CN108892718A

CN108892718A - A kind of Epinephelus coioides antimicrobial petide and its application

Info

Publication number: CN108892718A
Application number: CN201810711517.XA
Authority: CN
Inventors: 王维娜; 罗盛伟; 魏巍; 杨萍; 刘媛
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2018-07-03
Filing date: 2018-07-03
Publication date: 2018-11-27
Anticipated expiration: 2038-07-03
Also published as: CN108892718B

Abstract

The invention discloses a kind of Epinephelus coioides antimicrobial petides, antibacterial peptide is Epinephelus coioides CD59 albumen, its amino acid sequence is as shown in SEQ ID NO.1 or amino acid sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more amino acid and/or end modified and have same or higher active albumen.A kind of Epinephelus coioides antimicrobial petide provided by the invention can be applied to preparation fish especially grouper immune formulation or feed addictive, have broad application prospects the experiment proved that can inhibit the growth activity of vibrio alginolyticus；The present invention obtains the CD59 gene order of Epinephelus coioides for the first time, not only enrich the gene pool of grouper, and can be applied to preparation and reorganization albumen, antibacterial bacteriostatic additive and fish immunity preparation, new practical basis is provided for the physiologic immunity research of Epinephelus coioides.

Description

A kind of Epinephelus coioides antimicrobial petide and its application

Technical field

The invention belongs to genetic engineering research and development technology fields, and in particular to a kind of Epinephelus coioides antimicrobial petide and its application.

Background technique

Epinephelus coioides (Epinepheluscoioides) belong to Perciformes (Perciformes) , Sushi section (Serranidae), Epinephelinae (Epinephelinae), Epinephelus (Epinephelus) are that the reef of water warm is dwelt fish Class, the Indian Ocean and it is Pacific the torrid zone, subtropical seas it is widely distributed.Epinephelus is in one of rare seafood fish, meat Matter is delicious, and by liking for various regions consumer, economic value is huge.But due to facing to current climate change sharply Disaster and the quality problem seriously polluted, it is high so as to cause its pathogenicity rate and the death rate, the artificial of grouper is supported It grows and causes serious economic strike.Vibrio alginolyticus is common pathogeny bacterium, cultivates and passes through after the onset of grouper infection vibrio alginolyticus It is often used antibiotic to be handled, this not only brings secondary pollution also to bring food-safety problem to environment, to seriously make The about effective development of Grouper cultivating industry.Safely and effectively biological antibiosis agent can for Grouper cultivating industry for research and development Sustainable development has important practical usage and application value.

CD59 albumen belongs to 6 superfamily of leukocyte antigens, is glycoprotein widely distributed in a kind of body, has included 4- Three finger domains of 5 conservative disulfide bond.In addition, CD59 albumen, which includes one, can be incorporated into cellular phospholipid bilayer GPI anchoring domain.Although 6 superfamily member of leukocyte antigens is all containing conservative " CCXXXXCN " structural domain, only There is CD59 albumen that can regulate and control to complement system, and the disulfide bond pattern on CD59 albumen has also assisted in complement system Inhibiting effect.It is well known that the activation of complement signal all concentrates on the MAC component in downstream, but CD59 albumen can pass through It is directly bound directly with C8a or C9b, to effectively inhibit the process that C9 participates in C5b-9 assembling.In addition, nearest research It was found that CD59 albumen can be used as a multi-functional signaling molecule, immunity of organism is exercised non-dependent in complement pathway Immunoregulation effect, can directly participate in the activation of T cell, also can by regulation exocytosis albumen VAMP2 and Syntaxin-1, can also be by regulating and controlling the expression of DAF so as to cause the growth of tumour cell to adjust the secretion of insulin. Although some scholars are to zebra fish (Sun etc., 2013), bolti (Gan etc. 2015), Larimichthys crocea, rainbow trout and channel catfish In equal fish CD59 albumen have research (Liu et al, 2007；Papanastasiou et al,2007；Yeh et al, 2007), but Epinephelus coioides CD59 albumen is used to that vibrio alginolyticus growth to be inhibited temporarily to have not been reported.

Summary of the invention

The purpose of the present invention is to provide a kind of Epinephelus coioides antimicrobial petide and its applications.

