CN112961233A - Application of deep sea chitin peptide - Google Patents

Application of deep sea chitin peptide Download PDF

Info

Publication number
CN112961233A
CN112961233A CN202110226157.6A CN202110226157A CN112961233A CN 112961233 A CN112961233 A CN 112961233A CN 202110226157 A CN202110226157 A CN 202110226157A CN 112961233 A CN112961233 A CN 112961233A
Authority
CN
China
Prior art keywords
crustin
deep sea
application
chitin
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202110226157.6A
Other languages
Chinese (zh)
Other versions
CN112961233B (en
Inventor
孙黎
王玉建
管晓璐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Oceanology of CAS
Original Assignee
Institute of Oceanology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Oceanology of CAS filed Critical Institute of Oceanology of CAS
Priority to CN202110226157.6A priority Critical patent/CN112961233B/en
Publication of CN112961233A publication Critical patent/CN112961233A/en
Application granted granted Critical
Publication of CN112961233B publication Critical patent/CN112961233B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Insects & Arthropods (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of molecular biology, in particular to application of deep sea chitin (crustin). The deep sea crustin is applied to preparing a sterilization preparation. The deep sea crustin is a recombinant protein expressed in escherichia coli. The recombinant crustin is used for sterilization.

Description

Application of deep sea chitin peptide
Technical Field
The invention relates to the field of molecular biology, in particular to application of deep sea chitin (crustin).
Background
Crustin is a class of polypeptides that are predominantly present in crustaceans. Crustin generally has a molecular weight of 7-22kDa, contains a signal peptide at the N-terminal, a Multi-domain region (Multi-domain region) in the middle, and contains a highly conserved orotic acid protein domain (hey acid protein domain) with four disulfide bonds as the core at the C-terminal. Crustin is expressed in blood cells, liver pancreas, gill and intestine and responds immunologically upon bacterial or viral stimulation. Crustisn is capable of binding bacteria and has in vitro antibacterial activity. Furthermore, crustin is able to inhibit protease activity. However, crustin is derived from a plurality of sources, and different environments and species can cause mutual differences, so that the possibility of whether the crustin is related or not is unknown.
Disclosure of Invention
The invention aims to provide application of deep sea chitin (crustin).
In order to achieve the purpose, the invention adopts the technical scheme that:
an application of deep-sea crustin in preparing a bactericidal preparation.
The deep sea crustin is an amino acid sequence shown in SEQ ID No.1.
The deep sea crustin is a recombinant protein expressed in escherichia coli.
The bacteria are Bacillus wiedmannii, Streptococcus iniae or Micrococcus luteus.
The invention has the following advantages:
the crustin is derived from the Alnus japonicus in the deep sea extreme environment (a hot liquid jet in a Magnus basin) below kilometers, and the extreme environment in which the crustin lives has the characteristics of low temperature, high pressure, oligotrophism and the like, so that the Crustin has unique genome characteristics and physiological mechanisms different from terrestrial and shallow sea organisms; the identity with the known shallow sea crustacean crustin is only about 40-50 percent; meanwhile, the deep sea crustin-derived antibacterial agent can specifically kill bacillus cereus, streptococcus iniae or micrococcus luteus.
Drawings
FIG. 1 shows the killing effect of recombinant crustin protein on bacteria.
Detailed Description
The present invention will be further described with reference to the following examples. The examples are intended to illustrate the invention, but not to limit it in any way.
Example 1
Crustin (named Crus1) in the present invention is derived from the transcriptome of hot liquid-jet alvard shrimps in the deep-sea magnus basin, and is described in NCBI database under Genbank accession number: SRR 4342052; then, the sequence was obtained by sequence alignment using Protein BLAST, and the crustin sequence is the amino acid sequence shown in SEQ ID No.1.
SEQ ID No.1 of the sequence table is:
MMLVRVATVLLAVLVATTEASPSTPQDSRSCRYWCRKPDNAVYCCDFGNFPPIPVPVPEHKGVCPEVRPCPGIKFSPQLCPHDGHCKRNEKCCYDSCLEHHACKLASNA
