Antibacterial peptide humAMP43 as well as polynucleotide, expression vector, host cell, preparation method and application thereof

Abstract

The invention belongs to the technical field of peptide antibiotics, and discloses an antibacterial peptide humAMP43 and polynucleotide, an expression vector, a host cell, a preparation method and application thereof, the antibacterial peptide humAMP43 is a polypeptide composed of 12 amino acids, and the amino acid sequence of the antibacterial peptide humAMP43 is shown as SEQ ID NO.1. The invention further discloses a preparation method of the antibacterial peptide humAMP43. The polypeptide is obtained through deep learning model screening and experimental verification, and meanwhile, the invention provides a coding gene, a recombinant expression system and an adaptive solid-phase synthesis method of the polypeptide. The antibacterial peptide has a broad-spectrum efficient inhibition effect on gram-positive bacteria and gram-negative bacteria, and the antibacterial rate can reach 98.6%. The invention further provides application of the antibacterial peptide in preparation of antibacterial agents, antibacterial infection drugs, food preservatives and feed additives, and a new candidate molecule is provided for development and application of the antibacterial peptide in medicine, agriculture and food industries.

Description

Technical Field

[0001] This invention belongs to the field of peptide antibiotic technology, specifically an antimicrobial peptide humAMP43 and its polynucleotides, expression vector, host cell, preparation method and application. Background Technology

[0002] Bacterial contamination of food, plant diseases, and human infections pose significant threats to people's lives and production. Furthermore, the increasing resistance of microorganisms to antimicrobial drugs makes their prevention and control increasingly difficult. Traditional synthetic antimicrobial drugs, with their drug residues and toxic side effects, can no longer meet the needs. Therefore, there is an urgent need to develop safe and efficient antimicrobial agents in the food industry, livestock and poultry farming, pharmaceutical industry, and plant control.

[0003] Antimicrobial peptides, as an important component of the innate immune system, are considered one of the most promising alternatives to traditional antibiotics due to their broad-spectrum antimicrobial activity, rapid onset of action, and low likelihood of inducing bacterial resistance. Antimicrobial peptides typically achieve their bactericidal effect by disrupting the integrity of bacterial cell membranes; this physical mechanism makes it difficult for bacteria to develop resistance through single gene mutations. Therefore, discovering novel antimicrobial peptides with highly efficient antimicrobial activity is of significant practical importance for developing new antimicrobial agents or drugs for combating bacterial infections.

[0004] Currently, the main methods for obtaining antimicrobial peptides include direct extraction from organisms, recombinant expression via genetic engineering, and chemical synthesis. First, direct extraction from nature typically results in low yields and high costs, making it difficult to meet the needs of large-scale applications. Second, recombinant expression methods may face problems such as the expression product easily forming inclusion bodies, complex subsequent refolding processes, and low yields of active peptides. Finally, solid-phase synthesis in chemical synthesis methods, while offering advantages such as process control, ease of modification, and no sequence limitations, suffers from challenges. The amino acid sequence composition of different antimicrobial peptides directly affects coupling efficiency and the purity of the crude peptide product. Therefore, it is necessary to explore specific resin carriers, coupling systems, and cleavage reagents that are compatible with antimicrobial peptides. Inappropriate synthetic strategies may lead to incomplete synthesis of the target peptide chain, severe racemization, or improper side chain protection during cleavage, ultimately resulting in low yields and impaired product activity. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide humAMP43, its polynucleotides, expression vector, host cell, preparation method, and application. By constructing a peptide database for feature analysis and digital encoding, and using the PyTorch deep learning framework to build a model for prediction and screening, an antimicrobial peptide humAMP43 with inhibitory effects on various Gram-positive and Gram-negative bacteria with an inhibition rate of up to 98.6% is obtained. A suitable preparation method is also provided to provide candidate molecules for the development of novel antimicrobial agents.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides an antimicrobial peptide humAMP43, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: MWPLRVYTRKKR.

[0009] In a second aspect, the present invention provides a polynucleotide, wherein the polynucleotide is a polynucleotide H1 encoding the antimicrobial peptide humAMP43 or a polynucleotide H2 complementary to the polynucleotide H1.

[0010] Polynucleotides, including DNA and RNA, can be artificially synthesized using solid-phase synthesis technology.

[0011] Furthermore, the present invention also includes polypeptides or polypeptide analogs and derivatives, such as polynucleotide variants, encoding polypeptides having the same amino acid sequence as those of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, some polynucleotide variants are substitutions of polynucleotides that do not substantially alter the function of the polypeptide they encode.

