Preparation method and application of a pH-responsive Cecropin B antibacterial peptide

Abstract

The present invention discloses a preparation method and application of a pH-responsive Cecropin B antimicrobial polypeptide. The preparation method comprises the following steps: S1. reacting 2,3-dimethylmaleic anhydride and 1-hydroxybenzotriazole as raw materials in the presence of an acid-binding agent and a catalyst to obtain a ring-opening condensation product; and S2. grafting the ring-opening condensation product with cecropin B as raw materials, followed by purification and dialysis to obtain the pH-responsive Cecropin B antimicrobial polypeptide. The pH-responsive Cecropin B antimicrobial polypeptide can release the Cecropin B antimicrobial polypeptide at a specific point after long circulation, has high stability, and does not produce drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial technology, and in particular to a preparation method and application of a pH-responsive Cecropin B antibacterial polypeptide. Background Art

[0002] Haemophilus parasuis (HPS), also known as Glassella parasuis, belongs to the genus Haemophilus in the family Pasteurellaceae. It has 15 serotypes, of which serotypes 4 and 5 are the most prevalent in my country and are common in the upper respiratory tract of pigs. HPS is an opportunistic pathogen that can cause complications or secondary infections in porcine diseases such as blue ear disease, pseudorabies, parvovirus, circovirus, and classical swine fever, complicating epidemics and increasing economic losses. HPS can infect pigs of all ages, but clinically, it primarily affects pigs around weaning and during the nursery period, particularly piglets 4 to 8 weeks of age. Peak infection occurs in piglets 4 to 6 weeks of age during the nursery period, with an incidence rate generally ranging from 10% to 15%, and a mortality rate of 50% to 90%. With the global prevalence of HPS, it has caused significant harm and economic losses to the pig industry. Antimicrobial drugs such as sulfonamides, aminoglycosides, and macrolides are currently the mainstay of treatment for HPS. In recent years, due to the incomplete use of antimicrobial drugs, drug resistance and multidrug resistance of Haemophilus parasuis have been increasing year by year. A large number of basic and clinical studies have shown that most antimicrobial drugs are no longer effective in treating Haemophilus parasuis. Since the discovery and isolation of HPS resistant to sulfonamides in Switzerland and Denmark, we conducted resistance monitoring and analysis on 448 strains of HPS from 16 pig farms in various provinces in China from 2013 to 2017. We found that the resistance rates of Haemophilus parasuis to eight types of antibiotics were as follows: aminoglycoside antibiotics (21.0%-55.6%), macrolide antibiotics (26.6%-57.2%), lincomycin antibiotics (31.9%-86.7%), tetracycline antibiotics (9.0%-32.5%), polymyxin antibiotics (3.4%-54.4%), sulfonamide antibiotics (62.1%-88.0%), β-lactam antibiotics (8.4%-23.9%) and quinolone antibiotics (21.0%-48.9%). HPS isolates from Spain and the UK showed resistance to β-lactams, including cefquinome and ceftiofur, at a rate of 10%. HPS isolates from Germany showed significant resistance to aminoglycosides, such as streptomycin, with resistance rates reaching 60%. However, European isolates showed lower resistance to fluoroquinolones, with resistance to mabofloxacin at only 1.4%. This suggests that HPS resistance rates vary significantly by region, country, and drug dosage.

[0003] Antimicrobial peptides are small, biologically active peptides produced in organisms. Most of these active peptides possess strong alkalinity, thermal stability, and broad-spectrum antibacterial and antiviral properties. Their molecular weight ranges from 2,000 to 7,000, and they are composed of 20 to 60 amino acid residues. Thousands of antimicrobial peptides have been identified from microorganisms, plants, insects, arthropods, amphibians, mammals, and even humans. These peptides differ from traditional peptide antibiotics produced by microorganisms (including bacteria, fungi, and Streptomyces) in terms of their synthesis mechanism, amino acid composition, and mechanism of action. As pathogenic microorganisms become increasingly resistant to traditional antimicrobial drugs, antimicrobial peptides hold broad application prospects in veterinary clinical practice as candidate drugs and feed additives.

