Polypeptide composition with bacteriostatic activity and preparation method thereof

Abstract

The invention relates to the field of biological medicine, in particular to a polypeptide composition with bacteriostatic activity and a preparation method thereof.The polypeptide composition is prepared by wrapping polypeptide I with modified polyethylene glycol, then wrapping with nano-silver and finally modifying polypeptide II with the nano-silver. The outer layer is sequentially wrapped with spherical particles of modified polyethylene glycol, nano-silver and polypeptide II, and the polypeptide II and the nano-silver are promoted to be combined with a bacterial membrane and destroy the bacterial membrane through the non-specific electrostatic interaction of the polypeptide II and the nano-silver, so that the polypeptide II has relatively high antibacterial performance; the outer-layer polypeptide is degraded by proteolytic enzyme or plays a role with a bacterial membrane, so that the inner-layer polypeptide I wrapped by the modified polyethylene glycol is exposed, the antibacterial effect is further played, and the proteolytic stability of the polypeptide is improved; due to the spherical structure, the prepared polypeptide composition with the antibacterial activity has excellent antibacterial performance, meanwhile, the stability can be improved, and the polypeptide composition has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide composition with antibacterial activity and a preparation method thereof. Background Art

[0002] Antimicrobial peptides have multiple biological activities such as antibacterial and antifungal, antiviral, antiparasitic, and immunomodulatory. According to their physicochemical properties, antimicrobial peptides can be divided into cationic antimicrobial peptides and anionic antimicrobial peptides. Most antimicrobial peptides are cationic antimicrobial peptides, and only a few are neutral and anionic antimicrobial peptides. According to their structure, antimicrobial peptides can be divided into α-helical antimicrobial peptides, β-folded antimicrobial peptides, random coiled antimicrobial peptides, α-helical and β-folded antimicrobial peptides, and progressive cyclic antimicrobial peptides with more complex topological structures.

[0003] The mechanism of action of antimicrobial peptides can be divided into two categories: one is membrane-damaging mechanism; the other is non-membrane-damaging mechanism. Membrane-damaging antimicrobial peptides interact with the cell membrane of the target organism, causing membrane lysis, causing lesions on the membrane, and permeabilizing the phospholipid bilayer to cause cell death. Non-membrane-damaging antimicrobial peptides penetrate the cell membrane and their targets are organelles, proteins or nucleic acids.

[0004] The specificity of antimicrobial peptides in breaking membranes is extremely low, and it is difficult for bacteria to develop drug resistance through mutation. Antimicrobial peptides are considered to be the best antibiotic alternatives with the best development prospects and application potential due to their broad antimicrobial spectrum, variety, and wide range of options. However, antimicrobial peptides have problems such as poor stability, poor selectivity, long sequences, and high costs during clinical application.

[0005] In the prior art, a polypeptide is usually processed by deleting, inserting or replacing one or several amino acids to prepare a polypeptide composition. For example, patent document CN104327164B discloses a synthetic polypeptide and its use, which is a polypeptide with an amino acid sequence of QKKPVPIIYCNGTCQ, which is processed by deleting, inserting or replacing one or several amino acids to obtain a derivative polypeptide with the same biological function as the original polypeptide. The mixture of the two has antibacterial ability and can also promote the growth of bone cells, but the stability is poor.

[0006] Therefore, according to the above-mentioned related technologies, it is urgent to develop a polypeptide composition with antibacterial activity and a preparation method thereof. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a polypeptide composition with antibacterial activity and a preparation method thereof, so as to solve the problem of poor stability of the polypeptide composition in the prior art.

[0008] Based on the above purpose, the present invention provides a polypeptide composition with antibacterial activity and a preparation method thereof.

[0009] A polypeptide composition with antibacterial activity, wherein the polypeptide composition is a spherical particle with polypeptide I as the core and modified polyethylene glycol, nanosilver and polypeptide II wrapped in the outer layer in sequence;

[0010] The polypeptide I is obtained by mixing L-ZY13 and D-ZY13;

[0011] The two chain ends of the modified polyethylene glycol are respectively a thiol group and a carboxyl group;

[0012] The polypeptide II is obtained by mixing L-GW2 and D-GW2.

