CN111087449B

CN111087449B - Antibacterial peptide and preparation method and application thereof

Info

Publication number: CN111087449B
Application number: CN201911413001.8A
Authority: CN
Inventors: 戴鼎震; 陈俊红; 陈涛; 蒋加进; 方光远; 陆雨楠; 沙奕羽; 黄翔宇
Original assignee: Jinling Institute of Technology
Current assignee: Jinling Institute of Technology
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-05-14
Anticipated expiration: 2039-12-31
Also published as: CN111087449A

Abstract

The invention discloses an antibacterial peptide and a preparation method and application thereof, wherein the antibacterial peptide comprises an amino acid sequence: RRWQWRGSGRWQWRR are provided. It solves the following problems: (1) most of the existing antibiotic medicines generate drug resistance and can not effectively kill pathogenic bacteria; (2) the antibacterial peptide has limited natural resources, low antibacterial spectrum, limited yield and high preparation cost; (3) the antibacterial activity of the protein sample is improved. (4) The antibacterial activity of the polypeptide can be obviously improved by combining with other antibacterial preparations, so that the problems of high cost or environmental pollution of the existing polypeptide preparation technology are solved. The invention comprises an antibacterial peptide amino acid sequence and application thereof, and the product prepared by the invention can be used for preparing products resisting gram negative bacteria and gram positive bacteria and producing veterinary drugs, feed additives and preservatives.

Description

Antibacterial peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide, and a preparation method and application thereof.

Background

Antimicrobial peptides (AMPs) are also called host defense peptides, and are natural or artificially synthesized polypeptide substances with small molecular weight and certain antibacterial and immune effects. The special amino acid composition of the microbial cell makes the microbial cell have proper hydrophobicity and charge, so that the microbial cell can be combined with negatively charged substances on the surface of pathogenic microorganisms to further destroy the structure of a cell membrane, or can enter the interior of the cell to be combined with macromolecular substances such as nucleic acid, protein and the like, and finally the normal physiological function of the cell is disturbed and the cell is killed. The mechanism of action of antimicrobial peptides can be divided into two categories: membrane damage and intracellular damage. Among these, various factors such as amino acid sequence, lipid membrane, and peptide concentration control the biological activity of AMPs.

Long-term abuse of antibiotics has led to the emergence of drug-resistant pathogens such as superbacteria, viruses, fungi, and parasites, such that there is a lack of effective antibiotics for the treatment of these drug-resistant bacteria. The special action mechanism of the antibacterial peptide is different from that of antibiotics, so that drug-resistant pathogenic bacteria are not easily generated, and the antibacterial peptide is rapidly developed as a novel antibacterial drug.

The antibacterial peptide has the characteristics of thermal stability, good water solubility, no residue and difficult drug resistance generation, and different antibacterial peptides have different antibacterial effects on gram-negative bacteria and gram-positive bacteria such as escherichia coli, staphylococcus aureus, candida, pseudomonas aeruginosa, enterobacteria, serratia, proteus, streptococcus faecalis and the like. Therefore, the antibacterial peptide has wide application prospect in the product development fields of novel antibacterial drugs, food natural preservatives, animal feed additives and the like.

Many dietary protein molecules contain a large number of physiologically active peptides, which do not function in the original protein sequence, but can be released by digestion in the gastrointestinal tract or during food processing. The food protein-derived antibacterial peptide has the characteristics of high safety and good human body absorbability, can be directly added into food, and simultaneously has the in-vivo and in-vitro antibacterial effects.

The natural antibacterial peptide has limited resources, shows different antibacterial effects due to different amino acid sequences and molecular structures due to the limitation of the natural structure, has higher effective active dose, and has higher cost for the antibacterial peptide obtained by chemical synthesis. The polypeptide synthesized and prepared by the biotechnology method has the advantages of high activity and small influence of production on the environment. However, the prior art has the problems of low yield, high cost and the like when yeast and escherichia coli are used as genetic engineering bacteria for expression. And thus its practical application is greatly limited. Therefore, how to produce the antibacterial peptide in the microorganism conveniently, effectively and at low cost by using the genetic engineering technology becomes the key of the practical application.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing an antibacterial peptide aiming at the defects of the prior art.

