Cone snail α-helix, β-fold antimicrobial peptides and their preparation method and application

Abstract

The present invention discloses cone snail α-helical and β-pleated antimicrobial peptides, as well as their preparation and application. Three antimicrobial peptide precursor gene nucleic acid sequences, as shown in SEQ ID NOs. 1 to 3, were screened from a barrel-shaped cone snail multi-omics dataset. These three nucleic acid sequences were translated into amino acid sequences, and peptide sequences representing the antimicrobial active regions were designed with reference to known antimicrobial peptide sequences, resulting in three polypeptides represented by amino acids as shown in SEQ ID NOs. 7 to 9. These peptides were then directly synthesized using chemical methods to obtain the three cone snail antimicrobial peptides. The three cone snail antimicrobial peptides obtained have been shown to specifically inhibit the growth of Pichia pastoris or Escherichia coli, exhibiting good antimicrobial activity. These peptides can be used to prepare products with antimicrobial activity, demonstrating promising application prospects and economic value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to cone snail alpha-helix and beta-fold antimicrobial peptides, as well as preparation methods and applications thereof. Background Art

[0002] In recent years, the overuse of traditional antibiotics has led to the growing problem of bacterial resistance, making the search for new antibiotics increasingly challenging. Antimicrobial peptides (APs) originally refer to a class of polypeptides with antimicrobial activity induced by the body in animals and plants. These host defense peptides possess strong alkalinity, thermal stability, and broad-spectrum antimicrobial properties, making them a promising alternative to antibiotics. Their antimicrobial mechanism is as follows: most APs are cationic (positively charged) and amphipathic (hydrophilic and hydrophobic) α-helical / β-pleated peptide molecules. Due to their membrane permeability, cationic APs can bind to and interact with negatively charged cell membranes, altering the electrochemical potential across the membrane. This induction of membrane damage allows the penetration of larger molecules, such as proteins, disrupting cell morphology and membranes, ultimately leading to cell death. Therefore, compared to traditional antibiotics, they are less likely to induce resistance and hold great promise as therapeutic agents.

[0003] At present, it has been reported that more than 3,000 antimicrobial peptides have been identified from animals, fungi, plants and bacteria. For example, 15 venom short peptide sequences from 5 species of ants have been reported to have antimicrobial activity; 13 venom short peptide sequences from 7 species of bees have been reported to have antibody activity; 10 venom short peptide sequences from 9 species of snakes have been reported to have antibody activity; 55 active antimicrobial peptides have been found from more than 30 species of centipedes, etc.

[0004] Cone snail ( Cone Snail Cone snails, also known as cone snails, are carnivorous marine gastropod molluscs known for their beautiful shells and the unique toxins they secrete. There are currently over 1,000 registered species of cone snails, which prey on a variety of animals, including worms, snails, and fish, through their venom. Cone snail toxins are attractive new drug resources due to their novel chemical structure, strong biological activity, and high target selectivity. Over the past few decades, more than 70,000 natural cone snail toxins have been discovered and are widely used in pharmacology and neuroscience research.

[0005] However, compared with animals such as worms, snails, and fish, there are fewer studies on the antibacterial function of cone snail peptides. Currently, only a few related cone snail peptide sequences have been reported to have antibacterial functions, and research on the antibacterial application of these cone snail antimicrobial peptides is extremely rare. For example, one study reported that a conotoxin from the cone snail o1 superfamily can inhibit the growth of Mycobacterium tuberculosis, and another reported that an open-chain MVIIA structure from the cone snail ω-conotoxin can inhibit the growth of fungi. In addition, most of the existing antimicrobial peptides are obtained through blind screening, such as direct extraction from biological separation by experimental means, or cloning the obtained antimicrobial peptide gene sequence and introducing it into microbial culture, and then further separation, purification and identification through a series of experimental methods. For example, Chinese patent document CN115232850A discloses a method for preparing marine antimicrobial peptides, which involves freeze-drying and pulverizing marine biological materials, followed by enzymatic hydrolysis to separate and extract the antimicrobial peptides. Chinese patent document CN115057918A discloses in vitro recombinant expression and identification of novel antimicrobial peptides from American eel. The gene encoding the antimicrobial domain of a fungicidal protein from American eel is cloned, an expression vector is constructed, and the product is introduced into Escherichia coli for expression. The product is then purified using a chromatographic purification column and further identified. These methods not only involve numerous experimental procedures and are cumbersome, but are also time-consuming, inefficient, and costly, hindering the development, research, and application of antimicrobial peptides.

