CN110066330A - A kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application - Google Patents

A kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application Download PDF

Info

Publication number
CN110066330A
CN110066330A CN201910329721.XA CN201910329721A CN110066330A CN 110066330 A CN110066330 A CN 110066330A CN 201910329721 A CN201910329721 A CN 201910329721A CN 110066330 A CN110066330 A CN 110066330A
Authority
CN
China
Prior art keywords
glucan
binding protein
stichopus japonicus
gbp
imitative stichopus
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
CN201910329721.XA
Other languages
Chinese (zh)
Other versions
CN110066330B (en
Inventor
孔令明
雷一萱
丛海燕
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.)
Shandong University
Original Assignee
Shandong University
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 Shandong University filed Critical Shandong University
Priority to CN201910329721.XA priority Critical patent/CN110066330B/en
Publication of CN110066330A publication Critical patent/CN110066330A/en
Application granted granted Critical
Publication of CN110066330B publication Critical patent/CN110066330B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Peptides Or Proteins (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Animal Husbandry (AREA)
  • Food Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Oncology (AREA)

Abstract

The invention belongs to molecular biology field and gene engineering technology field, a kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application is specifically disclosed.The present invention obtains imitative stichopus japonicus Glucan binding protein A J-GBP using in-vitro recombination expression technology for the first time, and imitative stichopus japonicus glucan-binding protein (AJ-GBP) is preferably in sequence table SEQ ID NO.1 shown in amino acid sequence.The recombinant protein has agglutination to bacillus subtilis, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Curtobacterium luteum and Vibrio anguillarum, it is inhibited to bacillus subtilis, Escherichia coli, Curtobacterium luteum and Vibrio anguillarum, develop wide-spectrum antiseptic medicine object exploitation and in terms of have potential using value.

