Antibacterial peptide and application thereof

Abstract

The invention belongs to the technical field of biological application, and particularly relates to an antibacterial peptide and application thereof, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1. The invention provides an antibacterial molecule with a novel structure, the antibacterial molecule is composed of 28 amino acids and has no significant homology with reported mussel antibacterial peptide sequences such as Mytilin and Myticin, the antibacterial peptide enriches an antibacterial peptide molecular library, the theoretical isoelectric point of the polypeptide is up to 11.72, the net charge is + 4, the polypeptide has high positive charge density and a hydrophobic amino acid proportion of 53.6%, and the antibacterial peptide has a good application prospect. The amphiphilic structural characteristics are obviously better than those of the traditional antibacterial peptide, and the antibacterial peptide is endowed with the capability of efficiently targeting a bacterial membrane. Bacteriostatic experiments prove that the inhibition rate on escherichia coli reaches 60%, the inhibition rate on staphylococcus aureus is 20%, obvious concentration dependence and strain specificity are shown, the effect is accurate, and broad-spectrum drug resistance is not likely to be induced.

Description

Technical Field

[0001] This invention belongs to the field of bioapplication technology, specifically relating to an antimicrobial peptide and its application. Background Technology

[0002] Since the 1940s, antibiotics have been widely used in clinical practice. While these antibiotics, hailed as "miracle drugs," have successfully saved countless lives, the problem of bacterial resistance caused by their overuse has become a major global public health crisis. Taking *E. coli* as an example, its resistance rates to piperacillin, ceftriaxone, trimethoprim-sulfamethoxazole, levofloxacin, and ciprofloxacin all exceed 50%. Even more worrying is the continuous spread of pan-drug-resistant strains, leaving some infected cases without any effective antibacterial drugs. Therefore, developing novel antibacterial molecules that are less likely to induce resistance and have precise antibacterial effects has become an urgent need in the current research and development of anti-infective drugs. Summary of the Invention

[0003] The purpose of this invention is to provide an antimicrobial peptide that can efficiently target bacterial cell membranes and effectively circumvent the problem of broad-spectrum drug resistance that is easily generated by traditional antibiotics.

[0004] The technical solution adopted in this invention is: The present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The present invention also provides an application of the antimicrobial peptide, wherein the antimicrobial peptide is used to prepare an antimicrobial agent.

[0006] Preferably, the antibacterial agent targets Gram-positive and / or Gram-negative bacteria.

[0007] Preferably, the Gram-positive bacteria are Staphylococcus aureus; the Gram-negative bacteria are Escherichia coli.

[0008] Preferably, the antibacterial agent is prepared as follows: The antimicrobial peptide was diluted with PBS to a solution of 20 μmol / L to 320 μmol / L to obtain the antibacterial agent.

[0009] Preferably, the antibacterial agent is prepared as follows: The antimicrobial peptide was diluted to a solution of 320 μmol / L using PBS to obtain the antibacterial agent.

[0010] Preferably, the antibacterial agent further includes pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipients include at least one of fillers, flavoring agents, binders, disintegrants, antacids, and nutritional fortifiers.

[0012] Preferably, the filler comprises any one of microcrystalline cellulose, starch, lactose, and mannitol.

[0013] Preferably, the flavoring agent includes any one of steviol glycosides, aspartame, and sucrose.

[0014] Preferably, the adhesive comprises either starch paste or hydroxypropyl methylcellulose.

[0015] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0016] Preferably, the antacid agent includes either calcium carbonate or aluminum hydroxide.

