CN116024223A

CN116024223A - Conus alpha helix and beta sheet antibacterial peptide, and preparation method and application thereof

Info

Publication number: CN116024223A
Application number: CN202211581485.9A
Authority: CN
Inventors: 李蕊晗; 黄玉; 游欣欣; 石琼
Original assignee: Huizhou Dabaihui Modern Fisheries Application Research Institute
Current assignee: Huizhou Dabaihui Modern Fisheries Application Research Institute; Shenzhen Bgi Marine Research Institute
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-04-28

Abstract

The invention discloses alpha-helix and beta-sheet antibacterial peptide of conoid, a preparation method and application thereof, wherein three antibacterial peptide precursor gene nucleic acid sequences are screened from a plurality of sets of barreled conoid data, and are shown as SEQ ID NO.1 to SEQ ID NO. 3; translating the three nucleic acid sequences into amino acid sequences, and referring to the known antibacterial peptide sequences to design peptide sequences of antibacterial active areas to obtain polypeptides with three amino acids shown in SEQ ID NO.7 to SEQ ID NO. 9; and then, three conoid antibacterial peptides are obtained by direct synthesis through a chemical method. Proved by verification, the three conomical antibacterial peptides obtained by the invention can specifically inhibit the growth of pichia pastoris or escherichia coli, have good antibacterial activity, can be applied to prepare products with antibacterial activity, and have good application prospect and economic value.

Description

Conus alpha helix and beta sheet antibacterial peptide, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to alpha helix and beta sheet antibacterial peptide of conoids, and a preparation method and application thereof.

Background

In recent years, problems of bacterial resistance have become more serious due to abuse of conventional antibiotics, and finding new antibiotics has become more difficult. The antibacterial peptide (antimicrobial peptide) is a polypeptide substance with antibacterial activity, which is generated by induction in animals and plants, is host defense peptide, has the characteristics of strong alkalinity, thermal stability, broad-spectrum antibacterial property and the like, and is a biological resource with the most potential to replace antibiotics at present. The antibacterial mechanism of the antibacterial peptide is as follows: most of the antibacterial peptides are cationic (positively charged) and amphiphilic (hydrophilic and hydrophobic) alpha helix/beta sheet peptide molecules, and based on membrane permeability, the cationic antibacterial peptides can be combined with negatively charged cell membranes and interact with the negatively charged cell membranes to change electrochemical potential on the cell membranes, so that the cell membranes are induced to be damaged to permeate larger molecules such as proteins, and cell morphology and cell membranes are damaged, and finally cell death is caused. Therefore, compared with the traditional antibiotics, the traditional Chinese medicine composition is difficult to generate drug resistance and has good development prospect as a therapeutic drug.

Currently, it has been reported that over 3000 antimicrobial peptides have been identified from animals, fungi, plants and bacteria, such as: 15 venom oligopeptide sequences from 5 ants were reported to have antibacterial activity; 13 venom oligopeptide sequences from 7 bees were reported to have antibody activity; 10 venom oligopeptide sequences from 9 snakes were reported to have antibody activity; 55 active antibacterial peptides were found from more than 30 centipedes.

Conus majoris LCone Snail) The chicken heart snail is a meat-type marine gastropoda and is known for its beautiful special toxins secreted from the outer shell and the inner body. More than 1000 kinds of conus are currently registered in the book, and the conus prey on worms through venom,Snails or fish, etc. The conotoxin has novel chemical structure, strong biological activity and high targeting selectivity, so that the conotoxin is also an attractive new medicine resource. Over the last few decades, over 70,000 natural conotoxins have been found for widespread use in pharmacological and neuroscience research.

However, compared with animals such as worms, snails and fishes, the antibacterial function of the conomical polypeptides is less studied, only a few related conomical polypeptide sequences are reported to have the antibacterial function at present, and the antibacterial application research of the conomical antibacterial peptides is very rare. For example, one of the studies reported that a connoxin from the cono 1 superfamily could inhibit the growth of Mycobacterium tuberculosis and the other reported that an open-chain MVIIA structure from the cono omega-connoxin could inhibit the growth of fungi. In addition, most of the existing antibacterial peptides are obtained through blind screening, for example, the antibacterial peptides are directly separated and extracted from organisms through experimental means, or the obtained antibacterial peptide gene sequences are cloned and then introduced into microorganisms for culturing, and then the antibacterial peptides are further separated, purified and identified through a series of experimental means. For example, the preparation method of the marine organism antibacterial peptide disclosed in the Chinese patent document CN115232850A comprises the steps of freeze-drying and crushing marine organism materials, then carrying out enzymolysis, and separating and extracting the antibacterial peptide; in vitro recombinant expression and identification of novel American eel antibacterial peptide published by Chinese patent document CN115057918A, cloning an antibacterial domain gene of bactericidal protein from the American eel, constructing an expression vector, introducing into escherichia coli for expression, and further identifying after chromatographic purification by using a chromatographic purification column. The method has the advantages of more experimental procedures, complicated operation, long time consumption, low efficiency and high cost, and is not beneficial to development, research and application of the antibacterial peptide.

