CN106084024A - Litopenaeus vannamei antibacterial peptide CrustinA gene and the preparation and application of recombiant protein thereof - Google Patents
Litopenaeus vannamei antibacterial peptide CrustinA gene and the preparation and application of recombiant protein thereof Download PDFInfo
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Abstract
The invention provides a kind of can be applicable to fish and shrimp feed additive or disease-resistant drug exploitation the nucleotide sequence of Litopenaeus vannamei novel antimicrobial peptide gene C rustinA, expression of recombinant proteins carrier, the preparation method and applications of bacterial strain.The long 688bp of nucleotide sequence of described CrustinA, open reading frame 173 aminoacid of coding, speculating that molecular weight of albumen size is 18.5KDa, expression of recombinant proteins carrier pYE GAP α CrustinA is transformed into Pichia pastoris GS115, it is thus achieved that can stably express the recombiant protein bacterial strain of CrustinA.Expression strain and its recombinant antibacterial peptide albumen expressed prepared by the employing present invention have good antibacterial effect, can be applicable to produce Fish and shrimps antibacterials, vaccine or feed additive.
Description
Technical field
The invention belongs to biological gene engineering field, be specifically related to a kind of Litopenaeus vannamei antibacterial peptide-CrustinA gene
And the preparation and application of recombiant protein.
Background technology
Fishery is modern agriculture important component part, is human protein food important sources.During current fish production
Facing greatest problem is exactly disease problem: various antibacterials or virosis have often caused fish, shrimp mortality, thus give cultivation
Industrial belt carrys out tremendous economic loss.In order to prevent and control fish, shrimp disease occurs, and raiser has to use chemicals to enter in a large number
Row pond disinfection or in feedstuff add antibiotics.But use chemicals or antibiotics meeting in a large number
Bring serious ecological disruption and food-safety problem, it would therefore be highly desirable to exploitation can be applicable to the pollution-free, without residual of Modern Fishery
Stay, the most novel fisheries drug, reduce chemicals or the use of antibiotics.
Antibacterial peptide (Antimicrobial peptides) be again antimicrobial polypeptide or peptide antibiotic, is that biological cell is special
Determine gene code, through the class polypeptide that the induction of specific external condition produces.Antibacterial peptide is widely distributed in animal and plant body, is natural
The important component part of immune defense system, has that molecular weight is little, thermally-stabilised, a feature such as broad-spectrum antiseptic and non-immunogenicity.Anti-
Bacterium peptide is the most increasingly paid close attention to by people as the substitute of conventional antibiotic, is the active area of current International Academic research
One of.
Comparing with antibiotic, antibacterial peptide has following characteristics: not only can be with resisting gram-positive bacteria and negative bacterium, even to posting
Infested, fungus, virus also have resistant function；Molecular weight is little, is readily absorbed by, and bactericidal action is rapid, is not likely to produce drug resistance；
Antimicrobial concentration is little, and concentration unit is typically in μm ol/L level.Antibacterial peptide is resistant to high temperature during feed granulating, and scale fermentation is raw
Through high temperature enrichment process during product antibacterial peptide, can fully kill yeast thalline and be not resulted in antibacterial peptide inactivation, product is in popularization and application
After do not have the diffusion of engineering bacteria and cause Environmental and ecological problems, part antibacterial peptide has opposing pepsin and trypsin
Ability, therefore in terms of disease-resistant feed additive research and development, there is important application prospect.
Antibacterial peptide is prevalent in vertebrates or invertebrates.Different from vertebrates, invertebrates does not has
Antibody is the specific immune system of key character, resists cause of disease invasion and relies primarily on non-specific immune systems, and antibacterial peptide is made
Play an important role in resisting cause of disease phagocytic process for non-specific immune systems important component.Litopenaeus vannamei is as one
Important economic cultivated animals, has substantial amounts of cultivation in Asia and America.Litopenaeus vannamei belongs to invertebrates, its gene
Group is containing 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 recombiant protein of some gene is applied to the disease-resistant medicine of fish and shrimp
Thing is developed and obtains Patents.