The technical solution adopted by the present invention is that：

A kind of Epinephelus coioides antimicrobial petide, antibacterial peptide are Epinephelus coioides CD59 albumen, amino acid sequence such as SEQ ID Shown in NO.1 or amino acid sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more amino It is sour and/or end modified and there is same or higher active albumen.

SEQ ID NO.1：

MKRSLGICLVICSALIGLGSAIRCYSCKDYTASCTKQRECSYDDACLTLTERGGMTYRQCLKYSDCEYGRLSQMFPQ VSSFTFKCCNSDLCNSAPSSASMSVIGLLASAAVMWWCIH。

Encode the gene of above-mentioned Epinephelus coioides antimicrobial petide.

Further, the nucleotide sequence of gene is as shown in SEQ ID NO.2.

SEQ ID NO.2：

ATGAAGCGCTCCCTGGGGATCTGTCTGGTGATCTGCTCCGCTCTGATCGGACTGGGATCGGCCATCCGGTGTTACAG CTGTAAGGACTACACAGCCAGCTGCACCAAACAACGAGAGTGTAGCTATGACGATGCCTGTCTCACACTCACCGAGA GAGGTGGAATGACTTACCGTCAGTGTCTGAAGTACTCAGACTGTGAGTACGGCCGACTGTCCCAAATGTTCCCCCAG GTCTCCAGTTTCACCTTCAAGTGCTGCAACTCAGATCTGTGTAACTCCGCCCCCTCCTCTGCATCGATGTCTGTGAT TGGTCTTCTGGCCTCAGCGGCAGTCATGTGGTGGTGCATCCACTGA。

A kind of cloning vector, contains said gene.

A kind of expression vector, contains said gene.

The method for producing Epinephelus coioides antimicrobial petide, including above-mentioned expression vector is imported in host cell, expression obtains Epinephelus coioides antimicrobial petide.

Application of the above-mentioned Epinephelus coioides antimicrobial petide in preparation vibrio alginolyticus growth inhibitor.

A kind of aquatic livestock immune formulation, wherein containing above-mentioned Epinephelus coioides antimicrobial petide.

A kind of aquatic animal feed additive, wherein containing above-mentioned Epinephelus coioides antimicrobial petide.

The beneficial effects of the invention are as follows：

(1) the present invention provides a kind of Epinephelus coioides antimicrobial petides, the experiment proved that can inhibit the growth of vibrio alginolyticus Activity can be applied to preparation fish especially grouper immune formulation or feed addictive, have broad application prospects；

(2) present invention obtains the CD59 gene order of Epinephelus coioides for the first time, not only enriches the gene pool of grouper, And can be applied to preparation and reorganization albumen, antibacterial bacteriostatic additive and fish immunity preparation, exempt from for the physiology of Epinephelus coioides Epidemic disease research provides new practical basis；

(3) Epinephelus coioides CD59 nucleotides sequence is listed in and is expressed by recombinant strain, it can be with mass production grouper CD59 Recombinant protein greatly reduces production cost, has very high economic value.

Detailed description of the invention

Fig. 1 is the electrophoretic identification (M of the pcr amplification product of Epinephelus coioides CD59 gene ORF：DNA molecular amount standard； 1：Grouper CD59 gene ORF full length sequence PCR product)；

Fig. 2 is the electroresis appraisal of the pcr amplification product of grouper CD59 gene ORF ligase cut-grafting head (EcoRI, XhoI) Scheme (M：DNA molecular amount standard；1：Grouper CD59 gene ORF full length sequence PCR product with digestion connector)；

Fig. 3 is the electrophoretic identification (M of cloning vector pMD18-T-CD59 double digestion (EcoRI, XhoI)：DNA molecular amount mark It is quasi-；1：PMD18-T-CD59 plasmid；2：Double digestion pMD18-T-CD59 plasmid)；

Fig. 4 is the electrophoretic identification (M of expression vector pGEX4T-2-CD59 double digestion (EcoRI, XhoI)：DNA molecular amount Standard；1：PGEX4T-2-CD59 plasmid；2：Double digestion pGEX4T-2-CD59 plasmid)；