[0012] Thirdly, the present invention also provides an expression vector containing the aforementioned polynucleotides.

[0013] In this invention, the polynucleotide sequence of the antimicrobial peptide humAMP43 is inserted into a recombinant expression vector using genetic engineering techniques. The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0014] Fourthly, the present invention also provides a host cell containing the above-described expression vector.

[0015] In this invention, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Examples include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, and yeast.

[0016] The preferred host cell for this invention is Bacillus subtilis. When Bacillus subtilis is used as the host cell to express the antimicrobial peptide of this invention, it is not necessary to separate and purify the antimicrobial peptide. The Bacillus subtilis culture broth after induced expression can be directly used as a feed additive.

[0017] Fifthly, the present invention also provides the use of the aforementioned antimicrobial peptide humAMP43 or polynucleotide in the preparation of antimicrobial agents, antibacterial drugs, food preservatives or feed additives.

[0018] As a limitation, the aforementioned antimicrobial agents, antibacterial drugs, food preservatives, or feed additives are used to inhibit the growth of Gram-positive or Gram-negative bacteria;

[0019] The Gram-positive bacteria include Staphylococcus aureus, Bacillus subtilis, and Listeria.

[0020] The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, and Salmonella.

[0021] In a sixth aspect, the present invention also provides an antibacterial agent comprising the antimicrobial peptide humAMP43, which is an injection, ointment, spray or drop.

[0022] Furthermore, the antibacterial agent also includes a drug-acceptable carrier and excipients suitable for the corresponding dosage form. For example, in an injection, it may contain sterile solvents such as physiological saline and phosphate buffer, as well as necessary stabilizers; in an ointment, it may contain a matrix such as petrolatum, lanolin, or hydroxypropyl methylcellulose; and in a spray or drop, it may contain isotonic adjusters, pH buffers, and preservatives.

[0023] In a seventh aspect, the present invention also provides a method for preparing the antimicrobial peptide humAMP43, wherein, in accordance with the amino acid sequence shown in SEQ ID NO.1, amino acid monomers with protecting groups on the amino group are coupled to a solid support by a coupling reaction, the protecting groups of the amino acid monomers are removed to expose the free amino groups, and the amino acid monomers are sequentially coupled by a coupling reaction to synthesize a peptide chain.

[0024] The peptide chain was separated from the solid-phase support and purified to obtain the antimicrobial peptide humAMP43.

[0025] As a further limitation, the protecting group is an Fmoc protecting group;

[0026] The solid support is Rink amide MBHA resin or Wang resin.

[0027] The coupling reaction uses HBTU-HOBt-DIEA combined coupling agent, PyAOP-HOAt-DIEA combined coupling agent, or DIC-Oxyma Pure combined coupling agent.

[0028] Among them, the Fmoc protecting group is a 9-fluorenylmethoxycarbonyl protecting group, which protects the α-amino group in the solid-phase synthesis of peptides and can be gently removed by piperidine under alkaline conditions;

[0029] HBTU is O-benzotriazole-tetramethylurea hexafluorophosphate (CAS: 94790-37-1), HOBt is 1-hydroxybenzotriazole (CAS: 2592-95-2), DIEA is N,N-diisopropylethylamine (CAS: 7087-68-5); PyAOP is (7-azabenzotriazole-1-yl)oxytripyrrolidinylphosphine hexafluorophosphate (CAS: 156311-83-0), HOAt is 1-hydroxy-7-azabenzotriazole (CAS: 39968-33-7), DIC is N,N'-diisopropylcarbodiimide (CAS: 693-13-0), and Oxyma Pure is ethyl cyano(hydroxyimino)acetate (CAS: 3849-21-6).

[0030] As a further limitation, a deprotecting solution is used to remove the protecting group of the amino acid monomer, wherein the deprotecting solution is a piperidine N,N-dimethylformamide solution with a volume concentration of 20%~30%;

[0031] A cleavage fluid is used to separate the peptide chain from the solid support. The cleavage fluid is prepared by mixing trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5.

[0032] Trifluoroacetic acid (TFA), as a strong acid and solvent, is responsible for cleaving the peptide chain-resin linkage and removing the protecting groups of the amino acid side chains; triisopropylsilane (TIS), as a cation scavenger, can effectively capture the active cation intermediates generated during the cleavage process, prevent them from modifying the sensitive amino acid side chains, and thus protect the integrity of the target peptide.