[0013] Preferably, the amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val;

[0014] The mass ratio of L-ZY13 to D-ZY13 is 25-30:10-12.

[0015] Preferably, the amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp;

[0016] The mass ratio of L-GW2 to D-GW2 is 35-40:10-15.

[0017] Preferably, the particle size of the nanosilver is 15-25 nm.

[0018] Preferably, the number average molecular weight of the modified polyethylene glycol is 3500-4000 g / mol.

[0019] A method for preparing a polypeptide composition having antibacterial activity comprises the following steps:

[0020] Step A1. Add polypeptide I to a reaction bottle, add an organic solvent to swell for 15-20 minutes, add modified polyethylene glycol, mix and stir for 15-20 minutes, then add 1-hydroxybenzotriazole, 1,3-diisopropylcarbodiimide and 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3 hours, then add reagent P, stir and react at room temperature for 3-5 hours, filter and wash 3 times, rotary evaporate the filtrate, add ether to filter, centrifuge the precipitate, and vacuum dry for 3 hours to obtain a crude intermediate A;

[0021] Step A2. The crude intermediate A was dispersed in water and purified by preparative reverse-phase high performance liquid chromatography. The collected solution was concentrated by rotary evaporation and then freeze-dried for 3 hours to obtain pure intermediate A.

[0022] Step A3. Disperse the pure intermediate A in water, add nanosilver, and stir the reaction at room temperature for 8-10 hours to obtain intermediate B;

[0023] Step A4. Add polypeptide II to intermediate B, incubate at 35-40°C with an oscillation speed of 200-250 rpm for 10-12 hours under light-proof conditions, then centrifuge at 9000-10000 rpm for 30-35 minutes, then wash twice with water, freeze-dry, and obtain a polypeptide composition with antibacterial activity.

[0024] Preferably, the amount ratio of polypeptide I, organic solvent, modified polyethylene glycol, 1-hydroxybenzotriazole, 1,3-diisopropylcarbodiimide, 4-dimethylaminopyridine, reagent P and ether in step A1 is 35-42 g: 400-420 mL: 10-12 g: 20-25 g: 20-25 mL: 10-12 g: 400-420 mL: 380-400 mL;

[0025] The organic solvent in step A1 is a mixture of DMF and DCM in a volume ratio of 9:1;

[0026] The P reagent in step A1 is prepared by mixing trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 80-85:5:5:5:2-5.

[0027] Preferably, the usage ratio of the crude intermediate A and water in step A2 is 45-50 g:450-500 mL.

[0028] Preferably, the usage ratio of the pure intermediate A, water and nanosilver in step A3 is 40-45 g: 800-850 mL: 22-28 g.

[0029] Preferably, the usage ratio of polypeptide II and intermediate B in step A4 is 45-55 g:500-550 mL.

[0030] Beneficial effects of the present invention:

[0031] The present invention provides a polypeptide composition with antibacterial activity and a preparation method thereof, which has better antibacterial performance and takes into account stability. The polypeptide composition prepared by the present invention is a spherical particle with polypeptide I as the core, and the outer layer sequentially wraps modified polyethylene glycol, nanosilver and polypeptide II. Polypeptide I is a mixture of L-ZY13 and D-ZY13, wherein D-ZY13 has better antibacterial performance than L-ZY13. Polypeptide II is a mixture of L-GW2 and D-GW2. Natural polypeptides are generally L-type. Due to the configuration selectivity factor, D-GW2 is not easily specifically recognized by proteases, so D-GW2 has stronger stability.