Therefore, how to quickly and effectively deeply dig the antibacterial function of the cono polypeptides and obtain more cono antibacterial peptides, provides more choices for replacing the traditional antibiotic therapeutic drugs, and has important research significance and potential market value.

Disclosure of Invention

To solve the problems and achieve the aboveThe invention provides alpha helix and beta sheet antibacterial peptide of conoid, and a preparation method and application thereof, and combines with bioinformatics technology to prepare the antibacterial peptide from the conoid of barrel shapeConus betulinus) And (3) screening three antibacterial peptide precursor gene nucleic acid sequences in a plurality of groups of chemical data sets, manually screening and designing antibacterial activity areas of the conoid antibacterial peptide according to amino acid sequences translated from the three nucleic acid sequences, performing chemical synthesis, and obtaining three novel conoid antibacterial peptides after antibacterial activity verification and identification. The specific technical scheme is as follows:

first, the invention provides a method for producing the cone-shaped conoidConus betulinus) The nucleotide sequence of the three conoid antimicrobial peptide precursor genes screened by the multiple sets of the data sets is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

Secondly, the invention provides cono antimicrobial peptide precursor proteins based on the three cono antimicrobial peptide precursor genes, the amino acid sequences of which are determined according to codon coding rules and are respectively as follows:

the amino acid sequence of the cono antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.1 is shown as SEQ ID NO. 4;

the amino acid sequence of the cono antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.2 is shown as SEQ ID NO. 5;

the amino acid sequence of the cono antimicrobial peptide precursor protein determined based on the nucleic acid sequence of SEQ ID NO.3 is shown as SEQ ID NO. 6.

Thirdly, according to the determined conoid antibacterial peptide precursor protein amino acid sequence, combining with a known antibacterial peptide antibacterial activity polypeptide fragment, artificially screening and designing a conoid antibacterial peptide bacterial activity domain to obtain a conoid antibacterial peptide amino acid sequence, and synthesizing the conoid antibacterial peptide by a chemical method, wherein the designed conoid antibacterial peptide amino acid sequence is as follows:

the amino acid sequence of the conoid antibacterial peptide designed based on the conoid antibacterial peptide precursor protein with the amino acid sequence of EQ ID No.4 is shown in SEQ ID No. 7;

the amino acid sequence of the conoid antibacterial peptide designed based on the conoid antibacterial peptide precursor protein with the amino acid sequence of SEQ ID NO.5 is shown as SEQ ID NO. 8;

the amino acid sequence of the conoid antibacterial peptide designed based on the conoid antibacterial peptide precursor protein with the amino acid sequence of SEQ ID NO.6 is shown as SEQ ID NO. 9.

The invention further provides a preparation method of the conomical antibacterial peptide, which is used for preparing any one of the conomical antibacterial peptides; the method comprises the following specific steps:

1) Selecting multiple sets of conoid data, wherein the multiple sets of conoid data comprise a conoid genome, a transcriptome and a venom proteome data set;

2) Screening conoid antimicrobial peptide precursor gene sequences from the multiple sets of chemical data through homology comparison and activity simulation prediction, wherein the conoid antimicrobial peptide precursor gene sequences are shown as SEQ ID NO. 1-SEQ ID NO. 3;

3) Translating the screened antibacterial peptide precursor gene sequence into a corresponding conoid antibacterial peptide precursor protein sequence, wherein the sequences are shown in SEQ ID NO. 4-SEQ ID NO. 6;

4) Designing a potential mature active peptide region of the cono antimicrobial peptide precursor protein by referring to the antimicrobial peptide polypeptide fragments with known antimicrobial activity to obtain polypeptides with amino acid sequences shown as SEQ ID NO. 7-SEQ ID NO. 9;

5) Synthesizing the polypeptides shown in SEQ ID NO. 7-SEQ ID NO.9 by adopting a chemical synthesis method;

6) And (3) identifying the antibacterial effect of the synthesized polypeptide, thereby obtaining the cono polypeptide with antibacterial activity, namely the cono antibacterial peptide.