Fig. 5 be recombinant bacterial strain pGEX4T-2-CD59-BL21 expression product CD59 recombinant protein PAGE electrophoretic analysis figure and Western blot qualification figure (M：Protein Marker；1：PGEX4T-2-BL21 induced product；2：GST recombinant protein after purification； 3：The western blot qualification figure of GST recombinant protein after purification；4：PGEX4T-2-CD59-BL21 induced product；5：CD59 after purification Recombinant protein；6：The western blot qualification figure of CD59 recombinant protein after purification)；

Fig. 6 is pGEX4T-2-CD59 expression vector establishment schematic diagram；

Fig. 7 is Epinephelus coioides CD59 amino acid sequence and people CD59 Amino acid sequences alignment figure；

Fig. 8 is Epinephelus coioides CD59 Protein Epitopes predictive analysis results；

Fig. 9 is ELISA Binding experiment (the different words of Epinephelus coioides CD59 recombinant protein and vibrio alginolyticus under various concentration Matrix shows significant difference compared with the control group, P < 0.05)；

Figure 10 is influence (the different letter expressions under the processing of Epinephelus coioides CD59 recombinant protein to vibrio alginolyticus growth There are significant difference, P < 0.05 compared with the control group)；

Figure 11 is the influence formed under the processing of Epinephelus coioides CD59 recombinant protein to vibrio alginolyticus mushroom.

Specific embodiment

Below in conjunction with specific experiment, the present invention is further explained, it should be appreciated that following experiment be merely to illustrate the present invention and It is not used in and limits the scope of the invention.

Molecular biology experiment technology employed in following embodiment include PCR amplification, plasmid extract, plasmid conversion, DNA fragmentation connection, digestion, gel electrophoresis etc., unless otherwise specified, usually conventionally operate, for details, reference can be made to《Molecule Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang etc. is translated, 2002, Beijing：Science Press), or according to the normal condition proposed by manufacturer.

One, the ORF of Epinephelus coioides CD59 gene is obtained by design degenerate primer

According to conservative of the CD59 amino acid sequence in each species, degenerate primer is designed.First primer be CD59F1, sequence are：5 '-ATGAAGCACTCCCTGGGGAT (SEQ ID NO.3), Article 2 primer are CD59R1, and sequence is： 5 '-TCAGTGGATGCACCACCACA (SEQ ID NO.4), with M-MLV reverse transcriptase kit The Epinephelus coioides liver cDNA that (Promega, Madison, WI, USA) reverse transcription obtains is template, by touchdown Round pcr obtains pcr amplification product, is sequenced and is compared to it, be determined as CD59 gene, the length is 354bp (SEQ ID NO.2), and deduce its amino acid sequence (SEQ ID NO.1).PCR reaction condition is：94 DEG C of initial denaturation 4min；(1)94 DEG C denaturation 30s, anneal 72 DEG C of 30s, totally 5 circulation；(2) 94 DEG C of denaturation 30s, anneal 70 DEG C of 30s, extends 72 DEG C, totally 5 are followed Ring；(3) 94 DEG C of denaturation 30s, anneal 53 DEG C of 30s, extends 72 DEG C, totally 30 circulations；Last 72 DEG C are continued to extend 10min.PCR expands The electrophoretogram of volume increase object is shown in Fig. 1.

Two, bioinformatics method analyzes Epinephelus coioides CD59 protein sequence

Using two sequence analysis softwares of clustal-X and GeneDoc, by the amino acid sequence of Epinephelus coioides CD59 gene The amino acid sequence of column and people CD59 gene carries out sequence alignment, finds Epinephelus coioides CD59 protein sequence and mankind's CD59 egg Bai Xulie has higher homology (Fig. 7), and the epitope (Fig. 8) of Epinephelus coioides CD59 albumen is analyzed by software.

Three, the synthesis of Epinephelus coioides CD59 gene order

According to the cDNA sequence for the Epinephelus coioides CD59 gene analyzed, the primer at design synthesis upstream and downstream both ends.On Swimming primer is CD59F2, and sequence is：5'-CCGGAATTCCGATGAAGCACTCCCTGGGGAT(SEQ ID NO.5)；Draw in downstream Object is CD59R2, and sequence is：5'-CCGCTCGAGCTCAGTGGATGCACCACCACA(SEQ ID NO.6).It is obtained with reverse transcription Epinephelus coioides liver cDNA be template, the CD59 gene through PCR method amplification grouper, PCR reaction condition is：94 DEG C pre- It is denaturalized 3min；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 30 minutes, and totally 40 recycle；Last 72 DEG C extend 10 points Clock.The electrophoretic identification of pcr amplification product is shown in Fig. 2.

