CN106084024B - The preparation and application of litopenaeus vannamei antibacterial peptide-CrustinA gene and its recombinant protein - Google Patents
The preparation and application of litopenaeus vannamei antibacterial peptide-CrustinA gene and its recombinant protein Download PDFInfo
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Abstract
The present invention provides nucleic acid sequence, the preparation method and applications of recombinant protein expression vector, bacterial strain of a kind of litopenaeus vannamei novel antimicrobial peptide gene C rustinA that can be applied to fishes and shrimps feed addictive or disease-resistant drug exploitation.The long 688bp of the nucleic acid sequence of the CrustinA, open reading frame encodes 173 amino acid, supposition molecular weight of albumen size is 18.5KDa, and recombinant protein expression vector pYE-GAP α-CrustinA is transformed into Pichia pastoris GS115, obtains the recombinant protein bacterial strain that can stablize expression CrustinA.Recombinant antibacterial peptide albumen using expression bacterial strain prepared by the present invention and its expression has good antibacterial effect, can be applied to production fish and shrimps antibacterials, vaccine or feed addictive.
Description
Technical field
The invention belongs to biological gene engineering fields, and in particular to a kind of litopenaeus vannamei antibacterial peptide-CrustinA gene
And its preparation and application of recombinant protein.
Background technique
Fishery is modern agriculture important component, is human protein food important sources.During current fish production
Facing greatest problem is exactly disease problem: various bacteriums or virosis have often caused fish, shrimp mortality, thus to cultivation
Industry brings huge economic losses.In order to prevent and control fish, shrimp disease occurs, raiser have to largely to use chemicals into
Row pond disinfection adds antibiotics in feed.But largely use chemicals or antibiotics meeting
Bring serious ecological disruption and food-safety problem, it would therefore be highly desirable to develop can be applied to Modern Fishery it is pollution-free, without residual
It stays, safely and effectively Novel fishing medicine, reduces the use of chemicals or antibiotics.
Antibacterial peptide (Antimicrobial peptides) is called antimicrobial polypeptide or peptide antibiotic, is biological cell spy
Determine gene coding, a kind of polypeptide generated through the induction of specific external condition.Antibacterial peptide is widely distributed in animal and plant body, is natural
The important component of immune defense system has the characteristics that small molecular weight, thermostabilization, broad-spectrum antiseptic and non-immunogenicity.It is anti-
Bacterium peptide is just having been to be concerned by more and more people as the substitute of conventional antibiotic, is the active area of current International Academic research
One of.
Compared with antibiotic, antibacterial peptide has following characteristics: not only can be with resisting gram-positive bacteria and negative bacterium, or even to posting
Infested, fungi, virus also have resistant function；Molecular weight is small, is easy to be absorbed, and bactericidal effect is rapid, is not likely to produce drug resistance；
Antimicrobial concentration is small, and concentration unit is generally in a μm ol/L level.Antibacterial peptide is resistant to high temperature when feed granulating, scale fermentation life
Through high temperature enrichment process when producing antibacterial peptide, yeast thallus can be sufficiently killed without causing antibacterial peptide to inactivate, product is promoting and applying
Afterwards be not in the diffusion of engineering bacteria and lead to Environmental and ecological problems, part antibacterial peptide, which has, resists pepsin and trypsase
Ability, therefore it is with important application prospects in terms of disease-resistant feed additive research and development.
Antibacterial peptide is prevalent in vertebrate or invertebrate.Different from vertebrate, invertebrate does not have
Antibody is the specific immune system of important feature, resists cause of disease invasion and relies primarily on non-specific immune systems, antibacterial peptide is made
It plays an important role in resisting cause of disease phagocytic process for non-specific immune systems important component.Litopenaeus vannamei is as a kind of
Important economic cultivated animals have a large amount of cultivation in Asia and America.Litopenaeus vannamei belongs to invertebrate, gene
Group contains the genes such as a large amount of disease-resistant related genes, such as agglutinin, antibacterial peptide, lysozyme, peroxidase.In litopenaeus vannamei
Existing many disease-resistant relevant genes or molecule are found, and the recombinant protein of certain genes is applied to the disease-resistant medicine of fishes and shrimps
Object is developed and obtains related patents.