[0006] Therefore, how to quickly and effectively explore the antibacterial function of cone snail peptides and obtain more cone snail antibacterial peptides to provide more options for replacing traditional antibiotic treatment drugs has important research significance and potential market value. Summary of the Invention

[0007] In order to solve the above problems and achieve the above objectives, the present invention provides a cone snail α-helix, β-fold antimicrobial peptide and its preparation method and application, combined with bioinformatics technology, from barrel-shaped cone snail ( Conus betulinus ) A multi-omics dataset was used to screen three antimicrobial peptide precursor gene nucleic acid sequences. Based on the amino acid sequences translated from these three nucleic acid sequences, the antimicrobial active regions of cone snail antimicrobial peptides were manually screened and designed, and chemically synthesized. After antimicrobial activity verification and identification, three new cone snail antimicrobial peptides were obtained. The specific technical plan is as follows:

[0008] First, the present invention provides a method for preparing a barrel-shaped cone snail ( Conus betulinus ) Three cone snail antimicrobial peptide precursor genes identified and screened in the multi-omics data set, whose nucleic acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0009] Secondly, the present invention provides cone snail antimicrobial peptide precursor proteins based on the three aforementioned cone snail antimicrobial peptide precursor genes, whose amino acid sequences are determined according to the codon encoding rules and are as follows:

[0010] The amino acid sequence of the cone snail antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.4;

[0011] The amino acid sequence of the cone snail antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.2 is shown in SEQ ID NO.5;

[0012] The amino acid sequence of the cone snail antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.3 is shown in SEQ ID NO.6.

[0013] Again, the present invention provides a cone snail antimicrobial peptide that is synthesized by chemical methods based on the determined cone snail antimicrobial peptide precursor protein amino acid sequence, combined with known antimicrobial peptide antimicrobial activity polypeptide fragments, and artificially screened and designed cone snail antimicrobial peptide bacterial activity domains to obtain cone snail antimicrobial peptide amino acid sequences. The designed cone snail antimicrobial peptide amino acid sequence is:

[0014] The amino acid sequence of the cone snail antimicrobial peptide designed based on the cone snail antimicrobial peptide precursor protein with the amino acid sequence EQ ID NO.4 is shown in SEQ ID NO.7;

[0015] The amino acid sequence of the cone snail antimicrobial peptide designed based on the cone snail antimicrobial peptide precursor protein with the amino acid sequence of SEQ ID NO.5 is shown in SEQ ID NO.8;

[0016] The amino acid sequence of the cone snail antimicrobial peptide designed based on the cone snail antimicrobial peptide precursor protein with the amino acid sequence of SEQ ID NO.6 is shown in SEQ ID NO.9.

[0017] Therefore, the present invention provides a method for preparing a cone snail antimicrobial peptide, which is used to prepare any of the aforementioned cone snail antimicrobial peptides; the specific steps are as follows:

[0018] 1) Selecting a cone snail multi-omics dataset, wherein the multi-omics dataset includes a cone snail genome, transcriptome, and venom proteome dataset;

[0019] 2) screening cone snail antimicrobial peptide precursor gene sequences from the multi-omics dataset through homology comparison and activity simulation prediction, as shown in SEQ ID NO. 1 to SEQ ID NO. 3;

