CN113999296B

CN113999296B - Novel antibacterial polypeptide Spgillcin of scylla paramamosain177-189And applications thereof

Info

Publication number: CN113999296B
Application number: CN202111302626.4A
Authority: CN
Inventors: 王克坚; 洪筱; 王晓飞
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-11-01
Anticipated expiration: 2041-11-04
Also published as: CN113999296A

Abstract

The invention discloses a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177‑189And the use thereof, of formula C₈₀H₁₂₆N₂₆O₁₅S₁The amino acid sequence is shown as SEQ ID NO: 01. The invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177‑189Has obvious antibacterial effect on common pathogenic bacteria, has no cytotoxicity on normal scylla paramamosain blood cells and normal mammal cells such as mouse liver parenchyma cells and human normal liver cells, is derived from crustacean, can be used for aquatic feed additives, antibacterial drugs and the like, and has wide application prospect.

Description

Novel antibacterial polypeptide Spgillcin of scylla paramamosain177-189And applications thereof

Technical Field

The invention belongs to the technical field of marine molecular biology, and particularly relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189And applications thereof.

Background

Due to the lack of an adaptive immune system, invertebrates rely solely on the innate immune system against foreign invading pathogens. Antimicrobial peptides are an important part of the innate immune system and are believed to play a key role in protecting hosts from pathogenic microorganisms. The antibacterial peptide has multiple biological functions, including antibacterial, antifungal, antiviral, antitumor, antiparasitic, immunity regulating, etc. Currently, 3257 lines of antimicrobial peptides have been recorded in antimicrobial peptide databases (https:// wangped3. Com/main. Php).

After discovery of lysozyme (lysozyme) by Alexander Flemig in 1922, until 1981, cecropin (Cecropin), which was isolated from the cuphea leisurensis (Hyalophora cecropa), was considered to be the first actual antimicrobial peptide. Subsequently, more and more antibacterial peptides are found in various species and studied intensively. In 1996, the first antimicrobial peptide of crustaceans was discovered, which is derived from hemolymphocytes of common crabs (Carcinus maenas), and which is proline-rich in sequence and has antibacterial activity against both gram-negative and gram-positive bacteria. At present, many antibacterial peptides derived from Scylla paramamosain (Scylaparamosain) have been reported, such as cysteine-rich cationic antibacterial peptide Crustins, anti-lipopolysaccharide factors (Anti-1 ipolysopolysaccharide factors, ALFs) with a conserved LPS binding domain, and anionic antibacterial peptide Scygonadin with a reproductive immune protection function.

Nowadays, the antibacterial peptide and related products thereof are applied to the fields of food, medicine, livestock breeding, aquaculture and the like, and the research and development of the novel antibacterial peptide of the blue crabs provide a new idea for preventing and treating aquaculture diseases and screening antibacterial drugs.

Disclosure of Invention

The invention aims to provide a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189。

The invention also aims to provide the novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The use of (1).

One of the technical schemes of the invention is as follows:

novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189Of the formula C₈₀H₁₂₆N₂₆O₁₅S₁The amino acid sequence is shown as SEQ ID NO: 01.

The second technical scheme of the invention is as follows:

the novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The application of the compound in the preparation of antibacterial drugs, the antibacterial drug can be used for treating Staphylococcus epidermidis, staphylococcus aureus, listeria monocytogenes, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, pseudomonas fluorescens, aeromonas hydrophila, vibrio alginolyticus, vibrio parahaemolyticus, and Bacillus subtilis,Vibrio parahaemolyticus, vibrio harveyi and Vibrio fluvialis have inhibiting and killing effects.

The third technical scheme of the invention is as follows:

an antibacterial medicine contains the novel antibacterial polypeptide Spgillcin of Scylla paramamosain as the effective component_177-189。

In a preferred embodiment of the invention, the active ingredient of the invention is the novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189。

The fourth technical scheme of the invention is as follows:

the novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The aquatic feed additive has inhibiting and killing effects on Pseudomonas fluorescens, aeromonas hydrophila, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi and Vibrio fluvialis.