In litopenaeus vannamei or other shellfish, has been found to and announce there are many antibacterial peptide gene
In NCBI (US National Biotechnology Information center), but the present invention relates to antibacterial peptide gene CrustinA be this hair
Bright people has found and names for the first time, NCBI or pertinent literature still not about its nucleic acid sequence, recombinant protein research and
Application report.
Summary of the invention
The purpose of the present invention is to provide the systems of a kind of litopenaeus vannamei antibacterial peptide-CrustinA gene and its recombinant protein
Preparation Method and application, solve the problems, such as that current fishery disease is serious, ecological disruption caused by chemicals or antibiotics fishing medicine
And food medicament residue problem.
For achieving the above object, present invention employs following technical solutions:
A kind of litopenaeus vannamei antibacterial peptide, it is homologous with amino acid sequence at least 95% shown in SEQ ID NO:2 comprising having
The amino acid sequence of property.
A kind of gene C rustinA of above-mentioned litopenaeus vannamei antibacterial peptide, comprising having and nucleosides shown in SEQ ID NO:1
The nucleotide sequence of acid sequence at least 95% homology.
A kind of expression vector, for the gene C rustinA expression of nucleic acid sequence is connected to Yeast expression carrier pYE-
Between the EcoR I site and Xba I site of GAP α, and obtained expression vector pYE-GAP α-CrustinA.
A kind of preparation method of above-mentioned litopenaeus vannamei antibacterial peptide, including will the amino acid sequence as shown in SEQ ID NO:1
The DNA encoding sequence of column, which is cloned into expression vector, carries out protein expression, purifying, obtains litopenaeus vannamei antibacterial peptide-
CrustinA recombinant protein.
The detection and preparation method of a kind of above-mentioned CrustinA encoding gene and its variant, comprising the following steps: 1) extract
Litopenaeus vannamei total serum IgE obtains cDNA through mRNA purifying and reverse transcription；2) design primer, using PCR method amplification coding gene,
Obtain the encoding gene or its variant of the CrustinA；The primer is including the upstream primer as shown in SEQ ID NO:3 and such as
Downstream primer shown in SEQ ID NO:4.
A kind of above-mentioned litopenaeus vannamei antibacterial peptide is preparing the application in fishes and shrimps antibacterials, vaccine or feed addictive.
A kind of above-mentioned expression vector is preparing the application in fishes and shrimps antibacterials, vaccine or feed addictive.
Compared with the prior art, present invention has an advantage that
The present invention provides a kind of litopenaeus vannamei that can be applied to fishes and shrimps feed addictive or disease-resistant drug exploitation is novel
Nucleic acid sequence, the preparation method and applications of recombinant protein expression vector, bacterial strain of antibacterial peptide gene CrustinA.Institute of the present invention
The long 688bp of nucleic acid sequence of gene C rustinA is stated, open reading frame encodes 173 amino acid, thus it is speculated that molecular weight of albumen is big
Small is 18.5KDa, and recombinant protein expression vector pYE-GAP α-CrustinA is transformed into Pichia pastoris GS115, and acquisition can stablize table
Up to the recombinant protein bacterial strain of CrustinA.Using expression bacterial strain prepared by the present invention and the recombinant antibacterial peptide albumen tool of its expression
There is good antibacterial effect, can be applied to production fish and shrimps antibacterials, vaccine or feed addictive.
Detailed description of the invention
Fig. 1 is the Yeast expression carrier pYE-GAP α plasmid map that the present invention uses.
Fig. 2 is pYE-GAP α-crustin A plasmid enzyme restriction identification of the present invention.
Fig. 3 is that pYE-GAP α-crustin A electricity is turned yeast cells GS115 result by the present invention.
Fig. 4 is PCR identification positive colony bacterium of the present invention.
Fig. 5 is lab scale expression SDS-PAGE electrophoresis of the present invention.
Fig. 6 is crustin-A albumen WB qualification result of the present invention.
Fig. 7 is that the staphylococcus fungistatic effect of crustin-A albumen of the present invention is examined.
Fig. 8 is that the vibrio parahaemolytious fungistatic effect of crustin-A albumen of the present invention is examined.
Specific embodiment
One, litopenaeus vannamei antibacterial peptide: comprising having and at least 95% homology of amino acid sequence shown in SEQ ID NO:2
Amino acid sequence.
Two, the gene C rustinA of litopenaeus vannamei antibacterial peptide: comprising having and nucleotide sequence shown in SEQ ID NO:1
At least nucleotide sequence of 95% homology.