[0032] The nonspecific electrostatic interaction between the polypeptide provided by the present invention and nanosilver promotes the combination with the bacterial membrane and destroys the bacterial membrane, thereby increasing the antibacterial property; the outer layer polypeptide may be degraded by proteases or interact with the bacterial membrane to expose the polypeptide wrapped in the inner layer of modified polyethylene glycol, and compared with the polypeptide not modified with modified polyethylene glycol, the proteolytic stability of the polypeptide is significantly improved. Such a spherical structure enables the prepared polypeptide composition with antibacterial activity to have excellent antibacterial properties while also improving stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 It is a schematic diagram of the structure of the polypeptide composition with antibacterial activity in the present invention. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0036] The sources and properties of some of the raw materials used in the present invention are as follows:

[0037] Modified polyethylene glycol was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; nanosilver was purchased from Shanghai Chuangsai Technology Co., Ltd.; L-ZY13 and D-ZY13 were purchased from Shanghai Jier Biochemical Co., Ltd.; L-GW2 and D-GW2 were purchased from Anhui Guoping Pharmaceutical Co., Ltd.

[0038] Example 1: A polypeptide composition with antibacterial activity, comprising the following steps:

[0039] S1: Add 42g of polypeptide I to a reaction bottle, add 420mL of organic solvent to swell for 20min, add 12g of modified polyethylene glycol, mix and stir for 20min, then add 25g of 1-hydroxybenzotriazole, 25mL of 1,3-diisopropylcarbodiimide and 12g of 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3h, add 420mL of L reagent, stir and react at room temperature for 5h, filter and wash 3 times, rotary evaporate the filtrate, add 400mL of ether to filter, centrifuge the precipitate, and vacuum dry for 3h to obtain a crude intermediate A, in which two of the modified polyethylene glycol The side chain ends are thiol and carboxyl, respectively, the number average molecular weight of the modified polyethylene glycol is 3500 g / mol, polypeptide I is a mixture of 30 g L-ZY13 and 12 g D-ZY13, wherein the amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val, the organic solvent is a mixture of DMF and DCM in a volume ratio of 9:1, and reagent P is a mixture of trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 85:5:5:5:5;

[0040] S2: Disperse 50 g of crude intermediate A in 500 mL of water, purify by preparative reverse-phase high performance liquid chromatography, concentrate the collected solution by rotary evaporation, and then freeze-dry for 3 h to obtain pure intermediate A;

[0041] S3: Disperse 45 g of pure intermediate A in 850 mL of water, add 28 g of nanosilver, and react at room temperature with stirring for 8-10 h to obtain intermediate B, wherein the particle size of the nanosilver is 15 nm;

[0042] S4: Add 55g of polypeptide II to 550mL of intermediate B, incubate at 40°C with an oscillation speed of 250rpm for 12h under light-protected conditions, then centrifuge at 10000rpm for 35min, then wash twice with water, and freeze-dry to obtain a polypeptide composition with antibacterial activity, wherein polypeptide II is a mixture of 40gL-GW2 and 15gD-GW2, and the amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp.

[0043] Example 2: A polypeptide composition having antibacterial activity, comprising the following steps:

[0044] S1: Add 40g of polypeptide I to a reaction bottle, add 415mL of organic solvent to swell for 18min, add 11.5g of modified polyethylene glycol, mix and stir for 18min, then add 24g of 1-hydroxybenzotriazole, 24mL of 1,3-diisopropylcarbodiimide and 11.5g of 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3h, add 415mL of L-reagent, stir and react at room temperature for 4.5h, filter and wash 3 times, rotary evaporate the filtrate, add 395mL of ether to filter, centrifuge the precipitate, and vacuum dry for 3h to obtain a crude intermediate A, in which the modified polyethylene glycol The two chain ends of the alcohol are thiol and carboxyl, respectively. The number average molecular weight of the modified polyethylene glycol is 3600 g / mol. Peptide I is prepared by mixing 28 g L-ZY13 and 12 g D-ZY13, wherein the amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val. The organic solvent is prepared by mixing DMF and DCM in a volume ratio of 9:1. Reagent P is prepared by mixing trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 83:5:5:5:4.