[0033] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0034] (1) The present invention uses a deep learning model to screen and predict the antibacterial activity of more than 5 million peptide data from human proteome. The resulting antimicrobial peptide humAMP43 exhibits broad-spectrum and efficient inhibitory activity against a variety of pathogens, including Gram-positive and Gram-negative bacteria. Experimental verification shows that when the final concentration is 100 μg / mL, the highest inhibition rate can reach 98.6%, which is suitable for developing a new generation of safe and efficient antimicrobial agents.

[0035] (2) Based on the amino acid sequence characteristics of the antimicrobial peptide humAMP43, the present invention optimizes and establishes a solid-phase synthesis process using Rink amide MBHA resin or Wang resin as a carrier. The preparation method of the antimicrobial peptide humAMP43 has high yield, good biological activity and few by-products, providing a material basis for subsequent formulation research, activity evaluation and application in the fields of medicine, agriculture and food.

[0036] (3) This invention covers the polynucleotide sequence, expression vector, host cell and preparation method of antimicrobial peptide humAMP43, forming a complete system from molecular design, synthesis and preparation to application development, which is applicable to the development of new antimicrobial drugs, preservatives or biocontrol agents based on this antimicrobial peptide. Attached Figure Description

[0037] Figure 1 This is a high-performance liquid chromatography (HPLC) image of the antimicrobial peptide humAMP43.

[0038] Figure 2 This is the secondary mass spectrum of the antimicrobial peptide humAMP43.

[0039] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with those documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0043] Example 1: Antimicrobial peptide humAMP43

[0044] This embodiment describes the antimicrobial peptide humAMP43 and its screening method, as detailed below:

[0045] T1. Constructing a peptide database: Human proteome data was obtained from the NCBI database. Using a variety of protease digestion systems, including digestive system proteases, intracellular degradation systems, signal peptidases, and restriction proteolytic enzyme systems, human proteome data were simulated for enzyme digestion to construct a peptide database for predictive analysis. This database contains more than 51,301,260,000 peptide data from the human proteome.

[0046] T2. Characterization of Known Antimicrobial Peptides: Collect known antimicrobial peptide data, digitally encode the physicochemical properties of peptide sequences, including molecular weight, isoelectric point, amino acid composition, and charge; simultaneously perform homology analysis of peptide sequences, construct evolutionarily conserved domain maps, identify evolutionarily conserved amino acid residues and domains, and mine pattern features in the sequences.

[0047] T3. Deep Learning Model Construction and Prediction: A model is built based on the PyTorch deep learning framework. The model is trained using the feature data obtained in S2. Then, the model is applied to the peptide database constructed in T1 to predict antibacterial activity. The corresponding prediction probability and activity score are output for each peptide sequence fragment.

[0048] T4. Candidate Sequence Screening: The predicted results obtained in T3 are sorted and screened according to the activity score to obtain more than 30,000 candidate peptides with potential antibacterial activity.

[0049] T5. Wet Experiment Validation: The candidate peptides obtained in T3 were further screened in batches, and the corresponding peptides were artificially synthesized using a solid-phase synthesis method for antibacterial activity validation experiments. Among them, the peptides humAMP43, humAMP43B, humAMP43C, humAMP43D, humAMP43E, humAMP43F, humAMP43G, humAMP43H, humAMP43I, humAMP43J, humAMP43K, humAMP43L, humAMP43M, humAMP43N, humAMP43O, humAMP43P, humAMP43Q, humAMP43R, humAMP43S, humAMP43T, humAMP43U, humAMP43V, humAMP43W, humAMP43X, humAMP43Y, and humAMP43Z were serially diluted with MHB medium to concentrations of 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.50 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL, 3.91 μg / mL, 1.95 μg / mL, and 1.95 μg / mL, respectively. 0.98 μg / mL, 0.49 μg / mL, and 0.24 μg / mL were placed in culture tubes, labeled with the corresponding polypeptide number and concentration, and designated as concentration tubes. An equal volume of the Staphylococcus aureus test bacterial solution was added to each concentration tube, and the tubes were incubated at 35°C for 12 hours. Bacterial growth was then observed.

[0050] Compared with control tubes containing only bacterial suspension and culture medium, but no peptides, the lowest peptide concentration tube that was completely clear and without turbidity was identified as the minimum inhibitory concentration (MIC). The results are shown in Table 1. Only peptide humAMP43 exhibited antibacterial activity; humAMP43B to humAMP43Z showed no antibacterial activity against Staphylococcus aureus.

[0051] Table 1. Antibacterial activity and MIC results of the peptides against Staphylococcus aureus

[0052]

[0053] Through wet testing, a polypeptide with highly efficient antibacterial activity was finally identified and named antimicrobial peptide humAMP43. It is a polypeptide composed of 12 amino acids, and the amino acid sequence is shown in SEQ ID NO.1.

[0054] SEQ ID NO.1: MWPLRVYTRKKR.

[0055] Example 2: Preparation method of antimicrobial peptide humAMP43

[0056] This embodiment describes a solid-phase synthesis method for the antimicrobial peptide humAMP43, specifically including the following steps performed sequentially:

[0057] S1. Resin swelling and pretreatment: Weigh 0.1 mmol equivalent of Rink Amide MBHA resin into a solid-phase synthesis column, add 10 mL of dichloromethane (DCM) solution, and shake to swell for 30 min at room temperature. After removing the DCM, wash the resin three times with 10 mL of N,N-dimethylformamide (DMF).

[0058] S2. Removal of Fmoc protecting groups: Add 5 mL of DMF solution containing 20% ​​(v / v) piperidine to the reaction column, and shake at room temperature for 15 min to remove the Fmoc protecting groups on the resin. Discard the reaction solution and wash the resin 5 times with 10 mL of DMF.

[0059] S3. Amino acid coupling: Dissolve 0.4 mmol of Fmoc-amino acid monomer, 0.4 mmol of diisopropylcarbodiimide (DIC), and 0.4 mmol of Oxyma Pure in DMF. Activate at room temperature for 3 min, then add the activated solution to the reaction column and mix with the resin. Continue shaking at room temperature for 60 min. After the reaction, drain the coupling solution and wash the resin three times with DMF. After each round of coupling, sample the resin using ninhydrin or bromophenol blue detection to ensure complete reaction; if the test is positive, perform a second coupling.

[0060] S4. Cyclic synthesis: Repeat steps S2 and S3, sequentially linking each Fmoc-protected amino acid monomer according to the amino acid sequence from C-terminus to N-terminus of the antimicrobial peptide humAMP43 shown in SEQ ID NO.1.

[0061] S5. Final Deprotection and Resin Cleavage: After all amino acid sequences were assembled, final Fmoc removal was performed. The resin was washed with DCM and dried under vacuum. A cleavage buffer was prepared by mixing trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water in a volume ratio of 95:2.5:2.5. The dried resin was added to 10 mL of cooled cleavage buffer, and the reaction was carried out with shaking at room temperature for 3 h. After the reaction was completed, the mixture was filtered, and the filtrate (containing crude peptide) was collected and precipitated in cold diethyl ether. The white peptide precipitate was collected by centrifugation at 5000 rpm for 10 min at 4 °C.

[0062] S6. Crude Peptide Washing and Purification: The peptide precipitate was washed three times with cold anhydrous diethyl ether, collected by centrifugation, and vacuum dried to obtain the crude antimicrobial peptide humAMP43. The crude product was dissolved in an acetonitrile / water solution containing 0.1% TFA and purified by semi-preparative reversed-phase high-performance liquid chromatography. Chromatographic conditions were: C 18The column was 4.6 × 250 mm × 5 μm; mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution; the gradient elution program was: 20% B to 50% B, 30 min; 50% B to 100% B, 3 min; 100% B, hold for 5 min; 100% B to 20% B, 2 min; 20% B, hold for 10 min; the detection wavelength was 214 nm. The main peak fraction was collected, and after confirming a purity greater than 90% by analytical HPLC, the fractions were combined and lyophilized to obtain purified antimicrobial peptide humAMP43 white powder. The HPLC chromatogram is shown below. Figure 1 As shown.

[0063] S7. Product Identification: The purified product was dissolved in a 15% (v / v) acetonitrile aqueous solution and identified by electrospray ionization mass spectrometry. The mass spectrometry analysis results are as follows: Figure 2 As shown, the theoretical molecular weight is [M+H]. + The theoretical value was 1634.00 Da, while the measured value was 1634.10 Da, consistent with the theoretical result. The purity of the product was determined using analytical HPLC, and the results showed a purity greater than 90%.

[0064] This embodiment successfully prepared the antimicrobial peptide humAMP43 with high purity.

[0065] In other embodiments, a piperidine N,N-dimethylformamide solution with a volume concentration of 23%, 25%, 28%, or 30% is used as a deprotecting solution to remove the protecting groups of amino acid monomers. A cleaving fluid is prepared by mixing trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5, and is used to cleave and separate the peptide chain from the solid support.

[0066] Experimental Example: Determination of the antibacterial effect of the antimicrobial peptide humAMP43

[0067] The antimicrobial effects of the antimicrobial peptide humAMP43 against Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella were tested, and the minimum inhibitory concentration (MIC) was determined. Details are as follows:

[0068] (1) Experimental method for determining antibacterial rate

[0069] Six indicator bacteria—Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella—which had been cultured overnight, were adjusted to a concentration of 1×10⁻⁶ using MHB liquid medium. 6The working bacterial suspension was prepared at CFU / mL. Experimental groups were established in 96-well cell culture plates: experimental group, positive control group, and blank control group. Each group had three replicate wells to ensure reproducibility. Specifically, 20 μg of lyophilized antimicrobial peptide humAMP43 powder was mixed with MHB medium to prepare a 100 μL solution. In the experimental group, 100 μL of the working bacterial suspension and humAMP43 solution were added to each well to achieve a final concentration of 100 μg / mL. In the positive control group, 100 μL of the working bacterial suspension and 100 μL of MHB medium were added to each well. In the blank control group, 200 μL of sterile MHB medium was added to each well.

[0070] The 96-well plate was placed in a 37°C incubator and incubated statically for 18 hours. After incubation, the absorbance (OD) of each well was measured at 600 nm using a microplate reader. 600 ).

[0071] The formula for calculating the antibacterial rate is as follows:

[0072] Antibacterial rate = [1- (OD)] 实验组 - OD 空白组 ) / (OD 阳性对照组 - OD 空白组 )] × 100%.

[0073] (2) Method for determining the minimum inhibitory concentration

[0074] The lyophilized antimicrobial peptide humAMP43 powder was prepared into a solution using MHB medium and then serially diluted to create concentration gradients of 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.50 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL, 3.91 μg / mL, 1.95 μg / mL, 0.98 μg / mL, 0.49 μg / mL, and 0.24 μg / mL. Each solution was placed in a sterile test tube, and an equal volume of working bacterial suspension was added to each tube. The tubes were thoroughly mixed and incubated at 35°C for 12 hours, and bacterial growth was observed. The lowest peptide concentration in the tube, completely clear and without turbidity, was compared to a control tube containing only bacterial suspension and culture medium, and no visible peptide. This lowest peptide concentration was considered the minimum inhibitory concentration (MIC) of humAMP43 against this bacterium.

[0075] The results of the inhibition rate and minimum inhibitory concentration (MIC) determinations are summarized in Table 2. The experimental results show that the antimicrobial peptide humAMP43 exhibits significant broad-spectrum inhibitory activity against a variety of tested Gram-positive and Gram-negative bacteria. At a concentration of 100 μg / mL, the antimicrobial peptide achieved an inhibition rate of over 90% against all tested strains, with particularly outstanding antimicrobial performance against Staphylococcus aureus, reaching a maximum inhibition rate of 98.6%. The MIC determination results further validate that the antimicrobial peptide humAMP43 possesses highly efficient antimicrobial activity.

[0076] Table 2. Antibacterial effect of antimicrobial peptide humAMP43

[0077]

[0078] Example 3: Construction of a Polynucleotide Encoding the Antimicrobial Peptide humAMP43 and its Expression Vector This example provides the construction of a polynucleotide encoding the antimicrobial peptide humAMP43 and its expression vector. Based on the amino acid sequence of the antimicrobial peptide humAMP43, its encoding gene sequence was designed and chemically synthesized, named polynucleotide H1. Polynucleotide H1 was inserted into the multiple cloning site of the expression vector to construct a recombinant expression vector. Enzyme digestion and sequencing verification confirmed that the target gene was correctly inserted and maintained the correct reading frame.

[0079] In other embodiments, a polynucleotide H2 complementary to polynucleotide H1 was also synthesized, which can be used in genetic engineering experiments related to the antimicrobial peptide humAMP43.