Description

A kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application
Technical field
The invention belongs to molecular biology and gene engineering technology field, and in particular to a kind of imitative stichopus japonicus glucan binding domian egg It is white and its preparation method and application.
Background technique
Disclosing the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without certainty It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art Art.
Imitative stichopus japonicus (Apostichopus japonicus) belongs to Echinodermata (Echinodermata), Holothuroidea (Holothuroidea) , Parapet hand mesh (Aspidochirotida), Stichopodidae (Stichopodidae) are known as " marine ginseng " Good reputation, have very high nutritive value and pharmaceutical value.It is distributed more wide in China Shandong, Liaoning and Hebei surrounding waters It is general.In recent years, with the raising of people's social life condition and the enhancing of health perception, imitative stichopus japonicus market demand increases year by year Add, constantly cultivation scale is stimulated to increase, therefore imitative stichopus japonicus becomes the single maximum breed variety of the output value in China's culture fishery. But the disease problem encountered in breeding process is always the important restriction factor that puzzlement industry further develops.And use Drug is learned, such as chlorine dioxide, potassium permanganate, formalin and antibiotic treated, to varying degrees all to imitative stichopus japonicus There is certain toxic effect.Therefore carrying out disease prevention and cure using the means of biotechnology increasingly attracts people's attention.
Natural antimicrobial substance is widely present in animals and plants.Currently, the antibacterial material separated from animals and plants mainly has life Alkaloids, terpene, flavones, polyphenol, polysaccharide, organic acid, polypeptide, albumen etc., different active constituents, structure composition is different, resists Bacterium bacteriostasis mechanism is also different.Wherein, antibacterial protein is to be encoded to induce through external condition by a variety of biological cell specific genes One kind of generation has broad spectrum antibiotic, fungi, virus, protozoon, the macro-molecular protein for pressing down the effect of tumor killing cell isoreactivity. However, it is found by the inventors that it is more for the small molecule antibacterial peptide research for being relatively easily-synthesized acquisition at present, and to macromolecular antibacterial protein It studies less, is based particularly on imitative stichopus japonicus and prepares the research of dependent antimicrobial albumen and be rarely reported, be unfavorable for the matter of imitative stichopus japonicus product Measure the sound development of safety and relevant food industry.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of imitative stichopus japonicus glucan-binding protein and preparation method thereof and answers With.By to imitative stichopus japonicus β -1, the research of 3- glucan-binding protein, to find new imitative stichopus japonicus disease prevention and control, fine-variety breeding Strategy provides thinking, may advantageously facilitate China and imitates the health of apostichopus japonicus culture industry, sustainable development.
To achieve the above object, The technical solution adopted by the invention is as follows:
The first aspect of the invention provides a kind of imitative stichopus japonicus glucan-binding protein, the imitative stichopus japonicus glucan binding domian The amino acid sequence and SEQ ID NO.1 of albumin A J-GBP has 90% or more (containing 90%) sequence homology；It is furthermore preferred that institute The amino acid sequence for stating imitative stichopus japonicus Glucan binding protein A J-GBP is SEQ ID NO.1.
The second aspect of the invention provides the DNA molecular for encoding above-mentioned amino acid sequence.
Further, the present invention provide it is a kind of coding SEQ ID NO.1 shown in amino acid sequence DNA molecular have such as Nucleotide sequence shown in SEQ ID NO.2, or there is with SEQ ID NO.2 at least 90% sequence homology, and it being capable of table Up to the nucleotide sequence of amino acid sequence shown in SEQ ID NO.1.
The third aspect of the invention is provided containing the amino acid sequence for encoding the imitative stichopus japonicus glucan-binding protein Recombinant expression carrier, transgenic cell system or the transgenosis recombinant bacterium of DNA molecular.
The fourth aspect of the invention provides the DNA sequence dna, the recombinant expression carrier, transgenic cell system Or transgenosis recombinant bacterium is preparing the application in imitative stichopus japonicus Glucan binding protein A J-GBP.
The fifth aspect of the invention provides the preparation method of Glucan binding protein A J-GBP a kind of, including walks as follows It is rapid:
Using imitative stichopus japonicus cDNA as template, PCR amplification is carried out with primers F 1 and R1；
It is attached after PCR product is purified with coli expression carrier pEASY-E2 carrier, connection product conversion is big Enterobacteria, sequencing identification recon, obtains expression vector pEASY-AJ-GBP；