Four, the building of the cloning vector pMD18-T-CD59 of the sequence of CD59 containing Epinephelus coioides

Above-mentioned genetic fragment obtained is isolated and purified, then with pMD18-T (TAKARA) carrier according to the kit 4 DEG C of system of offer connect overnight (13h), and connection product is converted bacillus coli DH 5 alpha, through Amp+ resistance and blue hickie screening Positive colony out；Plasmid is extracted by the small extraction reagent kit of plasmid (Omega, USA), plasmid is verified and is sequenced by double digestion and tests Card, it was demonstrated that Epinephelus coioides CD59 sequence is cloned into carrier, is named as pMD18-T-CD59, and cloning vector converts Escherichia coli The obtained recombinant bacterial strain of DH5 α is named as pMD18-T-CD59-DH5 α.Using EcoRI, XhoI to cloning vector pMD18-T- CD59 carries out double digestion, and restriction enzyme mapping is shown in Fig. 3.

Five, the building of the expression vector pGEX4T-2-CD59 of the sequence of CD59 containing Epinephelus coioides

Cloning vector pMD18-T-CD59 through it is small mention plasmid kit (Omega, USA) extracting after, use restriction enzyme EcoRI and XhoI carries out double digestion, carries out agarose gel electrophoresis, and product is usedGel Extraction Kit is returned It receives, isolates and purifies the Epinephelus coioides CD59 sequence of about 354bp；

Vector plasmid pGEX4T-2 isolates and purifies the sheet of 3.6kb after restriction enzyme EcoRI and XhoI double digestion Section, the genetic fragment with the Epinephelus coioides CD59 of about 354bp is with 1:3 ratios mixing, with T4 ligase in 16 DEG C of connections overnight (about 15h).Then, CaCl is used₂Plasmid is transferred in bacillus coli DH 5 alpha by method, provides the conversion of Amp+ resistance in LB plate screening Son.Plasmid is extracted with standard method, the sequencing of Invitrogen company is sent to, sequencing result is compared, is angled tape lithosporic The gene order of fish CD59, and be properly inserted into expression vector pGEX4T-2, recombinant plasmid is named as pGEX4T-2-CD59. Plasmid construction process is shown in Fig. 7.PGEX4T-2-CD59 is carried to expression using EcoRI and XhoI and carries out double digestion, restriction analysis figure is shown in Fig. 4.

Six, the E. coli recombinant stain pGEX4T-2-CD59-BL21 structure of energy high efficient expression Epinephelus coioides CD59 albumen It builds

According to CaCl₂Above-mentioned obtained purpose recombinant plasmid is transferred to e. coli bl21, had with LB plate screening by method The transformant of Amp resistance, the recombinant bacterium are pGEX4T-2-CD59-BL21.

Seven, recombination Epinephelus coioides CD59 albumen is produced using Escherichia coli recombinant strain pGEX4T-2-CD59-BL21

Recombination BL21 monoclonal engineering bacteria is picked them separately, is inoculated in the LB culture medium containing AMP resistance, 37 DEG C, 200rpm culture expands strain and renews the fresh LB culture medium containing AMP resistance, works as OD₆₀₀Stop when to 0.8, and is added about 1mM IPTG induces 4h.7000rpm after induction, 4 DEG C of collection thallus.TEB buffer is added to suspend to thallus, is added suitable The lysozyme of amount, 4 DEG C are overnight, then again in ice bath ultrasonication to bright clear.Again with containing 30%Trition X-100's Buffer A is resuspended, then stands 30min on ice, and it is molten to albumen progress at room temperature that the buffer B containing urea is added Solution.According to the method for GST bind resin (Millipore), dissolved albumen is purified, as a result sees Fig. 5.