[0004] Cecropins are a class of cationic antimicrobial peptides discovered from the silkworm moth, Cecropin A, Cecropin B, Cecropin C, Cecropin D, Cecropin E, and Cecropin P1. Cecropin B exhibits the strongest antimicrobial activity. It consists of 35 amino acid residues and two highly folded α-helices separated by a hinge region and amidated at the C-terminus. Based on its structure, Cecropin B derivatives include Cecropin B1, Cecropin B2, and Cecropin B3. Studies have shown that these derivatives not only exhibit strong antimicrobial activity but also have anti-cancer effects.

[0005] Research on the bactericidal mechanism of Cecropin B has primarily focused on altering cell membrane permeability. Chen et al. observed, using transmission electron microscopy, that the cell membrane of Escherichia coli significantly swelled after exposure to Cecropin B. Immunogold staining did not reveal any cytoplasmic extrusion from the cell membrane, indicating that Cecropins primarily cause bacterial death by disrupting the cell membrane. As the antimicrobial activity of Cecropin B has gained increasing recognition, research on bacterial tolerance to it has also become increasingly important. Aquatic bacterial pathogens such as Vibrio anguillarum and Yersinia ruckeri can be induced to develop tolerance to Cecropin B. Studies have shown that the minimum inhibitory concentration (MIC) of Cecropin B against porcine Gram-negative bacteria such as HPS, Pasteurella multocida, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella choleraesuis, and Bordetella borreliosis ranges from 0.5 to 16 μg / mL, demonstrating significant antibacterial activity comparable to that of other α-helical antimicrobial peptides, moricin, or penicillin.

[0006] Although antimicrobial peptides have the advantage of low drug resistance as an alternative to antibiotics, HPS can still mutate from sensitive strains to resistant strains under the conditions of continuous induction of sub-MIC concentrations of CecropinB. In addition, peptide drugs are unstable and easily hydrolyzed, have low bioavailability in the body, and are easily metabolized. Summary of the Invention

[0007] In view of this, the present application provides a preparation method and application of a pH-responsive Cecropin B antimicrobial polypeptide, which can release Cecropin B antimicrobial polypeptide at a specific site after long circulation, has high stability and does not produce drug resistance.

[0008] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a method for preparing a pH-responsive Cecropin B antibacterial polypeptide, comprising the following steps:

[0010] S1. In the presence of an acid binding agent and a catalyst, 2,3-dimethylmaleic anhydride and 1-hydroxybenzotriazole are reacted to obtain a ring-opening condensation product;

[0011] S2. The ring-opening condensation product and cecropin B are used as raw materials for grafting reaction, followed by purification and dialysis to obtain pH-responsive cecropin B antibacterial polypeptide.

[0012] Preferably, the molar ratio of 2,3-dimethylmaleic anhydride to cecropin B is 1-20:1.

[0013] Preferably, the molar ratio of the catalyst to 2,3-dimethylmaleic anhydride is 0.8-1.5:1.

[0014] Preferably, in step S1, the pH value of the reaction is 7-10.

[0015] Preferably, in step S2, the dialysate used for dialysis is a phosphate buffer solution with a pH value of 7.4-9.

[0016] Preferably, in step S2, purification sequentially comprises the steps of reprecipitation and centrifugation to remove the supernatant.

[0017] Preferably, the acid binding agent is one or more of triethylamine and pyridine.

[0018] Preferably, the catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine.

[0019] In a second aspect, the present application provides a pH-responsive Cecropin B antibacterial polypeptide.

[0020] In a third aspect, the present application provides an application of a pH-responsive Cecropin B antimicrobial polypeptide in combating Haemophilus parasuis. The application pH range of the pH-responsive Cecropin B antimicrobial polypeptide is 5-7.4.

