CN101690492B - Protein nano complex promoting plant growth and preparation method and application thereof - Google Patents
Protein nano complex promoting plant growth and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a protein nano complex promoting plant growth and a preparation method and application thereof. A medical nano controlled-release system is adopted to couple a plant immunity activator Harpin protein with bioactivity with a carrier to prepare the nano complex. The Harpin protein with the bioactivity is obtained through separation and purification. Nanoparticles are prepared by PLGA, PVA and ultrasonic technology, and coat the Harpin protein to form the PLGA-protein nano complex carrying the Harpin protein. The protein nano complex prepared by the method comprises the following advantages that: (1) the bioavailability of the Harpin protein can be improved after the Harpin protein is coupled with the ultramicron carrier; (2) the Harpin protein is appropriately coated, and the protein can be protected; (3) the protein nano complex can promote a plant to absorb and utilize a protein medicament, the effect is strengthened or prolonged and obvious biological effects are generated; (4) the protein nano complex has simple and safe use; and (5) the protein nano complex has disease-resistant broad spectrum and does not pollute environment. The protein nano complex can be used for preventing and controlling plant diseases, promoting the growth of plants and improving crop yield.
Description
Technical field
The present invention relates to medicine and control of plant disease field, more specifically, the present invention relates to a kind of preparation method and application of protein nano complex of Promoting plant growth.Adopt the medicament nano controlled release system, bioactive plant immune activator Harpin albumen and carrier coupling will be arranged, the preparation nano particle.Said preparation can protect Harpin albumen, promotion plant absorb, strengthen or extend drug action medicine, can improve its bioavailability, produces obvious biology effect.This is the novel biopesticide that is used for control of plant disease of a kind of efficient, wide spectrum, safety.
Background technology
Plant disease, insect pest are the Main Agricultural disaster that threatens China's agricultural production and the national economic development for a long time always.Crops every year is the loss that causes of insect pest approximately 20% due to illness, and damage by disease and insect respectively accounts for half.Nearly 3000 kinds of the main plant disease of China is divided into bacillary, fungoid, Disease, and wherein virus disease is 900 kinds.For a long time, the control of plant disease depends on chemical bactericide always.But the life cycle of chemical bactericide is shorter and shorter, causes its pesticide resistance also more and more serious.In addition, chemical bactericide is helpless to many diseases.Therefore, pollution-free, the nonresistant biological bactericide of Development of Novel has become the task of top priority.
Harpin albumen is a kind of albumen that causes the pathogen Erwiniaamvlovora hrpN gene code of the rose fragrant plant fire blast such as apple, pear.Harpin albumen is that bacterium is to the pathogenic essential a kind of albumen of host plant, but can react by induced hypersensitivity on non-host plant, and inducible system resistance, be similar to immune response (the Wei Z.M. of the broad-spectrum disease resistance bacterium insect pest that higher mammal has, Laby R.J., Zumoff C.H., Bauer D.W., He S.Y., Collmer A., Beer S.V.1992, Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwiniaamylovora.Science 257:5066 85-8).receptors bind on Harpin albumen and plant cell membrane, many signal paths in activated plant, thereby Promoting plant growth, strengthen resistance against diseases (Lee, J., Klessig, D.F., N ü rnberger, T., 2001.A harpin binding site in tobacco plasmamembranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activatedprotein kinase activity.Plant Cell 13:1079-1093).Therefore, be widely used in crop protection with Harpin albumen as antibiotic bacteriostat, particularly monocotyledon and dicotyledon, comprise main crops and economic crops, vegetables etc.
Harpin albumen has attracted domestic and international scientist's concern to the good control efficiency of plant pest, at first the scholar of U.S. Cornell university found the antibacterial bacteriostatic function of Harpin albumen in 1992, and cooperate with the EDEN Bioscience company of the U.S., ratify by EPA (EPA) in 2000 at the Harpin of expression in escherichia coli albumen, trade name Messenger, approval number Reg.No.069834-002.Be called as " having started a new revolution in the biological and ecological methods to prevent plant disease, pests, and erosion field ".That Harpin albumen controlling plant diseases has is free from environmental pollution, do not produce the advantages such as resistance.
The 21 century that nanometer technology is generally acknowledged in the world the promising scientific research field of tool.The general character that nano material has is that granularity is minimum, surface area is very big.According to the research to nano material, its surface portion be atomic arrangement not only without long-range order but also without the amorphous layer of shortrange order, and therein heart position exist crystallization in order with the atom of periodically arranging.This special construction characteristics of nano particle have just caused special surface effect and the dimensional effect of nano material.In recent years, nanosecond science and technology have obtained the progress that attracts people's attention in the drug delivery field, develop the Nano medication of various ways.The size of Nano medication can obviously affect formulation and drug delivery mechanisms, for example grain diameter has the characteristic of Transdermal absorption at the Nano medication of 50-500nm, simultaneously, with the high degradation material of biocompatibility as auxiliary material, can also reach the effect (Shekunov that slow controlled release is put, B.Y., Chattopadhyay, P., Tong, H.Y., 2007.Particle sizeanalysis in pharmaceutics:principles, methods and applications.Pharm.Res.24 (2), 203-227).