[0020] 3) translating the screened antimicrobial peptide precursor gene sequence into the corresponding cone snail antimicrobial peptide precursor protein sequence, as shown in SEQ ID NO.4 to SEQ ID NO.6;

[0021] 4) referring to antimicrobial peptide fragments with known antimicrobial activity, potential mature active peptide regions of the cone snail antimicrobial peptide precursor protein were designed to obtain peptides with amino acid sequences as shown in SEQ ID NO. 7 to SEQ ID NO. 9;

[0022] 5) Synthesize the polypeptides represented by SEQ ID NO. 7 to SEQ ID NO. 9 using a chemical synthesis method;

[0023] 6) Identify the antibacterial effect of the synthesized polypeptide, thereby obtaining cone snail polypeptides with antibacterial activity, namely cone snail antimicrobial peptides.

[0024] As a preferred technical solution, in step 4), the polypeptide obtained by designing the potential mature active peptide region of the cone antimicrobial peptide precursor protein meets the following conditions:

[0025] a. It is a positively charged cation;

[0026] b. Its isoelectric point is greater than 8.0;

[0027] c. Its CTDD and PAAC values ​​are greater than 0.5;

[0028] d. It has α-helices and / or β-sheets;

[0029] e. The number of cysteines in its amino acid sequence is less than 5;

[0030] f. Its amino acid sequence length does not exceed 30 amino acids.

[0031] As a preferred technical solution, in step 6), the antibacterial effect of the synthesized polypeptide is identified by mixing the synthesized polypeptide with different microorganisms including experimental Escherichia coli and Pichia pastoris to verify its antibacterial activity.

[0032] In addition, the present invention also provides an application of a cone snail antimicrobial peptide, that is, the application of any one or more of the aforementioned cone snail antimicrobial peptides in the preparation of a product with antimicrobial activity.

[0033] Preferably, the product having antimicrobial activity can specifically inhibit the growth of Pichia pastoris and / or Escherichia coli.

[0034] In addition, the present invention provides a preparation for sterilization, wherein the preparation contains any one or more of the aforementioned cone antimicrobial peptides as an active ingredient.

[0035] Preferably, the preparation contains a cone snail antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.7 at a concentration of 917 μM; or contains a cone snail antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.8 at a concentration of 245 μM to 491 μM, preferably at a concentration of 245 μM and 491 μM; or contains a cone snail antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.9 at a concentration of 39 μM to 312 μM, preferably at a concentration of 78 μM, 39 μM, 156 μM, or 312 μM.

[0036] Beneficial effects of the present invention:

[0037] 1) The present invention mined three cone snail antimicrobial peptides from the cone snail multi-omics dataset and discovered for the first time their antimicrobial effects against two different fungi and bacteria, highlighting the antimicrobial potential of cone snail peptides. Among them, UBI_31-38 and CcAMP1-CB can inhibit the growth of the fungus Pichia pastoris, and YFGAP-CB shows inhibitory activity against Escherichia coli. This enriches the types and types of cone snail antimicrobial peptides and is expected to be used to prepare products with antimicrobial activity and replace antibiotics in the treatment of fungal or bacterial infections in clinical practice. It has good application prospects and economic value.

[0038] 2) This invention, for the first time, uses multi-omics dataset (genome, transcriptome, proteome) mining technology to mine and design multiple antimicrobial peptide sequences from cone snail multi-omics datasets at one time, and directly synthesizes antimicrobial peptide proteins through chemical methods. This effectively solves the problems of blind screening, low efficiency and high cost in the traditional antimicrobial peptide screening process, and provides a new research method for the development and utilization of cone snail genetic resources.

[0039] 3) The present invention combines bioinformatics technology to directly screen potential target sequences from the biological big data level, and combines activity simulation prediction with in vitro experimental verification methods, providing new ideas for the in-depth development of valuable biological genetic resources and a solid theoretical basis for the application of new marine antibacterial drugs in clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a three-dimensional modeling diagram of three structurally different antimicrobial peptides from barrel-shaped cone snails.