Above-mentioned expression vector pEASY-AJ-GBP is transferred to Trans BL21 (DE3) pLysS Competent cell, is screened Transformant is inoculated in the LB culture medium containing ampicillin by transformant, then pure with super filter tube with IPTG inducing expression Change recombinant protein to get Glucan binding protein A J-GBP.
The sixth aspect of the invention, provides the application of above-mentioned imitative stichopus japonicus glucan-binding protein, and the imitative stichopus japonicus Portugal is poly- Carbohydrate-binding protein AJ-GBP is used to prepare broad-spectrum antiseptic class pharmaceutical preparation or feed addictive.
Further, the imitative stichopus japonicus glucan-binding protein is used to prepare anti-bacillus subtilis, Staphylococcus aureus Bacterium, Escherichia coli, Pseudomonas aeruginosa, Curtobacterium luteum and Vibrio anguillarum pharmaceutical preparation or feed addictive.
The present invention has following advantageous effects:
The present invention obtains imitative stichopus japonicus Glucan binding protein A J-GBP using in-vitro recombination expression technology for the first time for the first time, warp Verification experimental verification, which has obvious inhibiting effect to gram-positive bacteria, Gram-negative bacteria, specifically, the recombination Albumen has bacillus subtilis, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Curtobacterium luteum and Vibrio anguillarum Agglutination, it is inhibited to bacillus subtilis, Escherichia coli, Curtobacterium luteum and Vibrio anguillarum, therefore developing Wide-spectrum antiseptic medicine object and feed addictive etc. have potential using value.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
Fig. 1 is the protein expression figure that detects of SDS-PAGE in the embodiment of the present invention 1, and 1 is MARKER1, molecular weight from Arrive greatly it is small be followed successively by 100kDa, 60kDa, 45kDa, 28kDa, 18kDa, 2 be MAKER2, and molecular weight is followed successively by from big to small 97.2kDa, 66.4kDa, 44.3kDa, 29kDa, 20.1kDa, 14.3kDa, 3 be egg in supernatant after inducing expression clasmatosis White, 4 be the albumen for not inducing thallus, and 5 be the albumen of solubilization of inclusion bodies after purification after inducing expression, and 6 be inducing expression Inclusion body protein afterwards, 7,8,9,10 be respectively first, second, third and fourth filtrate in ultra-filtration process.
Fig. 2 is agglutination figure of the imitative stichopus japonicus glucan-binding protein that provides of the embodiment of the present invention 2 to bacterium, wherein scheming 2 (1) are bacillus subtilis, and Fig. 2 (2) is staphylococcus aureus, and Fig. 2 (3) is Curtobacterium luteum, and Fig. 2 (4) is eel arc Bacterium, Fig. 2 (5) are Escherichia coli, and Fig. 2 (6) is Pseudomonas aeruginosa.
Fig. 3 is the imitative stichopus japonicus glucan-binding protein bacteriostatic activity figure that the embodiment of the present invention 3 provides, and wherein Fig. 3 (1) is withered Careless bacillus growth curve chart, Fig. 3 (2) are Curtobacterium luteum growth curve chart, and Fig. 3 (3) is raw for staphylococcus aureus Long curve graph, Fig. 3 (4) are Vibrio anguillarum growth curve chart, and Fig. 3 (5) is Escherichia coli Growth curve graph, and Fig. 3 (6) is Pseudomonas aeruginosa Growth curve chart.
Fig. 4 is that the imitative stichopus japonicus glucan-binding protein bacterium that the embodiment of the present invention 4 provides combines activity figure, and wherein 4A is bacterium The albumen that body combines, Fig. 4 B are the albumen of the 4th cleaning solution；It 1 is wherein MAKER in Fig. 4 A and Fig. 4 B, 2 be Escherichia coli, 3 It is staphylococcus aureus for bacillus subtilis, 4,5 be Vibrio anguillarum, and 6 be Curtobacterium luteum, and 7 be Pseudomonas aeruginosa.
Fig. 5 is the imitative stichopus japonicus glucan-binding protein glucan binding domian activity figure that the embodiment of the present invention 5 provides.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As previously mentioned, it is very few based on the research achievement that imitative stichopus japonicus prepares dependent antimicrobial albumen, therefore be unfavorable for imitative stichopus japonicus and produce The sound development of the quality safety and relevant food industry of product.
The study found that β -1,3- glucan-binding protein (β -1,3-glucan-binding proteins, abbreviation GBP), Also known as β -1,3- glucan identify albumen (beta-1,3-glucan recognition protein, abbreviation GRP), more with rouge Sugared glucan-binding protein (lipopolysaccharide and beta-1,3-glucan binding protein, abbreviation LGBP) belong to the different subtype of same gene.GBP has the non-own substance of identification, and phenol oxidase system before activating activates coagulation cascade System and activation lead to a series of functions such as cytotoxic factor/cell dissolution factor/antimicrobial factors release haemocyte threshing. And imitative stichopus japonicus glucan-binding protein antibacterial action is not yet had been reported that at present.Therefore the functional gene of imitative stichopus japonicus, system are excavated Standby biological antibiotic drug, helps to solve the problems, such as antibiotic resistant in current cultivation, has highly important theoretical and reality Meaning.