[0045] S2: Disperse 48 g of crude intermediate A in 480 mL of water, purify by preparative reverse-phase high performance liquid chromatography, concentrate the collected solution by rotary evaporation, and then freeze-dry for 3 h to obtain pure intermediate A;

[0046] S3: Disperse 44 g of pure intermediate A in 840 mL of water, add 26 g of nanosilver, and stir at room temperature for 9.5 h to obtain intermediate B, wherein the particle size of the nanosilver is 18 nm;

[0047] S4: Add 50 g of polypeptide II to 540 mL of intermediate B, incubate at 38°C with an oscillation speed of 240 rpm for 11.5 h under light-protected conditions, then centrifuge at 9800 rpm for 34 min, then wash twice with water, and freeze-dry to obtain a polypeptide composition with antibacterial activity, wherein polypeptide II is a mixture of 38 g L-GW2 and 12 g D-GW2, and the amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp.

[0048] Example 3: A polypeptide composition with antibacterial activity, comprising the following steps:

[0049] S1: Add 38g of polypeptide I to a reaction bottle, add 410mL of organic solvent to swell for 16min, add 11g of modified polyethylene glycol, mix and stir for 16min, then add 21g of 1-hydroxybenzotriazole, 21mL of 1,3-diisopropylcarbodiimide and 11g of 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3h, add 410mL of L reagent, stir and react at room temperature for 4h, filter and wash 3 times, rotary evaporate the filtrate, add 390mL of ether to filter, centrifuge the precipitate, and vacuum dry for 3h to obtain a crude intermediate A, in which two of the modified polyethylene glycol The side chain ends are thiol and carboxyl, respectively, the number average molecular weight of the modified polyethylene glycol is 3800 g / mol, polypeptide I is a mixture of 27 g L-ZY13 and 11 g D-ZY13, wherein the amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val, the organic solvent is a mixture of DMF and DCM in a volume ratio of 9:1, and reagent P is a mixture of trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 82:5:5:5:3;

[0050] S2: Disperse 46 g of crude intermediate A in 460 mL of water, purify by preparative reverse-phase high performance liquid chromatography, concentrate the collected solution by rotary evaporation, and then freeze-dry for 3 h to obtain pure intermediate A;

[0051] S3: Disperse 43 g of pure intermediate A in 830 mL of water, add 24 g of nanosilver, and stir at room temperature for 9 h to obtain intermediate B, wherein the particle size of the nanosilver is 22 nm;

[0052] S4: Add 48 g of polypeptide II to 510 mL of intermediate B, incubate at 36°C with an oscillation speed of 210 rpm for 10.5 h under light-protected conditions, then centrifuge at 9200 rpm for 31 min, wash twice with water, and freeze-dry to obtain a polypeptide composition with antibacterial activity, wherein polypeptide II is a mixture of 36 g L-GW2 and 12 g D-GW2, and the amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp.

[0053] Example 4: A polypeptide composition having antibacterial activity, comprising the following steps:

[0054] S1: Add 35g of polypeptide I to a reaction bottle, add 400mL of organic solvent to swell for 15min, add 10g of modified polyethylene glycol, mix and stir for 15min, then add 20g of 1-hydroxybenzotriazole, 20mL of 1,3-diisopropylcarbodiimide and 10g of 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3h, add 400mL of L-reagent, stir and react at room temperature for 3h, filter and wash 3 times, rotary evaporate the filtrate, add 380mL of ether to filter, centrifuge the precipitate, and vacuum dry for 3h to obtain a crude intermediate A, in which the chain ends of the modified polyethylene glycol on both sides are are thiol and carboxyl, respectively, the number average molecular weight of the modified polyethylene glycol is 4000 g / mol, polypeptide I is a mixture of 25 g L-ZY13 and 10 g D-ZY13, wherein the amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val, the organic solvent is a mixture of DMF and DCM in a volume ratio of 9:1, and reagent P is a mixture of trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 80-85:5:5:5:2-5;

[0055] S2: Disperse 45 g of crude intermediate A in 450 mL of water, purify by preparative reverse-phase high performance liquid chromatography, concentrate the collected solution by rotary evaporation, and then freeze-dry for 3 h to obtain pure intermediate A;

[0056] S3: Disperse 40 g of pure intermediate A in 800 mL of water, add 22 g of nanosilver, and react at room temperature with stirring for 8 h to obtain intermediate B, wherein the particle size of the nanosilver is 25 nm;

[0057] S4: Add 45 g of polypeptide II to 500 mL of intermediate B, incubate at 35°C with an oscillation speed of 200 rpm for 10 h under light-protected conditions, then centrifuge at 9000 rpm for 30 min, then wash twice with water, and freeze-dry to obtain a polypeptide composition with antibacterial activity, wherein polypeptide II is a mixture of 35 g L-GW2 and 10 g D-GW2, and the amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp.