[0041] Figure 2 This is the high performance liquid chromatogram of the synthesized antimicrobial peptide UBI_31-38 from the barrel-shaped cone snail.

[0042] Figure 3 This is the mass spectrum of the synthesized antimicrobial peptide UBI_31-38 from the barrel-shaped cone snail.

[0043] Figure 4 This is the high performance liquid chromatogram of the synthesized CcAMP1-CB antimicrobial peptide from the barrel-shaped cone snail.

[0044] Figure 5 This is the mass spectrum of the synthesized CcAMP1-CB antimicrobial peptide from the barrel-shaped cone snail.

[0045] Figure 6 This is the high performance liquid chromatogram of the synthesized barrel-shaped cone snail YFGAP-CB antimicrobial peptide.

[0046] Figure 7 This is the mass spectrum of the barrel-shaped cone snail YFGAP-CB antimicrobial peptide after synthesis.

[0047] Figure 8 This is a diagram showing the activity test effect of the barrel-shaped cone snail UBI_31-38 antimicrobial peptide inhibiting the growth of Pichia pastoris.

[0048] Figure 9 This is a diagram showing the activity test effect of the barrel-shaped cone snail CcAMP1-CB antimicrobial peptide inhibiting the growth of Pichia pastoris.

[0049] Figure 10 This is a diagram showing the activity test effect of the barrel-shaped cone snail YFGAP-CB antimicrobial peptide in inhibiting the growth of Pichia pastoris. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0051] It should be noted that the abbreviations used in the full-length amino acid sequences of the present invention are commonly used by those skilled in the art. Specifically, R is arginine (Arg), L is leucine (Leu), G is glycine (Gly), N is asparagine (Asn), C is cysteine ​​(Cys), T is threonine (Thr), V is valine (Val), M is methionine (Met), A is alanine (Ala), K is lysine (Lys), F is phenylalanine (Phe), S is serine (Ser), P is proline (Pro), E is glutamic acid (Glu), I is isoleucine (Ile), Q is glutamine (Gln), and W is tryptophan (Trp).

[0052] Example 1 is the acquisition of the precursor gene DNA and the coding precursor protein sequence of the cone snail antimicrobial peptide; Example 2 is the prediction of the active structure of the cone snail antimicrobial peptide; Example 3 is the activity test of the cone snail antimicrobial peptide.

[0053] Example 1 Obtaining the Precursor Gene DNA and Encoding Precursor Protein Sequence of Conus Antimicrobial Peptide

[0054] In this example, the Chinese barrel cone snail ( Conus betulinus) as the experimental research object. Using a homology alignment method, the antimicrobial peptide precursor gene coding sequence was identified from a multi-omics dataset of barrel cone snails and translated into the antimicrobial peptide precursor protein sequence. The specific process is as follows:

[0055] 1) Multi-omics dataset preparation

[0056] The multi-omics dataset in this example includes the genome, transcriptome, and venom proteome datasets of the barrel cone snail.

[0057] The NCBI accession number of the genome dataset is: GCA_016801955.1.

[0058] The transcriptome dataset includes transcriptome data from five specimens: Big (the venomous tube of a 10 cm large cone snail); Middle (the venomous tube of a 8.7 cm medium cone snail); Small (the venomous tube of a 6 cm small cone snail); Bulb (the venom sac of the aforementioned medium cone snail); and Normalized (the normalized dataset of the venomous tube of the aforementioned medium cone snail). The NCBI SRA database accession numbers for the transcriptome datasets are SRS1009725 to SRS1009729.

[0059] The PRIDE database accession number of the venom protein dataset is: PXD014892.

[0060] The protein-coding region of each transcript was predicted using TransDecoder software, and gene transcription levels were calculated using FPKM. All protein-coding sequences from the genome and transcriptome were translated into amino acids to construct a comprehensive protein set for Conus barrelii.