Three, expression vector pYE-GAP α-CrustinA: gene C rustinA expression of nucleic acid sequence is connected to Yeast expression load
It is obtained between the EcoR I site and Xba I site of body pYE-GAP α.
Four, the preparation method of litopenaeus vannamei antibacterial peptide: the DNA of the amino acid sequence as shown in SEQ ID NO:1 is compiled
Code sequence, which is cloned into expression vector, carries out protein expression, purifying, obtains litopenaeus vannamei antibacterial peptide-CrustinA and recombinates egg
It is white.
Five, the detection and preparation method of CrustinA encoding gene and its variant of the present invention: the following steps are included: 1) extracting
Litopenaeus vannamei total serum IgE obtains cDNA through mRNA purifying and reverse transcription；2) design primer, using PCR method amplification coding gene,
Obtain the encoding gene or its variant of the CrustinA；The primer is including the upstream primer as shown in SEQ ID NO:3 and such as
Downstream primer shown in SEQ ID NO:4.
Six, the purpose of the present invention is realized by following technological means:
(1) Total RNAs extraction: respectively from litopenaeus vannamei take optic stalk, the cheek, hepatopancrease, heart, stomach, intestines, rear coecum, muscle,
Nerve, epidermal tissue extract total serum IgE using RNA extracts kit after mixing.
(2) transcript profile is sequenced: transferring to sequencing company (Hua Da gene) to carry out transcript profile sequencing above-mentioned total serum IgE, to obtain
The transcription group information of the above-mentioned tissue of prawn is obtained, while obtaining correlated series label information (ESTs).
(3) acquisition of CrustinA partial nucleotide sequence (EST): the EditSeq journey in biosoftware DNAStar is used
Sequence searches PolyA tail, specific primer sequences and the overlay region of above-mentioned sequence, with the overlapping portion between SeqMan program removal sequence
Point, and splice and obtain full length cDNA sequence.Sequence uses BlastX (http://www.ncbi.nlm.nib.gov/ again after splicing
BLAST/ it) carries out homology search, compare analysis.The core with antibacterial peptide feature has been filtered out by Homology search and comparison
One of them is named as CrustinA by acid sequence.
(4) according to the est sequence of all shore prawn CrustinA, 5 '-RACE the acquisition of gene whole nucleotide sequence: are designed
With 3 '-RACE primers, is expanded by RACE and sequencing obtains the end CrustinA5 ' and 3 ' end unknown messages respectively, by 5 '
End and 3 ' terminal sequences carry out splicing and obtain complete CrustinA gene sequence information.
(5) acquisition of coded sequence: searching the opening code-reading frame (ORF) of sequence after splicing using EditSeq, reads open
Code area translates into amino acid sequence, while calculating the molecular weight and theory isoelectric point of albumen.With ammonia after Signa lP prediction translation
The signal peptide of base acid sequence and possible cleavage site.
(6) according to CrustinA sequence, CrustinA encoding histone zone amplication primer is designed, is respectively designed at the both ends of primer
Protectiveness base, by cloning site EcoR I and Xba I is connected into Yeast expression carrier pYE-GAP α, is transferred to Top10 clone
Bacterial strain.After digestion and sequence verification are errorless, and extract plasmid 10ug or more.Recombinant plasmid pYE- is linearized using AVr II
GAP α, electrotransformation Pichia pastoris GS115 select 5-10 plants of positive colonies, and are verified by PCR, select 1-2 plants of positive strains into
Row expression verifying.According to experiment be expected: in cell cultivation process, CrustinA albumen will secreting, expressing into culture medium, lead to
The expression for crossing SDS-PAGE electrophoresis detection and WB verifying purpose albumen has the expression for detecting target protein through analysis.
(7) purifying and antibacterial assay of CrustinA albumen.Finish red ferment for have proven to can to express CrustinA albumen
Female strain is inoculated into YPD culture medium (containing tryptone 2%, yeast extract 1%, glucose 2%), by the condition of culture of setting
After culture 96 hours, take supernatant spare bacterium solution 10000rpm centrifugation 2min.Supernatant samples are added to Ni-NTA chromatographic column
In, flow control collected penetrating component at 15ml/ hours or so, for the combination situation of SDS/PAGE analysis protein, with 5
The NTA-0Buffer of times Ni-NTA volume rinses the impurity that can not be adsorbed onto Ni-NTA, then with 5 times of Ni-NTA volume eluent mistakes
Column collects the eluent containing destination protein.Whether SDS-PAGE electrophoresis detection eluent contains destination protein or foreign protein,
It is concentrated using the protein solution that freeze drier will acquire, with protein quantification kit measurement protein concentration.