[0058] Comparative Example 1:

[0059] Compared with Example 1, this comparative example did not add D-ZY13 during the preparation of the polypeptide composition, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a polypeptide composition with antibacterial activity was obtained.

[0060] Comparative Example 2:

[0061] Compared with Example 1, this comparative example did not add D-GW2 during the preparation of the polypeptide composition, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a polypeptide composition with antibacterial activity was obtained.

[0062] Comparative Example 3:

[0063] Compared with Example 1, D-ZY13 and D-GW2 were not added during the preparation of the polypeptide composition in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a polypeptide composition with antibacterial activity was obtained.

[0064] Comparative Example 4:

[0065] Compared with Example 1, this comparative example did not add modified polyethylene glycol during the preparation of the polypeptide composition, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a polypeptide composition with antibacterial activity was obtained.

[0066] Comparative Example 5:

[0067] Compared with Example 1, nanosilver was not added during the preparation of the polypeptide composition in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a polypeptide composition with antibacterial activity was obtained.

[0068] Performance Test:

[0069] Antimicrobial activity test:

[0070] The polypeptide compositions prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were dissolved in PBS to prepare a polypeptide composition with a concentration of 10 μmol / L, and then the bacterial solution activated to the logarithmic phase was diluted with broth medium to an OD of 600 The concentration of the peptide composition is 0.1, wherein the bacteria in the bacterial solution are Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus respectively. After being mixed with the polypeptide composition in equal proportions, the antibacterial concentration of the polypeptide is detected in a 96-well plate. A positive control group without adding polypeptide and a blank control group without adding bacteria are set up. Each gradient has 3 replicate wells. The 96-well plate is placed in a 37°C incubator for static culture for 18-24 hours. The optical density value at 600 m, i.e., the OD value, is read using an enzyme reader to determine its minimum inhibitory concentration MIC.

[0071] Hemolysis test:

[0072] Take out the fresh sheep blood stored in the refrigerator at 4°C, draw 1 mL into a centrifuge tube, centrifuge for 5 minutes, pour off the upper plasma, white blood cells and platelets, then wash the red blood cells with PBS solution for 5 times, finally add 10 mL PBS to obtain pure red blood cells with a concentration of 10%, then dilute to 4% with PBS, dissolve the polypeptide composition prepared in Example 1-Example 4 and Comparative Example 1-Comparative Example 5 with PBS, and dilute the polypeptide composition with PBS to a concentration of 10 μmol / L, and mix it with 4% red blood cells in an equal proportion in a 1.5 mL centrifuge tube, place it in a constant temperature culture at 37°C for 4 hours, then centrifuge, add the supernatant to a 96-well plate, and measure its OD by an enzyme marker 540 , determine its hemolytic activity, use PBS and 0.1% TritonX-100 red blood cells as blank and positive controls, and calculate the hemolysis percentage using the following method:

[0073] Hemolysis rate (%) = (A-A0 / A t -A0)×100%

[0074] Wherein, A represents the absorption rate of the red blood cell supernatant with the polypeptide composition, A0 is the blank control, and A t It is a positive control.

[0075] Serum stability test:

[0076] 500 μL of serum was added to 2 mL of 1 mg / mL solution of the polypeptide composition prepared in Example 1 to Example 4 and Comparative Example 1 to Comparative Example 5, and placed in a constant temperature shaker at 37° C. After 5 hours, 100 μL of the reaction solution was taken out, and 200 μL of anhydrous ethanol was added to precipitate the protein in the serum. The mixed solution was allowed to stand at low temperature for 10 minutes and then centrifuged. The percentage of the concentration of the polypeptide composition prepared in Example 1 to Example 4 and Comparative Example 1 to Comparative Example 5 that was not decomposed in the serum after 5 hours was tested by high performance liquid chromatography.