Adopt controlling and releasing system with nanotechnology, utilize the plant immune activator Harpin protein nano complex of Harpin albumen development, have slow releasing pharmaceutical, prolong drug action time; Reach target and carry purpose; Guaranteeing to reduce dosage under pharmaceutically-active prerequisite, alleviating or avoid toxicity; Improve the characteristics such as medicine stability.This has great importance for improving the healthy of bioavailability, protection of the environment, guarantee China people.The Harpin protein nano complex is a kind of safe novel biopesticide, and the control that is used for plant disease has advantages of efficiently, wide spectrum, free from environmental pollution, has application prospect preferably.
Summary of the invention
The objective of the invention is to be to provide a kind of protein nano complex of Promoting plant growth.Comprise Harpin albumen and carrier in this nano complex.Harpin albumen can inducing plant disease resistance.Harpin albumen is by being combined with the acceptor HrBP1 of plant surface, the defensive enginery of activated plant self.Harpin albumen itself is to bacterium, virus, fungi, and nematode, insect be without killing or inhibitory action, but by be combined certain signal of rear generation with Plant accepter, stimulates or the immunologic mechanism of inducing plant self comes protective plant.Thereby Harpin has the resistant effect of wide spectrum, the generation of minimizing plant disease.Harpin can also promote the g and D of plant, and individuality and root amount, the precocity that promotes Seed germination, plant and solid as increasing plant improve output and the quality of crop.In addition, Harpin albumen can improve vegetables, the anti-ability of rotting, go mouldy of fruit, extends storage time.
Carrier in the Harpin protein nano complex is the ultra micron carrier, as wherein a kind of such as PLA, lactic acid-ethanol copolymer, Polyalkylcyanoacrylanano, polycaprolactone, Polyalkylcyanoacrylanano and polycaprolactone.Adopt the medicament nano controlled release system, bioactive plant immune activator Harpin albumen and carrier coupling will be arranged, the preparation protein nano complex.This protein nano complex can protect Harpin albumen, promotion plant absorb, strengthen or extend drug action medicine, can improve its bioavailability, produces obvious biology effect.
Another object of the present invention is to be to provide a kind of method for preparing described protein nano complex.Prepared protein nano complex average diameter of particles is 190.5nm, and in nano particle, 72.5% nano particle diameter is less than 225nm.Harpin albumen is positioned at nano complex inside.This Harpin protein nano complex has the distinctive character of controlling and releasing system with nanotechnology, makes it have many superiority aspect drug delivery: (1) but slowly-releasing Harpin albumen, thereby extend Harpin albumen action time; (2) can reach the purpose that target is carried; (3) can under the prerequisite that guarantees the effect of Harpin albumen, reduce dosage; (4) can improve the stability of Harpin albumen, be conducive to store; (5) can be in order to set up some new methods of administration.This is all that other delivery systems are incomparable.
Aspect the 3rd of the present invention, a kind of pharmaceutical composition is provided, said composition comprises the above-mentioned protein nano complex of effective dose.
Aspect the 4th of the present invention, provide the application in preparation prevention or treatment plant disease medicine of above-mentioned protein nano complex or pharmaceutical composition.
In order to achieve the above object, the present invention adopts following technical measures:
In one embodiment of the invention, provide a kind of Harpin of preparation method of protein.Employing contains the engineered strain E.coli BL21 (Chinese Typical Representative culture collection center C CTCC M202026) of harpin gene, and expression profile engineering restructuring Harpin albumen (rHrpZ albumen, rHrpZ).Centrifugal collection thalline suspends.The centrifugal collection supernatant of ultrasonic disruption.Adopt Ni-NTA Superflow post (Qiagen company product), according to the Qiagen operation manual purifying Harpin of company albumen.The Harpin albumen of PBS buffer solution (pH7.3) dialysis treatment purifying.Adopt SDS-PAGE electrophoresis and HPLC method, detect the purity of Harpin albumen.Adopt Bradford assay method, measure the Harpin protein concentration.
In one embodiment of the invention, provide a kind of method for preparing described Harpin protein nano complex.The method comprises the following steps:
A, PLGA (PLGA, lactide: glycolide=75: 25, molecular weight 40000) is dissolved in carrene, is made into 50mg/mL solution.0.5mL 20mg/mL Harpin albumen slowly is added drop-wise in 5mL 50mg/mL PLGA solution, uses simultaneously ultrasonic disruption instrument emulsification (50W processed 1 minute), form Water-In-Oil (W/O) emulsion.
B, water-in-oil emulsion is poured in 50mL 2%PVA (poly vinyl alcohol, molecular weight 70, the 000) aqueous solution immediately, ultrasonic emulsification (100W processed 2 minutes) forms the W/O/W emulsion.
C, the W/O/W emulsion is poured in distillation flask, carrene was removed in 40 ℃ of decompression distillation in 2 hours, solidified PLGA, consisted of the Harpin-PLGA protein nano complex, wherein wrapped up Harpin albumen.
The distilled water of D, use 40mL suspends the PLGA-Harpin nanoparticle that forms, centrifugal 20 minutes of 10000g, collecting precipitation.Precipitation is resuspended in distilled water, repeats this process 3 times to clean the outer PVA of Harpin-PLGA protein nano complex, is resuspended in distilled water the Harpin-PLGA protein nano complex standby at last.