[0017] Preferably, the nutritional fortifier includes any one of vitamin A, vitamin C, and vitamin D.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1. This invention provides a novel antimicrobial molecule composed of 28 amino acids, with a small molecular weight and easy synthesis. It shows no significant homology to previously reported mussel antimicrobial peptide sequences such as Mytilin and Myticin. The antimicrobial peptide of this invention enriches the antimicrobial peptide library. This peptide has a theoretical isoelectric point as high as 11.72 and a net charge of +4, possessing both high positive charge density and a 53.6% proportion of hydrophobic amino acids. Its amphiphilic structural characteristics are significantly superior to traditional antimicrobial peptides, endowing it with the ability to efficiently target bacterial membranes. Antibacterial experiments confirmed that it inhibits Escherichia coli by 60% and Staphylococcus aureus by 20%, exhibiting obvious concentration dependence and species specificity, with precise action and low likelihood of inducing broad-spectrum drug resistance.

[0019] The antimicrobial peptide of this invention has a secondary structure containing 35.71% α-helices, and its tertiary structure is highly similar to that of glyceraldehyde-3-phosphate dehydrogenase. Its key functional domains are highly conserved, and its structure exhibits strong stability. This peptide can be prepared in high purity through solid-phase chemical synthesis and has clear application prospects in the fields of anti-infective drug development and disease control in aquaculture. Attached Figure Description

[0020] Figure 1 This is a prediction graph of antimicrobial peptides.

[0021] Figure 2This is an amino acid sequence diagram of the candidate polypeptide McGAPDH; hydrophobic residues are shown in red, and proline and glycine are shown in blue.

[0022] Figure 3 The results show the predicted secondary structure of McGAPDH; A: Secondary structure of the antimicrobial peptide analyzed by SOPMA; B: Distribution characteristics of secondary structures at positions 5-25 of the target protein sequence: The schematic diagram above uses color coding to visually present the spatial arrangement of different secondary structures in the sequence: blue α-helix, purple β-sheet, green turn, and yellow random coil; The predicted curves below quantify the dynamic proportion changes of each structure along the sequence.

[0023] Figure 4 The following are the predicted results of protein tertiary structure: A: Glyceraldehyde-3-phosphate dehydrogenase model; B: Three-dimensional structural model of antimicrobial peptide McGAPDH; C: Laplace diagram of the tertiary structure of hGAPDH antimicrobial peptide; D: GMQE score of hGAPDH.

[0024] Figure 5 Phylogenetic tree constructed based on GAPDH amino acid sequence similarity.

[0025] Figure 6 The results are from multiple sequence alignment analysis of the GAPDH amino acid sequence.

[0026] Figure 7 This demonstrates the antibacterial activity of McGAPDH against Escherichia coli.

[0027] Figure 8 This demonstrates the antibacterial activity of McGAPDH against Staphylococcus aureus. Detailed Implementation

[0028] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0029] The inventive concept of this invention is as follows: Antimicrobial peptides (AMPs), small molecule polypeptides derived from the innate immune system of organisms, have attracted much attention due to their unique bactericidal mechanisms. These small molecule polypeptides, composed of 20-50 amino acids, are widely found in animals, plants, and microorganisms. They achieve bactericidal effects through multi-target actions such as rapidly disrupting bacterial cell membrane potential and inhibiting biofilm formation, making them less likely to induce bacterial resistance compared to traditional antibiotics. As a multifunctional innate immune molecule, antimicrobial peptides are gradually becoming an important solution to the global crisis of drug-resistant bacteria. Their mechanism of action breaks through the limitations of traditional antibiotics, not only achieving broad-spectrum bactericidal effects by disrupting bacterial cell membranes, forming ion channels, or interfering with lipid metabolism, but also exerting synergistic therapeutic effects by regulating host immune responses and neutralizing endotoxins. For example, copper single-atom-host defense peptide-based nanoreactors generate reactive oxygen species through Fenton-like catalysis, systematically disrupting the stress response system of methicillin-resistant Staphylococcus aureus (MRSA), while simultaneously promoting collagen fiber proliferation and angiogenesis at the site of infection.