[0077] Table 1 Summary of experimental data in Examples 1-4 and Comparative Examples 1-5

[0078]

[0079] Data Analysis:

[0080] As can be seen from Table 1, the polypeptide composition with antibacterial activity prepared by the present invention has better antibacterial performance while taking into account stability. The polypeptide composition prepared by the present invention is a spherical particle with polypeptide I as the core, and the outer layer sequentially wraps modified polyethylene glycol, nanosilver and polypeptide II. Polypeptide I is a mixture of L-ZY13 and D-ZY13, wherein D-ZY13 has better antibacterial performance than L-ZY13, and polypeptide II is a mixture of L-GW2 and D-GW2. Natural polypeptides are generally L-type. Due to the configuration selectivity factor, D-GW2 is not easily specifically recognized by proteases, so D-GW2 has stronger stability. The nonspecific electrostatic interaction between the polypeptide provided by the present invention and nanosilver promotes the combination with the bacterial membrane and destroys the bacterial membrane, thereby increasing the antibacterial property; the outer layer polypeptide may be degraded by proteases or interact with the bacterial membrane to expose the polypeptide wrapped in the inner layer of modified polyethylene glycol, and compared with the polypeptide not modified with modified polyethylene glycol, the proteolytic stability of the polypeptide is significantly improved. Such a spherical structure enables the prepared polypeptide composition with antibacterial activity to have excellent antibacterial properties while also improving stability.

[0081] In Comparative Example 1, since D-ZY13 was not added, it can be seen from Table 1 that the antibacterial performance decreased. This may be because the structure of D-ZY13 itself enables it to specifically interact with the bacterial cell wall, which can improve its antibacterial performance, while the stability was not greatly affected. This may be because there is no significant difference between D-ZY13 and L-ZY13 in serum stability and hemolytic activity. In Comparative Example 2, since D-GW2 was not added, it can be seen from Table 1 that the serum stability decreased. This may be because most proteases can only recognize L-type polypeptides, and proteases cannot recognize D-GW2 and thus cannot destroy The polypeptide has good stability; in Comparative Example 3, since no D-type polypeptide is added, it can be seen from Table 1 that the antibacterial performance and serum stability are both reduced. This may be because the spherical structure of the polypeptide composition, the outer polypeptide lacks D-GW2, which makes it easy for the protease to decompose L-GW2, aggravating the decomposition of the spherical structure, and the inner polypeptide lacks L-ZY13, which reduces the antibacterial performance. The two reduce the antibacterial performance and stability of the polypeptide composition; in Comparative Example 4, since no modified polyethylene glycol is added, it can be seen from Table 1 that the serum stability is significantly reduced, while the antibacterial performance does not change significantly. This It may be because of the lack of modified polyethylene glycol. There is no thiol group of modified polyethylene glycol to react with silver, and the carboxyl group to react with the polypeptide, so that the polypeptide composition becomes a spherical structure of polypeptide I and polypeptide II wrapped with nanosilver. This spherical structure exposes the polypeptide to the outside, which is easily hydrolyzed by protease, reducing its stability. The outer layer of nanosilver wraps all the polypeptides, so that it can interact with the bacterial membrane faster to destroy the bacterial membrane, which does not significantly change its antibacterial performance. At the same time, the larger the number average molecular weight of modified polyethylene glycol, the stronger its stability in serum, but the weaker its antibacterial performance; Comparative Example 5 due to Without the addition of nanosilver, it can be seen from Table 1 that the antibacterial performance is significantly reduced, while the stability in serum has no obvious change. This may be because the lack of nanosilver, the electrostatic attraction between the negatively charged microbial cell membrane and the positively charged or less negatively charged nanosilver, thereby destroying the integrity of the cells and losing the antibacterial effect of the nanosilver itself. The polypeptide composition becomes a spherical particle of two polypeptides wrapped in modified polyethylene glycol. This structure makes it impossible for the polypeptide to play an antibacterial role in time, resulting in a decrease in antibacterial performance, while the modified polyethylene glycol wrapped in the outer layer slightly increases its stability in serum.