[0021] The beneficial effects of the present application are as follows: the present invention adopts 2,3-dimethylmaleic anhydride to be grafted onto the side chain active amino group of Cecropin B polypeptide through a pH-sensitive amide bond, thereby changing the charge properties of the original polypeptide, enhancing its ability to escape the monitoring of the reticuloendothelial system, and giving it the ability of long circulation and targeted release, making it a highly potential new dosage form of responsive antibacterial polypeptide that does not produce drug resistance; the present invention uses pH-sensitive bonds to construct Cecropin B nanoparticles, which can accurately target and treat drug-resistant Haemophilus parasuis infections only when the pH is 5-7.4, have high stability and are not easily hydrolyzed, providing new ideas for the treatment of drug-resistant bacterial infections and providing new dosage forms for veterinary clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the molecular structural formula of Cecropin B;

[0023] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum (1HNMR) spectrum of pH-responsive Cecropin B antibacterial peptide (CBD);

[0024] Figure 3 The in vitro antibacterial activity of pH-responsive Cecropin B antibacterial peptide (CBD) is shown;

[0025] Figure 4 The cytotoxicity graph of pH-responsive Cecropin B antimicrobial peptide (CBD);

[0026] Figure 5 This is the pH-responsive in vitro release diagram of pH-responsive Cecropin B antibacterial peptide (CBD). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Antimicrobial peptides have attracted widespread attention in the pharmaceutical field due to their low active concentration, good biocompatibility, and environmental friendliness. However, their low circulation period, high degradability, and high production costs hinder their application in the treatment of drug-resistant bacteria. To address these challenges, peptide modification can be used to improve bioavailability. This strategy can be used to precisely target infections with drug-resistant strains or those with high MICs, thereby achieving the goal of treating drug-resistant bacterial infections.

[0029] As an emerging field of nanomedicine, there are many types of smart responsive nanocarriers, including pH-responsive, redox-responsive, and temperature-responsive carriers. These smart responsive polymers, as drug release systems, can react to certain environmental information at the lesion site and undergo corresponding changes. Since the controlled release of anticancer drugs from nanocarriers is very important in cancer treatment, and considering the weakly acidic environment of lysosomes and endosomes (pH 5-6), which is lower than that of blood (pH 7.4), pH-sensitive polymers can achieve controlled release of encapsulated drugs within cells. There are two main ways to use pH-sensitive polymers as carriers. One is to design them with pH-responsive chemical bonds such as acetal and hydrazone bonds. Under the weakly acidic conditions of lysosomes and endosomes, the pH-responsive chemical bonds of the micelles break, releasing the drug molecules, thereby achieving the therapeutic purpose. The other is to achieve pH responsiveness through the protonation and deprotonation of acidic or basic groups contained in the polymer, thereby achieving drug release.

[0030] Based on this, the present invention was created.

[0031] To compensate for the shortcoming of Cecropin B antimicrobial peptide that cannot achieve targeted release after long circulation, a preparation method for a new dosage form of self-assembled negative ion nanoparticles formed by bonding small molecules with pH-sensitive bonds was developed, thereby obtaining a responsive medicinal antimicrobial peptide with long circulation in the body, escape from reticuloendothelial system monitoring, and targeted targeting. This preparation method can be used to more rationally use antimicrobial peptides as a tool for drug resistance control, and at the same time provide certain thinking directions for the delivery and dosage form development of other antimicrobial peptides.

[0032] The present application provides a method for preparing a pH-responsive Cecropin B antibacterial polypeptide, comprising the following steps:

[0033] S1. In the presence of an acid-binding agent and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 2,3-dimethylmaleic anhydride and 1-hydroxybenzotriazole are reacted to obtain a ring-opening condensation product having the structural formula shown in Formula A, wherein R1 and R2 represent side chain groups of EDCl, R1 is (CH3)2-N-(CH2)3-, and R2 is -CH2-CH3. The reaction process is as follows:

[0034]

[0035] S2. The ring-opening condensation product was reacted with Cecropin B (CAS No. 203265-23-0, molecular formula Figure 1 As shown in FIG, a pH-responsive Cecropin B antimicrobial polypeptide is obtained by grafting the obtained polypeptide with the following formula:

[0036]

[0037] In terms of the selection of conditions in this scheme, the molar ratio of 2,3-dimethylmaleic anhydride to cecropin B is 1-20:1, and the molar ratio of the catalyst to 2,3-dimethylmaleic anhydride is 0.8-1.5:1. Within this range, 2,3-dimethylmaleic anhydride can be successfully grafted onto the side chain amino group of cecropin B; the solvents for the above reactions are all DMSO, and the acid-binding agent is one or more of triethylamine and pyridine. By adjusting the amount of the acid-binding agent, the pH value of the reaction in step S1 is controlled to 7-10 for the ring-opening of 2,3-dimethylmaleic anhydride; preferably, step S2 further includes adding a dehydrating agent, such as calcium chloride, to increase the reaction yield.