The fifth technical scheme of the invention is as follows:

an aquatic feed additive, the effective component of which comprises the novel antibacterial polypeptide Spgilllin of Scylla paramamosain_177-189。

The beneficial effects of the invention are:

1. the invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189Is composed of 13 amino acids and has a molecular formula of C₈₀H₁₂₆N₂₆O₁₅S₁The peptide has a molecular weight of about 1.7kDa, contains 5 positively charged amino acid residues, and is a cationic polypeptide having a positive charge according to the prediction that the isoelectric point of the peptide is 11.01 and the average coefficient of hydrophilicity is-0.554.

2. The invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189Has remarkable antibacterial effect on common pathogenic bacteria, and Spgillcin_177-189Has no cytotoxicity on normal Scylla paramamosain blood cells, normal mammalian cells such as mouse liver parenchymal cells and human normal liver cells.

3. The invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The antibacterial activity of (2) is not affected by high temperature and high salt ion concentration.

4. The invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189Has the activity of inhibiting the formation of biomembrane.

5. The invention relates to a novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189Is derived from crustacean, can be used as aquatic feed additive, can also be developed into antibacterial drugs and the like, and has wide application prospect.

Drawings

FIG. 1 shows the novel antibacterial polypeptide Spgillcin of Scylla paramamosain in example 3 of the present invention_177-189A map of the bactericidal kinetics of staphylococcus aureus, pseudomonas aeruginosa and aeromonas hydrophila; wherein the abscissa is time (min) and the ordinate is sterilization index (%).

FIG. 2 shows the novel antibacterial polypeptide Spgillcin of Scylla paramamosain in example 4 of the present invention_177-189A thermal stability profile of antimicrobial activity against staphylococcus aureus, pseudomonas aeruginosa, and aeromonas hydrophila; wherein the abscissa is time (h) and the ordinate is OD₆₀₀The value is obtained.

FIG. 3 shows the novel antibacterial polypeptide Spgillcin of Scylla paramamosain in example 5 of the present invention_177-189A plot of ion tolerance to antibacterial activity against staphylococcus aureus, pseudomonas aeruginosa, and aeromonas hydrophila; wherein the abscissa is time (h) and the ordinate is OD₆₀₀The value is obtained.

FIG. 4 shows Scylla paramamosain as a novel antibacterial polypeptide Spgillcin in example 6 of the present invention_177-189An experimental diagram for inhibiting the biofilm formation of staphylococcus aureus and pseudomonas aeruginosa; wherein the abscissa is Spgillcin_177-189The final concentration of (a), the ordinate is the biofilm formation rate.

FIG. 5 shows Scylla paramamosain as a novel antibacterial polypeptide Spgillcin in example 7 of the present invention_177-189And a transmission electron microscope observation picture of staphylococcus aureus and pseudomonas aeruginosa; control is sterile ultrapure water and the target strain are incubated for 1h, and the experimental group is Spgillcin with the final concentration of 12 mu M_177-189Incubating with the target strain for 1h (bar = 50)0nm)。

FIG. 6 shows the MTS-PMS method for detecting the novel antibacterial polypeptide Spgillcin of Scylla paramamosain in embodiment 8 of the invention_177-189Cytotoxicity test chart; wherein the abscissa is Spgillcin_177-189Protein concentration (. Mu.M) and cell proliferation rate (%) on the ordinate.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Preparation of (2)

The novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The amino acid sequence of (a) is:

Lys-Lys-Arg-Arg-Cys-Phe-Phe-Arg-His-Ile-Tyr-Val-Ala(SEQ ID NO：01)

the novel antibacterial polypeptide Spgillcin of the scylla paramamosain with the purity of more than 95 percent can be obtained by adopting a chemical solid-phase synthesis method_177-189. The novel antibacterial polypeptide Spgillcin of scylla paramamosain in the embodiment_177-189Entrusted Nanjing Kinsrui Biotechnology Ltd to synthesize by solid phase synthesis method, and provide detection information such as polypeptide molecular weight, HPLC, dissolution report, etc.