[0082] It should be understood by those skilled in the art that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0083] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide composition having antibacterial activity, characterized in that: The polypeptide composition is a spherical particle with polypeptide I as the core and modified polyethylene glycol, nanosilver and polypeptide II wrapped in the outer layer in sequence; The polypeptide I is obtained by mixing L-ZY13 and D-ZY13; The two chain ends of the modified polyethylene glycol are respectively a thiol group and a carboxyl group; The polypeptide II is obtained by mixing L-GW2 and D-GW2.

2. The polypeptide composition with antibacterial activity according to claim 1, characterized in that: The amino acid sequence of ZY13 is Val-Lys-Arg-Trp-Lys-Lys-Trp-Arg-Trp-Lys-Trp-Lys-Lys-Trp-Val; The mass ratio of L-ZY13 to D-ZY13 is 25-30:10-12.

3. The polypeptide composition with antibacterial activity according to claim 1, characterized in that: The amino acid sequence of GW2 is Gly-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Ile-Trp-Lys-Lys-Trp; The mass ratio of L-GW2 to D-GW2 is 35-40:10-15.

4. The polypeptide composition with antibacterial activity according to claim 1, characterized in that: The particle size of the nano silver is 15-25 nm.

5. The polypeptide composition with antibacterial activity according to claim 1, characterized in that: The number average molecular weight of the modified polyethylene glycol is 3500-4000 g / mol.

6. The polypeptide composition with antibacterial activity according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step A1. Add polypeptide I to a reaction bottle, add an organic solvent to swell for 15-20 minutes, add modified polyethylene glycol, mix and stir for 15-20 minutes, then add 1-hydroxybenzotriazole, 1,3-diisopropylcarbodiimide and 4-dimethylaminopyridine, stir and react at room temperature under nitrogen protection for 3 days, filter and wash 3 times, vacuum dry for 3 hours, then add reagent P, stir and react at room temperature for 3-5 hours, filter and wash 3 times, rotary evaporate the filtrate, add ether to filter, centrifuge the precipitate, and vacuum dry for 3 hours to obtain a crude intermediate A; Step A2. The crude intermediate A was dispersed in water and purified by preparative reverse-phase high performance liquid chromatography. The collected solution was concentrated by rotary evaporation and then freeze-dried for 3 hours to obtain pure intermediate A. Step A3. Disperse the pure intermediate A in water, add nanosilver, and stir the reaction at room temperature for 8-10 hours to obtain intermediate B; Step A4. Add polypeptide II to intermediate B, incubate at 35-40°C with an oscillation speed of 200-250 rpm for 10-12 hours under light-proof conditions, then centrifuge at 9000-10000 rpm for 30-35 minutes, then wash twice with water, freeze-dry, and obtain a polypeptide composition with antibacterial activity.

7. The polypeptide composition with antibacterial activity according to claim 6, characterized in that: In step A1, the amount ratio of polypeptide I, organic solvent, modified polyethylene glycol, 1-hydroxybenzotriazole, 1,3-diisopropylcarbodiimide, 4-dimethylaminopyridine, reagent P and ether is 35-42 g: 400-420 mL: 10-12 g: 20-25 g: 20-25 mL: 10-12 g: 400-420 mL: 380-400 mL; The organic solvent in step A1 is a mixture of DMF and DCM in a volume ratio of 9:1; The P reagent in step A1 is prepared by mixing trifluoroacetic acid, water, phenol, thioanisole and 1,2-dimercaptoethylene glycol in a volume ratio of 80-85:5:5:5:2-5.

8. The polypeptide composition with antibacterial activity according to claim 6, characterized in that: The usage ratio of the crude intermediate A and water in step A2 is 45-50 g: 450-500 mL.

9. The polypeptide composition with antibacterial activity according to claim 6, characterized in that: The usage ratio of the pure intermediate A, water and nanosilver in step A3 is 40-45 g: 800-850 mL: 22-28 g.

10. The polypeptide composition with antibacterial activity according to claim 6, characterized in that: The usage ratio of polypeptide II and intermediate B in step A4 is 45-55 g: 500-550 mL.

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