(8) the antibacterial effect analysis of CrustinA albumen.30uL logarithmic growth phase bacterium is taken, it is flat to be added to solid medium
Plate, mixing are applied on plate；It is sufficiently dipped into the CrustinA protein solution that concentration is (0.03mg/ml) with the sterilizing scraps of paper,
It then takes out and is labelled on plate, cultivate 12-20h.Sterile saline is set up as negative control, the same CrustinA of operating method
Protein Assav group sets up ampicillin and streptomysin drug sensitive test paper as positive control, directly drug sensitive test paper is taken to be labelled to
It is already coated on the plate of bacterium.
With reference to embodiments and its attached drawing is further non-limitingly described in detail technical solution of the present invention.
Method as used in the following examples is conventional method unless otherwise instructed, and specific steps can be found in:
" Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor).The primer and DNA sequence dna are synthesized by Hua Da gene (Shenzhen) Science and Technology Ltd..
PYE-GAP α carrier that the present invention uses, red yeast GS115 are purchased from Nanjing bronze object Bioisystech Co., Ltd, pYE-
The plasmid map difference of GAP α carrier is as shown in the figure；Oligo dT, various restriction enzymes and Taq enzyme are purchased from TaKaRa public affairs
Department, T4DNA ligase are purchased from NEB company, and plastic recovery kit and a small amount of extraction agent box of plasmid are purchased from OMEGA company, plasmid
A large amount of extraction agent boxes are purchased from Nucleo Bond company；DMEM high glucose medium, fetal calf serum are purchased from HyClone company；Turn
Transfection reagent Magetran is purchased from Origene company；Inverted microscope is Olympus product, and fluorescence inverted microscope is Nikon public
Take charge of product.SPF Penaeus Vannmei (10.2 ± 0.33g of weight) is derived from Guangxi Penaeus Vannmei country, aquatic products research institute seed multiplication farm.
PYE-GAP α plasmid (bronze object experiment saves), TOP10 bacterial strain (bronze object biology conservation), GS115 (are purchased from life company, bronze object is real
Test preservation), Protein Marker (self-control of bronze object biology), pvdf membrane (is purchased from U.S. Millipore company), and X-ray (is purchased from
Kodak), ECL developing solution (purchased from Chinese Puli's Lay company), the anti-His monoclonal antibody of mouse (self-control of bronze object biology laboratory),
Rabbit-anti mouse HRP secondary antibody (self-control of bronze object biology laboratory), Acr, Bis, Tris etc. (are purchased from Sigma company), and SDS (is purchased from
Amresco company), Tyrptone, Yeast Extract (being purchased from OXOID company), PCR reaction tube (are purchased from Fisher company),
0.22 μm of sterile filters and bag filter (being purchased from Millipore company), Ni2+IDA affinity chromatography glue (zoonbio company)
Agarose (is purchased from Shanghai genome company), and the small extraction reagent kit of DNA gel purification kit, plasmid (is purchased from AXYGEN company), SACI
(purchased from precious biology), conventional biochemical reagent is that domestic analysis is pure.Unless otherwise noted, other reagents that the present invention uses are city
Sell commodity.
The present invention is obtained by following steps:
(1) prawn sample of tissue: litopenaeus vannamei, average weight are about 12 grams.When sampling, other than hemolymph, also divide
Optic stalk, the cheek, hepatopancrease, heart, stomach, intestines, rear coecum, muscle, nerve, epidermis are not taken, are placed in RNAlater liquid (U.S. ANBIN
Company) in -80 DEG C save backup.
(2) Total RNAs extraction: total serum IgE is extracted using TRIzol reagent, referring to according to NanoDrop
The quality and integrality of 2100 Bioanalyzer Detection and Extraction total serum IgE of 1000spectrophotometer and Agilent,
Specific method is referring to specification.