As a preferred technical scheme, in step 4), the polypeptide obtained by designing the potentially mature active peptide region of the conomical antimicrobial peptide precursor protein meets the following conditions:

a. which 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. having an alpha helix and/or a beta sheet;

e. the number of cysteines in the amino acid sequence is less than 5;

f. the amino acid sequence is not longer than 30 amino acids.

In step 6), the antibacterial effect of the identified synthesized polypeptide is that the synthesized polypeptide is mixed and cultured with different microorganisms including experimental escherichia coli and pichia pastoris, and the antibacterial activity of the synthesized polypeptide is verified.

In addition, the invention also provides application of the conomical antibacterial peptide, namely application of any one or more of the conomical antibacterial peptides in preparation of products with antibacterial activity.

Preferably, the product with antibacterial activity is capable of specifically inhibiting the growth of pichia pastoris and/or escherichia coli.

In addition, the invention provides a preparation for sterilization, which contains any one or more of the conomical antibacterial peptides as the active ingredient.

Preferably, the preparation contains the conomical peptide with the concentration of 917 mu M and the amino acid sequence shown in SEQ ID NO. 7; or contains conoid antibacterial peptide with the amino acid sequence shown in SEQ ID NO.8 and the concentration of 245 mu M-491 mu M, preferably 245 mu M and 491 mu M; or contains conoid antibacterial peptide with the amino acid sequence shown as SEQ ID NO.9 at the concentration of 39 mu M-312 mu M, preferably 78 mu M, 39 mu M, 156 mu M and 312 mu M.

The invention has the beneficial effects that:

1) The invention digs out three kinds of conomical antibacterial peptides from the conomical data set, discovers the effect of resisting two different fungi and bacteria for the first time, emphasizes the antibacterial potential of the conomical polypeptide, wherein UBI_31-38 and cAMP1-CB can inhibit the growth of fungus pichia pastoris, YFGAP-CB shows the inhibiting activity on escherichia coli, enriches the variety and antibacterial variety of the conomical antibacterial peptides, and is expected to be applied to prepare products with antibacterial activity and replace antibiotics for treating fungus or bacterial infection in clinic, thereby having good application prospect and economic value.

2) According to the invention, a multi-group data set (genome, transcriptome and proteome) mining technology is adopted for the first time, a plurality of antibacterial peptide sequences are designed from the conoid multi-group data set in a one-time mining way, and the antibacterial peptide proteins are directly synthesized by a chemical method, so that the problems of blind screening, low efficiency and high cost in the traditional antibacterial peptide screening process are effectively solved, and a new research method is provided for development and utilization of conoid genetic resources.

3) The method combines bioinformatics technology, directly screens potential target sequences from a biological big data layer, combines activity simulation prediction and in-vitro experiment verification, provides a new thought for deep development of precious biological genetic resources, and provides a solid theoretical basis for applying novel marine antibacterial drugs to clinical treatment.

Description of the drawings:

FIG. 1 is a three-dimensional modeling diagram of three structurally different antimicrobial peptides of the barrel-shaped conoids.

FIG. 2 is a high performance liquid chromatogram of the synthesized barrel-shaped cono UBI_31-38 antimicrobial peptide.

FIG. 3 is a mass spectrum of the synthesized antibacterial peptide UBI_31-38.

FIG. 4 is a high performance liquid chromatogram of the synthesized barrel-shaped cono cAMP1-CB antimicrobial peptide.

FIG. 5 is a mass spectrum of the synthesized barrel-shaped cono cAMP1-CB antimicrobial peptide.

FIG. 6 is a high performance liquid chromatogram of the synthesized barrel-shaped cono YFGAP-CB antimicrobial peptide.

FIG. 7 is a mass spectrum of the synthesized antibacterial peptide YFGAP-CB of the cone-shaped cono.

FIG. 8 is a graph showing the effect of the antibacterial peptide UBI_31-38 on inhibiting the growth of Pichia pastoris.

FIG. 9 is a graph showing the effect of the antibacterial peptide CcAMP1-CB on the activity test of inhibiting the growth of Pichia pastoris.

FIG. 10 is a graph showing the effect of the antibacterial peptide YFGAP-CB on inhibiting the growth of Pichia pastoris.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments.

The abbreviations used in the full-length amino acid sequences of the present invention are abbreviations 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).