Eight, Epinephelus coioides CD59 proteantigen activity identification is tested

Immunological identification is carried out to recombination Epinephelus coioides CD59 albumen using western blot method.Wherein, primary antibody uses mouse Source GST monoclonal antibody (Novagen), secondary antibody use horse anti-mouse IgG-AP (Ding Guo biotech firm).As a result such as Fig. 5, display Source of mouse GST monoclonal antibody can identify the recombination Epinephelus coioides CD59 albumen by Bacillus coli expression, it was demonstrated that obtained egg White is recombination Epinephelus coioides CD59 albumen.

Nine, vibrio alginolyticus ELISA is tested

Vibrio alginolyticus is inoculated in LB culture medium, 37 DEG C, continuously cultivates in 200rpm to OD₆₀₀=0.8, it is used after being then centrifuged for PBS suspends again to strain, and adjusting bacteria concentration is 1 × 10⁷CFU ml-1, and above-mentioned vibrios is inoculated in 96 hole Elisa plates In.4 DEG C overnight after, 5% skim milk carries out closing 2h to Elisa plate, then after being cleaned with 0.5%Tween-20/PBS, 5 μ g/ml are separately added into, the GST albumen or CD59 albumen of 50 μ g/ml, 100 μ g/ml and 200 μ g/ml are incubated for 2h at room temperature.So After being cleaned afterwards with 0.5%Tween-20/PBS, respectively with source of mouse GST-tag primary antibody and horse anti-mouse HRP secondary antibody to Elisa plate into Row closing.200 μ l TMB solution are finally added in a dark environment, 2M sulfuric acid is then added, display reaction is terminated, and In OD₄₅₀Lower carry out reading Analysis.Calculation formula：Experimental group OD₄₅₀Reading/negative control group OD₄₅₀Reading, is as a result shown in Fig. 9.

As the result is shown：With GST control group it was found that, when be added CD59 protein concentration be gradually increasing when, with molten algae The combination ratio of vibrios gradually raises, and illustrates that Epinephelus coioides CD59 recombinant protein and vibrio alginolyticus have binding ability, and And there are dose relationships.

Ten, vibrio alginolyticus Cell suppression test

Vibrio alginolyticus is inoculated in LB culture medium, 37 DEG C, continuously cultivates in 200rpm to OD₆₀₀=0.8, it is used after being then centrifuged for PBS solution suspends again to strain, and adjusting bacteria concentration is 2 × 10⁵CFU ml^-1.With 49：Concentration is respectively 5 μ by 1 ratio The GST albumen or CD59 albumen of g/ml and 100 μ g/ml and vibrio alginolyticus carry out incubation at room temperature 2h, are then centrifuged for supernatant, use Tween-20/PBS cleans bacterium precipitating, and then above-mentioned bacterium mixture is inoculated in respectively in the LB culture medium of 10ml, 37 DEG C are incubated overnight, 4h the and 12h sample detection bacterium OD after culture₆₀₀, the result is shown in Figure 10.

As the result is shown：With PBS group and GST control group it was found that, by 100 μ g/ml CD59 albumen processing after molten algae Vibrios OD₆₀₀Substantially less than control group and PBS group illustrate that the CD59 protein concentration of 100 μ g/ml can effectively inhibit inoculation Concentration is 2 × 10⁵CFU ml^-1Vibrio alginolyticus growth.

11, vibrio alginolyticus mushroom forms Inhibition test

Algae vibrios is inoculated in LB culture medium, 37 DEG C, continuously cultivates in 200rpm to OD₆₀₀=0.8, PBS is used after being then centrifuged for Solution suspends again to strain, and adjusting bacteria concentration is 2 × 10⁵CFU ml^-1.With 49：Concentration is respectively 5 μ g/ml by 1 ratio Incubation at room temperature 2h is carried out with the GST albumen or CD59 albumen of 100 μ g/ml and vibrio alginolyticus, supernatant is then centrifuged for, uses Tween- 20/PBS cleans bacterium precipitating, is then respectively coated on LB plate, is incubated overnight, the result is shown in Figure 11.

The result shows that：With PBS group and GST control group it was found that, by 100 μ g/ml CD59 albumen processing after molten algae The bacterium colony of vibrios forms number and is substantially less than control group and PBS group, illustrates that the CD59 protein concentration of 100 μ g/ml can be effective It is 2 × 10 that ground, which inhibits inoculum density,⁵CFU ml^-1Vibrio alginolyticus growth.