(3) transcript profile library construction: the DNA fragmentation present in DnaseI digestion total serum IgE sample, reference kit (MRNA Isolation Systems) illustrate, mRNA is purified from total serum IgE.MRNA is random
Rear reverse transcription is interrupted into cDNA: by mRNA and interrupting reagent mixing, under heating conditions, and it is heavy to act on ethyl alcohol after a certain period of time
Method recycling in shallow lake interrupts product, configures the first chain synthesis reaction system and synthesizes cDNA.The end cDNA is repaired and is added for flat end, 3 '
Add (A) base, in flat 5 ' and 3 ' adjunction head of end cDNA, glue recycles the connection product of certain clip size.Use round pcr
Amplification has the DNA fragmentation of connector.After reaction, identification electrophoresis is carried out to PCR product, is purified with plastic recovery kit and is returned
It receives to target fragment, recovery product is dissolved in appropriate Elution Buffer, is marked, and so far library preparation is completed.
(4) transcript profile sequencing and gene identification: the litopenaeus vannamei library that will be built up exists according to sequencing procedures process
It is sequenced in 454GS-FLX system (Roche).Corresponding est sequence is obtained after being sequenced, and uses composite software
IAssembler (http://bioinfo, bti, cornell.edu/tool/iAssembler) assembles est sequence.
Function classification is carried out to all assembling sequences using GO (Gene Ontology), KEGG database.Categorization results, wherein one
Nucleic acid sequence CrustinA has antibacterial peptide feature, through ncbi database search, compares, without very high homology therewith in database
Nucleic acid sequence, therefore newfound nucleic acid sequence can be regarded as.
(5) CrustinA full length gene expands: according to all shore prawn CrustinA est sequences are obtained, design RACE draws
Object prepares PCR system and carries out the first round and the second wheel respectively using the 3 ' of CrustinA and 5 '-RACE-Ready cDNA as template
PCR amplification, volume increase object transfer to Hua Da Gene Tech. Company Limited to be sequenced；Sequencing result is carried out using DNAstar software
Analysis and splicing, to obtain the CrustinA gene comprising complete open reading frame (ORF).
(6) pYE-GAP α-crustinA: the carrier pYE-GAP α plasmid map that present example uses was as shown in Figure 1, should
Plasmid is cyclic annular double helix plasmid, and empty plasmid contains 3080 bases, is cried out containing a bleomycin screening-gene Zeocin, more
Foreign gene can be inserted in cloning site, and 6 histidine coding sequences, therefore expressed albumen are contained in multiple cloning sites upstream
Protein tag containing 6 histidines composition.
PYE-GAP α-crustinA plasmid construction is as shown in SEQ ID NO:6, using based on PAS (PCR-based
Accurate Synthesis) method synthesize gene crustinA, double digestion is connected to EcoR I of pYE-GAP α carrier
Between point (GAATTC) and Xba I site (TCTAGA)；The sequencing of picking positive clone molecule.
(7) pYE-GAP α-crustin-A plasmid enzyme restriction is identified: as shown in Fig. 2, in figure: M is nucleic acid molecular weight scale
Marker, the size of every band is respectively 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp from the bottom to top,
2000bp, 3000bp, 5000bp；1 is plasmid after digestion；2 be plasmid before digestion.
(8) plasmid is extracted and is linearized: 10uL linearization plasmid pYE-GAPa-crustin-A is added to equipped with 80uL
In the 1.5mLEP pipe of Pichia pastoris competence yeast cells, it is in 0.2cm electrotransformation cup that diameter is added to after mixing.Shock by electricity item
Part are as follows: voltage 1700V, time 8mS, electric shock 2 times.
(9) electricity turns yeast cells GS115: drawing 50ul, 100uL and 200uL linearization plasmid pYE- respectively
The mixed liquor of Lvcrustin-A-capsid is coated on 100ug/ml Zeocin antibiotic YPD plate, 30 degree of constant temperature incubations 48
Hour, bacterium colony is grown to plate, with the single bacterium grown on oese picking plate, it is linked into and is trained equipped with 10ml YPD liquid
It supports in base test tube (antibiotic Zeocin concentration 100ug/ml), 30 degree, 180rpm is incubated overnight, as a result as shown in Figure 3.