[0038] Specifically, in step S2, after the grafting reaction is completed, the product is purified and dialyzed. The purification sequentially includes the steps of reprecipitation and centrifugation to remove the supernatant. The specific steps are as follows:

[0039] S21. Dichloromethane and petroleum ether were added to the mixture of the grafting reaction product for reprecipitation, and a flocculent precipitate or the solution became turbid and then centrifuged;

[0040] S22. The supernatant of the product after centrifugation was removed, and the precipitate was dried using a rotary evaporator at a temperature of 30-40 ° C to dry the volatile solvent;

[0041] S23. Dissolve the precipitate obtained in S22 with a phosphate buffer having a pH of 7.4-9, and dialyze it with 100-300 times the volume of the buffer at room temperature for 1-7 days.

[0042] It is worth noting that in step S2, the dialysate used for dialysis is a phosphate buffer with a pH value of 7.4-9. An inappropriate pH value will cause the ligand on the polypeptide to be removed prematurely, causing the new drug to revert to the original drug. The pH value in this range can ensure a high product yield and a high grafting rate.

[0043] The following is an example of an acid-binding agent being triethylamine to illustrate the preparation mechanism of this scheme:

[0044] In dimethyl sulfoxide, an appropriate amount of triethylamine acts as an acid-binding agent to ring-open dimethylmaleic anhydride, and EDCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide) acts as a catalyst to connect to the carboxyl group after the ring opening. After the addition of HoBt (1-hydroxybenzotriazole), EDCl is replaced, making the amide condensation reaction conditions in step S2 milder. The side chain amino group of the lysine residue of cecropin B is more likely to attack the carboxyl end. At the same time, the steric hindrance of HoBt makes it difficult for the polypeptide to racemize, maintaining the conformation, thereby obtaining a pH-responsive cecropin B antibacterial polypeptide.

[0045] The present application provides a pH-responsive Cecropin B antibacterial polypeptide.

[0046] The present application provides an application of a pH-responsive Cecropin B antibacterial polypeptide in combating Haemophilus parasuis.

[0047] The present application will be further described below through specific examples.

[0048] Example 1

[0049] A method for preparing a pH-responsive Cecropin B antibacterial polypeptide comprises the following steps:

[0050] S1. 2,3-dimethylmaleic anhydride and triethylamine were dissolved in DMSO and stirred at room temperature to obtain a first solution; a DMSO solution of 1-hydroxybenzotriazole and a DMSO solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were mixed and then injected into the first solution, the pH value was adjusted to 8 using triethylamine, and the reaction was stirred at room temperature for 30 minutes to obtain a ring-opening condensation product;

[0051] S2. Inject the DMSO solution of Cecropin B polypeptide into the ring-opening condensation product obtained in S1, add anhydrous CaCl2, and stir in a water bath at 37°C for 24 hours to obtain a mixture;

[0052] S21. Add dichloromethane and petroleum ether to the mixture for reprecipitation. After thorough vortexing, let it stand at room temperature until flocculent precipitate appears or the solution becomes turbid.

[0053] S22. The product of S21 was placed in a high-speed refrigerated centrifuge, centrifuged, the supernatant was removed, and the precipitate was dried using a rotary evaporator at a temperature of 30-40 ° C to dry the volatile solvent;

[0054] S23. Dissolve the precipitate in step S22 with a phosphate buffer having a pH of 8, and dialyze it with 100-300 times the volume of the buffer at room temperature for 2 days, and then freeze-dry to obtain a pH-responsive Cecropin B antimicrobial polypeptide (CBD).