The technical problem to be solved by the invention is to provide a preparation method of the antibacterial peptide.

The technical problem to be finally solved by the invention is to provide the application of the antibacterial peptide, thereby providing a novel, efficient, nontoxic, nuisanceless and good-stability antibacterial preparation and changing the abuse condition of the current antibiotics and antiseptics.

The invention idea is as follows: selecting a plurality of antibacterial peptides with antibacterial activity in an Antibacterial Peptide Database (APD) by a bioinformatics method, comparing lysozyme, lactoferrin and other food-borne proteins by utilizing a BLAST program, and searching a similar polypeptide sequence with higher homology in a food protein nucleic acid database. Based on the analysis of the sequence homology, the potential polypeptide sequence with antibacterial activity is found out according to the structural function relationship of the antibacterial peptide, and the antibacterial peptide sequence is further designed artificially.

In order to solve the technical problem, the invention discloses an antibacterial peptide which comprises an amino acid sequence shown as SEQ ID NO. 1; SEQ ID NO. 1: RRWQWRGSGRWQWRR are provided.

Preferably, any one or two combinations of hydrophilic amino acid, hydrophobic amino acid, positive charge amino acid and negative charge amino acid are respectively connected on two sides of the amino acid sequence of SEQ ID NO. 1.

More preferably, any one of the following as shown in formula I:

(Y)_a(N)_b-RRWQWRGSGRWQWRR-(X)_a(M)_b

(X)_a(N)_b-RRWQWRGSGRWQWRR-(Y)_a(M)_b

(Y)_a(M)_b-RRWQWRGSGRWQWRR-(X)_a(N)_b

(X)_a(M)_b-RRWQWRGSGRWQWRR-(Y)_a(N)_b

Ⅰ；

wherein, X is hydrophobic amino acid such as F, I, W, L, A; y is a hydrophilic amino acid such as S, G, Y, T; m is a positively charged amino acid, such as R or K; n is a negatively charged amino acid, such as D or E; a and b represent different lengths of 5 to 15, respectively.

The antibacterial peptide is characterized in that amino acid sequences with positive and negative charges and hydrophilicity and hydrophobicity are added on two sides of the antibacterial peptide row, so that the antibacterial peptide row is better suitable for antibacterial preparations, feeds and food preservative additives to be used in oily and aqueous environments, and the antibacterial activity and the effective absorption utilization rate of the antibacterial peptide row are enhanced.

The preparation method of the antibacterial peptide is also within the protection scope of the invention.

The preparation method is a solid phase polypeptide synthesis method, and the linear peptide of the antibacterial peptide is synthesized according to the amino acid sequence of the antibacterial peptide.

Wherein, the preparation method can also be a genetic engineering method, and comprises the following steps:

(1) synthesizing a gene sequence according to the amino acid sequence of the antibacterial peptide, cloning the gene sequence into escherichia coli, transferring the gene sequence into escherichia coli host bacteria cells, and constructing a recombinant escherichia coli expression vector;

(2) transforming the recombinant escherichia coli expression vector constructed in the step (1) into a host cell BL21, performing IPTG induced expression, centrifuging to collect thalli, performing ultrasonic cracking, and centrifuging to collect inclusion body precipitates;

and (3) purifying the inclusion body precipitate collected in the step (2) to obtain the inclusion body.

Wherein, the gene engineering method is inserted into an expression vector of escherichia coli in a multi-copy mode, the final yield can reach more than 20g/L fermentation liquor, and the protein recovery rate is 70%.

The genetic engineering method specifically comprises the following steps of artificially designing peptide chain sequences of lysozyme, lactoferrin and other food-borne proteins to generate an antibacterial peptide chain, wherein the antibacterial peptide chain comprises the following steps:

(1) design of a gene sequence encoding a polypeptide:

HHHHHHSSSSGSSSS(DDDKBRRWQWRGSGRWQWRRORSSGSS)_P

wherein HHHHHHHH is a nickel chelating column purification label; SSSSGSSSS, SSGSS is a transition peptide, B, O represents (Y)_a(N)_b、(X)_a(M)_b、(X)_a(N)_bAnd (Y)_a(M)_bDDDK is intestinal stimulationThe enzyme cutting site, p is any number from 2 to 20 and represents a repeating sequence with the number of p, and a and b respectively represent 5 to 15 different lengths.