[0061] 2) Screening of antimicrobial peptide precursor gene DNA sequences from multi-omics datasets

[0062] A total of 2927 validated antimicrobial peptides from the public antimicrobial peptide database APD3 were used as reference sequences. First, an index library for sequence alignment was created using the annotated genes from the Conus barrelii genome using the makeblastdb command in the Blast software package (makeblastdb -dbtype nucl -parse_seqids -in Conus.gene.cds). Potential AMP precursor genes were then predicted from the genome using the TBLASTN algorithm (e-value less than 1e-5) in the Blast software. Sequences with an alignment rate greater than 50% were retained using the command tblastn -query APDeu.fa -db Conus.gene.cds -outfmt 6 -evalue 1e-5 -out APDeu.fa.m8. Then log in to the Windows visualization software AMPml software (2021, China, 2021SR0424886, https: / / github.com / flystar233 / AMPml) to upload the potential AMP precursor gene file, select the CTDD and PAAC modules, perform activity prediction on the segments containing potential antimicrobial peptide protein sequences in the retained sequences, and screen out the alignment sequences with a value greater than 0.5 in the CTDD and PAAC models.

[0063] Using the same alignment method as above, antimicrobial peptide precursor genes were predicted for the transcript datasets of the five transcriptomes.

[0064] Subsequently, an index library was constructed using the common antimicrobial peptide precursor proteins in the genomic and transcriptomic datasets as reference sequences. The proteomic data were aligned to this index library using Blastp software to obtain a final consensus set of antimicrobial peptide sequences across the three omics datasets. Sequences with an alignment length greater than 50% and an alignment rate greater than 80% were considered reliable.

[0065] Finally, after homology comparison and activity prediction, the DNA sequences of three potential antimicrobial peptide precursor genes were screened from genomic, transcriptomic, and proteomic datasets (shown as SEQ ID NOs. 1 to 3). Based on codon encoding rules, these antimicrobial peptide precursor genes were translated into corresponding precursor proteins, whose amino acid sequences are shown in SEQ ID NOs. 4 to 6, respectively.

[0066] Example 2 Prediction of the active structure of conus antimicrobial peptides

[0067] This study aimed to predict the active structures of cone snail antimicrobial peptides. By referencing known antimicrobial peptide sequences in the public antimicrobial peptide database APD3, we designed potential mature active peptide regions from the screened cone snail antimicrobial peptide precursor proteins. The resulting amino acid sequences of three barrel-shaped cone snail antimicrobial peptides are shown in SEQ ID NOs. 7 to 9, designated UBI_31-38, YFGAP-CB, and YFGAP-CB, respectively.

[0068] The ProtParam tool on the Expasy website (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical properties of the three antimicrobial peptides obtained from barrel-shaped cone snails. The physicochemical properties included molecular weight, isoelectric point, net charge value, and average hydrophilicity. The results are shown in Table 1.

[0069] Table 1. Prediction of physicochemical properties of three antimicrobial peptides from Conus barreli

[0070]

[0071] To ensure a high probability of peptide antimicrobial activity, the designed peptides all met the following conditions: a. the peptide was a positively charged cation; b. the isoelectric point was greater than 8.0; c. the CTDD and PAAC values ​​were greater than 0.5; d. it had an α-helical and / or β-pleated structure; e. the number of cysteines in the sequence was less than 5; and f. the sequence length did not exceed 30 amino acids.

[0072] Then, the three-dimensional structures of the three peptides were constructed using the software PEP-FOLD3, which is suitable for constructing the three-dimensional structure of short peptides. Figure 1 Based on the construction of the three-dimensional model, the structural characteristics of the three synthesized antimicrobial peptides are as follows: 1) UBI_31-38 has an α-helical structure; 2) YFGAP-CB has a mixed structure of α-helical and β-sheet; 3) CcAMP1-CB has a β-sheet structure.