In view of this, a kind of imitative stichopus japonicus glucan-binding protein is provided in the specific embodiment of the present invention, it is described The amino acid sequence and SEQ ID NO.1 of imitative stichopus japonicus Glucan binding protein A J-GBP has 90% or more sequence homology；More Preferably, the amino acid sequence of the imitative stichopus japonicus Glucan binding protein A J-GBP is SEQ ID NO.1.
In still another embodiment of the invention, the DNA molecular of above-mentioned amino acid sequence is provided.Due to the letter of codon And property, there may be the nucleotide sequences that many kinds can encode glucan-binding protein of the present invention.For code book The DNA molecular of the amino acid sequence of the imitative stichopus japonicus glucan-binding protein is invented, those skilled in the art can be easily It is manufactured and is synthesized using conventionally known method.Such as, by selecting to correspond to the amino acid for constituting designed amino acid sequence The codon of residue can be readily determined and provide the DNA of the amino acid sequence corresponding to imitative stichopus japonicus glucan-binding protein Molecule.
In still another embodiment of the invention, a kind of DNA for encoding amino acid sequence shown in SEQ ID NO.1 is provided Molecule has the nucleotide sequence as shown in SEQ ID NO.2, or has at least 90% homology with SEQ ID NO.2, and The nucleotide sequence of amino acid sequence shown in SEQ ID NO.1 can be expressed.
It should be noted that term " homologous " be primarily referred to as it is homologous in sequence, that is, be used to illustrate it is two or more Protein or DNA sequence dna ancestors having the same.Homologous sequence generally has similar function.The homology of protein and DNA Determine often through the similitude of their sequences, similitude is used to describe detection sequence and target during referring to sequence alignment Identical DNA base or the height of amino acid residue sequence proportion between sequence.
In still another embodiment of the invention, provide containing the amino for encoding the imitative stichopus japonicus glucan-binding protein Recombinant expression carrier, transgenic cell system or the transgenosis recombinant bacterium of the DNA molecular of acid sequence.
The recombinant expression carrier is that the DNA sequence dna is inserted into expression obtained in coli expression carrier to imitate The recombinant expression carrier of stichopus japonicus Glucan binding protein A J-GBP.
The coli expression carrier is pEASY-E2.
In still another embodiment of the invention, a kind of transgenosis recombinant bacterium is provided, the recombinant bacterium is will be described Recombinant expression carrier imports in Escherichia coli, and screening obtains transgenosis recombinant bacterium.
DNA sequence dna, the recombinant expression carrier, transgenic cell system or the transgenosis recombinant bacterium is in the imitative thorn of preparation Join the application in Glucan binding protein A J-GBP.
In still another embodiment of the invention, the preparation method of Glucan binding protein A J-GBP a kind of is provided, is wrapped Include following steps:
(1) using imitative stichopus japonicus cDNA as template, PCR amplification is carried out with primers F 1 and R1, for use；
(2) it is attached after PCR product is purified with coli expression carrier pEASY-E2 carrier, connection product conversion Escherichia coli, sequencing identification recon；
(3) above-mentioned expression vector pEASY-AJ-GBP is transferred to Trans BL21 (DE3) pLysS Competent cell, Transformant is screened, transformant is inoculated in the LB culture medium containing ampicillin, with IPTG inducing expression, then uses ultrafiltration Pipe purification of recombinant proteins to get arrive Glucan binding protein A J-GBP.
The primer is respectively as follows:
F1:5'-ATGACCGCCGGTGGTGGAGG-3'(SEQ ID NO.3)；
R1:5’-GCACCAGGATCGACGCTCAAG-3’(SEQ ID NO.4)。
In still another embodiment of the invention, the application of imitative stichopus japonicus glucan-binding protein, the imitative stichopus japonicus are provided Glucan binding protein A J-GBP is used to prepare broad-spectrum antiseptic class pharmaceutical preparation or feed addictive.
In still another embodiment of the invention, the imitative stichopus japonicus glucan-binding protein is provided and is used to prepare anti-withered grass Pharmaceutical preparation or the feeding of bacillus, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Curtobacterium luteum and Vibrio anguillarum Feed additives.
Further detailed description is done to the present invention below with reference to embodiment, embodiments of the present invention are not limited thereto.
Embodiment 1: the pronuclear recombination expression of the imitative code area stichopus japonicus AJ-GBP
Specific step is as follows:
1. the building of pronuclear recombination expression vector
The present invention uses the pEASY-E2 protokaryon of Beijing Quan Shijin biotechnology (TransGen Biotech) Co., Ltd Expression vector.Utilize whole code areas of specific primer PCR amplification AJ-GBP.PCR response procedures setting are as follows: 1. 94 1. pre- change Property 4min；2. 1. 94 are denaturalized 30s；3. 52 1. renaturation 30s；4. 1. 72 extend 1min；5. 1. 72 extend 10min eventually.2. by step 4. 3. carrying out 40 circulations.PCR product is subjected to purification and recovery, is connect with pEASY-E2 carrier.Connection product converts large intestine bar Bacterium Trans BL21 (DE3) pLysS, selects 5~10 single bacterium colonies and is inoculated in LB liquid medium, and sequencing company is sent to survey Sequence, to verify the correctness of reading frame, the specific primer is respectively as follows:
F1:5'-ATGACCGCCGGTGGTGGAGG-3'；
R1:5’-GCACCAGGATCGACGCTCAAG-3’。
The expression of recombinant protein: correct strain inoculated will be verified in the LB liquid medium of 50mL, be placed in shaken cultivation In case, 1. 37 cultivate 12~16 hours in 200rpm, then this culture solution is inoculated in the 500mL of new LB with the ratio of 1:100 In fluid nutrient medium, 1. 37 cultivate to OD600=0.6.IPTG is added, is allowed to final concentration and reaches 0.8mM, 1. 37 cultivate 5h.It collects Cell is resuspended after cleaning 3 times with PBS buffer solution, and 10000g is centrifuged 20min after clasmatosis, and precipitating inclusion body protein is taken to use Buffer A (5mM EDTA, 50mM Tris-HCL) is cleaned 2 times, Buffer B (5mM EDTA, 50mMTris-HCL, 2M urine Element) cleaning 2 times, use Buffer C (10mMTris-HCL, 0.1MNaH2PO4, 8M urea) dissolution after, with 0.45 μm of filter membrane mistake After filter, using Millipore10kd ultra-filtration centrifuge tube purification of recombinant proteins, the amino acid as shown in SEQ ID NO.1 can be obtained The albumen of sequence.
>AJ-GBP-aa
MTAGGGGNWEFQYYTNNRSNSYVRDNTLYIKPTLTSEKEGEAFLTSGTLNLWGASPADLCTGNNWWGCE RTGSFNNILNPIQSARLRTVNSFAFKHGRIEVFAQLPKGDWLWPAIWLLPKRNAYGGWPASGEIDLVESRGNRNLRA ADGTHVGAEQVGMTLHWGPYWPLNGYPMTHNAKNLPDGQTFGDGFHNYTLVWTADSLDFYLDGEPILERRSWC
Sequence signature:
Length: 219 amino acid
Type: amino acid
Chain: single-stranded
Topological structure: line style
Feature: feature: molecular weight 24.64kDa, isoelectric point 5.94.There are two protein kinase C phosphorylation sites for tool (S36EK, S83AR), three casein kinase i I phosphorylation sites (S36EKE, S55PAD, T186FGD), a β -1, the Portugal 3- Dextranase site (W127-E132-I133-D134), a β -1,3- polysaccharide connection identification motif (FHNYTLVWTADSLDFYLDG), a polysaccharide binding motif (VFAQLPKGDWLWPAIWLLP) and a dextranase motif (WPASGEIDLVES).In addition, AJ-GBP does not have the distinctive cell adhesion site RGD of shrimp β-GRP, and in amino acid sequence 107 positions find KGD.
Source: imitative stichopus japonicus
Embodiment 2: the Bacterial agglutinative activity analysis of the prokaryotic recombinant protein of the imitative code area stichopus japonicus AJ-GBP
To bacillus subtilis (Bacillus subtilis), staphylococcus aureus (Staphylococcus Aureus), Curtobacterium luteum (Curtobacteriumluteum) (G+Bacterium) and Vibrio anguillarum (Vibrio anguillarum), Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa) (G-Bacterium) agglutination experiment
Bacillus subtilis, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Curtobacterium luteum are inoculated into LB In fluid nutrient medium, 1. 37 cultivate to OD600Value to 0.6,1. cultivated 28 to OD using TCBS culture medium by Vibrio anguillarum600Value is extremely 0.6, then OD is diluted to sterile saline600Value is 0.001, and then 5000rpm is centrifuged 10 minutes collection bacterial sediments, It is washed with TBS buffer and is resuspended afterwards three times, bacteria concentration is made to be adjusted to 2 × 109A cell/mL.By 75 μ L recombinant protein (about 600 μ g/ Ml it) is separately added into 96 orifice plates, is then added 30 μ L bacterium solutions, be incubated at room temperature 1h, negative control group is by recombinant protein cow's serum Albumin (BSA) replaces.As a result, it has been found that recombinant protein can be aggregated above-mentioned 6 kinds of detections bacterium.
Experiment shows that gram-positive bacteria and Gram-negative bacteria can be effectively aggregated by recombinating AJ-GBP albumen.
Embodiment 3: the bacteriostatic activity analysis of imitative stichopus japonicus AJ-GBP prokaryotic recombinant protein
To staphylococcus aureus, bacillus subtilis, Curtobacterium luteum (G+Bacterium) and it is Vibrio anguillarum, Escherichia coli, green Purulence bacillus (G-Bacterium) Cell suppression test
Bacillus subtilis, staphylococcus aureus, Curtobacterium luteum, Pseudomonas aeruginosa and Escherichia coli are inoculated into LB In fluid nutrient medium, 1. 37 cultivate to OD600To 0.6, Vibrio anguillarum is inoculated into TCBS culture medium and 1. cultivates 28 to OD value600Value is extremely 0.6, it is spare after diluting 20 times with fresh culture.100 μ l bacterium solutions and 100 μ l weight are added in every hole in 96 porocyte culture plates Histone mixes, and 1. setting cultivation temperature cultivates 3h for 37 after, records an OD every 30min microplate reader600Value, about 7-8h Reach the growth platform phase, BSA is set as control group, counts OD600Value draws different groups of growth curve, and every group setting 3 parallel.
Experiment shows that recombinating AJ-GBP albumen is able to suppress Gram-negative bacteria, also has inhibition to make gram-positive bacteria With, but it is unobvious to the inhibiting effect of staphylococcus aureus and Pseudomonas aeruginosa.
Embodiment 4: the bacterium of the prokaryotic recombinant protein of the imitative code area stichopus japonicus AJ-GBP combines activity analysis
To staphylococcus aureus, bacillus subtilis, Curtobacterium luteum (G+Bacterium) and it is Vibrio anguillarum, Escherichia coli, green Purulence bacillus (G-Bacterium) Binding experiment