Litopenaeus vannamei or other Crustaceans, many antibacterial peptide genes have been had to have been found to and announce
On NCBI (US National Biotechnology Information center), but the antibacterial peptide gene CrustinA that the present invention relates to is this
A person of good sense finds first and names, NCBI or pertinent literature still not about its nucleotide sequence, the research of recombiant protein and
Application report.
Summary of the invention
It is an object of the invention to provide a kind of Litopenaeus vannamei antibacterial peptide-CrustinA gene and the system of recombiant protein thereof
Preparation Method and application, solve current fishery disease problem serious, the ecological disruption that chemicals or antibiotics fisheries drug cause
And food drug residue problem.
For achieving the above object, present invention employs following technical scheme:
A kind of Litopenaeus vannamei antibacterial peptide, comprises and has and aminoacid sequence at least 95% homology shown in SEQ ID NO:2
The aminoacid sequence of property.
Gene C rustinA of a kind of above-mentioned Litopenaeus vannamei antibacterial peptide, comprises and has and nucleoside shown in SEQ ID NO:1
The nucleotide sequence of acid sequence at least 95% homology.
A kind of expression vector, for being connected to Yeast expression carrier pYE-by described gene C rustinA expression of nucleic acid sequence
Between EcoR I site and the Xba I site of GAP α, and the expression vector pYE-GAP α-CrustinA obtained.
The preparation method of a kind of above-mentioned Litopenaeus vannamei antibacterial peptide, including by the aminoacid sequence as shown in SEQ ID NO:1
The DNA encoding sequence of row is cloned in expression vector and carries out protein expression, purification, it is thus achieved that and Litopenaeus vannamei antibacterial peptide-
CrustinA recombiant protein.
A kind of above-mentioned CrustinA encoding gene and the detection of variant thereof and preparation method, comprise the following steps: 1) extract
Litopenaeus vannamei total serum IgE, obtains cDNA through mRNA purification and reverse transcription；2) design primer, utilizes PCR method amplification coding gene,
Obtain encoding gene or its variant of described CrustinA；Described primer includes as shown in SEQ ID NO:3 forward primer and such as
Downstream primer shown in SEQ ID NO:4.
The application in preparing fish and shrimp antibacterials, vaccine or feed additive of a kind of above-mentioned Litopenaeus vannamei antibacterial peptide.
The application in preparing fish and shrimp antibacterials, vaccine or feed additive of a kind of above-mentioned expression vector.
Compared to prior art, present invention have an advantage that
The invention provides a kind of can be applicable to fish and shrimp feed additive or disease-resistant drug exploitation Litopenaeus vannamei novel
The nucleotide sequence of antibacterial peptide gene CrustinA, expression of recombinant proteins carrier, the preparation method and applications of bacterial strain.Institute of the present invention
State the long 688bp of nucleotide sequence of gene C rustinA, open reading frame 173 aminoacid of coding, thus it is speculated that molecular weight of albumen is big
Little it is transformed into Pichia pastoris GS115 for 18.5KDa, expression of recombinant proteins carrier pYE-GAP α-CrustinA, it is thus achieved that table can be stablized
Reach the recombiant protein bacterial strain of CrustinA.Expression strain and its recombinant antibacterial peptide albumen expressed prepared by the employing present invention have
There is good antibacterial effect, can be applicable to produce Fish and shrimps antibacterials, vaccine or feed additive.
Accompanying drawing explanation
Fig. 1 is the Yeast expression carrier pYE-GAP α plasmid map that the present invention uses.
Fig. 2 is that pYE-GAP α-crustin A plasmid enzyme restriction of the present invention is identified.
Fig. 3 is that pYE-GAP α-crustin A electricity is turned yeast cells GS115 result by the present invention.
Fig. 4 is that PCR of the present invention identifies positive colony bacterium.
Fig. 5 is that lab scale of the present invention expresses SDS-PAGE.
Fig. 6 is crustin-A albumen WB qualification result of the present invention.