(a) sequence characteristics:
● length: 109, effective length 21-109
● type: amino acid sequence
● chain type: single strand
● topology: linearity
(b) Molecular type: protein
(c) Signal peptide sequence: 1-20 amino acids
(d) Suppose that: whether or not
(e) Antisense: whether or not
(f) The initial sources were: deep sea Alwen shrimp
The molecular weight of the Crus1 is 13.4kDa, the N end contains a signal peptide, the middle part is a multi-domain region, and the C end contains a section of highly conserved orotic acid protein domain with four disulfide bonds as the core. However, the Alvard shrimp from which the protein is derived comes from deep sea environment, compared with shallow sea, the protein has the characteristics of low temperature, high pressure, oligotrophy and the like, and the identity with the known shallow sea crustacean crustin is only about 40-50%.
Example 2
Preparation of recombinant proteins of Crus1 and thioredoxin (Trx)
1) Construction of plasmid pEtCrus1 expressing recombinant protein of Crus 1:
according to the amino acid sequence of Crus1 obtained as described above, a sequence of Crus1 free of signal peptide was synthesized by Beijing Liuhua Dagenescience, Inc., and the corresponding signal peptide was removed, i.e., ATGATGCTGGTCCGTGTTG CGACCGTCTTGCTGGCA GTGCTGGTG GCGACGACGGAGGCA was removed, and ligated to expression vector pET28a (available from "Novagen, USA) to construct recombinant plasmid pEtCrus 1. DNA sequencing of the recombinant plasmid revealed that pEtCrus1 contains a gene encoding the sequence of Crus 1.
2) Induced expression and purification of recombinant Crus1 protein (rCrus1)
The above plasmid pEt was introducedCrus1 Escherichia coli BL21(DE3) (purchased from "Beijing Quanjin biol., Beijing) was transformed by a conventional method, cultured on LB solid medium containing kanamycin (50ug/ml) for 18 to 24 hours, and the transformant was picked and named as BL21/pEtCrus 1. BL21/pEtCrus1 was cultured overnight in LB liquid medium containing kanamycin (50 ug/ml); 5ml of overnight culture was added to 500ml of fresh LB liquid medium containing kanamycin (50ug/ml), and cultured at 37 ℃ with shaking at a rotation speed of 180rpm to OD600At 0.6, IPTG with a final concentration of 0.06mM is added, the culture is continued at 16 ℃ for 12-16h with shaking at a rotating speed of 120rpm, and then the culture is centrifuged at 6000g for 10min at 4 ℃, and the bacterial liquid is collected. Adding 30ml lysis solution, freezing at-80 deg.C for more than 2h, thawing at 4 deg.C, and ultrasonic crushing for 60min until the bacterial suspension becomes clear. The bacterial solution was centrifuged at 4 ℃ for 30min at 10000g, and the supernatant was recovered. The protein in the supernatant was recovered and purified using Ni-NTA protein purification resin (available from QIAGEN, Germany), and the purified protein was suspended in PBS buffer and named rCrus 1.
The lysate is 9.5mM NaH with final concentration2PO4、40mM Na2HPO40.5M NaCl and 8M urea, pH 9.5.
The PBS comprises the following components in percentage by weight: 0.8% NaCl, 0.02% KCl, 0.358% Na2HPO4.12H2O,0.024％NaH2PO4And the balance of distilled water.
The LB solid medium comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agarose
The LB liquid medium comprises the following components: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
3) Inducible expression and purification of recombinant Trx protein (rTrx)
pET32a (purchased from Novagen, USA) was transformed into E.coli BL21(DE3) by the method described above, cultured on LB solid medium containing ampicillin (100ug/ml) for 18-24 hours, and the transformant was picked and named BL21/pET32 a. BL21/pET32a was cultured overnight in LB liquid medium containing ampicillin (100 ug/ml); 5ml of overnight culture medium was added to the flask00ml of fresh LB liquid medium containing ampicillin (100ug/ml) was cultured at 37 ℃ with shaking at a rotation speed of 180rpm to OD600At 0.6, IPTG with a final concentration of 0.6mM is added, the culture is continued at 37 ℃ for 4-5h with shaking at a rotation speed of 180rpm, and then the culture is centrifuged at 6000g for 10min at 4 ℃, and the bacterial liquid is collected. Adding 30ml lysis solution, freezing at-80 deg.C for more than 2h, thawing at 4 deg.C, and ultrasonic crushing for 60min until the bacterial suspension becomes clear. The bacterial solution was centrifuged at 4 ℃ for 30min at 10000g, and the supernatant was recovered. The protein in the supernatant was recovered and purified using a Ni-NTA protein purification resin (available from QIAGEN, Germany), and the purified protein was suspended in PBS buffer and named rTrx.