Example 1 is a precursor gene DNA of cono antimicrobial peptide and the sequence encoding the precursor protein; example 2 is a conomical antibacterial peptide activity structure prediction; example 3 is an activity assay of conomical antimicrobial peptides.

EXAMPLE 1 preparation of precursor Gene DNA and the sequence encoding the precursor protein of Conus antibacterial peptide

This example selects Chinese barrel-shaped conoids from three markets in Hainan provinceConus betulinus) As an experimental study object. And identifying the encoding sequence of the antimicrobial peptide precursor gene from a plurality of sets of chemical data sets of the barrel-shaped conoids by adopting a homology comparison method, and translating the encoding sequence into an antimicrobial peptide precursor protein sequence. The specific process is as follows:

1) Multiple study data set preparation

The multiple sets of mathematical data in this example include genomic, transcriptomic, and venom proteomic data sets of the barrel conch.

Wherein the NCBI accession number of the genome dataset is: gca_016801955.1.

The transcriptome dataset included transcriptome data for 5 samples, respectively: big: a poison tube of an individual conoid with a body length of 10 cm; middle: a poison tube of a medium-sized individual conoid with a body length of 8.7 cm; small: a poison tube of a small individual conoid with a body length of 6 cm; bulb: the toxic capsule of the medium-sized individual conoids; normalized: the above-mentioned homogenized dataset of medium individual conotoxin tubes. The NCBI SRA database accession number for the transcriptome dataset is: SRS1009725 through SRS1009729.

The PRIDE database accession number for the venom protein dataset is: PXD014892.

The protein coding region of each transcript was predicted using TransDecoder software and the transcript level of the gene was calculated using FPKM values. All protein coding sequences from the genome and transcriptome are translated into amino acids, creating a comprehensive protein set of the barrel-shaped conoids.

2) Screening of antimicrobial peptide precursor Gene DNA sequences from multiple sets of chemical data

2927 verified antibacterial peptides in the public antibacterial peptide database APD3 are used as reference sequences. Firstly, establishing an index library for sequence comparison by using makeblastdb in Blast software package by utilizing genes annotated by barrel-shaped conoid genome, wherein the command is makeblastdb-dbtype nucleic-parameter_seqids-in Conus.gene.cds. The potential AMP precursor genes were then predicted from the genome by using TBLASTN algorithm (e value less than 1 e-5) in Blast software, with sequences with alignment ratios greater than 50% retained, commanded as TBLASTN-query APDeu.fa-db Conus.gene.cds-outfmt 6-value 1 e-5-out APDeu.fa.m8. And logging in windows visualization software AMPml software (2021, china,2021S R0424886, https:// gitub.com/flystar 233/AMPml) to upload potential AMP precursor gene files, selecting a CTDD and PAAC module, carrying out activity prediction on a section containing potential antibacterial peptide protein sequences in the reserved sequences, and screening out alignment sequences larger than 0.5 in the CTDD and PAAC models.

The same comparison method as described above was used to predict antimicrobial peptide precursor genes for the transcript datasets of the five transcriptomes.

An index library is then created using the antimicrobial peptide precursor protein common to both the genomic and transcriptome datasets as a reference sequence. The proteome data are compared to the index library by using Blastp software, and the final consensus antibacterial peptide sequence set of three histology data sets is obtained, wherein the sequences with the comparison length of more than 50% and the comparison rate of more than 80% are reliable sequences.

Finally, three potential antimicrobial peptide precursor genes are obtained through screening from genome, transcriptome and proteome data sets after homology comparison and activity simulation prediction, and DNA sequences are shown in SEQ ID NO.1 to SEQ ID NO. 3. According to the code rule, it is determined that the antimicrobial peptide precursor gene is translated into corresponding precursor protein, and the amino acid sequences of the precursor protein are respectively shown in SEQ ID NO.4 to SEQ ID NO. 6.

Example 2 prediction of the Activity Structure of Conus antibacterial peptides

The implementation is to predict the active structure of the conoid antibacterial peptide. The potential mature active peptide region of the conoid antimicrobial peptide precursor protein obtained by screening is designed by referring to known antimicrobial peptide polypeptide sequences in a public antimicrobial peptide database APD3, and the amino acid sequences of the three barrel-shaped conoid antimicrobial peptides obtained finally are shown in SEQ ID NO.7 to SEQ ID NO.9 and are respectively named UBI_31-38, YFGAP-CB and YFGAP-CB.