[0030] Thick-shelled mussels ( Mytilus coruscus Thick-shelled mussels are an important economic shellfish along the East China Sea coast of my country. In natural classification, they belong to the phylum Mollusca, class Bivalvia, order Heterostachidae, family Myriophylba, and genus Myriophyllum. They are mainly distributed in the temperate waters of the northwestern Pacific Ocean, especially along the Zhejiang coast, such as Zhoushan and Wenzhou, where large-scale thick-shelled mussel farming areas exist. China's farmed shellfish production reaches 15.7 million tons, accounting for 69% of the national aquaculture production, with thick-shelled mussels ranking among the top shellfish in terms of annual production. Currently, large-scale disease threats have emerged in the farming of representative shellfish such as mussels and clams, severely impacting the shellfish farming industry. In the long-term survival of a complex microbial environment, thick-shelled mussels rely on various antimicrobial peptides to build an immune defense. Mussels in different growth environments and at different developmental stages secrete different antimicrobial peptides to resist the invasion of exogenous pathogenic microorganisms. Different peptides exhibit diversity in antibacterial activity and physicochemical properties due to differences in structure and sequence. For example, the Mytilin family has broad-spectrum antimicrobial capabilities, inhibiting both bacteria and fungi. Thick-shelled mussel antimicrobial peptides exhibit enhanced membrane penetration due to their high hydrophobicity and cysteine-rich structure. Research on thick-shelled mussel antimicrobial peptides not only contributes to a deeper understanding of their immune defense mechanisms but also provides new ideas and directions for the development of novel antimicrobial peptide drugs and the healthy development of aquaculture.

[0031] This invention selects the thick-shelled mussel as the research object, and uses transcriptome sequencing technology to screen and identify its antimicrobial peptides, focusing on analyzing the structural and functional characteristics of the candidate peptide McGAPH. Total RNA was extracted from the thick-shelled mussel using the Trizol method, a transcriptome library was constructed and sequenced, transcripts were assembled using Trinity software, and candidate peptides such as McGAPH were screened using local BLAST alignment and CAMPR3 multi-algorithm prediction. Physicochemical analysis showed that McGAPH consists of 28 amino acids, with a theoretical isoelectric point of 11.72. The α-helix accounts for 35.71% of its secondary structure, and its tertiary structure is highly similar to glyceraldehyde-3-phosphate dehydrogenase, with a sequence identity of 73.15%. Antibacterial experiments showed that when the McGAPH concentration was 320 μmol / L, the inhibition rate against Gram-negative bacteria (Escherichia coli) reached 60%, while the inhibition rate against Gram-positive bacteria (Staphylococcus aureus) was approximately 20%, exhibiting concentration dependence and species specificity. Evolutionary analysis showed that the thick-shelled mussel and the purple mussel are closely related, and the McGAPDH sequence exhibits high conservation in key functional regions. This invention demonstrates that using high-throughput sequencing data and antimicrobial peptide databases for multiple sequence alignment is an efficient method for screening antimicrobial peptides, and antimicrobial experiments revealed the antimicrobial potential of McGAPDH from the thick-shelled mussel.

[0032] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0033] Example 1 An antimicrobial peptide and its applications are as follows: 1. Sample collection and weight data acquisition.

[0034] 1.1 Sample collection and preprocessing.

[0035] Sampling area: Ten adult thick-shelled mussels were collected from the Shengsi sea area of ​​Zhoushan City, preserved in ice boxes, and transported to the laboratory.

[0036] 1.2 Phenotypic Records: Measure the weight of each sample.

[0037] 1.3 Data Recording.

[0038] Phenotypic control and statistics: The mass data of each thick-shelled mussel were obtained within the range of 41±3g.

[0039] 1.4 RNA extraction from thick-shelled mussels.

[0040] Step 1: Liquid phase separation.

[0041] (1) Weigh 0.1g of tissue into a 1.5mL centrifuge tube, add 1mL of Trizol solution and two grinding beads, and grind at low temperature.

[0042] (2) After grinding, add 200 μL of chloroform, shake vigorously for 15 seconds and let stand for 5 minutes.

[0043] Step 2: RNA precipitation and resuspension.

