CN101690492A - 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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- CN101690492A CN101690492A CN200910272203A CN200910272203A CN101690492A CN 101690492 A CN101690492 A CN 101690492A CN 200910272203 A CN200910272203 A CN 200910272203A CN 200910272203 A CN200910272203 A CN 200910272203A CN 101690492 A CN101690492 A CN 101690492A
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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 that promotes the protein nano complex of plant growing.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, promote plant to the absorbing, strengthen or prolong drug action of medicine, can improve its bioavailability, produces tangible 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, due to illness the loss that causes of insect pest was about 20%, 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, viral 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, novel pollution-free, the nonresistant biological bactericide of development has become the task of top priority.
Harpin albumen is a kind of albumen that causes the pathogen Erwiniaamvlovora hrpN gene code of rose 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 on non-host plant, can react by induced hypersensitivity, 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 the plant cell membrane, many signal paths in the activated plant, thereby promotion plant growing, 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 HIN1independent 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, the scholar of U.S. Cornell university at first 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 advantages such as resistance.
The scientific research field that the 21 century that nanometer technology is generally acknowledged in the world has most future.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 is that atomic arrangement had not only had long-range order but also do not had 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 the 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 medicine field of sending, and develop the Nano medication of various ways.The size of Nano medication can obviously influence 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 the nano controlled release system, 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 the pharmaceutically-active prerequisite, alleviating or avoid toxicity; Improve characteristics such as medicine stability.This has great importance for improving bioavailability, protection environment, ensureing China's people health.The Harpin protein nano complex is a kind of safe novel biopesticide, and the control that is used for plant disease has efficiently, wide spectrum, advantage free from environmental pollution, has application promise in clinical practice.
Summary of the invention
The objective of the invention is to be to provide a kind of protein nano complex that promotes plant growing.Comprise Harpin albumen and carrier in this nano complex.Harpin albumen can inducing plant disease resistance.Harpin albumen is by combining the defensive enginery of activated plant self with the acceptor HrBP1 of plant surface.Harpin albumen itself is to bacterium, virus, fungi, and nematode, insect do not have to be killed or inhibitory action, but by with the plant receptors bind after produce certain signal, stimulate 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 precocious and solid as the individuality that increases plant and root amount, the sprouting that promotes seed, plant improved output and the quality of crop.In addition, Harpin albumen can improve vegetables, the anti-ability of rotting, go mouldy of fruit, prolongs 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, promote plant to the absorbing, strengthen or prolong drug action of medicine, can improve its bioavailability, produces tangible 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 72.5% nano particle diameter is less than 225nm in the nano particle.Harpin albumen is positioned at nano complex inside.This Harpin protein nano complex has the distinctive character of nano controlled release system, makes it have many superiority aspect medicine conveying: (1) but slowly-releasing Harpin albumen, thereby prolong 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, help storing; (5) can be in order to set up some new methods of administration.This all is 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 preparation Harpin method of protein.Employing contains the engineered strain E.coli BL21 (Chinese typical culture collection center C CTCC M202026) of harpin gene, and abduction delivering gene engineering reorganization 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 operation manual purifying Harpin of Qiagen 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.This method may further comprise the steps:
A, PLGA (poly-(lactic-co-glycolic acid) copolymer, lactide: glycolide=75: 25, molecular weight 40000) is dissolved in the carrene, is made into 50mg/mL solution.0.5mL 20mg/mL Harpin albumen slowly is added drop-wise in the 5mL 50mg/mL PLGA solution, uses ultrasonic disruption instrument emulsification (50W handled 1 minute) simultaneously, 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 handled 2 minutes) forms the W/O/W emulsion.
C, the W/O/W emulsion is poured in the distillation flask, carrene was removed in 40 ℃ of decompression distillation in 2 hours, solidified PLGA, constituted the Harpin-PLGA protein nano complex, wherein wrapped up Harpin albumen.
The distilled water of D, usefulness 40mL suspends centrifugal 20 minutes of 10000g, collecting precipitation with the PLGA-Harpin nanoparticle that forms.Precipitation is resuspended in the distilled water, repeats this process 3 times to clean the outer PVA of Harpin-PLGA protein nano complex, is resuspended in the distilled water 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,, 72.5% nano particle diameter is less than 225nm in the nano particle.