In one embodiment of the invention, measured the diameter of Harpin protein nano composite particle precursor.Measure with dynamic light scattering particle diameter detector, synthetic rHrpZ-PLGA nano complex average diameter of particles is 190.5nm,, in nano particle, 72.5% nano particle diameter is less than 225nm.
In one embodiment of the invention, Harpin protein nano complex envelop rate and rate of release have been measured.The rHrpZ-PLGA nano particle is about 62.3% to the envelop rate of rHrpZ, extracorporeal releasing test shows, have 51.0% rHrpZ to be released from the rHrpZ-PLGA particle within front 2 day time, and 92.7% rHrpZ discharge within 3 weeks from the rHrpZ-PLGA particle.Illustrate that the rHrpZ-PLGA particle has preferably slow controlled release and puts effect.
In one embodiment of the invention, measured Harpin protein nano complex activity.By detecting phenylalnine ammonialyase (phenylalanine ammonia lyase, PAL), measure Harpin protein nano complex active, determine Harpin protein nano complex to the inducing action of defensive Substance P AL in tobacco leaf, and compare with the effect of Harpin albumen.In test, spray for the first time rHrpZ albumen after PAL activity change amplitude obviously greater than for the second time, and spray that the impact on the PAL activity only limited in 14 hours after rHrpZ albumen.After spraying the rHrpZ-PLGA nano complex, although the PAL activity change is not sprayed rHrpZ albumen vary within wide limits for the first time, more than reaching for 2 weeks the perdurabgility of PAL increased activity effect.Result of the test shows, rHrpZ-PLGA nano complex (rHrpZ-PLGA NP) can help rHrpZ albumen to enter tobacco leaf, and can slowly progressively discharge rHrpZ albumen in the tobacco body, and constantly stimulate the PAL increased activity, reach the effect that extends timeliness.
In one embodiment of the invention, measured the impact of Harpin protein nano complex on the plant disease-resistant effect.But the expression of PR-5dB gene in Harpin albumen evoking tobacco, PR-5dB gene are a class plant course of disease related protein genes, and its transcriptional expression can help the invasion and attack of plant opposing pathogen.The expression of this gene is directly related with the plant disease-resistant ability.Difference according to the PR-5dB gene transcription level, estimate the drug effect of rHrpZ albumen and rHrpZ-PLGA NP (rHrpZ-PLGA nano complex), result shows, after spraying, can improve by rapid stimulation PR-5dB transcriptional level in rHrpZ albumen, similar with the variation alive of PAL enzyme, PR-5dB transcriptional level raising effect is held time shorter, is no more than 48 hours.And rHrpZ-PLGA NP can keep over 2 weeks the castering action of PR-5dB transcriptional level, drug effect is 5 times that spray with isoconcentration rHrpZ albumen, having realized continuing slow controlled release in tobacco puts, permanently effective plays a role, obviously improved the bioavilability of rHrpZ albumen, can avoid or reduce application times when use in the field, reduce human cost.
In one embodiment of the invention, measured Harpin protein nano complex Ralstonia solanacearum (Ralstonia solanacearum) has been infected attack protection effect.Ralstonia solanacearum is the pathogen of plant of Solanaceae bacterial wilt, as typical tobacco disease indigenous bacteria, detects the drug effect of rHrpZ albumen and rHrpZ-PLGA NP with Ralstonia solanacearum.Result of the test shows that rHrpZ albumen has improved the effect that tobacco opposing Ralstonia solanacearum infects.In the situation that equal effectively rHrpZ protein content, the drug effect of Harpin protein nano complex (rHrpZ-PLGA NP) is apparently higher than single rHrpZ albumen, and stimulating plant is built up resistance for a long time, promotes tobacco growing.
The invention still further relates to the application of Harpin protein nano complex in the medicine of preparation treatment or prevention bacterial-infection resisting.
The invention still further relates to the application of Harpin protein nano complex in the medicine of preparation treatment or prevention anti-fungal infection.
The invention still further relates to the application of Harpin protein nano complex in the medicine of preparation treatment or prevention viral infection resisting.
Advantage of the present invention is as follows:
(1) after Harpin albumen and the coupling of ultra micron carrier, can improve its bioavailability.
(2) Harpin albumen is suitably wrapped up, can protected protein matter.
(3) can promote plant to the absorbing of pharmaceutical grade protein, effect strengthens or extends, and produces obvious biology effect.
(4) use simple, safety.
(5) disease-resistant wide spectrum, free from environmental pollution.This protein nano complex can be used for the control of plant disease, the growth that promotes plant, raising crop yield.
Adopt controlling and releasing system with nanotechnology, the Harpin protein nano complex has approximately thing of slowly-releasing Harpin protein, extends Harpin pharmaceutical grade protein action time; Reach target and carry purpose; Under the prerequisite that guarantees the effect of Harpin pharmaceutical grade protein, reduce to Harpin protein pharmaceutical quantities; Improve the characteristics such as medicine stability.This has great importance for improving the healthy of bioavailability, protection of the environment, guarantee China people.
Description of drawings
Above and other objects of the present invention, characteristics and advantages can obtain the content shown in the detailed description and the accompanying drawings of the preferred embodiment of the present invention from following apparently, and in different views, identical reference symbol represents identical part.Accompanying drawing might not be shown to scale, and it focuses on illustrating enforcement of the present invention and effect.