[0073] Example 3 Activity test of cone snail antimicrobial peptides

[0074] First, the predicted conus antimicrobial peptide structure was synthesized using a standard amino acid peptide solid phase synthesis method. In this example, the chemical synthesis of the three antimicrobial peptides was custom-made by Shanghai Jier Biochemical Co., Ltd.

[0075] The synthesized peptide product was purified and its purity was tested. First, the synthesized peptide product was purified using a high performance liquid chromatography system (HPLC), and then eluted with an acetonitrile gradient of 1 mL / min. The results are as follows Figure 2 、 4, 6; then HPLC-MS / MS was used to determine the purity of UBI_31-38, CcAMP1-CB and YFGAP-CB polypeptide powders, and the results were as follows Figure 3 、 5 , 7. As can be seen, the purity of the synthesized peptides was above 95%, and the molecular weights of the synthesized linear peptides were 1090.28, 2036.31, and 3209.70 Daltons (Da), respectively. Finally, each peptide was stored in sterile deionized water at -80°C for activity testing.

[0076] In this example, the activity of three synthesized antimicrobial peptides was tested, and the specific operation was as follows:

[0077] First, prepare a PBS peptide solution. Dissolve 2 mg of each synthesized peptide in phosphate buffered saline (PBS) and perform multiple gradient dilutions to prepare peptide solutions of various concentrations for later use.

[0078] Among them, the concentrations of the prepared UBI_31-38 polypeptide PBS solutions include 917 μM, 458 μM, 229 μM, 114 μM, 57 μM, and 29 μM; the concentrations of the prepared YFGAP-CB polypeptide PBS solutions include 491 μM, 245 μM, 123 μM, 61.4 μM, 30.7 μM, and 15.4 μM; the concentrations of the prepared CcAMP1-CB polypeptide PBS solutions include 312 μM, 156 μM, 78 μM, and 39 μM, which were used for antibacterial experiments, with only microorganisms (0 μM), PBS buffer (PBS), and only polypeptides (indicated by the polypeptide name) as control groups.

[0079] In this example, the activity of three synthesized antimicrobial peptides was tested using experimental Escherichia coli and Pichia pastoris. Each strain was pretreated as follows: Each strain was cultured until the logarithmic growth phase, centrifuged, and washed three times with PBS to obtain a precipitate. All microbial precipitates were resuspended in PBS buffer (10⁴ CFU / mL) to obtain a test suspension for later use.

[0080] Bacterial inhibition test: Mix 10µL of the test bacterial suspension and 10µL of the peptide solution in PBS in a 200µL tube and incubate at 37°C for 2 hours. The incubation mixture is then transferred to a 96-well microtiter plate, with each well containing 200µL of LB medium. All wells of the microtiter plate are then placed in a 37°C incubator for continued incubation. During this time, OD600 values ​​are measured every half hour for a total of 20 hours (bacteria) or 48 hours (fungi) to generate growth curves for the test microorganisms. Three replicate wells are set for each peptide, and the experiment is repeated at least twice to obtain reliable results.

[0081] All data were statistically analyzed using SPSS (Statistical Package for Social Sciences) and GraphPadPrism software and expressed as mean ± standard deviation (n = 3). Paired sample t-test and multiple comparison methods were used to test the significance of differences between groups. The results are shown in Table 2 and Figures 8 to 10 shown

[0082] Table 2. Antimicrobial activities of three barrel-shaped cone snail antimicrobial peptides containing double α-helical structures

[0083]