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 in preceding 2 day time, from the rHrpZ-PLGA particle, to be released, and 92.7% rHrpZ discharge in 3 weeks from the rHrpZ-PLGA particle.Illustrate that the rHrpZ-PLGA particle has slow preferably 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 activity, determine the inducing action of Harpin protein nano complex, and compare with the effect of Harpin albumen to defensive Substance P AL in the tobacco leaf.In the test, spray for the first time rHrpZ albumen after PAL activity change amplitude obviously greater than for the second time, and spray that the influence to the PAL activity only limited in 14 hours behind the rHrpZ albumen.After spraying the rHrpZ-PLGA nano complex, though the PAL activity change is not for the first time sprayed rHrpZ albumen vary within wide limits, reach more than 2 weeks the perdurabgility of PAL increased activity effect.Result of the test shows that 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, constantly stimulates the PAL increased activity, reaches the effect that prolongs timeliness.
In one embodiment of the invention, measured of the influence of Harpin protein nano complex to the plant disease-resistant effect.But PR-5dB expression of gene in the 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.This expression of gene amount 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), the 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 above 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, when use in the field, can avoid or reduce application times, reduce human cost.
In one embodiment of the invention, measured Harpin protein nano complex Solanaceae Lei Er Salmonella (Ralstonia solanacearum) has been infected attack protection effectiveness.Solanaceae Lei Er Salmonella 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 Solanaceae Lei Er Salmonella.Result of the test shows that rHrpZ albumen has improved the effectiveness that tobacco opposing Solanaceae Lei Er Salmonella infects.Under the situation of 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 prolongs, and produces tangible 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 the nano controlled release system, the Harpin protein nano complex has slowly-releasing Harpin pharmaceutical grade protein, prolongs Harpin pharmaceutical grade protein action time; Reach target and carry purpose; Under the prerequisite that guarantees the effect of Harpin pharmaceutical grade protein, reduce and give Harpin protein pharmaceutical quantities; Improve characteristics such as medicine stability.This has great importance for improving bioavailability, protection environment, ensureing China's people health.
Description of drawings
Above and other objects of the present invention, characteristics and advantage obtain the content shown in the detailed description and the accompanying drawings of the preferred embodiment of the present invention from following with may be obvious that, and reference symbol identical in the different views is represented 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 E1-E5 from Ni-NTA Superflow post: the rHrpZ eluent M of fraction collection: 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
The anti-Solanaceae Lei Er Salmonella of tobacco is infected the effect collection of illustrative plates after Fig. 6 rHrpZ albumen, the rHrpZ-PLGA NP dispenser
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, these embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.In this patent present embodiment, (rHrpZ albumen rHrpZ) describes for example Harin albumen with gene engineering reorganization Harpin albumen.
The preparation of embodiment 1:Harpin protein
1.Harpin protein purification
Employing contains the engineered strain E.coli BL21 (Chinese typical culture collection center C CTCC M202026) of harpin gene, and abduction delivering gene engineering reorganization 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, add water and be settled to 1000ml with NaOH) in, 37 ℃, shaken cultivation 8-12h under the 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 ℃, shaken cultivation is when the OD600 value is about 0.6-0.8 under the 200r/min condition, adding inducer IPTG (isopropyl-, Promega company product) is 0.4mM to final concentration, 37 ℃ of following abduction deliverings, induced back 4 hours, 4000r/min collected thalline in centrifugal 20 minutes.
The thalline of collecting is resuspended among the 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 20mL Elute Buffer (50mmol/L NaH2PO40.5mol/L NaCl 250mmol/L imidazoles again, 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 the treated bag filter, puts into to fill 1000mL PBS buffer solution (20mmol/L NaH2PO4 pH7.4) dialyses in the beaker, dialyses 24 hours, changes dialysis PBS therebetween for 4 times.Albumen after the dialysis concentrates with polyethylene glycol (molecular weight is about 20000) and spends the night.More than operation is all carried out at 4 ℃.
2.Harpin the mensuration of lipidated protein
With reference to " molecular cloning ", detect with 10%SDS-PAGE, coomassie brilliant blue R250 dyeing detects the rHrpZ lipidated protein.The result as shown in Figure 1.
Adopt high pressure liquid chromatography (HPLC) to detect the purity of Harpin albumen.Flowing 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 at 20 ℃, opens uviol lamp and detects, and measures absorbing wavelength 280nm, flow velocity 1ml/min, it is steady to baseline to wash the C8 post with 70% methyl alcohol, uses 5% acetonitrile, and 95%PBS 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 PBS.The C8 post detects the rHrpZ purity of protein as shown in Figure 2.Through software analysis, on the collection of illustrative plates simple spike, confirm that the rHrpZ purity of protein of purifying reaches chromatographic purity.
