter of 9 cm. The thickness is about 1.5mm, placed horizontally to be solidified. Make circular holes with a diameter of 2.7 mm on the agar, add 10 μL of each purified WVC-II sample into the holes, use sterile water as a negative control, and penicillin sodium as a positive control. After adding the sample, put the plate in a refrigerator at 4°C. After the sample has fully diffused into the agar, place the plate upside down at 37°C and incubate overnight. The results of antibacterial experiments against Candida albicans (left), Staphylococcus aureus (middle) and Escherichia coli (right), in the figure: 1 indicates the antibacterial activity of penicillin sodium; 2 is the negative control dd H ₂ O; 3 is 1mg· Antibacterial activity of vespidin reverse analogs of mL ^-1 ). The bacteria tested in this experiment were Staphylococcus aureus. It can be seen from Figure 1 that the WVC-Ⅱ sample (No. 3 inhibition zone) has a good antibacterial effect.

(2) Determination of the minimum inhibitory concentration (MIC) of WVC-Ⅱ

MIC: Dilute the test strain to the logarithmic growth phase of the bacterial solution to about 5×10 ⁶ CFU·mL ^-1 , add it to a 96-well culture plate, add 90 μL of the bacterial solution to each well of the test well, and then add the doubly diluted Different concentrations of vespidin reverse analogue solution, 10 μL/well. The positive control is 100 μL/well bacterial solution, and the negative control is the corresponding 100 μL culture medium. Then culture at 37°C with slow shaking for about 16 hours, and measure OD630 with a microplate reader. The minimum concentration that inhibits bacterial growth is the MIC, and the results are shown in Table 3. It can be seen from Table 3 that the minimum inhibitory concentrations (Minimal inhibition concentration, MIC) of the vespidin reverse analog WVC-Ⅱ against Staphylococcus aureus, Escherichia coli and Candida albicans were 0.5, 1 and 0.5 μg· mL ^-1 , did not show hemolytic activity within the range of inhibitory concentration. It can be seen that WVC-Ⅱ has a good MIC value for Staphylococcus aureus, Escherichia coli and Candida albicans.

Table 3 MICs of reverse analogues of WVC-Ⅱ

(3) Hemolytic activity test

Human red blood cells were washed 4 times with PBS buffer (35 mM phosphate buffer, containing 150 mM NaCl, pH 7.4) and then suspended in PBS buffer to obtain red blood cell suspension (volume fraction 2.5%). Dissolve the reverse sequence analog of vespatin in PBS buffer to make a stock solution, take different volumes of peptide stock solution in 0.5mL of 2.5% red blood cell suspension, add PBS buffer to a final volume of 1mL, shake Mix evenly, measure color at 414nm, incubate at 37°C incubator for 1h, then centrifuge at 1200rpm for 15min, take supernatant and measure color at 414nm, take red blood cells suspended in PBS buffer as blank, and red blood cells suspended 100% hemolysis in TritonX100. Tests show that the prepared vespidin reverse analog has no obvious hemolytic activity.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Dali University

<120> A reverse sequence analogue of vespidin WVC-Ⅱ and its preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys His Arg Pro Xaa Gly Phe Ser Ala Pro Val Phe Leu Arg Pro Pro

1 5 10 15

Asp

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Lys Gly Arg Pro Xaa Gly Phe Ser Ala Pro Val Phe Leu Arg Pro Ile

1 5 10 15

Asp

Claims (10)

1. A wasp venom peptide anti-sequence analogue WVC-II, wherein the amino acid sequence of the wasp venom peptide anti-sequence analogue WVC-II is shown as SEQ ID NO:1 is shown in the specification; and X is hydroxyproline.

2. The method for preparing the wasp venom peptide anti-sequence analogue WVC-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 WVC-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 WVC-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 WVC-II after obtaining the reverse wasp venom peptide analog WVC-II in step 2).

10. Use of the reverse wasp venom peptide analog WVC-ii as claimed in claim 1 as an active ingredient in the preparation of a medicament for inhibiting staphylococcus aureus, escherichia coli and candida albicans.

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