Bacillus subtilis, staphylococcus aureus, Curtobacterium luteum, Pseudomonas aeruginosa and Escherichia coli are inoculated into LB In fluid nutrient medium, 1. 37 cultivate to OD600To 0.6, Vibrio anguillarum is inoculated into TCBS culture medium and 1. cultivates 28 to OD value600Value is extremely 0.6, then 5000rpm is centrifuged 10 minutes collection bacterial sediments, is washed with TBS buffer and is resuspended afterwards three times, is adjusted to bacteria concentration 3.5×108A cell/mL.200uL bacteria suspension and 200uL recombinant protein (about 600 μ g/ml) is taken to mix, concussion is incubated at room temperature 30min.5000rpm is centrifuged 10 minutes abandonings supernatant collection thallus, and after TBS washing thalline 4 times, the 4th cleaning solution and thallus divide Not Jia Ru albumen sample-loading buffer boiling water bath 10min, after supernatant is collected by centrifugation, carry out SDS-PAGE and Western Blotting testing goal band.
Experiment shows that recombinating AJ-GBP albumen has apparent combination activity to Gram-negative bacteria, gram-positive bacteria；And Recombination AJ-GBP albumen can effectively inhibit gram-positive bacteria, also there is apparent inhibiting effect to Gram-negative bacteria.
Embodiment 5: glucan, lipopolysaccharides, the peptide glycan of the prokaryotic recombinant protein of the imitative code area stichopus japonicus AJ-GBP, which combine, lives Property analysis
50 μ l are taken to be added in 96 orifice plates of high-affinity respectively the glucan, lipopolysaccharides, peptide glycan of 80 μ l/ml, 37 1. It dries overnight；1. next day 60 is incubated for 30min；The PBS that 200 μ l contain 5% skimmed milk power is added in every hole, 1. closes 2h in 37；It outwells Confining liquid, the PBST (20%Tween-20:PBS=1:100) that 200 μ l are added in every hole are washed four times, each 5min；Every hole is added 50 μ l albumen (the bis- times of gradient dilutions of PBS of albumen containing 1% skimmed milk power, totally six histone；Control group uses BSA)；Outwell egg White, the PBST (20%Tween-20:PBS=1:100) that 200 μ l are added in every hole is washed four times, each 5min；By primary antibody, (rat is anti- His-tag antibody) according to the dilution proportion of 1:300 in the PBS containing 1% skimmed milk power, 100 μ l, room temperature condition is added in every hole Lower reaction 2h；Outwell primary antibody, the PBST (20%Tween-20:PBS=1:100) that 200 μ l are added in every hole washes four times, every time 5min；By the rabbit-anti rat secondary antibody of horseradish peroxidase-labeled according to the dilution proportion of 1:3000 in containing 1% skimmed milk power In PBS, 100 μ l are added in every hole, react 2h under room temperature；Secondary antibody is outwelled, the PBST (20%Tween- of 200 μ l is added in every hole It 20:PBS=1:100) washes four times, each 5min；It is developed the color using the raw work EL-TMB colour reagent box in Shanghai, in microplate reader Light absorption value is read at 450nm.
Experiment shows that recombinating AJ-GBP albumen has apparent combination activity, and bond strength and protein concentration to glucan It is directly proportional, and activity is not combined significantly to lipopolysaccharides, peptide glycan.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art do not need The various modifications or changes that can be made are made the creative labor still in protection scope of the present invention.
SEQUENCE LISTING
<110>Shandong University
<120>a kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 219
<212> PRT
<213>artificial sequence
<400> 1
Met Thr Ala Gly Gly Gly Gly Asn Trp Glu Phe Gln Tyr Tyr Thr Asn
1 5 10 15
Asn Arg Ser Asn Ser Tyr Val Arg Asp Asn Thr Leu Tyr Ile Lys Pro
20 25 30
Thr Leu Thr Ser Glu Lys Glu Gly Glu Ala Phe Leu Thr Ser Gly Thr
35 40 45
Leu Asn Leu Trp Gly Ala Ser Pro Ala Asp Leu Cys Thr Gly Asn Asn
50 55 60
Trp Trp Gly Cys Glu Arg Thr Gly Ser Phe Asn Asn Ile Leu Asn Pro
65 70 75 80
Ile Gln Ser Ala Arg Leu Arg Thr Val Asn Ser Phe Ala Phe Lys His
85 90 95
Gly Arg Ile Glu Val Phe Ala Gln Leu Pro Lys Gly Asp Trp Leu Trp
100 105 110
Pro Ala Ile Trp Leu Leu Pro Lys Arg Asn Ala Tyr Gly Gly Trp Pro
115 120 125
Ala Ser Gly Glu Ile Asp Leu Val Glu Ser Arg Gly Asn Arg Asn Leu
130 135 140
Arg Ala Ala Asp Gly Thr His Val Gly Ala Glu Gln Val Gly Met Thr
145 150 155 160
Leu His Trp Gly Pro Tyr Trp Pro Leu Asn Gly Tyr Pro Met Thr His
165 170 175
Asn Ala Lys Asn Leu Pro Asp Gly Gln Thr Phe Gly Asp Gly Phe His
180 185 190
Asn Tyr Thr Leu Val Trp Thr Ala Asp Ser Leu Asp Phe Tyr Leu Asp
195 200 205
Gly Glu Pro Ile Leu Glu Arg Arg Ser Trp Cys
210 215
<210> 2
<211> 657
<212> DNA
<213>artificial sequence
<400> 2
atgaccgccg gtggtggagg aaactgggaa ttccagtact acaccaataa tcgcagcaac 60
agttacgtac gagacaacac cctctacatt aaacctaccc taacctccga gaaggaaggc 120
gaggcatttt taaccagtgg aacattgaac ctgtggggag cgtcacctgc tgacctctgt 180
accggaaaca attggtgggg ttgcgagaga accggaagtt tcaacaacat tctaaacccc 240
atacagtccg ccagactcag gacagttaac tctttcgctt tcaagcatgg acgaattgaa 300
gtctttgctc agttgcccaa aggagattgg ctttggccag caatttggct tctacccaaa 360
cgcaatgctt atggtggatg gcccgcatcc ggagagattg atctggttga atccagagga 420
aaccgaaact tgagagctgc agatggtacc cacgtcggag ctgagcaagt cggaatgacc 480
ttacattggg gaccctactg gcctctgaac ggctatccca tgacccataa tgccaagaac 540
ctaccagacg gacagacatt tggcgatggt ttccataact acacacttgt ctggactgct 600
gatagtctgg acttttatct agacggagaa ccaatccttg agcgtcgatc ctggtgc 657
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
atgaccgccg gtggtggagg 20
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
gcaccaggat cgacgctcaa g 21