3.Harpin the mensuration of protein content
(20mmol/L NaH2PO4, pH7.4) dialysis is 24 hours, changes dialysis PBS therebetween for 4 times with PBS for rHrpZ albumen behind the purifying.Albumen after dialysis is good concentrates with polyethylene glycol (molecular weight is about 20000).Adopt the Bradford method, the rHrpZ albumen after concentrating 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 is added successively in the enzyme mark microwell plate, supply 50 μ L with PBS.
(2) in every hole, add 200 μ L Bradford reagent working solutions (0.1% Coomassie brilliant blue G250,5% ethanol, 8.5% phosphoric acid).Behind vibration, the mixing, room temperature was placed 2 minutes.
(3) survey the OD570 value (λ=570nm) of each concentration of BSA albumen with microplate reader.With the BSA protein concentration is abscissa, is ordinate production standard curve with the OD570 value of each concentration of BSA albumen.
(4) use OD570 value, with the concentration of determining sample in the calibration curve with the quadrat method working sample.
The preparation of embodiment 2:Harpin protein nano complex
Take by weighing 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 the 5mL 50mg/mL PLGA solution (drips off in controlling 0.5 minute), use ultrasonic disruption instrument emulsification (50W handled 1 minute) to form Water-In-Oil (W/O) emulsion simultaneously, pour water-in-oil emulsion into 50mL 2% polyvinyl alcohol (poly vinyl alcohol immediately, PVA, molecular weight 70,000) in the aqueous solution.Ultrasonic emulsification (100W handled 2 minutes) forms the W/O/W emulsion.The W/O/W emulsion is poured in the distillation flask, and 40 ℃ of decompression distillation were removed carrene in 2 hours, made PLGA solidify to form nano particle, promptly constituted 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 the 40mL distilled water, centrifugal again, 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 the distilled water standby.
The mensuration of embodiment 3:Harpin protein nano complex particle diameter
The rHrpZ-PLGA nano particle of preparation is resuspended in the 10ml water, gets 3ml and joins in the quartz colorimetric utensil, and sample temperature is set at 20 ℃.Measure synthetic rHrpZ-PLGA nano complex particle diameter with dynamic light scattering particle diameter detector.Testing result shows that 72.5% nano particle diameter is less than 225nm in the nano particle of preparation, and average particulate diameter is 190.5nm.The nano particle diameter distribution as shown in Figure 3.
The mensuration of the mensuration 1.rHrpZ-PLGA nano complex envelop rate of embodiment 4:Harpin protein nano complex envelop rate and rate of release
The rHrpZ-PLGA nano particle is resuspended in the 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 the 50 μ l water, the Bradford method is measured the rHrpZ protein content.
Actual use rHrpZ quality * 100%rHrpZ-PLGA nano particle was about 62.3% to the envelop rate of rHrpZ when envelop rate=nano particle included rHrpZ quality/preparation.
2.rHrpZ-PLGA the mensuration of nano complex rate of release
RHrpZ-PLGA nano complex particle is resuspended in the PBE solution (20mM NaH2PO4,1mM EDTA pH7.4), slowly shook for 4 weeks sampling and measuring protein release efficiency under 25 ℃ on shaking table.With centrifugal 20 minutes of the nano granule suspension 10000g that obtains, collecting precipitation.Resolution of precipitate leaves standstill 1 hour with precipitating proteins at-20 ℃ in the acetone of precooling, 10000g is centrifugal 10 minutes subsequently, and precipitation is dissolved in the l mL distilled water after drying the complete acetone of volatilization.Protein solution is measured protein concentration with the Bradford method, and calculates rHrpZ-PLGA nano particle rate of release.
Release rate=(residual rHrpZ quality/nano particle initially seals the rHrpZ quality in the 1-nano particle) * 100%
The rHrpZ-PLGA nano particle is about 62.3% to the envelop rate of rHrpZ, extracorporeal releasing test shows, have 51.0% rHrpZ in preceding 2 day time, from the rHrpZ-PLGA particle, to be released, and 92.7% rHrpZ discharge in 3 weeks from the rHrpZ-PLGA particle.Illustrate that the rHrpZ-PLGA particle has slow preferably controlled release and puts effect.