Fig. 7 is the staphylococcus fungistatic effect inspection of crustin-A albumen of the present invention.
Fig. 8 is the vibrio parahaemolytious fungistatic effect inspection of crustin-A albumen of the present invention.
Detailed description of the invention
One, Litopenaeus vannamei antibacterial peptide: comprise and have and aminoacid sequence at least 95% homology shown in SEQ ID NO:2
Aminoacid sequence.
Two, gene C rustinA of Litopenaeus vannamei antibacterial peptide: comprise and have and nucleotide sequence shown in SEQ ID NO:1
The nucleotide sequence of at least 95% homology.
Three, expression vector pYE-GAP α-CrustinA: gene C rustinA expression of nucleic acid sequence is connected to yeast expression and carries
Obtain between EcoR I site and the Xba I site of body pYE-GAP α.
Four, the preparation method of Litopenaeus vannamei antibacterial peptide: the DNA of the aminoacid sequence as shown in SEQ ID NO:1 is compiled
Code sequence is cloned in expression vector and carries out protein expression, purification, it is thus achieved that Litopenaeus vannamei antibacterial peptide-CrustinA recombinates egg
In vain.
Five, CrustinA encoding gene of the present invention and the detection of variant thereof and preparation method: comprise the following steps: 1) extract
Litopenaeus vannamei total serum IgE, obtains cDNA through mRNA purification and reverse transcription；2) design primer, utilizes PCR method amplification coding gene,
Obtain encoding gene or its variant of described CrustinA；Described primer includes as shown in SEQ ID NO:3 forward primer and such as
Downstream primer shown in SEQ ID NO:4.
Six, it is an object of the invention to be realized by techniques below means:
(1) Total RNAs extraction: respectively from Litopenaeus vannamei take optic stalk, the cheek, hepatopancrease, heart, stomach, intestinal, rear coecum, muscle,
Neural, epidermal tissue, uses RNA to extract test kit and extracts total serum IgE after mixing.
(2) transcript profile order-checking: above-mentioned total serum IgE is transferred to order-checking company (Hua Da gene) carry out transcript profile order-checking, thus obtains
Obtain the transcript profile information of the above-mentioned tissue of prawn, obtain correlated series label information (ESTs) simultaneously.
(3) acquisition of CrustinA partial nucleotide sequence (EST): use the EditSeq journey in biosoftware DNAStar
Sequence searches PolyA tail, specific primer sequences and the overlay region of above-mentioned sequence, removes the overlapping portion between sequence by SeqMan program
Divide, and splicing obtains full length cDNA sequence.After splicing, sequence is again with BlastX (http://www.ncbi.nlm.nib.gov/
BLAST/) homology search, comparison analysis are carried out.The core with antibacterial peptide feature has been filtered out by Homology search and comparison
Acid sequence, by one of them named CrustinA.
(4) acquisition of gene whole nucleotide sequence: according to the est sequence of all shore prawn CrustinA, designs 5 '-RACE
With 3 '-RACE primers, expanded by RACE and check order obtain respectively CrustinA5 ' end and 3 ' end unknown messages, by 5 '
End and 3 ' terminal sequences carry out splicing and obtain complete CrustinA gene sequence information.
(5) acquisition of coded sequence: the opening code-reading frame (ORF) of sequence after using EditSeq lookup to splice, reads opening
Code district translates into aminoacid sequence, calculates the molecular weight of albumen and theoretical isoelectric point, IP simultaneously.With ammonia after Signa lP prediction translation
The signal peptide of base acid sequence and possible cleavage site.
(6) according to CrustinA sequence, design CrustinA encoding histone zone amplication primer, respectively design at the two ends of primer
Protectiveness base, is connected into Yeast expression carrier pYE-GAP α by cloning site EcoR I and Xba I, proceeds to Top10 clone
Bacterial strain.After enzyme action and sequence verification are errorless, and extract more than plasmid 10ug.Utilize AVr II linearisation recombiant plasmid pYE-
GAP α, electricity is converted Pichia pastoris GS115, selects 5-10 strain positive colony, and verified by PCR, selects 1-2 strain positive strain to enter
Row expresses checking.According to experiment expection: in cell cultivation process, CrustinA albumen will secreting, expressing in culture medium, logical
Cross SDS-PAGE electrophoresis detection and the expression of WB checking destination protein, have expression target protein being detected through analysis.