Claims (10)

1. a kind of imitative stichopus japonicus glucan-binding protein, which is characterized in that the ammonia of the imitative stichopus japonicus Glucan binding protein A J-GBP Base acid sequence and SEQ ID NO.1 have 90% or more sequence homology；Preferably, the imitative stichopus japonicus glucan-binding protein The amino acid sequence of AJ-GBP is SEQ ID NO.1.
2. encoding the DNA molecular of the amino acid sequence of imitative stichopus japonicus glucan-binding protein described in claim 1.
3. a kind of DNA molecular as claimed in claim 2, which is characterized in that the DNA molecular has such as SEQ ID NO.2 institute The nucleotide sequence shown, or there is at least 90% homology with SEQ ID NO.2, and express amino acid shown in SEQ ID NO.1 The nucleotide sequence of sequence.
4. recombinant expression carrier, transgenic cell system containing DNA molecular described in claim 2 or 3 or transgenosis recombination Bacterium.
5. a kind of recombinant expression carrier as claimed in claim 4, which is characterized in that by DNA described in claim 2 or 3 points Son is inserted into the recombinant expression carrier of the imitative stichopus japonicus Glucan binding protein A J-GBP of expression obtained in coli expression carrier, Wherein, the coli expression carrier is pEASY-E2.
6. a kind of transgenosis recombinant bacterium as claimed in claim 4, which is characterized in that the transgenosis recombinant bacterium is to want right Recombinant expression carrier described in asking 5 is imported in Escherichia coli to screen and be obtained.
7. DNA molecular as claimed in claim 2, recombinant expression carrier as claimed in claim 4, transgenic cell system turn base Because recombinant bacterium is preparing the application in imitative stichopus japonicus Glucan binding protein A J-GBP.
8. a kind of preparation method of Glucan binding protein A J-GBP described in claim 1, which is characterized in that preparation method is such as Under:
Using imitative stichopus japonicus cDNA as template, PCR amplification is carried out with primers F 1 and R1；
It is attached after PCR product is purified with escherichia coli vector pEASY-E2 carrier, connection product converts Escherichia coli, surveys Sequence identifies recon；
Expression vector pEASY-AJ-GBP is transferred to Trans BL21 (DE3) pLysS Competent cell, screens transformant, Transformant is inoculated in the LB culture medium containing ampicillin, with IPTG inducing expression, then purifies recombination with super filter tube Albumen is to get Glucan binding protein A J-GBP；
Wherein, the primer is respectively as follows: F1:5 '-ATGACCGCCGGTGGTGGAGG-3 '；R1:5'- GCACCAGGATCGACGCTCAAG-3’。
9. the application of imitative stichopus japonicus glucan-binding protein described in claim 1, which is characterized in that the imitative stichopus japonicus glucan knot Hop protein AJ-GBP is used to prepare broad-spectrum antiseptic class preparation or feed addictive.
10. the application of imitative stichopus japonicus glucan-binding protein described in claim 1, which is characterized in that the imitative stichopus japonicus glucan It is short that binding protein AJ-GBP is used to prepare anti-bacillus subtilis, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, gamboge The pharmaceutical preparation or feed addictive of dialister bacterium and Vibrio anguillarum.
CN201910329721.XA 2019-04-23 2019-04-23 Apostichopus japonicus glucan binding protein and preparation method and application thereof Active CN110066330B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910329721.XA CN110066330B (en) 2019-04-23 2019-04-23 Apostichopus japonicus glucan binding protein and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910329721.XA CN110066330B (en) 2019-04-23 2019-04-23 Apostichopus japonicus glucan binding protein and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN110066330A true CN110066330A (en) 2019-07-30
CN110066330B CN110066330B (en) 2020-06-09