Spgillcin_177-189The physicochemical parameters of (2) are shown in Table 1.

TABLE 1 novel antimicrobial polypeptide Spgillcin_177-189Physical and chemical parameters of

As can be seen from Table 1, spgillcin_177-189The cationic polypeptide has small molecular weight and strong hydrophobicity, and is positively charged.

Example 2 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The strains referred to in this example were: staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), listeria monocytogenes (Listeria monocytogenes), escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), acinetobacter baumannii (Acinetobacter baumannii), pseudomonas fluorescens (Pseudomonas fluorescens), aeromonas hydrophila (Aeromonas hydrophylla), vibrio harveyi (Vibrio harveyi), vibrio fluvialis (Vibrio fluvialis), vibrio alginolyticus (Vibrio algoliticus), vibrio parahaemolyticus (Vibrio parahaemolyticus). The strains are purchased from the strain preservation center of the institute of microbiology, china academy of sciences and are preserved and stored in the laboratory.

The specific implementation method comprises the following steps:

1. coating the preserved staphylococcus epidermidis, staphylococcus aureus, listeria monocytogenes, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, pseudomonas fluorescens and aeromonas hydrophila on a nutrient broth plate, and performing inverted culture at each appropriate temperature for overnight; vibrio alginolyticus, vibrio parahaemolyticus, vibrio fluvialis and Vibrio harveyi are coated on 2216 plate, and cultured upside down at 28 ℃ overnight. After the colonies grow up, 2-3 bacteria are selected to be singly cloned and inoculated to the corresponding slant culture medium for continuous culture for 12-16h.

2. The bacteria to be tested are washed down from the inclined plane by using sterile 10mM sodium phosphate buffer solution, and the concentration of the bacterial suspension is adjusted. The bacteria were diluted with a mixture of MH liquid medium and sodium phosphate buffer to a final concentration of 5X 10⁵cfu/mL。

3. The synthetic peptide powder was dissolved in sterile Milli-Q Water, filtered through a 0.22 μ M filter, diluted to 3, 6, 12, 24, 48, 96 μ M protein concentration by fold, and placed on ice or at 4 ℃ until use.

4. Antibacterial activity determination is carried out in a sterile 96-hole cell culture plate, a blank control group, a negative control group and an experimental group to be detected are respectively arranged, and each group is provided with three parallels:

(1) blank control group: 50 mul protein sample to be tested and 50 mul culture medium

(2) Negative control group: 50 μ L sterile Milli-Q Water and 50 μ L bacterial suspension

(3) Test groups: 50 mu L of protein sample to be detected and 50 mu L of bacterial suspension

5. Placing the 96-hole cell culture plate in an incubator with proper temperature, culturing for 18-24h, and observing the MIC result in the experimental group to be tested; and (3) blowing and uniformly mixing the experimental group to be detected, sucking a proper amount of bacterial liquid, dripping the bacterial liquid on a corresponding solid culture medium flat plate, performing inverted culture at a proper temperature for 1-2d, and observing an MBC result.

Spgillcin_177-189The results of the antibacterial activity of Spgillcin are shown in Table 2_177-189Has remarkable antibacterial activity to gram-positive bacteria and gram-negative bacteria.

TABLE 2 Spgillcin_177-189Antibacterial activity of

Note: MIC is the minimum inhibitory concentration (. Mu.M) and is indicated as a-b. a is the highest protein concentration of the thallus growth visible by naked eyes; b is the lowest protein concentration at which no growth of the bacterial cells is observed visually. MBC is the minimum bactericidal concentration (. Mu.M) and represents the lowest protein concentration capable of killing 99.9% of the bacterial cells.