[0044] (1) Centrifuge at 4℃ and 12000r / min for 10min, transfer the upper liquid to a new tube, add an equal volume of isopropanol, mix well, let stand for 10min, centrifuge again and discard the supernatant.

[0045] (2) The precipitate was washed with 200 μL of 75% ethanol and centrifuged. The supernatant was discarded and the precipitate was dried at room temperature for 10 min.

[0046] (3) Add 50 μL of DEPC water to the precipitate to dissolve the RNA precipitate, and determine the concentration by spectrophotometer and detect the purity by agarose gel electrophoresis.

[0047] 1.5 Reverse transcription of RNA from thick-shelled mussels.

[0048] (1) Prepare the genomic DNA removal reaction system.

[0049] Take 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, and 1 μg of total RNA, and bring the volume to 10 μL with RNase-Free ddH2O. Incubate the mixture at 42°C for 2 min to digest residual genomic DNA.

[0050] (2) Construction of reverse transcription system.

[0051] Pre-cooled Go Script TM After thawing the reverse transcription system, prepare 12 μL of RT-Mix, transfer it to a clean centrifuge tube, add 1 μg of the treated RNA sample, and finally add deionized water to bring the total volume to 20 μL.

[0052] The reaction program was set as follows: reverse transcription extension at 42℃ for 15 min, followed by heating at 70℃ for 15 min to complete enzyme inactivation and obtain cDNA.

[0053] The entire operation was performed in a sterile environment, with key steps conducted on ice to maintain system stability.

[0054] 1.6 PCR amplification.

[0055] PCR amplification was performed using cDNA obtained from reverse transcription of RNA as a template. The PCR reaction system is shown in Table 1. The PCR reaction program was: 94°C for 3 min; 94°C for 30 s, 54°C for 30 s, 72°C for 45 s, for 35 cycles; 72°C for 10 min. After the reaction, the amplified product was stored at 4°C.

[0056] Table 1 PCR reaction system 1.7 Library construction, sequencing and assembly.

[0057] (1) Systematic quality control was performed on the RNA samples extracted in the early stage of the experiment.

[0058] RNA concentration and purity were determined using a Nanodrop ND-2000 spectrophotometer, and RNA samples meeting quality standards were screened for library construction. Paired-end 150bp sequencing was performed on the Illumina HiSeq X Ten platform to ensure sufficient sequencing data output for subsequent analysis.

[0059] (2) Perform standardized quality control on the obtained raw data.

[0060] Low-quality reads were filtered using Fastp, and reads containing adapter sequences, N bases with a proportion >10%, or Phred scores ≤20 with a proportion ≥50% were removed to obtain high-quality clean data. Subsequently, FastQC was used to evaluate the sequencing data quality, including sequencing depth saturation analysis and sequence distribution randomness verification, to ensure that the data met the requirements for subsequent assembly.

[0061] Based on the preprocessed high-quality short reads, transcripts were assembled using Trinity v2.15.1 software, while a reference genome guidance strategy was integrated to optimize assembly efficiency. The longest transcript was selected as the gene representative by clustering, and the quality of the assembly results was validated.

[0062] 1.8 Local BLAST.

[0063] Based on the transcriptome assembly results of the thick-shelled mussel, a local BLAST was used to compare all obtained transcripts with an antimicrobial peptide database and screen for highly similar polypeptide coding sequences.

[0064] Operating steps: (1) Install the BLAST-2.4 software tool and configure the system working environment. Then, download the antimicrobial peptide sequence data from the antimicrobial peptide database APD3 to a local document to complete the data preparation. Integrate the transcript sequences into FASTA format files. Use the Makeblastdb command to format and index the above data to initially form a local database that can be searched by BLAST. APD3: https: / / aps.unmc.edu / AP / .

[0065] (2) Potential antimicrobial peptide genes were preliminarily identified through sequence similarity retrieval. This strategy effectively integrates public database resources with local bioinformatics analysis processes, ensuring the systematic and reproducible nature of antimicrobial peptide screening.