The physical and chemical properties of the three antibacterial peptides of the resulting barrel-shaped conoids, including molecular weight, isoelectric point, net charge value and average value of hydrophilicity, were predicted using the ProtParam tool of the Expasy website (https:// web. Expasy. Org/protParam /), and the results are shown in Table 1.

TABLE 1 prediction of physicochemical Properties of three antibacterial peptides of Conus pubescens

In order to ensure that the polypeptide has high possibility of having antibacterial activity, the designed polypeptide meets the following conditions: a. the polypeptide is a positive-charged cation; b. isoelectric point is greater than 8.0; c. CTDD and PAAC values greater than 0.5; d. having an alpha helical and/or beta sheet structure; e. the number of cysteines in the sequence is less than 5; f. the sequence is no more than 30 amino acids in length.

And then constructing three-dimensional structures of the three polypeptides by utilizing software PEP-FOLD3 suitable for constructing the three-dimensional structures of the short peptides, as shown in figure 1. Based on the construction of a three-dimensional model, three synthetic antibacterial peptides have the following structural characteristics: 1) Ubi_31-38 has an alpha helical structure; 2) YFGAP-CB has a mixed structure of alpha helices and beta sheets; 3) ccAMP1-CB has a beta sheet structure.

Example 3 Activity assay of Conus antibacterial peptide

Firstly, synthesizing the complete sequence of the conoid antibacterial peptide structure according to the predictive design by adopting a standard amino acid polypeptide solid-phase synthesis method. In this example, the chemical synthesis of the three antimicrobial polypeptides was custom-made by Shanghai Jier Biochemical Co.

The synthesized polypeptide product was purified and its purity was checked. The synthesized polypeptide product is purified by a High Performance Liquid Chromatography (HPLC) system, and then eluted by a gradient of 1mL/min acetonitrile, and the results are shown in figures 2, 4 and 6; the purity of the UBI_31-38, cAMP1-CB and YFGAP-CB polypeptide powders was then determined using HPLC-MS/MS, and the results are shown in FIGS. 3, 5, and 7. It can be seen that the purity of the synthesized polypeptides is above 95%, and the molecular weights of the synthesized linear peptides are 1090.28, 2036.31 and 3209.70 daltons (Da), respectively. Finally, each polypeptide is stored in sterile deionized water at-80 ℃ for activity test.

In this example, activity test was performed on three synthetic antimicrobial polypeptides, and the specific procedures were as follows:

first, a PBS polypeptide solution was prepared. And 2mg of each synthesized polypeptide is dissolved in phosphate buffer PBS, and subjected to gradient dilution for a plurality of times to prepare PBS polypeptide solutions with a plurality of concentrations for later use.

Wherein, the concentration of the prepared UBI_31-38 polypeptide PBS solution comprises 917 mu M, 458 mu M, 229 mu M, 114 mu M, 57 mu M and 29 mu M; the prepared YFGAP-CB polypeptide PBS solution comprises 491 mu M, 245 mu M, 123 mu M, 61.4 mu M, 30.7 mu M and 15.4 mu M; the prepared ccAMP1-CB polypeptide PBS solution concentrations include 312. Mu.M, 156. Mu.M, 78. Mu.M, 39. Mu.M, and are used for bacteriostasis experiments with only microorganisms (0. Mu.M), PBS Buffer (PBS), and only polypeptides (expressed as polypeptide names) as control groups.

In the embodiment, experimental escherichia coli and pichia pastoris are adopted to perform activity detection on three synthetic antibacterial polypeptides. Each strain is pretreated as follows: culturing each strain, centrifuging after each strain is cultured to logarithmic phase, eluting for 3 times by using PBS, and collecting precipitate; all obtained microbial pellets were resuspended in PBS buffer (104 CFU/mL) to obtain a detection bacterial suspension for later use.

Bacteriostasis test: mixing 10 mu L of detection bacterial suspension and 10 mu L of PBS polypeptide solution sample in a 200 mu L tube, and standing and culturing at 37 ℃ for 2 hours. The cultured mixture was then transferred to 96-well enzyme-labeled wells, each well containing 200. Mu.L of LB medium. All the wells of the ELISA plate were placed in a microorganism incubator at 37℃for continuous cultivation. During this period, OD600 values were measured every half hour for a total of 20 hours (bacteria) or 48 hours (fungi) for plotting the growth curve of the detected microorganisms. Three parallel wells were set up for each polypeptide and the experiment was repeated at least twice to obtain reliable results.