Example 3 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Kinetics curve of sterilization

Selecting staphylococcus aureus, pseudomonas aeruginosa and aeromonas hydrophila as bacteria to be detected, and carrying out pair synthesis on the synthetic peptide Spgillcin_177-189The bactericidal kinetics of (a) were determined.

1-fold MBC (Staphylococcus aureus 12. Mu.M, pseudomonas aeruginosa 12. Mu.M, aeromonas hydrophila 24. Mu.M) Spgillcin was added_177-189After incubating with the bacteria for a certain time, taking 5 mu L of suspension to dilute into 500 mu L of NaPB, sucking 50 mu L of suspension after mixing uniformly, coating the suspension on a nutrient broth plate, and performing inverted culture for 18-24h at a proper temperature for clone counting. The samples incubated with sterile MillQ water and bacterial suspension for 0h were used as positive controls, 5. Mu.L of clones coated on nutrient broth plates with the same dilution factor were defined as 100%, and the bactericidal index was the percentage of the number of clones in the experimental group incubated for a certain period of time relative to the number of clones in the positive controlShown (results see figure 1). Spgillcin_177-189When the strain is incubated with staphylococcus aureus for 3min, the sterilization index reaches 50 percent, and when the strain is incubated for 45min, the sterilization index reaches 100 percent; when the culture medium is incubated with pseudomonas aeruginosa for 40min, the bactericidal index reaches 50 percent, and when the culture medium is incubated for 120min, the bactericidal index reaches 100 percent; the bacterial resistance is incubated with aeromonas hydrophila for about 10 min, the bactericidal index reaches 50 percent, and the bactericidal index reaches 100 percent after incubation for 180 min.

Example 4 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Antimicrobial active thermal stability

Selecting Staphylococcus aureus as gram-positive bacteria representative, and Pseudomonas aeruginosa and Aeromonas hydrophila as gram-negative bacteria representative, and treating Spgillcin_177-189The heat stability of the antibacterial activity was measured. The specific procedure was similar to the antimicrobial activity assay described in example 2. Modulation of the synthetic peptide Spgillcin_177-189The concentration of MBC (12 mu M of staphylococcus aureus, 12 mu M of pseudomonas aeruginosa and 24 mu M of aeromonas hydrophila) is 1 time, and the MBC is respectively placed on ice after water bath for 10 min, 20min and 30min in boiling water at the temperature of 100 ℃ for standby. The synthetic peptide Spgillcin_177-189Or respectively incubating sterile Milli-Q Water with the bacteria to be detected for 24h, and measuring OD with enzyme-labeling instrument at 0, 12 and 24h₆₀₀Value of (c) (see figure 2 for results). The results show that Spgillcin_177-189Has good antibacterial activity against Staphylococcus aureus, pseudomonas aeruginosa and Aeromonas hydrophila after water bath in 100 deg.C boiling water for 10, 20, 30min.

Example 5 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Tolerance to antimicrobially active ions

Selecting Staphylococcus aureus, pseudomonas aeruginosa and Aeromonas hydrophila to Spgillcin_177-189The tolerance of the antibacterial active ions was determined. Modulation of the synthetic peptide Spgillcin_177-189The concentration was 1-fold higher than that of MBC (Staphylococcus aureus 12. Mu.M, pseudomonas aeruginosa 12. Mu.M, aeromonas hydrophila 24. Mu.M), and the mixture was kept on ice for further use. The specific procedure was similar to the determination of the antibacterial activity described in example 2, and NaCl was added to the medium at different concentrations to give final concentrations of 0, 10, 20, 40, 80, 160mM NaCl. Mixing Spgillcin_177-189Or respectively incubating sterile Milli-Q Water with the bacteria to be detected for 24h, and measuring OD with enzyme labeling instrument at 0, 12 and 24h₆₀₀Value of (d) (see figure 3 for results). The results show that Spgillcin_177-189Can inhibit the growth of staphylococcus aureus, pseudomonas aeruginosa and aeromonas hydrophila in NaCl (0-160 mM) with different concentrations, and has good ion tolerance.