Fig. 1 .10%SDS-PAGE detects rHrpZ protein purification purity collection of illustrative plates
CL:rHrpZ crude extract FT:rHrpZ crude extract leakage liquid from the Ni-NTA Superflow post
E1-E5: the rHrpZ eluent M of fraction collection: middle molecular weight protein matter Marker (Beijing TIANGEN)
Fig. 2: HPLC C8 post detects rHrpZ purity of protein collection of illustrative plates
Fig. 3 dynamic light scattering particle diameter detector is measured rHrpZ-PLGA nano complex particle diameter
Fig. 4 rHrpZ-PLGA nano complex is induced the PAL determination of activity
A: after spraying rHrpZ for the first time, tobacco PAL activity change
B: after spraying rHrpZ for the second time, tobacco PAL activity change
C: after spraying the rHrpZ-PLGA nano particle, tobacco PAL activity change
Fig. 5 rHrpZ-PLGA nano complex induces the PR-5dB genetic transcription to measure
A: after spraying rHrpZ, the PR-5dB gene transcription level changes
B: after spraying rHrpZ-PLGA NP, the PR-5dB gene transcription level changes
After Fig. 6 rHrpZ albumen, rHrpZ-PLGA NP dispenser, the Resistance In Tobacco Ralstonia solanacearum infects the effect collection of illustrative plates
A sprays the rHrpZ-PLGA NP that contains 20ug/mL rHrpZ
B sprays 20ug/mL rHrpZ albumen
C sprays PBS
Embodiment
By in conjunction with following specific embodiment, illustrate the present invention.What however, it should be understood that is that these embodiment only are not used in for explanation the present invention and limit the scope of the invention.In this patent the present embodiment, (rHrpZ albumen rHrpZ) describes for example Harin albumen with gene engineering restructuring Harpin albumen.
The preparation of embodiment 1:Harpin protein
1.Harpin protein purification
Employing contains the engineered strain E.coli BL21 (Chinese Typical Representative culture collection center C CTCC M202026) of harpin gene, and expression profile engineering restructuring Harpin albumen (rHrpZ albumen, rHrpZ).The bacterial classification that picking is preserved with glycerine, be inoculated in LB liquid nutrient medium (the LB liquid nutrient medium 1L: peptone 10g that contains 200 μ g/mL ampicillins, dusty yeast 5g, NaCl 10g, be dissolved in the 800ml distilled water, regulate pH to 7.0 with NaOH, add water and be settled to 1000ml) in, 37 ℃, shaken cultivation 8-12h under 200r/min.Be inoculated in fresh 2YT liquid nutrient medium (the peptone 16g that contain 200 μ g/mL ampicillins by 4% amount next day, dusty yeast 10g, NaCl 5g, be dissolved in the 800ml distilled water, regulate pH to 7.0 with NaOH, add water and be settled to 1000ml) in, at 37 ℃, when under the 200r/min condition, shaken cultivation to OD600 value is about 0.6-0.8, adding inducer IPTG (isopropyl-β-D-thiogalactoside, Promega company product) is 0.4mM to final concentration, 37 ℃ of lower abduction deliverings, induced rear 4 hours, 4000r/min collected thalline in centrifugal 20 minutes.
The thalline of collecting is resuspended in Lysis Buffer (50mmol/L NaH2PO4 0.5mol/LNaCl 20mmol/L imidazoles, pH8.0), ultrasonic disruption (200W, 10 minutes).12000rpm collects supernatant after centrifugal 20 minutes be the rHrpZ protein crude extract.
The rHrpZ protein crude extract passes 2mL Ni-NTA Superflow post (Qiagen company product) twice with 1mL/min speed, Lysis Buffer with 50 bed volumes cleans the post bed with same speed, use again 20mL Elute Buffer (50mmol/L NaH2PO40.5mol/L NaCl 250mmol/L imidazoles, pH8.0) wash-out is collected destination protein rHrpZ.
Will be through Ni
++The rHrpZ protein purification sample of post wash-out, collection is packed in treated bag filter, puts into to fill 1000mL PB S buffer solution (20mmol/L NaH2PO4, pH7.4) beaker and dialyse, and dialyses 24 hours, changes dialysis PBS therebetween for 4 times.Albumen after dialysis spends the night with polyethylene glycol (molecular weight is about 20000) is concentrated.Above operation is all carried out at 4 ℃.
2.Harpin the mensuration of lipidated protein
With reference to " molecular cloning ", detect coomassie brilliant blue R250 staining examine rHrpZ lipidated protein with 10%SDS-PAGE.Result as shown in Figure 1.
Adopt high pressure liquid chromatography (HPLC) to detect the purity of Harpin albumen.Mobile phase is set to: A water; B methyl alcohol; The C acetonitrile; D 50mM PBS pH6.0.After the exhaust of Agilent 1100HPLC system pipeline, the C8 post is installed, column temperature is set as 20 ℃, opens uviol lamp and detects, and measures absorbing wavelength 280nm, flow velocity 1ml/min, wash the C8 post with 70% methyl alcohol steady to baseline, use 5% acetonitrile, 95%PB S balance pillar is to baseline values, open injection annulus, sample introduction 20 μ L.During wash-out, 0-5min, 5% acetonitrile, 95%PBS; 6-30min, 5-50% acetonitrile linear gradient, all the other are PB S.The C8 post detects the rHrpZ purity of protein as shown in Figure 2.Through software analysis, be simple spike on collection of illustrative plates, confirm that the rHrpZ purity of protein of purifying reaches chromatographic purity.