All data were counted using SPSS (Statistical Package for Social Sciences) and GraphPad Prism software and expressed as mean ± standard deviation (n=3). The significance of the differences between groups was tested using paired sample t-test and multiple comparisons. The results are shown in Table 2 and FIGS. 8 to 10

TABLE 2 antibacterial Activity of three barrel-shaped Conus antibacterial peptides containing double alpha helical structures

The identification shows that all three polypeptides show a certain antibacterial and bacteriostatic effect, wherein, UBI_31-38 and cAMP1-CB can inhibit the growth of fungus Pichia pastoris, and YFGAP-CB shows inhibitory activity on escherichia coli. And UBI_31-38 shows long-term antibacterial property after culturing for 30 hours under the condition of the concentration of the polypeptide PBS solution (917 mu M) at high concentration and gradually strengthening the antibacterial effect; in the ccAMP1-CB antibacterial experiment, after the culture is carried out for 30 hours, the antibacterial effect of each polypeptide PBS solution has an antibacterial effect at the concentration, and after the culture is carried out for 40 hours in an experimental group at high concentration (491 mu M and 245 mu M), the antibacterial effect is obvious, and the antibacterial effect and the concentration of the antibacterial effect are proved to have a certain positive correlation. YFGAP-CB shows excellent ability to inhibit the growth of E.coli at each concentration after 10 hours of culture. Therefore, the three polypeptides identified and screened in the barrel-shaped conoids are verified to have different antifungal and antibacterial capacities, and can be regarded as three novel antibacterial peptides, and three novel conoids are also successfully obtained.

The three structurally different barrel-shaped conomical peptides can be applied to preparing products with antibacterial activity, including various bactericidal preparations or products capable of replacing antibiotics, and have the characteristics of green and safe properties and good industrial production value and economic value.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the embodiments are to be considered in all respects as illustrative and not restrictive. Furthermore, it should be understood that, although the present disclosure describes embodiments, this description is not intended to include only one embodiment, and those skilled in the art should understand that the present disclosure is not limited to the embodiments described herein, and that the embodiments described in the examples may be combined appropriately to form other embodiments that will be understood by those skilled in the art.

Claims

1. The conoid antibacterial peptide precursor gene is characterized in that: the nucleic acid sequence is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

2. The conoid antibacterial peptide precursor protein is characterized in that: the nucleic acid sequence of which is the cono antimicrobial peptide precursor gene according to claim 1, determined based on the codon encoding rule:

3. The conoid antibacterial peptide is characterized in that: the antibacterial peptide is obtained by manually screening and designing an antibacterial activity domain based on the amino acid sequence of the conoid antibacterial peptide precursor protein of claim 2 to obtain a conoid antibacterial peptide sequence and synthesizing the conoid antibacterial peptide sequence by a chemical method; the conoid antibacterial peptide sequence obtained by manual screening design is as follows:

4. A preparation method of conoid antibacterial peptide is characterized by comprising the following steps: the method is used for preparing any one of the conomical antimicrobial peptides in claim 3; the method comprises the following specific steps:

5. The method for preparing conomical peptide according to claim 4, wherein: in the step 4), the polypeptide obtained by designing the potential mature active peptide region of the conoid antimicrobial peptide precursor protein meets the following conditions:

a. which 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. having an alpha helix and/or a beta sheet;

e. the number of cysteines in the amino acid sequence is less than 5;

f. the amino acid sequence is not longer than 30 amino acids.

6. The method for preparing conomical peptide according to claim 4, wherein: in the step 6), the antibacterial effect of the identified synthesized polypeptide is that the synthesized polypeptide is mixed and cultured with different microorganisms including experimental escherichia coli and pichia pastoris, and the antibacterial activity of the synthesized polypeptide is verified.

7. The application of the conoid antibacterial peptide is characterized in that: use of any one or more cono antimicrobial peptides according to claim 3 for the preparation of a product with antimicrobial activity.

8. The use according to claim 7, characterized in that: the product with antibacterial activity can specifically inhibit the growth of pichia pastoris and/or escherichia coli.

9. A formulation for use in sterilization, characterized in that: the preparation contains any one or more conomical antibacterial peptides of claim 3 as an active ingredient.

10. The bactericidal formulation of claim 9 wherein: the preparation contains conoid antibacterial peptide with the concentration of 917 mu M and the amino acid sequence shown as SEQ ID NO. 7; or contains the conoid antibacterial peptide with the concentration of 245 mu M to 491 mu M and the amino acid sequence shown as SEQ ID NO. 8; or contains conoid antibacterial peptide with the concentration of 39 mu M-312 mu M and the amino acid sequence above as shown in SEQ ID NO. 9.