The mensuration of embodiment 5:Harpin protein nano complex activity
(phenylalanine ammonia lyase is that plant is connected elementary metabolism and phenylpropyl alcohol alkanes metabolic regulation single step reaction enzyme with microorganism PAL) to phenylalnine ammonialyase, is the key enzyme and the rate-limiting enzyme of the metabolism of phenylpropyl alcohol alkanes.Phenylpropyl alcohol alkanes metabolic pathway is important approach very in the 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 multiple secondary metabolites such as flavonoids, lignin, and these products play an important role in growth and development of plant, disease-resistant, degeneration-resistant reaction.(Dong Yanzhen, the progress of plant phenylalnine ammonialyase gene.The biotechnology circular, 2006, supplementary issue, 31-33)
Phenylalnine ammonialyase is the enzyme that plays an important role in the plant defense, by detecting PAL, measure Harpin protein nano complex activity, determine the inducing action of Harpin protein nano complex, and compare with the effect of Harpin albumen to defensive Substance P AL in the tobacco leaf.
1. sample treatment
Get the tobacco plant of 9 strain health, be divided into 3 groups, every group 3 strain, 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) PBS buffer solution.
After sprinkling, gather the tobacco leaf sample, weigh, place the EP pipe ,-20 ℃ of preservations are standby.
2. the active mensuration of phenylalnine ammonialyase (PAL)
Get about 0.2-0.8g blade, cut little, put into the mortar of ice precooling respectively, adding 3-5mL precooling contains 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 following 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 the thermostat water bath, puts into ice bath immediately, adds 0.25ml 5mol/L HCl cessation reaction.Every kind of reactant liquor is got 3ml in cuvette, with the absorbance under the UV-120-02 type ultraviolet specrophotometer survey 290nm wavelength.Each repeats 3 times, averages.
PA L 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 influence to the PAL activity only limited in 14 hours behind the 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 also is 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 the cell wall permeability to descend simultaneously.Because the decline of cell wall permeability makes albumen mass-energy still less have an effect by the receptors bind on cell wall and the cell membrane, obviously reduces than the drug effect of spraying for the first time so spray same concentration rHrpZ albumen for the second time.And after spraying the rHrpZ-PLGA nano complex, though the PAL activity change is not for the first time sprayed rHrpZ albumen vary within wide limits, reach more than 2 weeks the perdurabgility of PAL increased activity effect.Result of the test shows that 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, constantly stimulates the PAL increased activity, reaches the effect that prolongs timeliness.
Embodiment 6:Harpin protein nano complex is to the influence of plant disease-resistant effect
But PR-5dB expression of gene in the 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.This expression of gene amount 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 among the embodiment 5, spray rHrpZ albumen and rHrpZ-PLGA NP sample respectively, 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, and room temperature left standstill 15 minutes;
(2) 12000g is centrifugal 15 minutes;
(3) get the upper strata water;
(4) adding equal-volume isopropyl alcohol room temperature (identical below 20-25 ℃) left standstill 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 the relative amount of PR-5dB, be confidential reference items with actin gene transcription level at plant interior expression.According to actin gene and PR-5dB gene design primer (table 1), primer is given birth to worker company by Shanghai and is synthesized.
Table 1Sequences of gene-specific primers used in RT-PCR analysis
3.cDNA it is synthetic
RNA after the 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
Oligo(dT)
20(10pmol/μL) 1μl
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.In aseptic PCR pipe, add following component, aspirate mixing with liquid-transfering gun.Behind 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, the dyeing of bromination second heavy stone used as an anchor, uviol lamp is observed amplification down.Gel after the electrophoresis end through digital image-forming and analysis, is write down the difference of each band amount.With 1D gray analysis software analysis band gray value, the band gray value is directly proportional with 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
Result's demonstration can improve by rapid stimulation PR-5dB transcriptional level after rHrpZ albumen sprays, and similar with the variation alive of PAL enzyme among the embodiment 5, PR-5dB transcriptional level raising effect is held time shorter, is no more than 48 hours.And rHrpZ-PLGA NP can keep above 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, when use in the field, can avoid or reduce application times, reduce human cost.
Embodiment 7:Harpin protein nano complex infects the attack protection test to the anti-Solanaceae Lei Er Salmonella of plant
Solanaceae Lei Er Salmonella (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 tremendous loss.As typical tobacco disease indigenous bacteria, detect the drug effect of rHrpZ albumen and rHrpZ-PLGA NP with Solanaceae Lei Er Salmonella.
