CN110240638A - A kind of antibacterial peptide and its application using bioinformatics method building - Google Patents
A kind of antibacterial peptide and its application using bioinformatics method building Download PDFInfo
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- CN110240638A CN110240638A CN201910494930.XA CN201910494930A CN110240638A CN 110240638 A CN110240638 A CN 110240638A CN 201910494930 A CN201910494930 A CN 201910494930A CN 110240638 A CN110240638 A CN 110240638A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Abstract
A kind of antibacterial peptide and its application using bioinformatics method building.The present invention provides a kind of novel antibacterial peptides, antibacterial peptide gene is obtained using the transcript profile data combination bioinformatics method of the small wheat SN6306 that lays down, by converting Bacillus coli expression destination protein, the antifungal activity of this antibacterial peptide is separately verified by the methods of PDA Plating, wheat leaf blade smearing.The antibacterial peptide can be used for preparing the inhibitor of anti-Fusarium graminearum, resist powdery mildew of wheat, be a kind of raw material with the huge antimycotic inhibitor of potential utility value.The antibacterial peptide that this patent provides can replace traditional chemical pesticide, prevent and treat wheat and the fungal disease of other crops.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to it is a kind of using bioinformatics method building antibacterial peptide and
It is applied.
Background technique
Wheat is important one of the crops in China, and mainly as cereal crops.Wheat scab and wheat powdery mildew
It is the important disease for seriously affecting Huang-Huai-Hai area of wheat yield and quality of wheat.Current mainstream control measure is still with chemical prevention
It is main.The application of pesticide exacerbates environmental pollution, and pathogen is easy to cause to generate drug resistance.Antibacterial peptide is in organism to thin
Bacterium and fungi have the small-molecular peptides of inhibitory or killing effect, and the composition of the natural defending system of pathogen invasion is resisted in many organisms
Part can sterilize rapidly because of its special mechanism of action and not easily lead to drug resistance, and anti-with wide spectrum to phytopathogen
Property, become a kind of natural drug (swallow dawn kingfisher etc., 2017) with development potential.
The biological activity of antibacterial peptide includes antibacterium, antimycotic and adjust immune function.
Most of antibacterial peptides, which can press down, kills gram-positive bacteria and Gram-negative bacteria, the different types of antibacterial of separate sources
Some differences of the antibacterial activity of peptide.It is most of at present to think that antibacterial mechanisms are antibacterial peptide and bacterial cell membrane interaction
(Bechinger etc., 2017；Tossi etc., 2012).Positively charged cationic antibacterial peptide and gramnegative bacterium are negatively charged
The surface molecular membrane phospholipid of lotus generates electrostatic, is incorporated on bacterial cell membrane, then the hydrophobic section of antibacterial peptide causes membrane structure to become
Change (Ageitos etc., 2016).
Constantly upgrade as microorganism leads to the problem of drug resistance to conventional antibiotic, people increasingly pay close attention to anti-fungus peptide
Potentiality (Epand etc., 1999) as new antibiotic.Anti-fungus peptide (Antifungal peptide, AFP) is antibacterial peptide
A subset in (Antimicrobial peptide, AMP), AFP can be divided into linear peptides, form amphipathic hydrophobic helices
Peptide, β-piece peptide, alpha-helix and β-piece hybrid peptide, the peptide rich in specific amino acids and modified cyclic peptide and lipopeptid (Nguyen
Deng 2011；Tam etc., 2015；Hamley etc., 2015).Antibacterial peptide 3 kinds of hypothesis main to the resistance mechanism of fungi at present: first is that
Antibacterial peptide inhibits the synthesis of the important composition ingredients of cell walls such as chitin, glucan, hinders the formation of fungal cell wall；Second is that
Antibacterial peptide and cell membrane interaction destroy cell membrane, content are made to leak；Third is that antibacterial peptide acts on the fungal cells such as mitochondria
Device is finally reached and presses down antifungal purpose.
Antibacterial peptide not only has antibacterial action, can also adjust immune system, such as chemotactic, immune cell activated and adjusting are scorching
Disease.Antibacterial peptide fowlicidin-1 (6-26) has very strong adjusting immune system, directly recruitment neutrophil leucocyte, activation huge
The ability of phagocyte, enhancement antigen specificity adaptive immune response finds neutrophilia after peptide is injected into mouse peritoneum
Granulocyte is by specific chemotactic, fowl-1(6-26) with enhance antigen-specific immune response after ovalbumin co-administered
General trend, can be used as vaccine adjuvant use (Bommineni etc., 2014).
Wheat and wild kindred plant produce antibacterial peptidyl abundant in long-term disease-resistant evolutionary process in genome
Cause carries out the excavation of novel antimicrobial peptide using bioinformatics binding molecule cytobiology technology, finds efficient anti-plant
The novel antimicrobial peptide of disease has important practical value.