(7) purification of CrustinA albumen and antibacterial assay.To have proven to express the complete red ferment of CrustinA albumen
Female strain is inoculated into YPD culture medium (containing tryptone 2%, yeast extract 1%, glucose 2%), by the condition of culture set
After cultivating 96 hours, bacterium solution 10000rpm is centrifuged 2min, and to take supernatant standby.Supernatant samples is added to Ni-NTA chromatographic column
In, flow speed control, at about 15ml/ hour, collects penetrating component, for the combination situation of SDS/PAGE analysing protein, with 5
The NTA-0Buffer of times Ni-NTA volume rinses the impurity that cannot be adsorbed onto Ni-NTA, then by 5 times of Ni-NTA volume eluent mistakes
Post, collects the eluent containing destination protein.Whether SDS-PAGE electrophoresis detection eluent contains destination protein or foreign protein,
Freezer dryer is utilized to be concentrated by the protein solution of acquisition, with protein quantification kit measurement protein concentration.
(8) the antibacterial effect analysis of CrustinA albumen.Take 30uL exponential phase antibacterial, join solid medium and put down
Plate, mixing is applied on flat board；The CrustinA protein solution that concentration is (0.03mg/ml) fully it is dipped into the sterilizing scraps of paper,
Then take out and be labelled on flat board, cultivate 12-20h.Set up sterile saline as negative control, the same CrustinA of operational approach
Protein Assav group, sets up ampicillin and streptomycin drug sensitive test paper as positive control, directly takes drug sensitive test paper and be labelled to
Already coated with on the flat board of antibacterial.
Below in conjunction with embodiment and accompanying drawing thereof, technical solution of the present invention is further non-limitingly described in detail.
Method used in following embodiment is conventional method if no special instructions, and concrete 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 are synthesized by Hua Da gene (Shenzhen) Science and Technology Ltd..
The 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 of GAP α carrier is the most as shown in the figure；It is public that oligo dT, various restricted enzyme and Taq enzyme are purchased from TaKaRa
Department, T4DNA ligase is purchased from NEB company, and glue reclaims test kit and plasmid a small amount of extraction agent box purchased from OMEGA company, plasmid
A large amount of extraction agent boxes are purchased from Nucleo Bond company；DMEM high glucose medium, hyclone are purchased from HyClone company；Turn
Transfection reagent Magetran is purchased from Origene company；Inverted microscope is Olympus product, and fluorescence inverted microscope is that Nikon is public
Department's product.SPF Penaeus vannamei (body weight 10.2 ± 0.33g), takes from Guangxi Aquatic product institute Penaeus vannamei country seed multiplication farm.
PYE-GAP α plasmid (bronze object experiment preserves), TOP10 bacterial strain (bronze object biology conservation), (purchased from life company, bronze object is real for GS115
Test preservation), Protein Marker (self-control of bronze object biology), pvdf membrane (purchased from Millipore company of the U.S.), X-ray (is purchased from
Kodak), ECL nitrite ion (purchased from Puli Lay company of China), mouse-anti His monoclonal antibody (self-control of bronze object biology laboratory),
Rabbit anti-Mus HRP bis-anti-(self-control of bronze object biology laboratory), Acr, Bis, Tris etc. (purchased from Sigma company), SDS (is purchased from
Amresco company), Tyrptone, Yeast Extract (purchased from OXOID company), PCR reaction tube (purchased from Fisher company),
0.22 μm sterile filters and bag filter (purchased from Millipore company), Ni2+IDA affinity chromatograph glue (zoonbio company)
Agarose (purchased from Shanghai genome company), the little extraction reagent kit of DNA gel purification kit, plasmid (purchased from AXYGEN company), SACI
(purchased from precious biological), routine biochemistry reagent is domestic analytical pure.Unless otherwise noted, other reagent 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 is about 12 grams.During sampling, in addition to hemolymph, also divide
Do not take optic stalk, the cheek, hepatopancrease, heart, stomach, intestinal, rear coecum, muscle, nerve, epidermis, be placed in RNAlater liquid (U.S. ANBIN
Company) in-80 DEG C save backup.