3.Harpin the mensuration of protein content
RHrpZ albumen after purifying is changed dialysis PBS therebetween for 4 times with PBS (20mmol/L NaH2PO4, pH7.4) dialysis 24 hours.Albumen after dialysis is good is concentrated with polyethylene glycol (molecular weight is about 20000).Adopt the Bradford method, the rHrpZ albumen after concentrated is measured protein concentration with TIANGEN Bradford determination of protein concentration kit (TIANGEN company product), and adjusting the rHrpZ protein concentration with PBS at last is 20mg/mL.
The concrete operations of Bradford method are as follows:
(1) 0,1,2,3,4,5,6 μ L 1mg/ml bovine serum albumin(BSA) (BSA) standard liquids are added in enzyme mark microwell plate successively, supply 50 μ L with PBS.
(2) add 200L Bradford reagent working solution (0.1% Coomassie brilliant blue G250,5% ethanol, 8.5% phosphoric acid) in every hole.After vibration, mixing, room temperature was placed 2 minutes.
(3) survey the OD570 value (λ=570nm) of each concentration of BSA albumen with microplate reader.Take the BSA protein concentration as abscissa, take the OD570 value of each concentration of BSA albumen as ordinate production standard curve.
(4) with the OD570 value of same method working sample, use the concentration of determining sample in calibration curve.
The preparation of embodiment 2:Harpin protein nano complex
Take 250mg PLGA (poly d, l-lactide-co-glycolide, lactide: glycolide=75: 25, molecular weight 40000, Shandong wild goose tail feather chemical industry Co., Ltd product), be dissolved in the 5mL carrene, be made into 50mg/mL solution.0.5mL 20mg/mL rHrpZ slowly is added drop-wise in 5mL 50mg/mL PLGA solution (drips off in controlling 0.5 minute), use simultaneously ultrasonic disruption instrument emulsification (50W processed 1 minute) to form Water-In-Oil (W/O) emulsion, pour immediately water-in-oil emulsion into 50mL 2% polyvinyl alcohol (poly vinyl alcohol, PVA, molecular weight 70,000) in the aqueous solution.Ultrasonic emulsification (100W processed 2 minutes) forms the W/O/W emulsion.The W/O/W emulsion is poured in distillation flask, and 40 ℃ of decompression distillation were removed carrene in 2 hours, made PLGA solidify to form nano particle, namely consisted of rHrpZ-PLGA nano complex (rHrpZ-PLGA NP).
With the rHrpZ-PLGA nano complex particle suspension that forms, centrifugal 20 minutes of 10000g, collecting precipitation.RHrpZ-PLGA nano complex particle is resuspended in 40mL distilled water, more centrifugal, repeat this process 3 times, to clean the outer PVA of nano particle.At last, the rHrpZ-PLGA protein nano complex of acquisition is suspended from distilled water standby.
The mensuration of embodiment 3:Harpin protein nano complex particle diameter
The rHrpZ-PLGA nano particle of preparation is resuspended in 10ml water, gets 3ml and joins in quartz colorimetric utensil, and sample temperature is set as 20 ℃.Measure synthetic rHrpZ-PLGA nano complex particle diameter with dynamic light scattering particle diameter detector.Testing result shows, in the nano particle of preparation, 72.5% nano particle diameter is less than 225nm, and average particulate diameter is 190.5nm.The nano particle diameter distributes as shown in Figure 3.
The mensuration of embodiment 4:Harpin protein nano complex envelop rate and rate of release
1.rHrpZ-PLGA the mensuration of nano complex envelop rate
The rHrpZ-PLGA nano particle is resuspended in 100l water, add the 1ml cold acetone, mixing,-20 ℃ freezing 1 hour, centrifugal 10 minutes of 20000g, precipitation is used the cold acetone washed twice, centrifugal, be deposited under 37 ℃ acetone is volatilized totally, be dissolved in 50 μ l water, the Bradford method is measured the rHrpZ protein content.
Actual use rHrpZ quality * 100% when envelop rate=nano particle includes rHrpZ quality/preparation
The rHrpZ-PLGA nano particle is about 62.3% to the envelop rate of rHrpZ.
2.rHrpZ-PLGA the mensuration of nano complex rate of release
RHrpZ-PLGA nano complex particle is resuspended in (20mM NaH2PO4,1mM EDTA, pH7.4) in PBE solution, slowly shook for 4 weeks sampling and measuring protein release efficient under 25 ℃ on shaking table.With centrifugal 20 minutes of the nano granule suspension 10000g that obtains, collecting precipitation.Be precipitated and dissolved in the acetone of precooling ,-20 ℃ standing 1 hour with precipitating proteins, 10000g is centrifugal 10 minutes subsequently, the precipitation dry the volatilization complete acetone after, be dissolved in 1mL distilled water.Protein solution is measured protein concentration with the Bradford method, and calculates rHrpZ-PLGA nano particle rate of release.
Release rate=(in the 1-nano particle, residual rHrpZ quality/nano particle initially seals the rHrpZ quality) * 100%
The rHrpZ-PLGA nano particle is about 62.3% to the envelop rate of rHrpZ, extracorporeal releasing test shows, have 51.0% rHrpZ to be released from the rHrpZ-PLGA particle within front 2 day time, and 92.7% rHrpZ discharge within 3 weeks from the rHrpZ-PLGA particle.Illustrate that the rHrpZ-PLGA particle has preferably slow controlled release and puts effect.
The mensuration of embodiment 5:Harpin protein nano complex activity
Phenylalnine ammonialyase (phenylalanine ammonia lyase, PAL) is that plant is connected primary metabolite and phenylpropyl alcohol alkanes metabolic regulation single step reaction enzyme with microorganism, is key enzyme and the rate-limiting enzyme of the metabolism of phenylpropyl alcohol alkanes.Phenylpropyl alcohol alkanes metabolic pathway is very important approach in plant metabolism, and all materials that contain phenylpropyl alcohol alkane skeleton are all directly or indirectly generated by this approach.The metabolism of phenylpropyl alcohol alkanes can generate the multiple secondary metabolites such as flavonoids, lignin, and these products play an important role in the growing of plant, disease-resistant, degeneration-resistant reaction.(Dong Yanzhen, the progress of Plant Phenylalanine Ammonia-Lyase Gene.The biotechnology circular, 2006, supplementary issue, 31-33)
Phenylalnine ammonialyase is the enzyme that plays an important role in plant defense, by detecting PAL, measure Harpin protein nano complex active, determine Harpin protein nano complex to the inducing action of defensive Substance P AL in tobacco leaf, and compare with the effect of Harpin albumen.
1. sample treatment
Get the tobacco plant of 9 strain health, be divided into 3 groups, every group of 3 strains, and spray with 3 groups of samples respectively.These 3 groups of samples are respectively: (1) 10 μ g/mL rHrpZ protein solution sample; The rHrpZ-PLGA nano complex sample of (2) 10 μ g/mL rHrpZ; (3) PB S buffer solution.
After sprinkling, gather the tobacco leaf sample, weigh, be placed in the EP pipe ,-20 ℃ save backup.
2. the active mensuration of phenylalnine ammonialyase (PAL)
Get approximately 0.2-0.8g blade, cut little, put into respectively the mortar of ice precooling, add the 3-5mL precooling to contain boric acid (the 0.1mol/L boric acid of 5mmol/L mercaptoethanol, pH8.8) buffer solution adds the 0.05g polyvinylpyrrolidone, and ice bath grinds pulping, 4 ℃ of centrifugal 25min of lower 4800r/min, supernatant is the PAL enzyme extract.
PAL enzyme reaction system (3.1ml) alive: 0.1ml enzyme liquid; 1ml 0.02mol/L phenyl alanine; 2ml distilled water
Contrast does not add substrate (enzyme extract), adds 0.1ml distilled water.Reactant liquor is put 30 ℃ of insulation 30min in thermostat water bath, puts into immediately ice bath, adds 0.25ml 5mol/L HCl cessation reaction.Every kind of reactant liquor is got 3ml in cuvette, with the absorbance under UV-120-02 type ultraviolet specrophotometer survey 290nm wavelength.Each repeats 3 times, averages.
The PAL enzymic activity=(Δ OD290nm * VT)/(0.01 * WF * VS * t)
Light absorption value change under the 290nm wavelength in reaction time
ΔOD290nm
Change
VT extracts PAL enzyme liquid cumulative volume (ml)
WF fresh weight (g)
VS takes PAL enzyme liquid long-pending (ml) when measuring
The t reaction time (min)
The PAL enzyme is lived and is changed as accompanying drawing 4.As can be seen from Fig. 4, spray for the first time rHrpZ albumen after PAL activity change amplitude obviously greater than for the second time, and spray that the impact on the PAL activity only limited in 14 hours after rHrpZ albumen.This is that PAL is active to rise because rHrpZ albumen can stimulate.And PAL is the key enzyme that lignin forms, and is also the rate-limiting enzyme of its formation, rises because PAL is active, and lignin forms fast, causes plant cell wall to be developed to secondary cell wall fast by primary cell wall, causes simultaneously the cell wall permeability to descend.Due to the decline of cell wall permeability, make albumen mass-energy still less have an effect by the receptors bind on cell wall and cell membrane, obviously reduce so spray for the second time the drug effect that same concentration rHrpZ protein ratio sprays for the first time.And after spraying the rHrpZ-PLGA nano complex, although the PAL activity change is not sprayed rHrpZ albumen vary within wide limits for the first time, more than reaching for 2 weeks the perdurabgility of PAL increased activity effect.Result of the test shows, rHrpZ-PLGA nano complex (rHrpZ-PLGA NP) can help rHrpZ albumen to enter tobacco leaf, and can slowly progressively discharge rHrpZ albumen in the tobacco body, and constantly stimulate the PAL increased activity, reach the effect that extends timeliness.
The impact of embodiment 6:Harpin protein nano complex on the plant disease-resistant effect
But the expression of PR-5dB gene in Harpin albumen evoking tobacco, PR-5dB gene are a class plant course of disease related protein genes, and its transcriptional expression can help the invasion and attack of plant opposing pathogen.The expression of this gene is directly related with the plant disease-resistant ability, can be used as the important evidence of research Harpin albumen drug effect.
Measuring tobacco pathogenesis-related proteins PR-5dB gene transcription level with the reverse transcription PCR semi-quantitative method changes.Adopt and measure the identical method of PAL enzymic activity in embodiment 5, spray respectively rHrpZ albumen and rHrpZ-PLGA NP sample, and establish the contrast of PBS sample.
1. the extraction of the total RNA of tobacco
Tobacco plant is after rHrpZ albumen, rHrpZ-PLGA NP nano complex and PBS sample treatment, and the blade that takes a morsel extracts total RNA as follows with Trizol reagent.
(1) sample adds chloroform (200 μ l/mlTrizol) 100 μ l, thermal agitation 30 seconds, standing 15 minutes of room temperature;
(2) 12000g is centrifugal 15 minutes;
(3) get the upper strata water;
(4) added equal-volume isopropyl alcohol room temperature (identical below 20-25 ℃) standing 30 minutes;
(5) 12000g is centrifugal 10 minutes, removes supernatant;
(6) add 75% ethanol (1ml/mlTrizol), 500 μ l, precipitation suspends;
(7) 8000g, 4 ℃ of centrifugal 5min remove supernatant, repeating step 6,7 twice, drying at room temperature;
(8) with 20 μ L RNA Free H
2The O dissolution precipitation.
(9) RNA formaldehyde-agarose electrophoresis is identified.
2. design of primers is with synthetic
In order to contrast PR-5dB at the relative amount of plant interior expression, take the transcriptional level of actin gene as internal reference.According to actin gene and PR-5dB gene design primer (table 1), primer is synthetic by Shanghai living work company.
Table 1Sequences of gene-specific primers used in RT-PCR analysis
3.cDNA synthetic
RNA after dissolving prepares the reverse transcription system by following proportioning, with TOYOBO company reverse transcription kit, the mRNA reverse transcription is formed cDNA.
The reverse transcription system component:
5 * reverse transcription solution, 4 μ L
dNTPs(10mM) 2μL
Reverse transcriptase 1 μ L
Oligo(dT)
20(10pmol/μL) 1μl
RNA enzyme inhibitor 1 μ l
Total RNA X μ l
Water 11-X μ l
The reverse transcription condition:
42 ℃ 20 minutes
99 ℃ 5 minutes
4 ℃ 5 minutes
4.PCR reaction
The cDNA of reverse transcription gained as template, is carried out pcr amplification.Add following component in aseptic PCR pipe, aspirate mixing with liquid-transfering gun.After the centrifugal 10sec of 1000rpm, the PCR pipe is placed on the PCR instrument.
The PCR reaction system:
10×PCR buffer(La) 2.5μl
dNTPs(2.5mM) 1μl
MgCl
2(25mM) 1.5μl
Upstream primer (20 μ M) 2 μ l
Downstream primer (20 μ M) 2 μ l
Taq archaeal dna polymerase 1 μ l
Mend aseptic ddH
2O to 25 μ l
The PCR reaction condition:
94℃ 2min；
94℃ 30s
57℃ 30s
72 ℃ of 45s, 28 times;
72℃ 5min
4℃ 15min。
Get 5 μ l PCR products electrophoresis on 1.0% Ago-Gel, ethidium bromide dyeing is observed amplification under uviol lamp.Gel after electrophoresis is finished records the difference of each band amount through digital image-forming and analysis.With 1D gray analysis software analysis band gray value, the band gray value is directly proportional to the PCR product quality.The PR-5dB band gray value of every kind of sample is obtained the relative expression quantity of PR-5dB in this sample divided by this sample actin gene magnification band gray value, to reduce experimental error.According to PR-5dB relative expression quantity in each group test, draw PR-5dB gene transcription level changing trend diagram, as accompanying drawing 5.
According to the difference of PR-5dB gene transcription level, estimate the drug effect of rHrpZ albumen and rHrpZ-PLGA NP (rHrpZ-PLGA nano complex), calculate drug effect with following formula:
Drug effect=TG-AUC-initial p R-5dB relative expression quantity * duration of efficacy
The result demonstration can improve by rapid stimulation PR-5dB transcriptional level after rHrpZ albumen sprays, and similar with the variation alive of PAL enzyme in embodiment 5, PR-5dB transcriptional level raising effect is held time shorter, is no more than 48 hours.And rHrpZ-PLGA NP can keep over 2 weeks the castering action of PR-5dB transcriptional level, drug effect is 5 times that spray with isoconcentration rHrpZ albumen, having realized continuing slow controlled release in tobacco puts, permanently effective plays a role, obviously improved the bioavilability of rHrpZ albumen, can avoid or reduce application times when use in the field, reduce human cost.
Embodiment 7:Harpin protein nano complex infects the attack protection test to the Genes For Plant Tolerance Ralstonia solanacearum
Ralstonia solanacearum (Ralstonia solanacearum) is the pathogen of plant of Solanaceae bacterial wilt, and bacterial wilt is the plant of Solanaceae important diseases that extensively shows effect in worldwide, produces to crops and brings huge loss.As typical tobacco disease indigenous bacteria, detect the drug effect of rHrpZ albumen and rHrpZ-PLGA NP with Ralstonia solanacearum.