Summary of the invention
Reported antibacterial peptide can destroy membrane structure mostly, and gram-positive bacteria and Gram-negative bacteria are killed in suppression, promote
It is immune to eliminate infection, protect host to encroach on from cause of disease.Antibacterial peptide is from a wealth of sources, has hair in animals and plants and microbial body
It is existing but less to the antibacterial peptide research report of resist powdery mildew of wheat and head blight at present.
This patent obtains antibacterial peptide gene using the transcript profile data combination bioinformatics method of the small wheat SN6306 that lays down,
That is W662 is separately verified by converting Bacillus coli expression destination protein by the methods of PDA Plating, wheat leaf blade smearing
The antifungal activity of this antibacterial peptide.The antibacterial peptide that this patent provides can replace traditional chemical pesticide, prevent and treat wheat and other
The fungal disease of crop.
The present invention is achieved by the following technical solutions:
The present invention provides a kind of antibacterial peptide, the amino acid sequence of the antibacterial peptide is as shown in sequence table SEQ ID No.2.
The cDNA sequence of antibacterial peptide gene provided by the invention is as shown in sequence table SEQ ID No.1.
Antibacterial peptide provided by the invention can be used for preparing antimycotic inhibitor.
Antibacterial peptide provided by the invention can be used for preparing the inhibitor of anti-Fusarium graminearum.
Antibacterial peptide provided by the invention can be used for preparing the inhibitor of resist powdery mildew of wheat.
The beneficial effects of the present invention are: the present invention provides a kind of novel antibacterial peptide, which can be used for preparing
Anti- Fusarium graminearum, resist powdery mildew of wheat inhibitor, be a kind of there is the huge antimycotic inhibitor of potential utility value
Raw material.
Detailed description of the invention
Fig. 1 is the cDNA sequence of W662 gene and the amino acid sequence of coding.
Fig. 2 is the signal peptide analysis of the amino acid sequence of W662 albumen.
Fig. 3 is the transbilayer helix prediction of W662 albumen.
Fig. 4 is 12% SDS-PAGE electrophoretic analysis pET-32a (+) destination protein expression；Wherein: M is pre-mixed egg
White marker object (width)；1, control is not induced；2: induction overnight；3, the precipitating after ultrasonication；4, the supernatant after ultrasonication；
5-7, the cleaning solution in purification process；8, the destination protein of purifying；9, the destination protein of digestion.
Fig. 5 is 12% SDS-PAGE electrophoretic analysis W662 destination protein expression；Wherein: M, pre-mixed proteins label
Object (width) (Broad)；1, control is not induced；2: induction overnight；3, the precipitating after ultrasonication；4, the supernatant after ultrasonication；
5-7, the cleaning solution in purification process；8, the destination protein of purifying；9, the destination protein of digestion.
Fig. 6 is protein quantification standard curve.
Fig. 7 is Western Blot detection pET-32a (+), Y2944, Y5468 and W662 protein expression；Wherein: M,
Blue Plus II Protein Marker(14-120 kDa)；1, pET-32a (+) albumen；2, the pET-32a after digestion
(+) albumen；3, Y2944 albumen；After 4, Y2944 fusion protein digestions；5, Y5468 albumen；After 6, Y5468 fusion protein digestions；
7, W662 albumen；After 8, W662 fusion protein digestions.
Fig. 8 is that Fusarium graminearum infects wheat leaf blade figure, wherein A: joined pET-32a (+) label protein；B: it is added
Y2944 albumen；C: it joined Y5468 albumen；D: it joined W662 albumen.
Fig. 9 is to feel head blight region length of blade on statistics wheat leaf blade.
Figure 10 is that powdery mildew infects YN15 wheat leaf blade, wherein A: having smeared pET-32a (+) label protein；B: it smears
Y2944 albumen；C: it has smeared Y5468 protein D: having smeared W662 albumen.
Figure 11 is to count the ratio that powdery mildew region and the blade gross area are felt on wheat leaf blade.
Figure 12 is the powdery mildew mycelia distribution situation after blade dyeing, wherein A: having smeared pET-32a (+) label protein
Blade；B: the blade of Y2944 albumen has been smeared;C: the blade of Y5468 albumen has been smeared;D: W662 albumen has been smeared
Blade.
Specific embodiment
The bioinformatic analysis method that embodiment 1 utilizes finds antibacterial peptide
Utilize antibacterial peptide database APD(http: //aps.unmc.edu/AP/main.php) in existing antibacterial peptide data with
The small wheat SN6306 blade transcript profile sequencing data of laying down for the Powdery Mildew induction that this seminar has completed is compared, and analyzes small
To 1 antibacterial peptide gene, i.e., antibacterial peptide encoding gene sequence in wheat is excavated in transcript profile dataW662.Analyze W662
Gene order, amino acid sequence, and analyze the primary structure (https: //web.expasy.org/ of W662 albumen
Protparam/), secondary structure (https: //npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl page=
Npsa_sopma.html), signal peptide (http://www.cbs.dtu.dk/services/SignalP/) and transbilayer helix
(http://www.cbs.dtu.dk/services/TMHMM-2.0/) etc..
W662Gene is the small wheat SN6306 leaf of laying down using existing antibacterial peptide sequence and the Powdery Mildew induction of the website APD
The novel antibacterial peptide gene found in piece transcript profile sequencing data comparison result, cDNA overall length are respectively 246bp, amino acid encoding
Number is respectively 81 (Fig. 1).W662Albumen has signal peptide, and the cleavage site of signal peptide is respectively between 32-33 amino acid.
It is learnt by the comparison result on wheat database URGI, W662 gene is located on 5B chromosome.W662 does not have conserved structure
Domain.By the website ProtParam and SOPMA, isoelectric point, molecular weight, hydrophobicity and the fat coefficient and egg of albumen are predicted
White alpha-helix, beta sheet, extended chain and irregular curling etc., and alpha-helix is crimped onto proportion in albumen with irregular
Larger (table 1).According to the prediction in the website Signal P4.1, it was demonstrated that this albumen all contains signal peptide (Fig. 2).By TMHMM
2.0 websites predict protein topology structure, it was found that W662 albumen may be secretory protein (Fig. 3).
The structural analysis of 1 W662 albumen of table
The verifying of the antibacterial functions of 2 antibacterial peptide of embodiment
No.1 method
The building of No.1.1 antibacterial peptide prokaryotic expression carrier, is transferred to expression bacterial strain
By what is excavated from small wheat SN6306 transcript profile data of laying downW662Gene order carries out signal peptide prediction, and removes signal
The coded sequence of peptide.According to Escherichia coli to the expression Preference pair of codonW662Carry out codon optimization, sequence both ends point
It is not added toNcoI restriction enzyme site andEcoRI restriction enzyme site transfers to Jinan Bo Shang Bioisystech Co., Ltd to carry out gene chemical synthesis,
And withNcoI andEcoRI is that cloning site is connected into carrier pET-32a (+).
PET-32a (+) plasmid built is transferred to expression bacterial strain BL21(DE3) plysS competent cell.
(1) thermal shock method convertsE. coliBL21(DE3) plysS competent cell
1) it takes 50 μ L to be stored in the competent cell of -80 DEG C of refrigerators, 10 μ L recombinant plasmid dnas is added, mix rapidly, ice bath
30min(is operated in superclean bench).
2) ice bath terminates, by mixture in 42 DEG C of metal baths heat shock 90s, set 1 ~ 2min on ice rapidly.
3) 800 μ LLB fluid nutrient mediums will be added in the reaction tube equipped with mixture, mixes, in 200rpm, 37 DEG C of constant temperature
Shaking table is incubated for 40 ~ 60min.
4) reaction tube 12000 × g in centrifuge, of short duration centrifugation inhale and abandon 600 μ L supernatants, and remainder resuspension is added drop-wise to
On LB solid medium containing corresponding antibiotic, uniformly the bacterium of conversion is coated on agar plate with sterile spreading rod.(in
It is operated in superclean bench)
5) plate forward direction places 15 ~ 30min, is placed in 37 DEG C of constant incubators and is inverted culture 16h.
(2) picking single colonie
Single colonie on picking plate is added 5mL and contains in the LB liquid medium of corresponding antibiotic, mixes, shakes in 37 DEG C of constant temperature
Swing device 200rpm shaken cultivation 16h.To the end of cultivating, saves bacterium and take part bacterium solution sequence verification.
The inducing expression of No.1.2 antimicrobial peptide protein
(1) pET-32a(+ will be contained) BL21(DE3 of plasmid) and BL21(DE3 containing pET-W662 plasmid) plysS according to
5% volume ratio is added 30mL and contains in the self-induction culture medium of 100mg/mL ampicillin, in 37 DEG C of constant temperature oscillators,
200rpm vibrates 5h, then oscillator temperature is adjusted to 25 DEG C, continues with 10 ~ 16h of 200rpm constant temperature oscillation.
(2) SDS-PAGE detects inducible protein expression
1) match glue
Table 2 matches glue component table
2) sample treatment
13000 × g of bacterium solution after taking 1mL to induce is centrifuged 10min, abandons supernatant, and the protein extract that 100 uL are added is resuspended.It will
Centrifuge tube is placed in 30min in 95 DEG C of metal baths.
3) SDS-PAGE electrophoresis
Sample point sample after taking 10uL to heat starts SDS-PAGE electrophoresis.
Deposition condition: after beginning voltage 110V, 30 ~ 40min of electrophoresis, voltage is set as 120V, continues 100 ~ 120min of electrophoresis,
Until the Coomassie brilliant blue instruction band in swimming lane is moved at the 1cm of separation gel bottom, stop electrophoresis.
4) gel imaging
According to the operating procedure on Coomassie brilliant blue protein adhesive rapid dye liquor (Beijing Suo Laibao Science and Technology Ltd) specification
It carries out gel-colored.
Application method is as follows:
The preparation of working solution takes 100mL solution B, and 2mL solution A is added, and mixes, this is working solution.
It is put into container 1. the PAGE glue (by taking 8cm × 10cm size as an example) after electrophoresis is removed, 50mL deionization is added
Water stops after being heated to boiling, and continuation is shaken 5 minutes on decolorization swinging table, discards aqueous solution.
2. 25mL rapid dyeing working solution is added, 30 ~ 60s of fluidized state is kept after being heated to boiling, it is subsequent to stop heating
Continue 5 ~ 10min of shake on decolorization swinging table, discard dyeing liquor (protein band should be visible at this time).
3. about 50mL deionized water is added, 30 ~ 60s of fluidized state is kept after being heated to boiling, is continued after stopping heating
5 ~ 10min is shaken on Tuo Se Yao bed, changing water can be completed decoloration, observe result.
Points for attention:
1. the quality of water is extremely important, and quality is better, and sensitivity is higher, if research has shown that with originally in the cleaning step before dyeing
Water heating cleaning, dyeing effect and sensitivity are only identical as conventional methanol coomassie brilliant blue staining.
2. Shi Keyong tap water cleaning of decolourizing.
3. repeating decolorization process to the dyeing glue for obtaining no background or putting glue in water overnight.
4. the PAGE glue dyed through this product, the contracting degree of rising of glue less than 5%, the glue after dyeing can place the several months in water and
Without obvious decoloration.
5. 5min is shaken in continuation on decolorization swinging table every time after heating, dyeing effect can be enhanced.
6. a dyeing liquor has slight erosion, band gloves is needed to operate.Gel after dyeing is taken a picture using gel imaging system,
Observe result.
The purifying of No.1.3 antimicrobial peptide protein
The purifying of No.1.3.1 antimicrobial peptide protein
According to Beaver BeadsTMIt is pure that specification on His-tag Protein Purification kit carries out albumen
Change, operating process is as follows:
(1) magnetic bead pre-processes:
1) castor magnetic bead product is placed on eddy blending machine and is mixed well, take 5mL suspension containing magnetic beads to be centrifuged in 15mL with pipettor
Guan Zhong carries out Magnetic Isolation, abandons supernatant, centrifuge tube is removed from magnetic separator.
2) 5 mL Binding Buffer are added into the above-mentioned centrifuge tube equipped with magnetic bead, spin upside down centrifuge tube number
It is secondary, so that magnetic bead is suspended again；Magnetic Isolation is carried out, supernatant is removed.Repeated washing 2 times.(note: during Magnetic Isolation, it is
The loss of magnetic bead in use is reduced, after solution becomes clarification, centrifuge tube lid is covered tightly, keeps centrifuge tube still in magnetism
On separator, hand-held magnetic separator and centrifuge tube are spun upside down for several times, make clear solution rinse centrifuge tube cover it is remaining
Magnetic bead stands a moment, solution is made to become clarification again；It is the same below.)
(2) target protein is in conjunction with magnetic bead
1) with the thallus of 10 mL Binding Buffer suspension 2g weight in wet bases, after being crushed and being cracked, the as thick egg of target
White sample is added in the centrifuge tube equipped with pretreatment magnetic bead, and centrifuge tube is placed in eddy blending machine oscillation 15s.
2) centrifuge tube is placed on rotary mixer, 20 ~ 30min(is if desired, can be at 2-8 DEG C for room temperature rotation mixing
Low temperature environment under, rotating mixed 1h prevents target protein from degrading).
3) centrifuge tube is placed on magnetic separator and carries out Magnetic Isolation, removal supernatant is into new centrifuge tube in case of after
Continuous detection removes centrifuge tube from magnetic separator and carries out subsequent wash step.
(3) magnetic bead washs
1) 10 mL Washing Buffer are added into the centrifuge tube equipped with magnetic bead, gently overturns centrifuge tube for several times, makes magnetic bead
Again it suspends, Magnetic Isolation removes cleaning solution into new centrifuge tube, in case sample detection.Repeat this step 1 time.
2) 10mL Washing Buffer is added to the centrifuge tube that magnetic bead is housed, so that magnetic bead is suspended again, magnetic bead is hanged
Liquid is transferred to new centrifuge tube (avoiding non-specific adsorption protein contamination target protein on former centrifugation tube wall), and Magnetic Isolation is moved
Supernatant is to cleaning solution collecting pipe out.
(4) target protein elutes
1) user can change elution volume adjustment target protein concentration as needed, and 2 ~ 10 mL Elution Buffer are added,
Gently overturning centrifuge tube for several times, makes magnetic bead suspend, Magnetic Isolation, collects eluent into new centrifuge tube, the mesh as purified
Mark protein sample.
2) if desired, can repeat the above steps 1 time, sample is collected into new centrifuge tube, to detect target protein
Whether elute completely.
(5) magnetic bead post-processes
1) 5 mL Elution Buffer are added in the centrifuge tube equipped with magnetic bead, spin upside down centrifuge tube for several times, makes magnetic bead
It suspends, Magnetic Isolation removes supernatant.
2) it repeats the above steps 2 times.
3) 5mL ddH is added in centrifuge tube2O spins upside down centrifuge tube for several times, magnetic bead is made to suspend, Magnetic Isolation, removal
Supernatant.
4) it repeats the above steps 2 times.
5) Storage Buffer is added makes total volume 5mL into magnetic bead, and being stored in 2-30 DEG C, (long-term preservation is set
In 2-8 DEG C), it can be used for the purifying of same protein next time.
(6) magnetic bead regenerates
Magnetic bead is used continuously more than three times, and the ability of combining target albumen may be substantially reduced, it is proposed that carries out magnetic bead regeneration
Processing.By taking 5mL 10% (v/v) suspension containing magnetic beads as an example, magnetic bead regenerative operation is described in detail:
1) suspension containing magnetic beads are subjected to Magnetic Isolation, remove supernatant, centrifuge tube is removed from magnetic separator, added in centrifuge tube
Enter 5mL ddH2O spins upside down centrifuge tube for several times, magnetic bead is made to suspend again, Magnetic Isolation, removes supernatant.
2) 5 mL Stripping Buffer are added, spins upside down centrifuge tube for several times, magnetic bead is made to suspend again, room temperature rotation
Turn mixing 5min, Magnetic Isolation removes supernatant.Repeat this step 1 time.
3) 5mL ddH is added2O spins upside down centrifuge tube for several times, magnetic bead is made to suspend again, Magnetic Isolation, removes supernatant
Liquid repeats this step 2 time.
4) alkali process: 5 mL Beads Washing Buffer are added, spins upside down centrifuge tube for several times, makes magnetic bead again
It suspends, room temperature rotation mixing 5min, Magnetic Isolation removes supernatant.5 mL ddH are added2O spins upside down centrifuge tube for several times,
Magnetic bead is set to suspend again, Magnetic Isolation removes supernatant.It repeats ddH2O washing step 3 ~ 5 times, until cleaning solution is in neutrality.
5) 5 mL Recharge Buffer are added, spins upside down centrifuge tube for several times, magnetic bead is made to suspend again, room temperature rotation
Turn mixing 20min, Magnetic Isolation removes supernatant.
6) 5mL ddH is added2O spins upside down centrifuge tube for several times, magnetic bead is made to suspend again, Magnetic Isolation, removes supernatant
Liquid.This step 4 time or more is repeated, guarantees that nickel ion removal is complete.
7) Storage Buffer is added makes total volume 5mL into magnetic bead, and being stored in 2-30 DEG C, (long-term preservation is set
In 2-8 DEG C).
(7) PAGE gel electrophoresis detection protein purification situation is contaminated using Coomassie brilliant blue protein adhesive rapid dye liquor
Glue observes result.
The dialysis of No.1.3.2 antimicrobial peptide protein
Bag filter equipped with albumen after purification is placed in the sodium radio-phosphate,P-32 solution for filling 33mM, is placed on 4 DEG C of magnetic stirring apparatus
It dialyses, every 4h replaces a dialyzate, dialyses altogether one day night.Albumen after dialysis is sub-packed in 2mL centrifuge tube, is protected
In the presence of -80 DEG C of refrigerators.
The measurement of No.1.3.3 antimicrobial peptide protein concentration
Determination of protein concentration, operation are carried out according to the operating method on simple protein quantification kit (Bradford) specification
Method is as follows:
(1) prepare protein standard solution
Protein standard solution, which is diluted to, makes final concentration of 0.22mg/ml.Protein standard substance should use identical with testing protein sample
Solution dilution.
(2) protein quantification
Protein quantification operation is carried out according to Easy Protein Quantitativr Kit (Bradford) kit specification.
Bovine serum albumin(BSA) (BSA) standard solution (0.22mg/mL) is diluted according to following table
Table 3 dilutes bovine serum albumin(BSA) (BSA) standard solution
The standard sample of each volume is added in ELISA Plate, the Coomassie brilliant blue dye that 200 μ L are added to 20 μ L is mended with sterile water
Liquid is stored at room temperature 10 ~ 20min, and with light absorption value of the microplate reader measurement each sample at 595nm, record reading repeats this operation 3
It is secondary, guarantee that data are accurate.Using the light absorption value of the sample without containing BSA as blank control, protein concentration standard curve is drawn.
Protein concentration is calculated according to the method described above, and the protein concentration such as obtained is not within standard curve, it is proposed that dilute sample is surveyed again
It is fixed.
Points for attention:
Coomassie brilliant G-250 and quartz colorimetric utensil can produce strong combination, and glass or plastic cuvette can be used；
It is measured by the sequence of protein concentration from low to high, does not clean cuvette repeatedly, water quality will affect measurement result；
The light absorption value reacted within 5 ~ 20min is most stable, is kept for the reaction time of each pipe consistent, preferably surveyed within the same time
Amount guarantees accuracy of reading；
The drafting of standard curve can be divided into 2 ~ 3 groups, do operation repetitive, to obtain more accurate result.
No.1.3.4 Western Blot detection
(1) sample 5ul is taken, successively loading, carries out SDS-PAGE electrophoresis, runs 30min under first 100V voltage and glue is concentrated, to sample
Running under into separation gel, then 120V voltage to electrophoresis terminates.
(2) pvdf membrane impregnates 30s in 100% methanol；According to " sponge-filter paper-gel-pvdf membrane-filter paper-sponge "
Sequence is successively put to anode into transferring film slot by cathode, pours into transferring film buffer；Transferring film 2h, constant current 150mA under 200V voltage.
(3) after transferring film, pvdf membrane is put into 1 × TBST buffer and is washed 3 times, each 5min.
(4) pvdf membrane is placed in room temperature in the confining liquid containing 5% skimmed milk power and closes 1h.
(5) pvdf membrane is taken out from confining liquid, is put into 1 × TBST buffer and is washed 3 times, each 5min；Use confining liquid
It dilutes primary antibody (His, 1:2000), film is put into primary antibody dilution, is placed in 4 DEG C of incubation 2h of shaking table.
(6) pvdf membrane is taken out from primary antibody, is put into 1 × TBST buffer and washs 3 times, each 5min；With containing 5%
The confining liquid of skimmed milk power dilutes secondary antibody (sheep anti mouse, 1:1000)；Film is put into secondary antibody diluent, is placed in shaking table incubation at room temperature
1h。
(7) pvdf membrane is taken out, is put into 1 × TBST buffer and washs 3 times, each 5min.
(8) the A liquid of ECL developer solution and B liquid are mixed according to the ratio of 1:1, film is made to come into full contact with developer solution.
(9) gel imaging analysis.
The functional analysis of No.1.3.5 W662 albumen inhibition fungi
The influence that No.1.3.5.1 albumen develops Fusarium graminearum mycelia
The influence that excised leaf bacterination process verifying W662 albumen develops Fusarium graminearum mycelia
1) inducible protein is expressed
The step of step is with aforementioned identical experiment.
2) SDS-PAGE electrophoresis detection protein expression situation
The step of step is with aforementioned identical experiment.
3) ultrasonic disruption cell
Supernatant is abandoned in centrifugation, collects the Escherichia coli induced overnight, and bacterium is resuspended with the ratio that 5mL sterile water is added in 1g weight in wet base thallus
Body, with maximum power 50W, broken time 5s, intermittent time 3s ultrasonic disruption cell under cryogenic conditions, until thallus is clarified
It is not sticky.Crude protein supernatant is collected by centrifugation, and is stored in -20 DEG C of refrigerators.
4) protein quantification
According to 2.6.4.3(2) method to carry out crude protein quantitative.
5) influence that excised leaf bacterination process verifying W662 albumen develops Fusarium graminearum mycelia
The order of method reference opening allows etc. (2016), chooses the wheat leaf blade of several leaves wholeheartedly, cuts among the blade that length is 5cm
The circular wound that diameter is 1mm2 is caused with punch or pipettor gun point in part among blade upper surface.It will be by the 5 thick eggs of μ L
Albumen made of white and 1 μ L Fusarium graminearum conidium liquid-spore mixed liquor is added dropwise on the circular wound of central vane.
Both ends of the blade is inserted into moisturizing identification plate, makes blade is domed to stand on identification plate, is in round hole at the top of arch.Finally,
Identification plate is placed in 25 DEG C of constant incubator culture 3d.Culture terminates, and observes the sense bacterium situation at round hole position, analyzes albumen pair
The resistance of head blight.
Influence of the No.1.3.5.2 W662 albumen to melon infected with powdery mildew fungus wheat leaf blade
(1) destination protein purified smears YN15(tobacco grower 15) wheat leaf blade
The long YN15 wheat leaf blade centre part 5cm to a leaf wholeheartedly is chosen, and marks the region with marker.Take part pure
The destination protein of -80 DEG C of purifying is stored in after change, (digestion condition: every 300 μ L concentration is 241 ~ 252 μ g/ through enterokinase digestion
The recombinant enterokinase of 0.06U is added in the purifying protein of mL, the constant temperature digestion 16h in 25 DEG C of metal baths) after, it is added final concentration of
0.025% tween after mixing, picks albumen with cotton swab and is applied to leaf marking region, blade tow sides will be smeared
It is even.Each albumen does multiple repetitions.By the YN15 wheat smeared be placed in 23 DEG C of temperature, illumination 16h/d incubator in.
(2) the susceptible situation of YN15 wheat leaf blade is analyzed based on Photoshop software
Referring to the method for (2016) such as Li Renhui.It is equal to white powder according to the ratio that white powder infects region and normal green leaf area
The pixel ratio for infecting region and normal green leaf area, with the YN15 wheat leaf blade after mobile phone camera shooting albumen smearing 5d
Image respectively obtains pixel and the two picture that white powder infects region and normal green leaf area on Photoshop software
The total pixel of the sum of element, i.e. blade.The pixel in region and the ratio of the total pixel of blade are infected by comparing white powder, analyze albumen dialogue
Powder germ infects the influence of wheat leaf blade.
(3) the susceptible situation of microscopically observation YN15 wheat leaf blade
1) decoloration, dyeing of YN15 wheat leaf blade
By wheat leaf blade to be seen destainer soaked overnight, rinsed 2 times with aqua sterilisa, be put into dyeing 15 in dyeing liquor ~
30min, i.e. observable multiple with sterile water repeated flushing, or deposit in and save in liquid, it at most can be reserved for 3d.
2) the susceptible situation of microscopically observation YN15 wheat leaf blade
Blade to be seen is taken out, open and flat is laid on glass slide, is placed on microscopical objective table, in 4 times of object microscopic observation leaves
Piece is simultaneously taken pictures.
Test results and analysis
The expression and purifying of No.2.1 W662 albumen
The standby recombinant plasmid transformed BL21(DE3 completed of corporation) plysS is expressed into bacterial strain, after being sequenced, by sequence verification
The correctly bacterial strain self-induction protein expression containing recombinant plasmid.Self-induction protein expression condition are as follows: in 200rpm, 37 DEG C of constant temperature
It, will when the expression bacterial strain containing recombinant plasmid is 0.6 ~ 1 in logarithmic growth to OD600 value in self-induction culture medium in oscillator
Oscillator temperature is down to 25 DEG C, and lactose enters the expression of cell inducible protein.After to inducing expression, part thallus is taken to be added suitable
Protein extract is measured, 12% SDS-PAGE electrophoresis is carried out, detects protein expression situation.Through preliminary electrophoresis detection to purpose egg
After white expression high in thallus supernatant, thallus, ultrasonication are collected, the broken crude protein supernatant that will be collected by centrifugation passes through
The castor magnetic beads for purifying destination protein of Specific adsorption His label protein.12% SDS-PAGE electrophoresis detection albumen table is carried out again
It reaches and purification effect.Go out pET-32a (+) according to DNAMAN software prediction, W662 destination protein molecular weight is respectively 22kD, 25
The molecular weight of albumen of kD, the size and prediction of observing the purpose band of Figure 4 and 5 are almost the same, show that destination protein is expressed as
Function, purification effect are good.The dense of pET-32a (+), W662 purifying destination protein is measured according to the protein quantification standard curve of Fig. 6
Degree is 252 μ g/mL, 253 μ g/mL respectively, measures pET-32a (+), W662 crude protein concentration is 540 μ g/mL, 471 μ g/ respectively
mL。
Destination protein after purification and the destination protein after enterokinase digestion are detected by Western blot, as a result
It shows stripe size and is expected consistent.Because the destination protein after digestion is separated with His label protein, so without Faxian on pvdf membrane
The hybridization signal of destination protein after showing digestion, the only hybridization signal (Fig. 7) of the His label protein of destination protein leading portion.
The functional verification of No.2.2 albumen
The influence that No.2.2.1 albumen develops Fusarium graminearum mycelia
Excised leaf bacterination process verifies protein function.
Crude protein and Fusarium graminearum conidiospore suspension are mixed by a certain percentage, take 2 μ L albumen spore mixing drops
It is added on the through hole at YN15 wheat leaf section center.The blade handled well is domed to be placed on moisturizing identification plate, is trained at 25 DEG C
Support closing culture 3 days in case.By observing as it can be seen that pET-32a (+) albumen has been added dropwise in the wheat leaf blade ratio for being added to W662 albumen
Blade it is susceptible light (Fig. 8, Fig. 9).
Influence of the No.2.2.2 albumen to melon infected with powdery mildew fungus wheat leaf blade
By the enterokinase digestion of destination protein after purification, using 0.025% tween as surfactant, it is applied to a leaf one
The YN15 wheat leaf blade middle section of the heart, tow sides smoothen repeatedly, it is ensured that albumen comes into full contact with blade.The wheat being disposed is pacified
It sets and is inoculated with Powdery Mildew in constant temperature white powder incubator.After 5 days, observe the wheat leaf blade for having smeared W662 albumen do not feel bacterium or
It is susceptible light, and smeared the blade of unloaded body protein and do not smear albumen region it is susceptible heavy, it was demonstrated that W662 albumen has inhibition white
Powder germ infects the effect (Figure 10) of wheat leaf blade.
The leaf area pixel and the total picture of blade of the infection powdery mildew after albumen is smeared are calculated by Photoshop software
The ratio between element compares the susceptible degree of blade.It can be seen that the blade for having smeared unloaded body protein is susceptible serious, susceptible region and full leaf section
Ratio reaches 13%, and the blade for having smeared W662 albumen is only 1.67%., powdery mildew is inhibited to infect effect preferably (Figure 11).
By wheat leaf blade decoloration, dyeing and microscopically observation as it can be seen that having smeared the leaves infected white powder of destination protein
Germ has relatively smeared unloaded body protein and non-application area is light, and mycelia is few.Show W662 albumen to inhibition melon infected with powdery mildew fungus
Effect it is obvious (Figure 12).
Purpose egg is proved through gene chemical synthesis, vector construction, albumen pronucleus expression, the test of PDA plate and excised leaf test
The white development for being able to suppress Fusarium graminearum mycelia, inhibits infecting for Powdery Mildew.
Sequence table
<110>Shandong Agricultural University
<120>a kind of antibacterial peptide and its application using bioinformatics method building
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 246
<212> DNA
<213>wheat (Triticum aestivum)
<400> 1
atggcttctg ccggccgtcg tcccacggtg ctccagcaga tcgctctctt cctcgtcgtc 60
gccgcggtga tcatgaacag ctccgtctgc cttggagccg ctggccacga cgccactgta 120
gtaggcactg gtagcaacga ccctaaccac cctgcttttc cgtcgccgcc tggtaaaccc 180
tacaccggtc gtccgtgcag caaaatttac ggctgtaatg taccaccggc aggtggccag 240
ccctaa 246
<210> 2
<211> 81
<212> PRT
<213>wheat (Triticum aestivum)
<400> 2
Met Ala Ser Ala Gly Arg Arg Pro Thr Val Leu Gln Gln Ile Ala Leu
1 5 10 15
Phe Leu Val Val Ala Ala Val Ile Met Asn Ser Ser Val Cys Leu Gly
20 25 30
Ala Ala Gly His Asp Ala Thr Val Val Gly Thr Gly Ser Asn Asp Pro
35 40 45
Asn His Pro Ala Phe Pro Ser Pro Pro Gly Lys Pro Tyr Thr Gly Arg
50 55 60
Pro Cys Ser Lys Ile Tyr Gly Cys Asn Val Pro Pro Ala Gly Gly Gln
65 70 75 80
Pro
Claims (5)
1. a kind of antibacterial peptide, it is characterised in that: the amino acid sequence of the antibacterial peptide is as shown in sequence table SEQ ID No.2.
2. a kind of antibacterial peptide according to claim 1, it is characterised in that: the cDNA sequence of the antibacterial peptide gene such as sequence
Shown in table SEQ ID No.1.
3. a kind of application of antibacterial peptide according to claim 1, it is characterised in that: the antibacterial peptide is used to prepare antimycotic
Inhibitor.
4. a kind of application of antibacterial peptide according to claim 5, it is characterised in that: the antibacterial peptide is used to prepare anti-cereal
The inhibitor of sickle-like bacteria.
5. a kind of application of antibacterial peptide according to claim 5, it is characterised in that: the antibacterial peptide is used to prepare anti-wheat
The inhibitor of powdery mildew.
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| EP0602098A1 (en) * | 1991-09-02 | 1994-06-22 | Zeneca Limited | Biocidal proteins |
| US20070044171A1 (en) * | 2000-12-14 | 2007-02-22 | Kovalic David K | Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
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