(2) Total RNAs extraction: use TRIzol reagent to extract total serum IgE, with reference to according to NanoDrop
The quality of 1000spectrophotometer and Agilent 2100 Bioanalyzer Detection and Extraction total serum IgE and integrity,
Concrete grammar is with reference to description.
(3) transcript profile library construction: with DnaseI digestion total serum IgE sample present in DNA fragmentation, reference reagent box (MRNA Isolation Systems) illustrate, mRNA is purified from total serum IgE.MRNA is random
Interrupt rear reverse transcription and become cDNA: by mRNA with interrupt reagent mixing, under conditions of heating, and ethanol sinks after acting on certain time
Shallow lake method reclaims and interrupts product, configures the first chain synthesis reaction system and synthesizes cDNA.Being flat end by cDNA end reparation, 3 ' add
Adding (A) base, add joint at flat end cDNA 5 ' and 3 ', glue reclaims the connection product of certain clip size.Use round pcr
Expand the DNA fragmentation with joint.After reaction terminates, carry out PCR primer identifying electrophoresis, reclaim kits with glue and return
Receiving purpose fragment, reclaim product and be dissolved in appropriate Elution Buffer, carry out labelling, so far prepared by library.
(4) transcript profile order-checking and gene recognition: the Litopenaeus vannamei library that will build up, exists according to sequencing procedures flow process
Check order in 454GS-FLX system (Roche).After order-checking, obtain corresponding est sequence, use composite software
Est sequence is assembled by iAssembler (http://bioinfo, bti, cornell.edu/tool/iAssembler).
Use GO (Gene Ontology), KEGG data base that all assembling sequences are carried out function classification.Categorization results, wherein one
Nucleotide sequence CrustinA has antibacterial peptide feature, through ncbi database search, comparison, does not has very high homology the most therewith in data base
Nucleotide sequence, therefore can regard as newfound nucleotide sequence.
(5) CrustinA full length gene amplification: according to obtaining all shores prawn CrustinA est sequence, design RACE draws
Thing, with 3 ' and the 5 '-RACE-Ready cDNA of CrustinA as template, preparation PCR system carries out the first round and second respectively and takes turns
PCR expands, and volume increase thing transfers to Hua Da Gene Tech. Company Limited to check order；Utilize DNAstar software that sequencing result is carried out
Analyze and splicing, thus obtain the CrustinA gene comprising complete open reading frame (ORF).
(6) the carrier pYE-GAP α plasmid map that pYE-GAP α-crustinA: present example uses is as it is shown in figure 1, be somebody's turn to do
Plasmid is ring-type Double helix plasmid, and empty plasmid contains 3080 bases, cries out containing a bleomycin screening-gene Zeocin, many
Cloning site may be inserted into exogenous gene, and 6 histidine coding sequence, therefore expressed albumen are contained in multiple clone site upstream
Protein tag containing 6 histidine compositions.
PYE-GAP α-crustinA plasmid construction, as shown in SEQ ID NO:6, uses based on PAS (PCR-based
Accurate Synthesis) method synthetic gene crustinA, double digestion is connected to the EcoR I position of pYE-GAP α carrier
Between point (GAATTC) and Xba I site (TCTAGA)；Picking positive colony checks order.
(7) pYE-GAP α-crustin-A plasmid enzyme restriction is identified: as in figure 2 it is shown, 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 enzyme action；2 is plasmid before enzyme action.
(8) plasmid extraction and linearisation: 10uL linearization plasmid pYE-GAPa-crustin-A is joined equipped with 80uL
In the 1.5mLEP pipe of Pichia sp. competence yeast cells, join a diameter of 0.2cm electricity after mixing and convert in cup.Electric shock bar
Part is: voltage 1700V, time 8mS, electric shock 2 times.
(9) electricity turns yeast cells GS115: draw 50ul, 100uL and 200uL linearization plasmid pYE-respectively
The mixed liquor of Lvcrustin-A-capsid is coated on 100ug/ml Zeocin antibiotic YPD flat board, 30 degree of constant temperature culture 48
Hour, treat that flat board grows bacterium colony, with single bacterium of growth on inoculating loop picking flat board, it is linked into and trains equipped with 10ml YPD liquid
Supporting in base test tube (antibiotic Zeocin concentration 100ug/ml), 30 degree, 180rpm incubated overnight, result is as shown in Figure 3.
(10) PCR identifies positive colony bacterial strain: selects 6 strain positive colonies, extracts genomic DNA (numbered 123 respectively
45 6), using 5 ' pGAP priming and 3 ' AOX1priming primers that genes of interest is carried out PCR qualification, result shows all
For the positive.PCR identifies positive colony, it is contemplated that stripe size about 1.0K..As shown in Figure 4, in figure: M is DNA Marker, from
Under be respectively 100,250,500,750,1000,2000bp to upper each band；1-6 is positive colony PCR band.
(11) lab scale is expressed: crustin-A albumen size 18.5KD, PI=8.71, aminoacid SEQ ID NO:5 after translation
Shown in.Take the positive expression strain (No. 3 bacterial strains) of above-mentioned qualification to access equipped with 9ml YPD (antibiotic Zeocin 200ug/mL)
Test tube in, 30 degree, 220rpm cultivate after 24h, take 500uL and be linked into 50ml YPD (antibiotic Zeocin 200ug/mL) liquid
In body culture medium, 30 degree, 220rpm cultivation, every 0h, sample from culture medium when 24h, 48h, 72h, 96h, 10000rpm, 2min
Centrifugal supernatant of collecting, use SDS-PAGE electrophoresis detection result is as shown in Figure 5.In figure: Lane M:protein marker；
Lane 1:pGAPZaA--capsid converts GS115 strain culturing 0 hour；Lane 2:pGAPZaA--capsid converts GS115
Strain culturing 24 hours；Lane 3:pGAPZaA--capsid converts GS115 strain culturing 48 hours；Lane 4:pGAPZaA--
Capsid converts GS115 strain culturing 72 hours；Lane 5:pGAPZaA--capsid converts GS115 strain culturing 96 hours.
Showing 24h through analysis result, 48h, 72h, 96h bacterial strain all has the positive, selects a strain to carry out bacterial strain amplification culture, destination protein bar
Band is as shown by arrows.
(12) WB (Western blot) identifies: taking step (11) supernatant samples and carry out SDS-PAGE electrophoresis, electrophoresis is tied
After bundle, gel is cut out to suitable size, balance 20min with transferring film buffer.Filter paper several piece and pvdf membrane is cut out by gel size
One piece, immerse 10min in transferring film buffer.24 layers are stacked in order by positive pole to negative pole in membrane-transferring deviceFilter paper, pvdf membrane,
Gel, 24 layersFilter paperAnd Accurate align, Glass rod carefully gets rid of bubble.Placement switches on power after completing, constant current 20V constant voltage or
30mA constant current transfer 1-2h.After transfer terminates, pvdf membrane is taken out, observe whether pre-dyed marker shifts well.Take with tweezers
Go out pvdf membrane, be soaked in PBS-T liquid rinsing 3 times, each 5min.Taking out pvdf membrane to be put in confining liquid, room temperature is steadily shaken
2h.Then pvdf membrane is cleaned three times with PBST, each 5min.Film is put in plastic bag, labelling is carried out in the front of film, then
Dropping has diluted in bag one resists, and adds 6-Histag monoclonal antibody, hatches 1h for 37 DEG C.Take the film out, clean with PBS-T
Three times, each 5min.In the same way by pvdf membrane sheep anti-mouse igg-HRP antibody incubation 1-2h.After two anti-effects, take out
Film cleans three times with PBS-T, each 3-5min.Then film is put in plastic bag, drip appropriate DAB nitrite ion lucifuge and develop the color extremely
There is target protein band clearly to occur, terminate reaction with ddH2O, film is placed on filter paper, takes pictures after film parches or seal up for safekeeping.
Crustin-A albumen WB qualification result as shown in Figure 6, is verified through Western blot, and the albumen of purification is by anti-his
Antibody recognition, stripe size is consistent with expection, it was demonstrated that crustin-A expresses and purification success.In figure: Lane M:protein
marker；Lane 1 blank；The crustin-A protein sample of Lane 2-5 variable concentrations.
(13) CrustinA recombinant protein purification: step (11) bacterium solution 5000rpm × 10min is centrifuged, takes supernatant and enter
Row protein purification.Concrete grammar: be fixed on support by chromatographic column, closes the medicated cap of chromatographic column；Slight mixing Ni-NTA resin
Filler (containing isopyknic 30% ethanol in filler), takes 4-5ml and loads in chromatographic column, stand and treat the whole natural subsidence of filler extremely
After Di Bu, unclamp medicated cap and allow 30% ethanol flow out, then balance pillar once with the buffer B of 8 times of column volumes；Toward balancing
Pillar add about 8 times of column volumes protein solution, adjust medicated cap coutroi velocity, the most naturally mistake post；With 8 times of posts
Volume buffer C rinses pillar 2 times, collects effluent.With 1 times of column volume with buffer D eluting pillar 4 times, collect
Effluent；With the buffer E eluting pillar 4 times of 1 times of column volume, collect effluent；Take and penetrate liquid and each gradient effluent enters
Row SDS-PAGE electrophoresis, analyzes recombinant protein distribution situation in each pipe.Use the dense of protein quantification kit measurement albumen
Degree.
(14) CrustinA recombiant protein bacteriostatic experiment: take 30uL exponential phase antibacterial, joins solid medium and puts down
Plate, mixing is applied on flat board；The CrustinA protein solution that concentration is (0.03mg/ml) fully it is dipped into the sterilizing scraps of paper,
Then take out and be labelled on flat board, cultivate 20h.Setting up sterile saline as negative control, operational approach is with CrustinA egg
White experimental group, sets up ampicillin and streptomycin drug sensitive test paper as positive control, directly takes drug sensitive test paper and be labelled to
On coated germy flat board.
Using Fructus Vitis viniferae ball and vibrio alginolyticus as experimental strain respectively, identify fungistatic effect by Bactericidal test, result is divided
The most 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 substantially have bacteriostasis.
It should be pointed out that, to those of ordinary skill in the art, under the premise without departing from the principles of the invention, also may be used
To make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (7)
1. a Litopenaeus vannamei antibacterial peptide, it is characterised in that: comprise and have with aminoacid sequence shown in SEQ ID NO:2 at least
The aminoacid sequence of 95% homology.
2. gene C rustinA of Litopenaeus vannamei antibacterial peptide described in a right 1, it is characterised in that: comprise and have and SEQ ID
The nucleotide sequence of the homology of nucleotide sequence at least 95% shown in NO:1.
3. an expression vector, it is characterised in that described expression vector is by the gene C rustinA core described in claim 2
Acid expressed sequence is connected between EcoR I site and the Xba I site of Yeast expression carrier pYE-GAP α, and the expression obtained
Carrier pYE-GAP α-CrustinA.
4. the preparation method of Litopenaeus vannamei antibacterial peptide described in a claim 1, it is characterised in that include such as SEQ ID
The DNA encoding sequence of the aminoacid sequence shown in NO:1 is cloned in expression vector and carries out protein expression, purification, it is thus achieved that all receive
Shore Ch-penaedin-CrustinA recombiant protein.
5. CrustinA encoding gene described in a claim 2 and the detection of variant thereof and preparation method, it is characterised in that
Comprise the following steps: 1) extract Litopenaeus vannamei total serum IgE, obtain cDNA through mRNA purification and reverse transcription；2) design primer, utilizes
PCR method amplification coding gene, it is thus achieved that the encoding gene of described CrustinA or its variant；Described primer includes such as SEQ ID
Forward primer shown in NO:3 and as shown in SEQ ID NO:4 downstream primer.
6. Litopenaeus vannamei antibacterial peptide described in a claim 1 is in preparing fish and shrimp antibacterials, vaccine or feed additive
Application.
7. expression vector application in preparing fish and shrimp antibacterials, vaccine or feed additive described in a claim 3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446032A (en) * | 2017-07-03 | 2017-12-08 | 汕头大学 | A kind of antibacterial peptide and its application |
| CN108976298A (en) * | 2018-07-25 | 2018-12-11 | 汕头大学 | It is a kind of from the anti-WSSV peptide LvHcS52 of hemocyanin in shrimp Litopenaeus vannamei and its application |
| CN109627310A (en) * | 2019-01-10 | 2019-04-16 | 汕头大学 | A kind of word line bow crab antibacterial protein extracting method and its food fresh keeping application |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654656A (en) * | 2005-01-14 | 2005-08-17 | 中国科学院海洋研究所 | FenneropenaeusChinensis Crustin gene and coded protein and cloning method therefor |
| CN101525617A (en) * | 2009-03-09 | 2009-09-09 | 中国科学院海洋研究所 | Eriocheir sinensis Crustin-1 gene and in-vitro recombination expression |
| CN102816815A (en) * | 2012-03-30 | 2012-12-12 | 浙江海洋学院 | Yeast fermentation production method of recombinant antibacterial peptide rCrustinI |
-
2016
- 2016-06-12 CN CN201610417577.1A patent/CN106084024B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654656A (en) * | 2005-01-14 | 2005-08-17 | 中国科学院海洋研究所 | FenneropenaeusChinensis Crustin gene and coded protein and cloning method therefor |
| CN101525617A (en) * | 2009-03-09 | 2009-09-09 | 中国科学院海洋研究所 | Eriocheir sinensis Crustin-1 gene and in-vitro recombination expression |
| CN102816815A (en) * | 2012-03-30 | 2012-12-12 | 浙江海洋学院 | Yeast fermentation production method of recombinant antibacterial peptide rCrustinI |
Non-Patent Citations (4)
| Title |
|---|
| 张艳艳等: "凡纳滨对虾crustin-like基因的克隆及在副溶血弧菌感染条件下的表达分析", 《西北农林科技大学学报（自然科学版）》 * |
| 杜志强: "凡纳对虾crustin 1 抗菌肽基因的克隆与性质研究", 《四川师范大学学报( 自然科学版)》 * |
| 杜志强等: "凡纳对虾crustin2基因的序列及结构分析", 《湖北农业科学》 * |
| 黎铭等: "凡纳滨对虾CrustinⅠ基因的克隆与序列分析", 《广西农业科学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446032A (en) * | 2017-07-03 | 2017-12-08 | 汕头大学 | A kind of antibacterial peptide and its application |
| CN107446032B (en) * | 2017-07-03 | 2020-05-15 | 汕头大学 | Antibacterial peptide and application thereof |
| CN108976298A (en) * | 2018-07-25 | 2018-12-11 | 汕头大学 | It is a kind of from the anti-WSSV peptide LvHcS52 of hemocyanin in shrimp Litopenaeus vannamei and its application |
| CN108976298B (en) * | 2018-07-25 | 2021-09-10 | 汕头大学 | anti-WSSV peptide LvHcS52 derived from litopenaeus vannamei hemocyanin and application thereof |
| CN109627310A (en) * | 2019-01-10 | 2019-04-16 | 汕头大学 | A kind of word line bow crab antibacterial protein extracting method and its food fresh keeping application |
| 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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