Example 6 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189Inhibiting bacterial biofilm formation

Selecting staphylococcus aureus and pseudomonas aeruginosa as target strains, and detecting Spgillcin by using crystal violet staining method_177-189Inhibiting the formation of bacterial biofilm. First, the target bacterial suspension is adjusted to OD₆₀₀=0.003, 50. Mu.L each of the antimicrobial peptide Spgillcin_177-189Mixing with bacterial suspension to 96-well plate to make antibacterial peptide Spgillcin_177-189The final concentrations were 6. Mu.M, 3. Mu.M, 1.5. Mu.M and 0.75. Mu.M, while negative controls were set, and left to stand in an incubator at 37 ℃ for 24 hours. Subsequently, the cells were washed twice with 1 × PBS and fixed with formaldehyde for 15min. Removing formaldehyde, dyeing with 0.1% (w/v) crystal violet for 15min, washing off flooding with ultrapure water, adding 95% ethanol, and shaking at room temperature for 30min. Finally, the OD was measured with a microplate reader₆₀₀Value of (c) (see figure 4 for results). The results show that Spgillcin is present in the stage of attachment of Staphylococcus aureus to the solid surface_177-189The addition of (0.75. Mu.M, 1.5. Mu.M, 3. Mu.M, 6. Mu.M) can significantly reduce the adhesion amount of Staphylococcus aureus, and has concentration dependence; while in the process of forming the pseudomonas aeruginosa biofilm, only the higher concentration Spgillcin is formed_177-189Can significantly reduce the amount of adhesion of Pseudomonas aeruginosa by treatment (6. Mu.M). It can be seen that Spgillcin_177-189Can inhibit the biofilm formation of staphylococcus aureus and pseudomonas aeruginosa, and has better inhibiting effect on the biofilm formation of staphylococcus aureus.

Example 7 novel antibacterial polypeptide Spgillcin of Scylla paramamosain_177-189The staphylococcus aureus and the pseudomonas aeruginosa are selected as strains to be detected after the action with the bacteria and the observation of a transmission electron microscope, and the preparation steps of the transmission electron microscope sample are as follows:

1. staphylococcus aureus or Pseudomonas aeruginosa were picked up and cultured in 20mL of nutrient broth, at 37 deg.C, 200rpm, overnight.

2. Adjusting the concentration of the bacteria to be detected to OD₆₀₀=1.0, centrifuge, remove supernatant, add 1mL Spgillcin_177-189The peptides were synthesized to a final concentration of 12. Mu.M and incubated for 1h at room temperature.

3. 5000g,5min, adding 600. Mu.L of 2.5% glutaraldehyde for resuspension, and fixing at 4 ℃ overnight.

4. 5000g,5min, 1mL PBS heavy suspension of thalli, again centrifugal.

5. The supernatant was aspirated off and the cells were resuspended in 15. Mu.L PBS and added to a cast agar model.

6. 5000g,5min, absorbing water on the liquid surface of the thallus, adding 2% molten agar solution, and standing on ice until solidification.

7. The agar blocks containing the bacteria were cut to the size of rice grains, 1mL of PBS resuspended the agar blocks, the supernatant aspirated, and resuspended and fixed with 600. Mu.L of 2.5% glutaraldehyde and allowed to stand overnight at 4 ℃.

The supernatant was aspirated, 1mL PBS was resuspended and washed on agar blocks for 2 times, and transmission electron microscopy was performed after the subsequent embedding, slicing and other steps (see FIG. 5. The results show that in the control group, staphylococcus aureus had high internal electron density, intact internal and external membrane structure, smooth external membrane surface, unchanged cell morphology, 1 xMBC Spgillcin_177-189After the treatment, the inside of the cells was foamed, the shapes and boundaries of the inner and outer membranes were blurred, some of the cells were directly crushed, and the contents were discharged. In the same way, in the control group, the electron density in the pseudomonas aeruginosa is high, the inner and outer membrane structures are complete and the boundary is clear; spgillcin via 1 xMBC_177-189After the treatment, the periplasmic space between the inner and outer membranes of the thallus is enlarged, the cell morphology is changed, and the surface of some thallus has wrinkles, and the contents are discharged.