The above experiment shows：Epinephelus coioides CD59 albumen has the binding ability with vibrio alginolyticus, and presents agent Magnitude relation, while with the raising of protein concentration, the growth of vibrio alginolyticus can be effectively inhibited.It can be seen that recombinant protein CD59 can have potentiality to be exploited in terms of antibacterial bacteriostatic, it can also be used to prepare aquatic livestock immune formulation or feed addictive.

SEQUENCE LISTING

<110>South China Normal University

<120>A kind of Epinephelus coioides antimicrobial petide and its application

<130> 2018.6.29

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 117

<212> PRT

<213> Epinepheluscoioides

<400> 1

Met Lys Arg Ser Leu Gly Ile Cys Leu Val Ile Cys Ser Ala Leu Ile

1 5 10 15

Gly Leu Gly Ser Ala Ile Arg Cys Tyr Ser Cys Lys Asp Tyr Thr Ala

20 25 30

Ser Cys Thr Lys Gln Arg Glu Cys Ser Tyr Asp Asp Ala Cys Leu Thr

35 40 45

Leu Thr Glu Arg Gly Gly Met Thr Tyr Arg Gln Cys Leu Lys Tyr Ser

50 55 60

Asp Cys Glu Tyr Gly Arg Leu Ser Gln Met Phe Pro Gln Val Ser Ser

65 70 75 80

Phe Thr Phe Lys Cys Cys Asn Ser Asp Leu Cys Asn Ser Ala Pro Ser

85 90 95

Ser Ala Ser Met Ser Val Ile Gly Leu Leu Ala Ser Ala Ala Val Met

100 105 110

Trp Trp Cys Ile His

115

<210> 2

<211> 354

<212> DNA

<213> Epinepheluscoioides

<400> 2

atgaagcgct ccctggggat ctgtctggtg atctgctccg ctctgatcgg actgggatcg 60

gccatccggt gttacagctg taaggactac acagccagct gcaccaaaca acgagagtgt 120

agctatgacg atgcctgtct cacactcacc gagagaggtg gaatgactta ccgtcagtgt 180

ctgaagtact cagactgtga gtacggccga ctgtcccaaa tgttccccca ggtctccagt 240

ttcaccttca agtgctgcaa ctcagatctg tgtaactccg ccccctcctc tgcatcgatg 300

tctgtgattg gtcttctggc ctcagcggca gtcatgtggt ggtgcatcca ctga 354

<210> 3

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 3

atgaagcact ccctggggat 20

<210> 4

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 4

tcagtggatg caccaccaca 20

<210> 5

<211> 31

<212> DNA

<213>It is artificial synthesized

<400> 5

ccggaattcc gatgaagcac tccctgggga t 31

<210> 6

<211> 30

<212> DNA

<213>It is artificial synthesized

<400> 6

ccgctcgagc tcagtggatg caccaccaca 30

Claims

1. a kind of Epinephelus coioides antimicrobial petide, antibacterial peptide is Epinephelus coioides CD59 albumen, amino acid sequence such as SEQ ID Shown in NO.1 or amino acid sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases one or more amino It is sour and/or end modified and there is same or higher active albumen.

2. encoding the gene of Epinephelus coioides antimicrobial petide described in claim 1.

3. gene according to claim 2, nucleotide sequence is as shown in SEQ ID NO.2.

4. a kind of cloning vector contains gene described in claim 2 or 3.

5. a kind of expression vector contains gene described in claim 2 or 3.

6. the method for producing Epinephelus coioides antimicrobial petide, including expression vector described in claim 5 is imported in host cell, Expression obtains Epinephelus coioides antimicrobial petide.

7. application of the Epinephelus coioides antimicrobial petide described in claim 1 in preparation vibrio alginolyticus growth inhibitor.

8. a kind of aquatic livestock immune formulation, wherein containing Epinephelus coioides antimicrobial petide described in claim 1.

9. a kind of aquatic animal feed additive, wherein containing Epinephelus coioides antimicrobial petide described in claim 1.