Solanaceae Lei Er Salmonella spends the night with following medium culture.
Regulate pH value to 7.2 with NaOH.
Get centrifugal 5 minutes of the Solanaceae Lei Er Salmonella 12000rpm of 1mL incubated overnight, thalline is resuspended in the 1mL distilled water.Get tobacco 3 strains of growing way equalization, Solanaceae Lei Er Salmonella suspension is injected into tobacco in the stem of Tu0.5cmChu with the dosage of every strain 5 μ L.Simultaneously, three strain tobaccos spray PBS, 20ug/mLrHrpZ protein solution respectively, 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 PBS after 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 effectiveness that tobacco opposing Solanaceae Lei Er Salmonella infects.Under the situation of 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 and preparation method and application that promotes plant growing
＜130〉a kind of protein nano complex and preparation method and application that promotes plant growing
<140>2009102722035
<141>2009-09-23
<160>4
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
＜213〉synthetic
<400>1
agtaagcaac?tgggacgata 20
<210>2
<211>20
<212>DNA
＜213〉synthetic
<400>2
ccactaagga?cgatgtttcc 20
<210>3
<211>20
<212>DNA
＜213〉synthetic
<400>3
actttgatgg?tgctggtaga 20
<210>4
<211>19
<212>DNA
＜213〉synthetic
<400>4
gtaggcatct?ccaatggga 19
Claims (9)
1. a protein nano complex that promotes plant growing is characterized in that, comprises Harpin albumen and carrier in this nano complex.
2. carrier according to claim 1 is characterized in that: this carrier is the ultra micron carrier, and the ultra micron carrier is PLA, lactic acid-ethanol copolymer, Polyalkylcyanoacrylanano, polycaprolactone, Polyalkylcyanoacrylanano or polycaprolactone.
3. the described a kind of method for preparing protein nano complex of claim 1 is characterized in that this method may further comprise the steps:
(1) with PLGAlactide: glycolide=75: 25, molecular weight 40000 is dissolved in the carrene, be made into 50mg/mL solution, 0.5mL 20mg/mL Harpin albumen slowly is added drop-wise in the 5mL50mg/mL PLGA solution, handles forming Water-In-Oil W/O emulsion in 1 minute simultaneously with ultrasonic disruption instrument emulsification 50W;
(2) water-in-oil emulsion is poured in the 50mL 2%PVA aqueous solution, ultrasonic emulsification 100W handles and formed the W/O/W emulsion in 2 minutes;
(3) the W/O/W emulsion is poured in the 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 the distilled water, repeats this process 3 times to clean the outer PVA of nano particle, at last nano particle is resuspended in the distilled water standby.
4. as the preparation method of a kind of protein nano complex as described in the claim 3, it is characterized in that described PLGA is a nanoemulsion.
5. a pharmaceutical composition is characterized in that said composition comprises the described protein nano complex of claim 1 of effective dose.
6. described a kind of protein nano complex of claim 1 or the application of the described pharmaceutical composition of claim 5 in the controlling plant diseases medicine.
7. the application of the described a kind of protein nano complex of claim 1 in the medicine of preparation treatment or prevention bacterial-infection resisting.
8. the application of the described a kind of protein nano complex of claim 1 in the medicine of preparation treatment or prevention anti-fungal infection.
9. the application of the described a kind of protein nano complex of claim 1 in the medicine of preparation treatment or prevention viral infection resisting.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107987134A (en) * | 2018-01-26 | 2018-05-04 | 上海国礼生物科技有限公司 | A kind of 3-protein d hw1 and its encoding gene dhw1 and application |
| CN115843794A (en) * | 2022-12-13 | 2023-03-28 | 贵州大学 | Nano system of plant protein-based drug-encapsulated molecules, preparation method and application |
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| CN116801734A (en) * | 2020-08-11 | 2023-09-22 | 塞拉治疗有限责任公司 | Green closed-loop biowaste refining process for producing intelligent active extracts and delivery system for its use |
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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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107987134A (en) * | 2018-01-26 | 2018-05-04 | 上海国礼生物科技有限公司 | A kind of 3-protein d hw1 and its encoding gene dhw1 and application |
| CN115843794A (en) * | 2022-12-13 | 2023-03-28 | 贵州大学 | Nano system of plant protein-based drug-encapsulated molecules, preparation method and application |
| 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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