(10) PCR identifies positive colony bacterial strain: selecting 6 plants of positive colonies, extracting genomic DNA respectively, (number is 123
45 6), carry out PCR identification using 5 ' the pGAP AOX1priming primer pair target gene of priming and 3 ', as the result is shown
For the positive.PCR identifies positive clone molecule, it is contemplated that stripe size about 1.0K..As shown in figure 4, in figure: M is DNA Marker, from
Under to upper each band be respectively 100,250,500,750,1000,2000bp；1-6 is positive clone molecule PCR band.
(11) lab scale is expressed: crustin-A albumen size 18.5KD, PI=8.71, amino acid SEQ ID NO:5 after translation
It is shown.The expression bacterial strain (No. 3 bacterial strains) of the above-mentioned identification positive is taken to access equipped with 9ml YPD (antibiotic Zeocin 200ug/mL)
Test tube in, 30 degree, 220rpm culture for 24 hours after, take 500uL to be linked into 50ml YPD (antibiotic Zeocin 200ug/mL) liquid
In body culture medium, 30 degree, 220rpm culture, every 0h, for 24 hours, 48h, 72h, when 96h are sampled from culture medium, 10000rpm, 2min
Supernatant is collected by centrifugation, it is as shown in Figure 5 using SDS-PAGE electrophoresis detection result.In figure: Lane M:protein marker；
Lane 1:pGAPZaA--capsid is converted GS115 strain culturing 0 hour；Lane 2:pGAPZaA--capsid converts GS115
Strain culturing 24 hours；Lane 3:pGAPZaA--capsid is converted GS115 strain culturing 48 hours；Lane 4:pGAPZaA--
Capsid is converted GS115 strain culturing 72 hours；Lane 5:pGAPZaA--capsid is converted GS115 strain culturing 96 hours.
It is analyzed as the result is shown for 24 hours, 48h, 72h, 96h bacterial strain have the positive, select one plant of progress bacterial strain amplification culture, destination protein item
Band is as shown by arrows.
(12) WB (Western blot) is identified: step (11) supernatant samples being taken to carry out SDS-PAGE electrophoresis, electrophoresis knot
Gel is cut out to suitable size after beam, balances 20min with transferring film buffer.Filter paper several piece and pvdf membrane are cut out by gel size
One piece, immerse 10min in transferring film buffer.24 layers are stacked in order to cathode by anode in membrane-transferring deviceFilter paper, pvdf membrane,
Gel, 24 layersFilter paperAnd Accurate align, glass bar carefully exclude bubble.Place after the completion of power on, constant current 20V constant pressure or
30mA constant current transfers 1-2h.After transfer, pvdf membrane is taken out, whether observation pre-dyed marker shifts well.It is taken with tweezers
Pvdf membrane out is soaked in PBS-T liquid and rinses 3 times, each 5min.It takes out pvdf membrane to be put into confining liquid, room temperature is steadily shaken
2h.Then pvdf membrane is cleaned three times with PBST, each 5min.Film is put into plastic bag, the front of film is marked, then
The primary antibody diluted is added dropwise into bag, 6-Histag monoclonal antibody, 37 DEG C of incubation 1h are added.It takes the film out, is cleaned with PBS-T
Three times, each 5min.In the same way by pvdf membrane sheep anti-mouse igg-HRP antibody incubation 1-2h.After secondary antibody effect, take out
Film is cleaned three times with PBS-T, each 3-5min.Then film is put into plastic bag, appropriate DAB developing solution is added dropwise and is protected from light colour developing extremely
There is clearly target protein band appearance, is terminated and reacted with ddH2O, film is placed on filter paper, takes pictures or seals up for safekeeping after film parches.
Crustin-A albumen WB qualification result through Western blot as shown in fig. 6, verify, and the albumen of purifying is by anti-his
Antibody identification, stripe size are consistent with expection, it was demonstrated that crustin-A is expressed and purified successfully.In figure: Lane M:protein
marker；1 blank control of Lane；The crustin-A protein sample of Lane 2-5 various concentration.