Taking the antibacterial peptide from food protein as a starting point, selecting a plurality of antibacterial peptides with antibacterial activity in an Antibacterial Peptide Database (APD) by a bioinformatics method, comparing lysozyme, lactoferrin and other food-derived proteins by utilizing a BLAST program, and designing brand-new antibacterial peptides and combinations thereof on the basis.

(2) Artificially synthesizing the base sequence of the polypeptide protein coding gene, and cloning the base sequence to an escherichia coli expression vector to construct a recombinant escherichia coli expression vector.

Transforming the expression vector into a host cell E.Col1BL21(DE3), carrying out IPTG induced expression, then centrifugally collecting thalli, carrying out ultrasonic lysis, centrifugally collecting inclusion body precipitate; after the inclusion bodies are washed, the inclusion bodies are fully dissolved by 2mmol/L DTT, 8mol/L urea and pH7.8, and then the inclusion bodies are filtered by an ultrafiltration membrane to collect filtrate for later use.

Nickel NTA Sepharose FF pre-packed column was equilibrated to baseline with equilibration buffer and equilibrated to baseline with 8M urea-2 mmol/L DTT-equilibration buffer. And (3) loading the ultrafiltrate, washing by using a washing solution, eluting by using an elution buffer solution, collecting an elution peak, desalting an elution sample by using Sephadex G-25 under the condition of 50mM Tris buffer solution pH7.5, and collecting elution peak protein.

And (3) carrying out enzyme digestion on the desalted sample by using enterokinase and trypsin respectively, and purifying the enzyme-digested sample by using an ion exchange/desalting column and a C-18 reversed-phase HPLC (high performance liquid chromatography) preparative column to obtain the target polypeptide sample.

The application of the antibacterial peptide is also within the protection scope of the invention, wherein the antibacterial peptide is used as a bacteriostatic agent.

The antibacterial peptide has a wide application prospect in the field of development of novel antibacterial drugs, food natural preservatives, animal feed additives and other products.

Preferably, the antibacterial peptide has a good antibacterial effect on pseudomonas aeruginosa; wherein, the pseudomonas aeruginosa is ATCC 27853.

A polypeptide or protein containing the antibacterial peptide is also within the protection scope of the invention.

A composition comprising the antibacterial peptide and a pharmaceutically acceptable carrier is also within the protection scope of the invention.

The application of the composition in bacteriostatic agents is also within the protection scope of the invention.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the invention provides a novel antibacterial peptide sequence, which has a considerable inhibiting effect on common escherichia coli, salmonella and bacillus subtilis, also has a good antibacterial effect on first-line drug-resistant bacteria pseudomonas aeruginosa and staphylococcus aureus, has a wide antibacterial spectrum, and can replace an antiseptic and an antibiotic under an effective dose. The application of the compound in the fields of medical treatment, food health care, food processing and the like can help to change the abuse condition of antibiotics and antiseptics.

(2) The invention also provides a biosynthesis process which is simple in process and can be prepared in a large scale, so that the biosynthesis process can be widely applied to the fields of medical treatment, food production, animal breeding and the like, the health of people is guaranteed, the environmental pollution caused by antibiotics and the like is reduced, and the sustainable development of the society is promoted.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: obtaining thallus containing expression recombinant protein by fermentation

Constructing recombinant gene engineering bacteria BL pET30a ABP-1 of the antibacterial peptide ABP-1:

the amino acid sequence of the antibacterial peptide ABP-1 is as follows:

HHHHHHSSSSGSSSS(DDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSS)₁₀wherein HHHHHHHH is a nickel chelating column purification label; SSSSGSSSS, SSGSS is transition peptide, and DDDDK is enterokinase enzyme cutting site.

The nucleotide sequence of the antibacterial peptide ABP-1 is artificially synthesized and coded according to the codon preference of escherichia coli and is shown as SEQ ID NO. 2. Wherein catatg and ggtacc are the restriction sites of restriction enzymes NdeI and KpnI.