[0066] 1.9. Antimicrobial peptide prediction.

[0067] For candidate antimicrobial peptide sequences obtained by local BLAST sequence alignment and screening, the CAMPR3 platform, which integrates multiple algorithms, is used to predict whether they are antimicrobial peptides. CAMPR3 outputs prediction results based on four algorithms: support vector machine, random forest classifier, artificial neural network, and discriminant analysis classifier.

[0068] CAMPR3 platform: http: / / www.camp3.bicnirrh.res.in / predict / hii.php.

[0069] 1.10. Identification of antibacterial activity.

[0070] The selected McGAPDH candidate peptides were chemically synthesized using a solid-phase method. McGAPDH was synthesized by Shanghai Xinhao Biotechnology Co., Ltd. The purity of the peptides was identified by liquid chromatography and mass spectrometry, and the purity was >95%.

[0071] The antimicrobial activity of antimicrobial peptides was determined using the inhibition curve method. The experimental procedure for the inhibition curve method is as follows: (1) Prepare LB medium; dilute the bacterial suspension cultured to the logarithmic growth phase with LB medium in a serial dilution until each mL of bacterial suspension contains 1×10⁻⁶ cells / mL. 4 ~1×10 5 The test bacteria were all purchased from Shanghai Luwei Technology Co., Ltd., and included one Gram-negative bacterium and one Gram-positive bacterium. The Gram-negative bacterium was *Escherichia coli* (E. coli). Escherichia coli LWCC1033); Gram-positive bacteria are Staphylococcus aureus ( Staphylococcus aureus (LWCC1010).

[0072] (2) Prepare 20 μg / mL of the antibiotics ampicillin and kanamycin sulfate as positive controls for Staphylococcus aureus and Escherichia coli; The antimicrobial peptide McGAPDH was diluted with PBS to a concentration gradient of 20 μmol / L, 40 μmol / L, 80 μmol / L, 160 μmol / L, and 320 μmol / L.

[0073] (3) Add 80 μL of bacterial culture and 20 μL of antimicrobial peptide solution of different concentrations to each well of a 96-well cell culture plate; set PBS as the negative control.

[0074] (4) After measuring the initial OD600 value, the bacteria were incubated at 37℃ for 18 hours. The final OD600 value was then measured, and the bacterial growth value and growth percentage were calculated. Finally, an inhibition curve was plotted with the concentration of the antimicrobial peptide McGAPH as the x-axis and the growth percentage as the y-axis. The experiment required control of bacterial activity and sterilization conditions.

[0075] (5) Each experiment is repeated three times to reduce experimental error, verify the reliability of the experiment and eliminate the influence of random factors.

[0076] 2. Results.

[0077] This invention combines Figures 1 to 8 Based on the experimental data, the sequence characteristics and antibacterial function of the antimicrobial peptide McGAPDH are further explained as follows: 2.1 Sequence screening and source confirmation.

[0078] McGAPDH sequences were obtained through transcriptome sequencing combined with local BLAST screening. For example... Figure 2 As shown, the sequence is 28 amino acids in length, with specific sequence information as shown in SEQ ID NO.1. It contains 15 hydrophobic amino acids and 5 positively charged amino acids, with a total net charge of +4 and a theoretical isoelectric point of 11.72. It shows a 64.52% similarity to the known antimicrobial peptide hGAPDH(2-32) (accession number AP02017) in the APD3 database.

[0079] SEQ ID NO. 1: LKVGINGFGRIGRLVMRAALDKGVSVVA.

[0080] 2.2 Validation of antimicrobial peptide prediction.

[0081] Figure 1 The CAMPR3 platform prediction results show that the probability of identifying it as an antimicrobial peptide is 94.2% with Support Vector Machine, 98.45% with Random Forest classifier, 98.8% with Discriminant Analysis classifier, and also 98.8% with Artificial Neural Network. The results from the four algorithms are highly consistent, confirming its antimicrobial peptide attribute.