Family

ID=67368523

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910329721.XA Active CN110066330B (en) 2019-04-23 2019-04-23 Apostichopus japonicus glucan binding protein and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110066330B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112707960A (en) * 2020-12-28 2021-04-27 集美大学 Penaeus vannamei beta-1, 3-glucan binding protein antibacterial peptide
CN114907468A (en) * 2022-06-10 2022-08-16 东莞理工学院 Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106749588A (en) * 2016-12-07 2017-05-31 山东大学 A kind of imitative stichopus japonicus peptidoglycan recognition protein with bactericidal activity and its preparation method and application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106749588A (en) * 2016-12-07 2017-05-31 山东大学 A kind of imitative stichopus japonicus peptidoglycan recognition protein with bactericidal activity and its preparation method and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FRANCISCO VARGAS-ALBORES ET AL.: "Beta glucan binding protein and its role in shrimp immune response", 《AQUACULTURE》 *
ZHANG,X., ET AL.: "GenBank Accession：PIK59610.1", 《GENBANK》 *
徐帅妹等: "变形链球菌葡聚糖结合蛋白研究进展", 《口腔医学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112707960A (en) * 2020-12-28 2021-04-27 集美大学 Penaeus vannamei beta-1, 3-glucan binding protein antibacterial peptide
CN114907468A (en) * 2022-06-10 2022-08-16 东莞理工学院 Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof
CN114907468B (en) * 2022-06-10 2023-05-23 东莞理工学院 Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof

Also Published As

Publication number Publication date
CN110066330B (en) 2020-06-09

Similar Documents

Publication Publication Date Title
CN101801995B (en) Antibiotic peptides
CN111748018B (en) Biocompatible antibacterial peptide with self-assembly potential, and preparation method and application thereof
CN103773774A (en) Anthropogenic insect-resistant gene and anti-Cry1Ab toxin idiotype single-chain antibody coded by anthropogenic insect-resistant gene as well as application of antibody
JP6810470B2 (en) Silk fiber containing fusion protein and method for producing the silk fiber
CN110066330A (en) A kind of imitative stichopus japonicus glucan-binding protein and its preparation method and application
CN111171123B (en) Plant immune activation protein PsPII1 and application thereof
CN106749588B (en) A kind of imitative stichopus japonicus peptidoglycan recognition protein and its preparation method and application with bactericidal activity
CN111533786A (en) Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof
CN110903383A (en) Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof
CN114181321B (en) Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein and preparation method and application thereof
Zhang et al. In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum
Ip et al. Understanding the transition from water to land: Insights from multi-omic analyses of the perivitelline fluid of apple snail eggs
CN112778401B (en) Caprylic acid acylation modified antibacterial peptide and application thereof
CN108026155A (en) New antimicrobial peptide, their variation and purposes
CN108314718A (en) A kind of house fly antibiotic peptide MAF-1A peptides aggressiveness, its encoding gene and its expression and application
Ma et al. Two Novel Duck Antibacterial Peptides, Avian $\beta $-Defensins 9 and 10, with Antimicrobial Activity
CN108003233A (en) Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application
Lv et al. Antibacterial activities and mechanisms of action of a defensin from manila clam Ruditapes philippinarum
DE102014101663A1 (en) Synthetic artificial peptides with antimicrobial activity
CN106632692A (en) AMP (antimicrobial peptide) fusion protein as well as preparation method and application thereof
KR101601364B1 (en) A method for designing antimicrobial peptides for reducing the hemolysis thereof
CN108752455A (en) A kind of recombination and preparation of mycophylaxin and its application
CN110305887A (en) Anti-fungus peptide Drosomycin, preparation method and applications
CN110317253B (en) Chicken-derived antibacterial peptide and preparation method thereof
CN108546250B (en) Cyclic-L-glutamic acid-L-leucine and application thereof

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