[0055] The molar ratio of 2,3-dimethylmaleic anhydride to cecropin B is 10:1, and the molar ratio of the catalyst to 2,3-dimethylmaleic anhydride is 1:1.

[0056] The product of this scheme was identified, and the results were as follows Figure 2 As shown, there are corresponding nuclear magnetic resonance hydrogen species peaks of CB (cecropin B) and DA (2,3-dimethylmaleic anhydride) on CBD, indicating that the binding is successful.

[0057] Example 2

[0058] A method for preparing a pH-responsive Cecropin B antibacterial polypeptide comprises the following steps:

[0059] S1. 2,3-dimethylmaleic anhydride and triethylamine were dissolved in DMSO and stirred at room temperature to obtain a first solution; a DMSO solution of 1-hydroxybenzotriazole and a DMSO solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were mixed and then injected into the first solution, the pH value was adjusted to 10 using triethylamine, and the reaction was stirred at room temperature for 30 minutes to obtain a ring-opening condensation product;

[0060] S2. Inject the DMSO solution of Cecropin B polypeptide into the ring-opening condensation product obtained in S1, and stir in a water bath at 37°C for 24 h to obtain a mixture;

[0061] S21. Add dichloromethane and petroleum ether to the mixture for reprecipitation. After thorough vortexing, let it stand at room temperature until flocculent precipitate appears or the solution becomes turbid.

[0062] S22. The product of S21 was placed in a high-speed refrigerated centrifuge, centrifuged, the supernatant was removed, and the precipitate was dried using a rotary evaporator at a temperature of 30-40 ° C to dry the volatile solvent;

[0063] S23. Dissolve the precipitate in step S22 with phosphate buffer at pH 7.4, and dialyze it with 100-300 times the volume of the buffer at room temperature for 2 days, and then freeze-dry to obtain pH-responsive Cecropin B antibacterial polypeptide.

[0064] The molar ratio of 2,3-dimethylmaleic anhydride to cecropin B is 1:1, and the molar ratio of the catalyst to 2,3-dimethylmaleic anhydride is 0.8:1.

[0065] Example 3

[0066] A method for preparing a pH-responsive Cecropin B antibacterial polypeptide, wherein the other steps are the same as those in Example 1, except that the molar ratio of 2,3-dimethylmaleic anhydride to cecropin B is 20:1, the molar ratio of the catalyst to 2,3-dimethylmaleic anhydride is 1.5:1, and the pH value in step S23 is 9.

[0067] Evaluation and Testing

[0068] The pH-responsive Cecropin B antimicrobial polypeptide (CBD) prepared in Example 1 was used as the research object to study the antimicrobial activity, cytotoxicity test and pH response performance against Haemophilus parasuis. The specific test methods are as follows:

[0069] Antibacterial activity: After resuscitation, Haemophilus parasuis (HPS) was subcultured twice in TSA medium. Single colonies were picked and cultured in TSB broth with shaking for 16 to 24 hours until the logarithmic growth phase. The bacterial solution was diluted with TSB broth to a McFarland turbidity of 0.5 using the McFarland turbidimetric method. The bacterial solution concentration at this time was approximately 1×108 CFU / mL. The solution was then diluted 10-fold with TSB broth to a bacterial concentration of 1×10 7 CFU / mL. Cecropin B, CBD, and 2,3-dimethylmaleic anhydride (DA) were diluted serially with TSB broth to 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 ppm, then incubated with bacteria. The cells were cultured in a 37°C, 5% CO2 incubator. The results were observed after about 24 hours and the data were recorded. Figure 3 shown.