Example 8 novel antibacterial polypeptide Spgillein of Scylla paramamosain_177-189Cytotoxicity assays

Selecting normal scylla paramamosain blood lymphocyte, human normal liver cell (L02) and mouse liver parenchymal cell: (AML 12), novel antibacterial peptide Spgillcin for scylla paramamosain_177-189Cytotoxicity was measured. The specific implementation steps are as follows:

1. collecting Eriocheir sinensis blood lymphocytes, human normal liver cells and mouse liver parenchymal cells with good growth state, and adjusting cell concentration to 1 × 10⁵And (4) uniformly blowing the cells per mL, adding 100 mu L of cell suspension into each hole of a 96-hole cell culture plate, and placing the cell suspension in an incubator at a proper temperature to culture more than 80% of the cells attached to the wall.

2. The medium was carefully aspirated and Spgillcin was added at various concentrations (6. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, 96. Mu.M)_177-189The culture medium is placed in an incubator at a proper temperature for 24 hours.

3. Adding 20 mu L of MTS-PMS solution, incubating for 2h in dark place, and measuring OD by using an enzyme-labeling instrument₄₉₂Value, evaluation of Spgillcin_177-189Cytotoxicity (results see figure 6). The results show that Spgillcin was present at different concentrations_177-189After 24h of co-incubation with normal cells, the cell activity has no significant difference, and Spgillcin is proved_177-189Has no cytotoxicity to normal scylla paramamosain blood cells, mouse liver parenchymal cells and human normal liver cells.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Sequence listing

<110> university of mansion

<120> novel antibacterial polypeptide Spgillcin177-189 of scylla paramamosain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Scylla paramamosain

<400> 1

Lys Lys Arg Arg Cys Phe Phe Arg His Ile Tyr Val Ala

1 5 10

Claims

1. Novel antibacterial polypeptide Spgillcin of scylla paramamosain_177-189The method is characterized in that: the molecular formula is C₈₀H₁₂₆N₂₆O₁₅S₁The amino acid sequence is shown as SEQ ID NO:01, shown in the figure.

2. The novel antibacterial polypeptide Spgillcin of scylla paramamosain as claimed in claim 1_177-189The application in preparing antibacterial drugs is characterized in that: the antibacterial drug has inhibiting and killing effects on Staphylococcus epidermidis, staphylococcus aureus, listeria monocytogenes, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, pseudomonas fluorescens, aeromonas hydrophila, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi and Vibrio fluvialis.

3. An antibacterial drug, which is characterized in that: the effective component of the novel antibacterial polypeptide Spgillcin of Scylla paramamosain as claimed in claim 1_177-189。

4. The antibacterial agent according to claim 3, characterized in that: the effective component of the novel antibacterial polypeptide Spgillcin of Scylla paramamosain as claimed in claim 1_177-189。

5. The novel antibacterial polypeptide Spgillcin of Scylla paramamosain as claimed in claim 1_177-189The application of the compound feed additive in preparing aquatic feed additives is characterized in that: the aquatic feed additive has inhibiting and killing effects on Pseudomonas fluorescens, aeromonas hydrophila, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi and Vibrio fluvialis.

6. An aquatic feed additive, which is characterized in that: the effective component of the scylla paramamosain comprises the novel antibacterial polypeptide Spgillcin of claim 1_177-189。

7. An aquaculture feed additive according to claim 6 wherein: the novel antibacterial polypeptide Spgillcin of Scylla paramamosain as claimed in claim 1 as the effective component_177-189。