[0082] 2.3 Structural Feature Analysis.

[0083] Figure 3A shows the secondary structure predicted by SOPMA: α-helix with 10 amino acids, accounting for 35.71%; extended chain with 10 amino acids, accounting for 35.71%; and random coil with 8 amino acids, accounting for 28.57%. Figure 3 B further indicates that the α-helix is ​​enriched at positions 5 to 25, forming an alternating helical-coil conformation.

[0084] Figure 4 The homology modeling results show that the sequence identity is 73.15% using glyceraldehyde-3-phosphate dehydrogenase as a template. Figure 4 The C-Laplace diagram shows that over 90% of the residues have a reasonable conformation. Figure 4 The GMQE score of 0.91 in D confirms the reliability of the three-level structure model.

[0085] 2.4. Evolutionary conservation analysis.

[0086] Figure 5 Phylogenetic trees show that the thick-shelled mussel is most closely related to the purple mussel, and the GAPDH sequence is highly conserved in key functional regions.

[0087] Figure 6 Multiple sequence alignment revealed the N-terminal basic amino acid clusters (K2, R9, R... 13 ) and C-terminal hydrophobic region (L 21 V 22 A 23 L 24 It is highly conserved in 13 species.

[0088] 2.5. Antibacterial activity verification.

[0089] Figure 7 Data shows that the inhibition rate of McGAPDH against E. coli increases with increasing concentration: When McGAPDH concentration was 20 μmol / L, the growth percentage was 0.95 ± 0.02, with an inhibition rate of 5%. When McGAPDH concentration was 160 μmol / L, the growth percentage decreased to 0.65 ± 0.04, with an inhibition rate of 35%. When McGAPDH concentration was 320 μmol / L, the growth percentage reached 0.40 ± 0.03, with an inhibition rate of 60%. The differences between each concentration group and the negative control were significant (P < 0.05). The growth percentage of the positive control group (20 μg / mL kanamycin sulfate) was less than 0.05, therefore McGAPDH achieved complete inhibition of *Escherichia coli*.

[0090] Figure 8 Data shows that McGAPDH has a weak inhibitory effect on Staphylococcus aureus: At concentrations of 20 μmol / L to 160 μmol / L, the growth percentage remained at 0.85–0.95, with an inhibition rate of less than 15%; at 320 μmol / L, the growth percentage decreased to 0.80 ± 0.05, with an inhibition rate of 20%.

[0091] 3. Conclusion.

[0092] The above results indicate that McGAPDH has a concentration-dependent inhibitory effect on Gram-negative and Gram-positive bacteria in the range of 20 μmol / L to 320 μmol / L, and its inhibitory effect on Escherichia coli is significantly better than that on Staphylococcus aureus, providing experimental evidence for its application as an antibacterial agent.

[0093] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

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

1.

2. The application of the antimicrobial peptide as described in claim 1, characterized in that, The antimicrobial peptide is used to prepare antimicrobial agents.

3. The application as described in claim 2, characterized in that, The antibacterial agent targets Gram-positive and / or Gram-negative bacteria.

4. The application as described in claim 3, characterized in that, The Gram-positive bacteria is Staphylococcus aureus; The Gram-negative bacterium is Escherichia coli.

5. The application as described in claim 2, characterized in that, The method for preparing the antibacterial agent is as follows: The antimicrobial peptide was diluted with PBS to a solution of 20 μmol / L to 320 μmol / L to obtain the antibacterial agent.

6. The application as described in claim 5, characterized in that, The method for preparing the antibacterial agent is as follows: The antimicrobial peptide was diluted to a solution of 320 μmol / L using PBS to obtain the antibacterial agent.

7. The application as described in claim 2, characterized in that, The antibacterial agent also includes pharmaceutically acceptable excipients.

8. The application as described in claim 7, characterized in that, Pharmaceutically acceptable excipients include at least one of fillers, flavoring agents, binders, disintegrants, antacids, and nutritional fortifiers.

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