[0084] Identification revealed that all three peptides exhibited significant antibacterial activity. UBI_31-38 and CcAMP1-CB both inhibited the growth of the fungus Pichia pastoris, while YFGAP-CB exhibited inhibitory activity against Escherichia coli. Furthermore, at a high peptide concentration (917 μM) in PBS, the antibacterial effect of UBI_31-38 gradually increased after 30 hours of incubation, demonstrating long-lasting antibacterial activity. In the CcAMP1-CB antibacterial assay, antibacterial activity was observed at all peptide concentrations after 30 hours of incubation, with the highest concentrations (491 μM and 245 μM) showing a particularly pronounced antibacterial effect after 40 hours of incubation, demonstrating a positive correlation between antibacterial activity and concentration. YFGAP-CB exhibited excellent inhibition of E. coli growth at all concentrations after 10 hours of incubation. Therefore, it was verified that the three peptides identified and screened from the barrel-shaped cone snail have different antifungal and antibacterial abilities and can be regarded as three new antimicrobial peptides. Three new cone snail antimicrobial peptides were also successfully obtained.

[0085] The three structurally different barrel-shaped cone snail antimicrobial peptides can be used to prepare products with antibacterial activity, including various bactericidal preparations, or used to prepare products that can replace antibiotics. They are green and safe and have good industrial production value and economic value.

[0086] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from all perspectives. In addition, it should be understood that although this specification is described in terms of implementation methods, it does not contain only one technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should read the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Cone antimicrobial peptide, characterized in that: The antimicrobial peptide is based on the nucleic acid sequence shown in SEQ ID NO.1, which is translated into a precursor protein sequence shown in SEQ ID NO.4 according to codon coding rules, and then the antimicrobial activity domain is artificially screened and designed to obtain the amino acid sequence shown in SEQ ID NO.7, which is then synthesized by chemical methods.

2. A method for preparing a cone snail antimicrobial peptide, characterized in that: The method is used to prepare the cone snail antimicrobial peptide according to claim 1; the specific steps are as follows: 1) Selecting a cone snail multi-omics dataset, wherein the multi-omics dataset includes a cone snail genome, transcriptome, and venom proteome dataset; 2) screening the cone snail antimicrobial peptide precursor gene sequence from the multi-omics dataset through homology comparison and activity simulation prediction, as shown in SEQ ID NO. 1; 3) translating the screened antimicrobial peptide precursor gene sequence into the corresponding cone snail antimicrobial peptide precursor protein sequence, as shown in SEQ ID NO.4; 4) referring to antimicrobial peptide fragments with known antimicrobial activity, the potential mature active peptide region of the cone snail antimicrobial peptide precursor protein was designed to obtain a polypeptide with the amino acid sequence shown in SEQ ID NO.7; 5) Synthesize the polypeptide represented by SEQ ID NO. 7 using a chemical synthesis method; 6) Identify the antibacterial effect of the synthesized polypeptide, thereby obtaining cone snail polypeptides with antibacterial activity, namely cone snail antimicrobial peptides.

3. The method for preparing the conus antimicrobial peptide according to claim 2, wherein: In step 4), the polypeptide obtained by designing the potential mature active peptide region of the cone snail antimicrobial peptide precursor protein meets the following conditions: a. It is a positively charged cation; b. Its isoelectric point is greater than 8.0; c. Its CTDD and PAAC values ​​are greater than 0.5; d. It has α-helices and / or β-sheets; e. The number of cysteines in its amino acid sequence is less than 5; f. Its amino acid sequence length does not exceed 30 amino acids.

4. The method for preparing the conus antimicrobial peptide according to claim 2, wherein: In step 6), the antibacterial effect of the synthesized polypeptide is identified by mixing the synthesized polypeptide with different microorganisms including experimental Escherichia coli and Pichia pastoris to verify its antibacterial activity.

5. The application of cone snail antimicrobial peptide is characterized by: The use of the cone snail antimicrobial peptide according to claim 1 in the preparation of a product with antimicrobial activity; the product with antimicrobial activity can specifically inhibit the growth of Pichia pastoris and / or Escherichia coli.

6. A preparation for sterilization, characterized in that: The preparation contains the cone antimicrobial peptide according to claim 1 as an active ingredient.

7. The bactericidal preparation according to claim 6, characterized in that: The preparation contains the cone snail antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO. 7, with a concentration of 917 μM.

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