The nucleotide sequence was digested with restriction enzymes NdeI and KpnI, and inserted into the multiple cloning site NdeI and KpnI of E.coli expression vector pET30a to construct ABP-1 expression vector, which was then inserted into CaCl₂The recombinant strain BL21 ABP-1 is constructed by transferring the recombinant strain into a host bacterium cell BL21 of escherichia coli by a transformation method, and is preserved by glycerol for later use.

The identified glycerol frozen strain (the constructed recombinant antibacterial peptide gene engineering bacteria BL21-ABP-1) is selected and inoculated in 100mL LB culture solution containing 100mg/L kanamycin, and cultured at 220r/min at 37 ℃ for overnight. Adding the bacterial liquid into 5 1000mL shake flask fermentation medium containing 100mg/L kanamycin culture solution according to the volume ratio of 1/100, culturing at 37 ℃ until logarithmic phase, and inoculating into a 50L fermentation tank for fermentation when A600 reaches 1.0.

The specific process parameters and process control are as follows:

the inoculation amount of BL21-ABP-1 is 15 percent, namely the initial OD value is less than 0.3, the temperature is 37 ℃, and the pH value is controlled to be 7.0-7.2 by ammonia water and 30 percent phosphoric acid.

2. The initial stirring speed is 150rpm, the tank pressure is 0.01MPa, the temperature is 37 ℃, and the dissolved oxygen is 100 percent. Fermenting for 1-3 hours until the OD value reaches 3.

Feeding after the OD value reaches 3, wherein the feeding speed is 0.1-0.2mL/min/L, the stirring speed is 200-900 rpm, the ventilation volume is 0.5-3 vvm, the dissolved oxygen level is maintained between 20-40%, the specific growth rate is controlled to be less than 1, and the OD rises at a constant speed, so that the stirring speed, the ventilation volume and the dissolved oxygen are controlled.

The method adopts a continuous feeding fermentation process, feeding is kept as long as the dissolved oxygen and specific growth rate are within a set range, and if the growth rate is too high, the feeding speed and the rotation number are reduced, even the feeding is stopped.

4. Fermenting until OD value reaches 10-20, cooling to 25-30 ℃, keeping constant feed supplement speed at 0.5mL/min/L, and carrying out IPTG induction (final concentration is 0.6-1 mmol/L) until fermentation is finished (more than 4 hours).

During the fermentation process, the fermentation tank is regulated and controlled. The temperature for induction is 36 ℃ when the culture is carried out for 4 hours. By the process, more than 100g/L of bacterial sludge can be finally obtained, and the expression quantity of the recombinant protein is more than 30 g/L.

Wherein, the recombinant protein is antibacterial peptide ABP-1, and the amino acid sequence is as follows:

HHHHHHSSSSGSSSS(DDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSS)₁₀i.e. by

HHHHHHSSSSGSSSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSS

Wherein HHHHHHHH is a nickel chelating column purification label;SSSSGSSSS、SSGSSis a transition peptide, DDDDK is an enterokinase enzyme cutting site, Y: tyr, E: glu, F: phe, K: lys, R: arg balances charge and hydrophobicity on both sides of the polypeptide, and R is also a cleavage site for trypsin.

The formula of each stage of fermentation medium is as follows:

(1) the first seed culture medium is the same as LB culture medium

(2) Culture medium in the tank: 10g/L of yeast extract; typton15 g/L; NaCl 4 g/L; 2.5g/L of glycerol; the solvent is water.

Adding after autoclaving: the inorganic salt mother liquor after sterile filtration has the following final concentration of each component: k2HPO43 g/L; KH2PO41 g/L; MgSO4 & 7H2O 6 g/L; CaC120.013 g/L; VB10.005g/L

Feeding: typton 150 g/L; yeast Extract 100 g/L; 300g/L of glycerol.

Example 2: purification of the protein of interest

After the induction expression of the bacterial cells IPTG obtained in the step 4 of the example 1, the bacterial cells collected by centrifugation are cracked by ultrasonic waves, and the inclusion body sediment is collected by centrifugation. The inclusion bodies are respectively washed by washing solution (50mmol/L phosphate buffer solution, 1 percent Triton 100, 2mol/L urea and pH7.8), centrifuged for 20min at more than 10000r/min, the inclusion body precipitate is collected, and then the inclusion body precipitate is dissolved fully by extraction solution (50mmol/L phosphate buffer solution, 8mol/L urea and 2mmol/L DTT, pH7.8) and collected by ultrafiltration membrane with molecular weight of 5 ten thousand for later use.