(13) CrustinA recombinant protein purification: by step (11) bacterium solution 5000rpm × 10min be centrifuged, take supernatant into
Row protein purification.Specific method: chromatographic column is fixed on bracket, closes the cap of chromatographic column；It is slight to mix Ni-NTA resin
Filler (contains 30% isometric ethyl alcohol) in filler, 4-5ml is taken to be fitted into chromatographic column, stands to filler whole natural subsidence extremely
Behind bottom, unclamps cap and 30% ethyl alcohol is allowed to flow out, it is then primary with the buffer B balance pillar of 8 times of column volumes；It is past to balance
Pillar be added the protein solutions of about 8 times of column volumes, adjusting cap subset control flow speed, mistake column naturally under the effect of gravity；With 8 times of columns
Volume is rinsed pillar 2 times with buffer C, collects efflux.It is eluted pillar 4 times, is collected with the use buffer D of 1 times of column volume
Efflux；It is eluted pillar 4 times with the buffer E of 1 times of column volume, collects efflux；Take penetrate liquid and each gradient efflux into
Row SDS-PAGE electrophoresis analyzes recombinant protein distribution situation in each pipe.Use the dense of protein quantification kit measurement albumen
Degree.
(14) CrustinA recombinant protein bacteriostatic experiment: taking 30uL logarithmic growth phase bacterium, and it is flat to be added to solid medium
Plate, mixing are applied on plate；It is sufficiently dipped into the CrustinA protein solution that concentration is (0.03mg/ml) with the sterilizing scraps of paper,
It then takes out and is labelled on plate, cultivate 20h.Sterile saline is set up as negative control, operating method is the same as CrustinA egg
White experimental group sets up ampicillin and streptomysin drug sensitive test paper as positive control, directly drug sensitive test paper is taken to be labelled to
On coated germy plate.
It uses grape ball and vibrio alginolyticus as experimental strain respectively, fungistatic effect is identified by Bactericidal test, is as a result divided
Not as shown in FIG. 7 and 8, in figure: A:crustin-A；NC: saline control.Compared with negative control, crustin-A is to Portugal
Grape coccus and vibrio parahaemolytious obviously have bacteriostasis.
It should be pointed out that those of ordinary skill in the art, without departing from the principle of the present invention, may be used also
To make several improvements and modifications, these modifications and embellishments are also considered to be within the scope of the present invention.
Claims (7)
1. a kind of litopenaeus vannamei antibacterial peptide, it is characterised in that: amino acid sequence is amino acid sequence shown in SEQ ID NO:2.
2. a kind of gene C rustinA of litopenaeus vannamei antibacterial peptide described in claim 1, it is characterised in that: nucleotide sequence
For nucleotide sequence shown in SEQ ID NO:1.
3. a kind of expression vector, which is characterized in that the expression vector is by gene C rustinA core as claimed in claim 2
Sour expressed sequence is connected between the EcoR I site and Xba I site of Yeast expression carrier pYE-GAP α, and obtained expression
Carrier pYE-GAP α-CrustinA.
4. the preparation method of litopenaeus vannamei antibacterial peptide described in a kind of claim 1, which is characterized in that including will be such as SEQ ID
The DNA encoding sequence of nucleotide sequence shown in NO:1, which is cloned into expression vector, carries out protein expression, purifying, obtains all receive
Shore Ch-penaedin-CrustinA recombinant protein.
5. a kind of detection and preparation method of CrustinA encoding gene as claimed in claim 2, which is characterized in that including following
Step: 1) extracting litopenaeus vannamei total serum IgE, obtains cDNA through mRNA purifying and reverse transcription；2) design primer is expanded using PCR method
Increase encoding gene, obtains the encoding gene or its variant of the CrustinA；The primer include as shown in SEQ ID NO:3 on
Swim primer and the downstream primer as shown in SEQ ID NO:4.
6. litopenaeus vannamei antibacterial peptide is in preparing fishes and shrimps antibacterials, vaccine or feed addictive described in a kind of claim 1
Application.
7. expression vector described in a kind of claim 3 is preparing the application in fishes and shrimps antibacterials, vaccine or feed addictive.
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| CN107446032B (en) * | 2017-07-03 | 2020-05-15 | 汕头大学 | Antibacterial peptide and application thereof |
| CN108976298B (en) * | 2018-07-25 | 2021-09-10 | 汕头大学 | anti-WSSV peptide LvHcS52 derived from litopenaeus vannamei hemocyanin and application thereof |
| CN109627310B (en) * | 2019-01-10 | 2021-12-10 | 汕头大学 | Method for extracting antimicrobial protein of Chinese mitten crab and food preservation application thereof |
| CN115572325A (en) * | 2022-10-26 | 2023-01-06 | 江苏海洋大学 | Preparation method and application of exopalaemon carinicauda antibacterial peptide and recombinant protein thereof |
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