Example 3
Bactericidal application of rCrus1
Step 1) preparation of the bacterial suspension
Streptococcus iniae (Streptococcus iniae) was cultured in TSB medium (purchased from Bomb organisms, Qingdao), and Micrococcus luteus (Micrococcus luteus), Bacillus wiedmannii and Vibrio harveyi (Vibrio harveyi) were cultured to OD in LB liquid medium600At 0.8, and then centrifuged (5000g, 4 ℃,10 min), the cells were collected and suspended in PBS to a final concentration of 106cfu/ml, namely the bacterial suspension.
The streptococcus iniae G26 is preserved in China general microbiological culture Collection center (CGMCC), and the address is as follows: west road No.1, north chen, chaoyang, beijing, No. 3, zip code, 100101. The preservation number is: CGMCC No.1984, preservation date 2007.3.22; and is described in the patent application No. 201210255992.3.
The micrococcus luteus is purchased from China general microbiological culture Collection center (CGMCC), the addresses of which are as above, and the preservation numbers are as follows: CGMCC No. 1.8591.
The Bacillus wiedmannii SR52 is preserved in China Center for Type Culture Collection (CCTCC) with the address: wuhan university. The preservation number is: CCTCC M2019788, preservation date 2019.10.9.
The Vibrio harveyi strain is a well-known strain, and is described in Zhang, W., and L.Sun.2007.cloning, characterization and molecular application of a beta-agarase gene from Vibrio sp.strain V134.appl.environ.Microbiol.73:2825-2831 in the following documents.
Step 2) sterilization detection of rCrus1
200 μ l of the Bacillus wiedmannii, Streptococcus iniae, Micrococcus luteus and Vibrio harveyi suspensions obtained in the step 1) above were placed in 1.5ml centrifuge tubes, respectively, and rCrus1, rTrx or PBS (control) was added to the bacterial suspension to a final concentration of 20 μ M, and each group was paralleled to detect the bactericidal activity (see Table 1).
TABLE 1
Figure BDA0002956316280000041
As can be seen from Table 1, rCrus1 obtained by the method has certain bactericidal capacity on Bacillus cereus, Streptococcus iniae or Micrococcus luteus.
Then, the experimental group and the control group obtained by aiming at the rCrus1 pair of bacillus cereus, streptococcus iniae or micrococcus luteus are incubated for 2 hours at room temperature, and the bacterial suspension is diluted by PBS gradient and coated on an LB solid culture medium plate. Colonies were counted after 18-20 hours incubation at 28 ℃. The results show that the survival numbers of Bacillus wiedmannii, streptococcus iniae and micrococcus luteus were significantly reduced after incubation with rCrus1, whereas the control protein rTrx had no effect on the survival of Bacillus wiedmannii, micrococcus luteus and streptococcus iniae (fig. 1).
The bactericidal effect of rCrus1 was detected as seen in fig. 1. Bacillus wiedmannii (A1), Streptococcus iniae (Streptococcus iniae) (B1) and Micrococcus luteus (Micrococcus luteus) (C1) were incubated with rCrus1, rTrx or PBS (control) for 2h and then plated on LB solid medium plates. The survival of the bacteria was observed after 18-20 hours of incubation at 28 ℃ and the number of bacteria (colony forming units, CFUs) was counted. Data maps were generated from CFUs (A2, B2, and C2). The experimental data are the results of three parallel experiments. P < 0.01. These results indicate that the rCrus1 of the present invention has significant bacterial killing ability against bacillus cereus, streptococcus iniae, or micrococcus luteus.
Sequence listing
<110> oceanographic institute of Chinese academy of sciences
<120> application of deep sea chitin peptide
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 89
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Ser Pro Ser Thr Pro Gln Asp Ser Arg Ser Cys Arg Tyr Trp Cys Arg
1 5 10 15
Lys Pro Asp Asn Ala Val Tyr Cys Cys Asp Phe Gly Asn Phe Pro Pro
20 25 30
Ile Pro Val Pro Val Pro Glu His Lys Gly Val Cys Pro Glu Val Arg
35 40 45
Pro Cys Pro Gly Ile Lys Phe Ser Pro Gln Leu Cys Pro His Asp Gly
50 55 60
His Cys Lys Arg Asn Glu Lys Cys Cys Tyr Asp Ser Cys Leu Glu His
65 70 75 80
His Ala Cys Lys Leu Ala Ser Asn Ala
85