Nickel Sepharose FF or nickel NTA Sepharose FF pre-packed columns were equilibrated to baseline with equilibration buffer, and 8M urea-2 mmol/L DTT-equilibration buffer pH7.8 was used to equilibrate to baseline.

The ultrafiltrate was loaded at 0.5mL/mL bed volume min, and unadsorbed samples were washed off with 50mM phosphate buffer pH7.5 containing 0.5M NaCl and 2mmol/L DTT containing 20mM imidazole equilibration buffer at a flow rate of 1-2mL/mL bed volume min. Elution was performed to baseline using 50mM phosphate buffer containing 0.5M NaCl, ph7.5, and elution buffer containing 500mM imidazole, at a flow rate of 1-2mL/mL bed volume min. Sephadex G-25 was desalted using 50mM Tris buffer pH7.5 and the eluted peak protein was collected.

Desalting the sample, collecting eluted peak protein, and performing enzyme digestion with enterokinase and trypsin. According to the protein concentration of the sample, precooling the sample to 4 ℃, and adding protease at a ratio of 1: 400-4000 for enzyme digestion. And (4) after the enzyme digestion is finished, passing through a nickel sepharose FF chromatographic column again, and obtaining filtrate which is the target protein of the antibacterial peptide.

And (3) detection: (1) measuring the protein concentration of the product by using bovine serum albumin as a reference standard substance by using a Coomassie brilliant blue method, and finally obtaining 20g/L of target protein by volume conversion; (2) the protein recovery was about 70% calculated as 30g/L inclusion body protein per liter (Coomassie Brilliant blue method).

Example 3:

the antimicrobial peptide ABP-1 sample prepared in example 2 was inoculated into LB medium and sterilized by filtration to prepare an antimicrobial peptide stock solution of 2000. mu.g/mL. Then, the solution was diluted to obtain 1000. mu.g/mL and 200. mu.g/mL of the antimicrobial peptide solution, respectively.

The prepared antibacterial peptide solution is transferred and cultured to escherichia coli ATCC25922, bacillus subtilis ATCC9372, salmonella ATCC14028, pseudomonas aeruginosa ATCC27853 and staphylococcus aureus ATCC 25923.

Specifically, PCR tubes were grouped according to different concentrations of antimicrobial peptide (ABP-1), target bacteria inhibition, sodium diacetate control and kana control, as shown in Table 1. Respectively adding 100 mu L of the prepared antibacterial peptide ABP-1 with different concentrations and 100 mu L of the prepared escherichia coli ATCC25922, bacillus subtilis ATCC9372, salmonella ATCC14028, pseudomonas aeruginosa ATCC27853 and staphylococcus aureus ATCC25923, culturing in a shaking way at the temperature of 37 ℃ and 200rpm for 14-18 h with the total volume of 200 mu L and the OD value of 0.05.

150 mu L of the cultured bacterial liquid is taken from each bacterial liquid and added into a 96-well plate, and the light absorption value of the bacterial liquid at 630nm is measured by a microplate reader. The respective bacteriostatic ratios were expressed by the degree of decrease in OD630 values (calculation formula: bacteriostatic ratio ═ absorbance of pure bacterial liquid-absorbance of each sample)/absorbance of pure bacterial liquid × 100%), and the bacteriostatic ratio results are shown in table 1 below.

TABLE 1

As can be seen from Table 1, compared with sodium diacetate, the antibacterial effect of the antimicrobial peptide (ABP-1) on Escherichia coli ATCC25922, Salmonella ATCC14028, Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 is improved at a low concentration (100. mu.g/mL); the antibacterial effect of the antibacterial protein (ABP-1) on pseudomonas aeruginosa ATCC27853 and staphylococcus aureus ATCC25923 is also improved at high concentration (500 mu g/mL); particularly, sodium diacetate has no bacteriostatic effect on pseudomonas aeruginosa ATCC27853, while the bacteriostatic rate of the antimicrobial protein (ABP-1) is 57 percent at low concentration and is more 98 percent at high concentration. In addition, both the low concentration and the high concentration of the antimicrobial protein (ABP-1) were highly elevated over kanamycin (ABP-1). In general, the prepared antibacterial protein (ABP-1) has certain antibacterial effect on the strains, wherein the antibacterial effect on pseudomonas aeruginosa ATCC27853 is the highest. Moreover, the antibacterial peptide is a polypeptide substance, can be degraded in vivo, is nontoxic and harmless, is applied to the fields of medical treatment, food health care, food processing and the like instead of an antiseptic and an antibiotic, has a good application prospect, and can help to change the abuse condition of the current antibiotic and the antiseptic.

The present invention provides a novel antibacterial peptide, a preparation method and application thereof, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Jinling science and technology institute

<120> antibacterial peptide, preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Antimicrobial Peptide (Antimicrobial Peptide)

<400> 1

Arg Arg Trp Gln Trp Arg Gly Ser Gly Arg Trp Gln Trp Arg Arg

1 5 10 15

<210> 2

<211> 1005

<212> DNA

<213> antimicrobial peptide (ABP-1)

<400> 2

catatgcatc atcatcatca tcactcctcc tcctccggct cctcctcctc cgacgatgac 60

gataaatatt atgaggagag aagatggcaa tggagaggct ccggcagatg gcaatggaga 120

agattttttt tcaaaaaaag atcctccggc tcctccgacg atgacgataa atattatgag 180

gagagaagat ggcaatggag aggctccggc agatggcaat ggagaagatt ttttttcaaa 240

aaaagatcct ccggctcctc cgacgatgac gataaatatt atgaggagag aagatggcaa 300

tggagaggct ccggcagatg gcaatggaga agattttttt tcaaaaaaag atcctccggc 360

tcctccgacg atgacgataa atattatgag gagagaagat ggcaatggag aggctccggc 420

agatggcaat ggagaagatt ttttttcaaa aaaagatcct ccggctcctc cgacgatgac 480

gataaatatt atgaggagag aagatggcaa tggagaggct ccggcagatg gcaatggaga 540

agattttttt tcaaaaaaag atcctccggc tcctccgacg atgacgataa atattatgag 600

gagagaagat ggcaatggag aggctccggc agatggcaat ggagaagatt ttttttcaaa 660

aaaagatcct ccggctcctc cgacgatgac gataaatatt atgaggagag aagatggcaa 720

tggagaggct ccggcagatg gcaatggaga agattttttt tcaaaaaaag atcctccggc 780

tcctccgacg atgacgataa atattatgag gagagaagat ggcaatggag aggctccggc 840

agatggcaat ggagaagatt ttttttcaaa aaaagatcct ccggctcctc cgacgatgac 900

gataaatatt atgaggagag aagatggcaa tggagaggct ccggcagatg gcaatggaga 960

agattttttt tcaaaaaaag atcctccggc tcctcctaag gtacc 1005

Claims

1. An antibacterial peptide, characterized in that the amino acid sequence of the antibacterial peptide is HHHHHHSSSSGSSSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSSDDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSS.

2. The method for producing an antibacterial peptide according to claim 1, wherein the linear peptide of the antibacterial peptide is synthesized by the amino acid sequence thereof by a solid phase peptide synthesis method.

3. The method for preparing the antibacterial peptide according to claim 1, which is a genetic engineering method comprising the steps of:

(1) synthesizing a gene sequence according to the amino acid sequence of the antibacterial peptide, cloning the gene sequence into escherichia coli, and constructing a recombinant escherichia coli expression vector;

(3) and (3) purifying the inclusion body precipitate collected in the step (2) to obtain the inclusion body.

4. The use of the antimicrobial peptide of claim 1, wherein said antimicrobial peptide is used in the preparation of a bacteriostatic agent.

5. The use of claim 4, wherein the antimicrobial peptide is used in the preparation of a medicament for treating infectious diseases caused by gram-positive bacteria.

6. The use of claim 4, wherein the antimicrobial peptide is used in the preparation of a medicament for treating infectious diseases caused by gram-negative bacteria.

7. A polypeptide comprising the amino acid sequence of the antimicrobial peptide of claim 1.

8. A protein comprising the amino acid sequence of the antimicrobial peptide of claim 1.

9. A composition comprising the antimicrobial peptide of claim 1 and a pharmaceutically acceptable carrier.