Ralstonia solanacearum spends the night with following medium culture.
Regulate pH value to 7.2 with NaOH.
Get centrifugal 5 minutes of the Ralstonia solanacearum 12000rpm of 1mL incubated overnight, thalline is resuspended in the 1mL distilled water.Get tobacco 3 strains of growing way equalization, with the dosage of every strain 5 μ L, the Ralstonia solanacearum suspension is injected into tobacco in the stem of Tu0.5cmChu.Simultaneously, three strain tobaccos spray respectively PBS, 20ug/mLrHrpZ protein solution, contain the rHrpZ-PLGA NP of 20ug/mL rHrpZ, observe 10 days sequela situations.Tobacco growing and incidence such as accompanying drawing 6.
The tobacco of spraying afterwards PBS in 10 days obviously falls ill, and blade is crispaturaed, color and luster is dim heavy, and the tobacco leaf growing way of sprinkling rHrpZ albumen is better, color and luster is comparatively bright; And the tobacco growing way of spraying rHrpZ-PLGA NP is very vigorous, and leaf color is vivid.Result of the test shows that rHrpZ albumen has improved the effect that tobacco opposing Ralstonia solanacearum infects.In the situation that equal effectively rHrpZ protein content, the drug effect of Harpin protein nano complex (rHrpZ-PLGA NP) is apparently higher than single rHrpZ albumen, and stimulating plant is built up resistance for a long time, promotes tobacco growing.SEQUENCE LISTING
＜110〉Wuhan University
＜120〉a kind of protein nano complex of Promoting plant growth and preparation method and application
＜130〉a kind of protein nano complex of Promoting plant growth and preparation method and application
<140>2009102722035
<141>2009-09-23
<160>4
<170>PatentIn version 3.1
<210>1
<211>20
<212>DNA
＜213〉artificial synthetic
<400>1
<210>2
<211>20
<212>DNA
＜213〉artificial synthetic
<400>2
<210>3
<211>20
<212>DNA
＜213〉artificial synthetic
<400>3
<210>4
<211>19
<212>DNA
＜213〉artificial synthetic
<400>4
gtaggcatct ccaatggga 19
Claims (9)
1. the protein nano complex of a Promoting plant growth, is characterized in that, comprises Harpin albumen and carrier in this nano complex.
2. the protein nano complex of a kind of Promoting plant growth according to claim 1, it is characterized in that: described carrier is the ultra micron carrier, and the ultra micron carrier is PLA, lactic acid-ethanol copolymer, Polyalkylcyanoacrylanano, polycaprolactone, Polyalkylcyanoacrylanano or polycaprolactone.
3. the preparation method of the protein nano complex of a kind of Promoting plant growth as claimed in claim 1, is characterized in that, preparation process is as follows:
(1) with PLGAlactide: glycolide=75: 25, molecular weight 40000 is dissolved in carrene, be made into 50mg/mL solution, 0.5mL20mg/mLHarpin albumen slowly is added drop-wise in 5mL50mg/mLPLGA solution, processes simultaneously forming Water-In-Oil W/O emulsion in 1 minute with ultrasonic disruption instrument emulsification 50W;
(2) water-in-oil emulsion is poured in the 50mL2%PVA aqueous solution, ultrasonic emulsification 100W processes and formed the W/O/W emulsion in 2 minutes;
(3) the W/O/W emulsion is poured in distillation flask, carrene was removed in 40 ℃ of decompression distillation in 2 hours, solidified PLGA, and preparation PLGA-Harpin nanoparticle wherein wraps up Harpin albumen;
(4) with the distilled water of 40mL, the PLGA-Harpin nanoparticle that forms is suspended, centrifugal 20 minutes of 10000g, collecting precipitation, precipitation is resuspended in distilled water, repeats this process 3 times to clean the outer PVA of nano particle, at last nano particle is resuspended in distilled water standby.
4. a kind of preparation method of protein nano complex as claimed in claim 3, is characterized in that, described PLGA is nanoemulsion.
5. a pharmaceutical composition, is characterized in that said composition comprises the protein nano complex claimed in claim 1 of effective dose.
6. a kind of protein nano complex claimed in claim 1 or the application of pharmaceutical composition claimed in claim 5 in the controlling plant diseases medicine.
7. the application of a kind of protein nano complex claimed in claim 1 in the medicine of preparation treatment or prevention bacterial-infection resisting.
8. the application of a kind of protein nano complex claimed in claim 1 in the medicine of preparation treatment or prevention anti-fungal infection.
9. the application of a kind of protein nano complex claimed in claim 1 in the medicine of preparation treatment or prevention viral infection resisting.
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| CN115843794B (en) * | 2022-12-13 | 2024-03-01 | 贵州大学 | Nanometer system of plant protein-based drug-encapsulated molecules and preparation method and application thereof |
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| CN1468963A (en) * | 2002-07-19 | 2004-01-21 | 深圳市武大万德福基因工程有限公司 | Construction and application of gene engineering strain of Harpin protein expressing colibacillus |
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| WO2022036010A1 (en) * | 2020-08-11 | 2022-02-17 | Cellacure Llc | Green closed loop bio-waste refining process for producing smart active extracts and delivery systems for their application |
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