[0070] CCk-8 assay: RAW264.7 cells in the logarithmic phase were collected and suspended in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. 4 The cells were seeded at a density of 100 μg / mL in a 96-well culture plate, 200 μL of cell suspension was added to each well (the edge wells were filled with sterile PBS), and the cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. The cell culture medium in the culture plate was discarded, and 200 μL of DMEM basal culture medium containing 128, 32, 8, and 2 μg / mL of CBD and Cecropin B was added to each well. Each concentration was repeated 3 times. At the same time, a blank group (containing DMEM culture medium, CCk-8) and a control group (containing cells, DMEM culture medium, and CCk-8) were set up. After incubation in a 37°C, 5% CO2 cell culture incubator for 24 hours, a 10% mass fraction CCk-8 solution was added to each well and cultured for 1 hour. The cells were placed on a horizontal shaker and shaken at low speed for 10 minutes to fully dissolve the crystals. The absorbance (OD) value of each well was measured at 450 nm using an enzyme-linked immunosorbent assay. Three parallel sets were set up and the average value was taken. The cell viability was calculated according to the following formula:

[0071] Cell survival rate (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The results are as follows Figure 4 shown.

[0072] pH response performance study:

[0073] Using phosphate buffer as the solvent, UV-visible spectrophotometry was performed on the ring-opened 2,3-dimethylmaleic anhydride at a full wavelength scan from 190 to 900 nm. The maximum wavelength was 205.3 nm, indicating minimal interference at this wavelength. Using phosphate buffer as a blank control, corresponding concentrations of ring-opened 2,3-dimethylmaleic anhydride were dissolved in a release matrix and diluted to concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 ppm, respectively. Real-time absorbance was measured at 205 nm by HPLC, and a linear regression was performed using peak area as the ordinate and mass concentration as the abscissa to plot a standard curve.

[0074] A 1000MW dialysis bag containing 2mL of buffer was completely immersed in 38mL of phosphate buffer at pH 5, 6, and 7, respectively. CBD was added at a concentration of 1000ppm in the buffer as Cecropin B, and the mixture was shaken at a constant temperature of 37°C. 1mL of sample was taken at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 36h, and 1mL of liquid was added to the release medium. The dialysate was centrifuged, filtered, and the real-time absorbance was measured by high-performance liquid chromatography. The integrated peak area was used to calculate the concentration of the standard curve to obtain the cumulative release percentage, and a release curve was plotted as shown in the following figure: Figure 5 shown.

[0075] According to the test results, Figure 3 The results showed that for the same bacterial species, CBD and CB showed similar minimum inhibitory concentrations, while DA had no inhibitory effect and did not interfere with the results. Figure 4 For cell survival test, verify the toxicity of drugs to cells. Figure 3 Compared with the experimental results of , when the concentration is more than ten times the minimum inhibitory concentration, the cell survival rate is still greater than 80%, which means that the drug toxicity is negligible; Figure 5 To study pH responsiveness and release efficiency, the cumulative release rate was calculated by the dialysis bag method, and it was found that as the pH continued to decrease, the drug release rate accelerated.

[0076] In summary, the pH-responsive Cecropin B antibacterial peptide of this scheme can act on most Gram-negative bacteria, has a fast effect, does not react with bacterial life activities, and basically cannot produce drug resistance.

[0077] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a pH-responsive Cecropin B antimicrobial polypeptide, characterized in that: The following steps are involved: S1. In the presence of an acid binding agent and a catalyst, 2,3-dimethylmaleic anhydride and 1-hydroxybenzotriazole are reacted to obtain a ring-opening condensation product; S2. The ring-opening condensation product and cecropin B are reacted with each other for amide reaction, and then purified and dialyzed to obtain a pH-responsive Cecropin B antimicrobial polypeptide; in step S2, the dialysate for dialysis is a phosphate buffer solution having a pH value of 7.4-9; The ring-opening condensation product structural formula is: 。 2. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: The molar ratio of the 2,3-dimethylmaleic anhydride to the cecropin B is 1-20:

1.

3. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: The molar ratio of the catalyst to the 2,3-dimethylmaleic anhydride is 0.8-1.5:

1.

4. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: In step S1, the pH value of the reaction is 7-10.

5. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: In step S2, purification sequentially includes the steps of reprecipitation and centrifugation to remove the supernatant.

6. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: The acid binding agent is one or more of triethylamine and pyridine.

7. The method for preparing the pH-responsive Cecropin B antimicrobial polypeptide according to claim 1, wherein: The catalyst is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine.

8. A pH-responsive Cecropin B antimicrobial polypeptide prepared by the method according to any one of claims 1 to 7.

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