[0080] Example 4: Construction of Genetically Engineered Bacteria Expressing the Antimicrobial Peptide humAMP43 This example provides host cells expressing the antimicrobial peptide humAMP43. The recombinant expression vector constructed in Example 3 was transformed into *Escherichia coli* BL21(DE3) competent cells using a heat shock method. The transformed bacterial culture was plated on LB agar containing kanamycin (50 µg / mL) and incubated overnight at 37°C. Single colonies were picked for expansion culture, and plasmids were extracted for PCR and enzyme digestion verification, successfully obtaining genetically engineered bacteria containing the target expression vector.

[0081] Example 5: Application of antimicrobial peptide humAMP43 in the preparation of antimicrobial spray

[0082] This embodiment provides a specific application of the antimicrobial peptide humAMP43 in the preparation of antimicrobial agents. 10 mg of the antimicrobial peptide humAMP43 synthesized in Example 2 was weighed and dissolved in 10 mL of sterile phosphate buffer to prepare a 1 mg / mL peptide solution. 0.1 mL of Tween-80 was added to this solution as a dispersant, and sterile physiological saline was added to bring the volume to 100 mL. The solution was then filtered through a 0.22 µm filter membrane for sterilization to obtain an antimicrobial spray. This spray can be used for antimicrobial treatment of skin or environmental surfaces.

[0083] In other embodiments, the antimicrobial peptide humAMP43 is formulated with pharmaceutical excipients into a lyophilized powder for injection, used in the preparation of formulations for treating bacterial infections. Alternatively, the antimicrobial peptide humAMP43 is mixed with an ointment base to form a topical antimicrobial ointment for local antibacterial treatment of skin infections. The antimicrobial peptide humAMP43 or its polynucleotides can also be used to prepare food preservatives or feed additives.

[0084] In other embodiments, the solid support is Rink amide MBHA resin or Wang resin; the coupling agent used in the coupling reaction is HBTU-HOBt-DIEA combined coupling agent, PyAOP-HOAt-DIEA combined coupling agent or DIC-Oxyma Pure combined coupling agent, and the other steps are the same as in Example 2, all of which can produce the antimicrobial peptide humAMP43.

[0085] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications to equivalent embodiments. However, any simple modifications, equivalent changes and modifications made to the above embodiments without departing from the technical essence of the claims of the present invention shall still fall within the scope of protection of the claims of the present invention.

Claims

1. An antimicrobial peptide humAMP43, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, The polynucleotide is a polynucleotide H1 encoding the antimicrobial peptide humAMP43 as described in claim 1, or a polynucleotide H2 complementary to the polynucleotide H1.

3. An expression carrier, characterized in that, It contains the polynucleotide described in claim 2.

4. A host cell, characterized in that, It contains the expression vector as described in claim 3.

5. The use of the antimicrobial peptide humAMP43 of claim 1 or the polynucleotide of claim 2 in the preparation of antimicrobial agents, antibacterial drugs, food preservatives or feed additives.

6. The application according to claim 5, characterized in that, The antibacterial agents, antibacterial drugs, food preservatives, or feed additives are used to inhibit the growth of Gram-positive or Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus, Bacillus subtilis, and Listeria. The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, and Salmonella.

7. An antibacterial agent, characterized in that, The antimicrobial agent contains the antimicrobial peptide humAMP43 and is available as an injection, ointment, spray, or drop.

8. A method for preparing the antimicrobial peptide humAMP43 according to claim 1, characterized in that, Following the amino acid sequence shown in SEQ ID NO.1, amino acid monomers with protecting groups were coupled to a solid support via a coupling reaction. The protecting groups of the amino acid monomers were then removed, exposing the free amino groups. The amino acid monomers were then sequentially linked via another coupling reaction to synthesize a peptide chain. The peptide chain was separated from the solid-phase support and purified to obtain the antimicrobial peptide humAMP43.

9. The method for preparing the antimicrobial peptide humAMP43 according to claim 8, characterized in that, The protecting group is an Fmoc protecting group; The solid support is Rink amide MBHA resin or Wang resin. The coupling reaction uses HBTU-HOBt-DIEA combined coupling agent, PyAOP-HOAt-DIEA combined coupling agent, or DIC-Oxyma Pure combined coupling agent.

10. The method for preparing the antimicrobial peptide humAMP43 according to claim 8 or 9, characterized in that, The protecting groups of amino acid monomers are removed using a descaling solution, wherein the descaling solution is a 20%~30% (v / v) N,N-dimethylformamide solution of piperidine. A cleavage fluid is used to separate the peptide chain from the solid support. The cleavage fluid is prepared by mixing trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5.

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