Claims (4)

1. The application of deep sea crustin is characterized in that: the deep sea crustin is applied to preparing a sterilization preparation.
2. Use according to claim 1, characterized in that: the deep sea crustin is an amino acid sequence shown in SEQ ID No.1.
3. Use according to claim 1 or 2, characterized in that: the deep sea crustin is a recombinant protein expressed in escherichia coli.
4. Use according to claim 1, characterized in that: the bacteria are Bacillus wiedmannii, Streptococcus iniae or Micrococcus luteus.
CN202110226157.6A 2021-03-01 2021-03-01 Application of deep sea chitin peptide Active CN112961233B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110226157.6A CN112961233B (en) 2021-03-01 2021-03-01 Application of deep sea chitin peptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110226157.6A CN112961233B (en) 2021-03-01 2021-03-01 Application of deep sea chitin peptide

Publications (2)

Publication Number Publication Date
CN112961233A true CN112961233A (en) 2021-06-15
CN112961233B CN112961233B (en) 2022-09-13

Family

ID=76276169

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110226157.6A Active CN112961233B (en) 2021-03-01 2021-03-01 Application of deep sea chitin peptide

Country Status (1)

Country Link
CN (1) CN112961233B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140130A (en) * 2010-11-12 2011-08-03 中国科学院海洋研究所 Antibacterial peptide and use thereof
CN102586260A (en) * 2012-03-13 2012-07-18 中国科学院海洋研究所 PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene
CN105859863A (en) * 2016-06-12 2016-08-17 广西壮族自治区水产科学研究院 Litopenaeus vannamei antimicrobial peptide-CrustinB gene and preparation and application of recombinant protein of gene
CN107937406A (en) * 2017-11-30 2018-04-20 宁波大学 A kind of application of Portunus trituberculatus Miers novel C rustin genes and its recombinant protein
CN109134635A (en) * 2018-08-07 2019-01-04 内蒙古科技大学 A kind of antibacterial peptide and the preparation method and application thereof with composite bio-active

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140130A (en) * 2010-11-12 2011-08-03 中国科学院海洋研究所 Antibacterial peptide and use thereof
CN102586260A (en) * 2012-03-13 2012-07-18 中国科学院海洋研究所 PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene
CN105859863A (en) * 2016-06-12 2016-08-17 广西壮族自治区水产科学研究院 Litopenaeus vannamei antimicrobial peptide-CrustinB gene and preparation and application of recombinant protein of gene
CN107937406A (en) * 2017-11-30 2018-04-20 宁波大学 A kind of application of Portunus trituberculatus Miers novel C rustin genes and its recombinant protein
CN109134635A (en) * 2018-08-07 2019-01-04 内蒙古科技大学 A kind of antibacterial peptide and the preparation method and application thereof with composite bio-active

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PREMRUETHAI SUPUNGUL等: "Cloning, expression and antimicrobial activity of crustinPm1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon", 《DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY》 *
任海波等: "三疣梭子蟹抗菌肽Scygonadin基因的克隆与序列分析", 《生物学杂志》 *

Also Published As

Publication number Publication date
CN112961233B (en) 2022-09-13

Similar Documents

Publication Publication Date Title
Sandiford et al. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci
Ong et al. The response of ferritin to LPS and acute phase of Pseudomonas infection
Okamoto et al. Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis
WO2008016240A1 (en) Antimicrobial protein specific to staphylococcus aureus
CN112301021A (en) Enlysin and perforin composition for resisting escherichia coli phage expression and preparation method and application thereof
CN111235119B (en) Preparation and application of fusion antibacterial protein
CN107201372B (en) Penaeus monodon peroxide reductase 1 coding gene sequence and application thereof
Su et al. Differential expression, molecular cloning, and characterization of porcine beta defensin 114
Ma et al. Identification and characterization of a novel antibacterial peptide, avian β-defensin 2 from ducks
Fang et al. Cloning and expression of human beta-defensin-2 gene in Escherichia coli
Hu et al. Identification and characterization of two LBP/BPI genes involved in innate immunity from Hyriopsis cumingii
CN112961233B (en) Application of deep sea chitin peptide
Xie et al. Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G 4
Luo et al. Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides)
CN110452895B (en) Lysozyme from bacteriophage and gene and application thereof
Yang et al. A bactericidal permeability-increasing protein (BPI) from manila clam Ruditapes philippinarum: Investigation on the antibacterial activities and antibacterial action mode
Oh et al. Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities
Peng et al. Tissue distribution, expression, and antimicrobial activity of Anas platyrhynchos avian β-defensin 6
CN107653255B (en) Penaeus monodon peroxidase reductase gene Prx5 and application thereof
Anju et al. A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression
KR20200056194A (en) Antimicrobial peptide with increased antimicrobial activity and antimicrobial composition comprising the same as effective component
Huang et al. Characterization of the BPI-like gene from a subtracted cDNA library of large yellow croaker (Pseudosciaena crocea) and induced expression by formalin-inactivated Vibrio alginolyticus and Nocardia seriolae vaccine challenges
CN114134131B (en) Application of deep sea g-type lysozyme
CN107760689A (en) Suppress iron-binding protein gene Cq Ferritin of WSSV infection and preparation method and application
CN113332418B (en) Application of fish GCSF3

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant