CN104561073B - The screening technique of compound is infected using 2-Cys Prx as the anti-Plant nematode of target spot - Google Patents
The screening technique of compound is infected using 2-Cys Prx as the anti-Plant nematode of target spot Download PDFInfo
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Abstract
A kind of screening technique infecting compound using 2 Cys types peroxiredoxins as the anti-Plant nematode of target spot, this method include building the recombinant expression plasmid pET41b DdPrx of the 2 Cys Prx genes containing Ditylenchus destructor；Recombinant expression plasmid is transferred to BL21 (DE3) competent cell, by IPTG Fiber differentiations to stablize expression recombination Dd PRX albumen；Candidate compound and recombination DdPrx albumen are incubated altogether, detect whether candidate compound has in vitro inhibitory activity to recombination DdPrx albumen according to thiocyanate-ferric.This method have many advantages, such as directly, it is easy, quick, can be not only used for manual screening, and can be used for the screening of extensive high-throughput inhibitor.This method can be used to research and develop the anti-Plant nematode of screening and infect drug.
Description
Technical field
The present invention relates to plant nematode field of pesticides, and in particular to 2-Cys type peroxiredoxins
(Peroxiredoxins, Prx) i.e. 2-Cys Prx are that the anti-Plant nematode of target spot infects the screening technique of compound.
Background technology
Plant nematode is to cause one of important pathogen object of plant disease.In the world, plant pathogeny line insect
Generation with harm with the development of agriculture and forestry and the evolution of tillage and cultivation system getting worse, it has also become Agricultural Bio-disasters
Relay the third-largest disaster after insect, plant disease.It is reported that the nematode for seriously endangering China's agriculture, woods, industrial crops etc. reaches
More than 100 kinds.Plant nematode is about every year 125000000000 dollars to the production loss of whole world crops.Prevention nematode at present
Method be mainly to be used in combination what some still ratified to use now by the selection of cultivation technique, shift of crops, resistant variety
Chemical nematicides.
Nematicide is born along with the getting worse of eelworm harm, although these chemical agents are to control of nematode
Positive effect is played, but the pharmacy variety of the existing prevention nematodiasis in China is few, available property is not strong, related nematicide
Research and development, either in theoretical research and technological development, very weak part, nematicidal all or in pesticide field
Active constituent and mechanism of action in terms of research it is less.Because of environmental pressure, the hair of the chemical prevention and control method of plant nematode
Exhibition is by stern challenge.It is imperative to develop novel, less toxic, efficient nematicide.Plant nematode antioxidant system mechanism
Further investigation provides theoretical foundation and new point of penetration for the development and utilization of drug target.
Biology generally generates active oxygen (reactiveoxygen species, ROS) during aerobic metabolism, in order to support
The damage of anti-ROS, biology form glutathione peroxidase (glutathione in long-term evolutionary process
Peroxudase, GPx), catalase (catalase, CAT), superoxide dismutase (superoxide dismutase,
SOD), the various antioxidant systems such as peroxiredoxin (peroxiredoxins, Prxs).It is excessive endogenous to remove in time
Property activity chalcogen and its ambient enviroment include the exogenous ROS that host generates damage, equally there is also each in Plant nematode body
Kind antioxidant system is allowed to maintain a normal level.For example, during the phytopathy original infects host, plant is logical
It crosses and generates peroxide to resist the invasion of pathogen, the pathogens such as nematode then remove its murder by poisoning by secreting antioxidase.
As a member in antioxidase family, peroxiredoxin gene is widely present in prokaryotes and eukaryon life
In object.The Prx functions having now been found that make body from DNA damage, albuminous degeneration and lipid peroxide in addition to keeping ROS balances
Outside the injuries such as change, a variety of biologies such as Apoptosis, proliferation, differentiation, Intracellular signals transmission and gene expression regulation are also participated in
Function.As that studies Prxs deepens continuously, using Prxs as potential drug, vaccine or diagnosis target just by increasingly
More concerns and research.
Three drug, vaccine or diagnosis target aspects are concentrated mainly on to the research of Prxs applications in recent years, wherein with Prx
Research is more in terms of carrying out vaccine development and the relationship of Prx and tumour as antigen.In the research using Prx as antigen, state
Outer is even more the report for having related patents, and e.g., Wuchereria malayi (Brugia malayi) Prx6 has applied for U.S. as vaccine antigen
State's patent (U.S.Patent 6,352,836)；Leishmania donovani (L.donovani) Prx1a is as a kind of vaccine antigen
Candidate has applied for United States Patent (USP) (U.S.Patent 7795406), and carries out prevention Plant nematode medicine by target of Prx
The report of object development is also fewer at present.
Invention content
The purpose of the present invention is in view of the above shortcomings of the prior art, provide a kind of be used for 2-Cys types peroxide also
Protoenzyme is that target spot carries out the recombinant expression plasmid that anti-Plant nematode infects screening compound, which is pET41b-
DdPrx。
Present invention simultaneously provides a kind of compound is infected by the anti-Plant nematode of target spot of 2-Cys types peroxiredoxin
Screening technique, this method comprises the following steps：
A, the recombinant expression plasmid pET41b-DdPrx of the Prx genes of 2-Cys containing Ditylenchus destructor is built；
B, recombinant expression plasmid is transferred to BL21 (DE3) competent cell, by IPTG Fiber differentiations to stablize expression weight
Group Dd-PRX albumen；The recombination Dd-PRX albumen is Ditylenchus destructor 2-Cys Prx and pET-41b fusion proteins.
The IPTG inducing culturing conditions are：18-37 DEG C of temperature, a concentration of 0.2-1.4mmol/L of IPTG.
C, candidate compound and recombination DdPrx albumen are incubated altogether, whether candidate compound is detected according to thiocyanate-ferric
There is higher in vitro inhibitory activity to recombination DdPrx albumen.
The total incubation refers to that will recombinate DdPrx albumen the reaction containing 10 μm of ol/L-40 μm of ol/L candidate compounds is added
In buffer solution, 30min is incubated under 30 DEG C of water bath conditions, wherein the reaction buffer is the 1 × PBS of the DTT containing 10mmol/L
Buffer solution, pH 7.0.
The compound to recombination DdPrx albumen with higher inhibitory activity obtained according to screening technique provided by the invention
Verified for bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3-, which has the activity that anti-Plant nematode infects, and can resist
Plant nematode infects, and can be used to prepare anti-Plant nematode and infect drug.
The present invention also provides application of the above-mentioned screening technique in researching and developing anti-Plant nematode and infecting drug.
The screening method in vitro of the present invention is designed based on thiocyanate-ferric, and principle is that Prx albumen can be catalyzed H2O2
It is converted into H2O and O2, remaining H2O2Then by Fe2+It is oxidized to Fe3+, Fe3+The red complex formed again with thiocyanate ion
In A475There is maximum light absorption value at place to measure micro H2O2Variable quantity.And 2-Cys Prx inhibitor can be substantially reduced recombination
DdPrx proteins carries H2O2Activity.For the screening of 2-Cys Prx inhibitor, have many advantages, such as directly, it is easy, quick, both may be used
For manual screening, and it can be used for the screening of extensive high-throughput inhibitor.And the Vivo Studies on Screening method of verification of the present invention is
Had based on rice root-knot nematode and infects rice root this characteristic design, if candidate compound has the activity of anti-nematode infection,
Then rice root-knot nematode infects the root knot that rice root generates and will significantly reduce compared with the control.The method is not required to the i.e. available meat of dyeing
Eye observation, is convenient for the activity change of the anti-nematode infection of real-time tracking candidate compound.
Description of the drawings
Fig. 1 is that DdPrx reading frames are cloned and electrophoresis result figure is identified in digestion.
In figure, M1：5000 marker, 1：PCR product, 2：PET-41b is through XhoI single endonuclease digestions, and 3：PET41b-DdPrx is passed through
XhoI single endonuclease digestions, 4：PET41b-DdPrx is through NcoI single endonuclease digestions, and 5：PET41b-DdPrx is through XhoI and NcoI double digestions, M2：
15000 marker。
Fig. 2 is the influence for inducing IPTG concentration and temperature to recombinating DdPrx expressing quantities.
In figure：M:Instant protein molecular weight standard (low)；
A is the recombination DdPrx expressing quantities of different IPTG concentration.Wherein, 1：IPTG a concentration of 1.4mmol/L, 2：
IPTG a concentration of 1.0mmol/L, 3：IPTG a concentration of 0.6mmol/L, 4：IPTG a concentration of 0.2mmol/L, 5：IPTG is a concentration of
0mmol/L；
BL21 (DE3) competent cell expression recombination DdPrx albumen results when B is 18 DEG C.Wherein, 1：There is IPTG to induce
The supernatant of carrying out ultrasonic bacteria breaking, 2：Have IPTG induce carrying out ultrasonic bacteria breaking full bacterium solution, 3：The full bacterium solution of no IPTG inductions；
BL21 (DE3) competent cell expression recombination DdPrx albumen results when C is 25 DEG C.Wherein, 1：There is IPTG to induce
The supernatant of carrying out ultrasonic bacteria breaking, 2：Have IPTG induce carrying out ultrasonic bacteria breaking full bacterium solution, 3：The full bacterium solution of no IPTG inductions；
BL21 (DE3) competent cell expression recombination DdPrx albumen results when D is 37 DEG C.Wherein, 1：No IPTG inductions
Full bacterium solution, 2：Have IPTG induce full bacterium solution, 3：There is the supernatant of the carrying out ultrasonic bacteria breaking of IPTG inductions.
Fig. 3 is to give birth to the small beaker of rice seedling to be put into culture figure in the transparent plastic case equipped with appropriate moistening fine sand.
Fig. 4 is that two bromomethyl quinolines of 1,4- dioxos -2,3- of various concentration are compareed with medicament, acetone control root knot is grown
Tendency chart.
In figure, medicament A is bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3-, and comparison medicament B is bis- (bromomethyl) quinolines of 2,3-
Quinoline.
Specific implementation mode
The clone of 1 Ditylenchus destructor 2-Cys Prx genes of embodiment
Specific primer design, sense primer are carried out according to Ditylenchus destructor cDNA full length sequences in GeneBank
N-PRXF：5’-CCATGGTACCCAAGTTTGAGACCATC-3 ' (SEQ ID No.1), underscore are NcoI restriction enzyme sites；Under
Swim primer X-PRXR：3’-ATAAAGCCGTTCGTCTTCGAGCTC-5 ' (SEQ ID No.2), underscore are XhoI digestions position
Point.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Using the Ditylenchus destructor cDNA prepared by reverse transcription method as template, PCR expansions are carried out with above-mentioned primer
Increase, reaction system be 5 × GXL PCR Buffer, 10 μ l, 4 μ l of dNTP Mixture, 1 N-PRXF μ l, 1 X-PRXR μ l,
0.5 μ l of Prime STAR GXL, 1 cDNA μ l and H2O 32.5μl.Reaction condition is：94℃5min；94 DEG C of 30s, 53 DEG C
30s, 72 DEG C of 1min, 35 cycles；72℃10min.The structure of 2 recombinant expression plasmid of embodiment
The PCR product obtained in embodiment 1 is connected into pEASY-Blunt cloning vectors and is transferred to Trans1-T1, takes 200 μ
On the LB tablets of the kanamycins containing 50ug/ml, 37 DEG C are incubated overnight l bacterium solutions.Next day chooses single bacterium colony and carries out bacterium solution culture, through bacterium
The positive colony of liquid PCR verifications send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced, sequencing result DNAMAN
(7.0.2.176) carries out sequence alignment with known Ditylenchus destructor Prx cDNA, as a result unanimously.
Prx-pEASY-Blunt recombinant clones plasmid and pET-41b expression plasmids is double through NcoI and XhoI (NEB) respectively
Digestion, glue recycle PRX genes and pET-41b expression plasmids, connect overnight at 4 DEG C by T4 ligases (the full formula gold in Beijing) step
It connects.2 μ l connections reaction products being added to BL21 (DE3) competent cell after 100 μ l thaw, (full formula gold biotechnology is limited
Products) in, ice bath 30min；42 DEG C of water-bath 60-90s；Ice bath 2-3min immediately；Often pipe is added without Ampicillin
LB liquid medium 890ml makes bacterium activity recovery with 150-200rmp/min shake culture 1h at 37 DEG C；Take 500 μ l bacterium solutions
It is uniformly coated on LB tablets.Positive colony is selected, and carries out digestion identification (Fig. 1).The results show that recombination Prx-pET-41b
(DdPrx) Prokaryotic expression vector construction success can be used for recombinating the expression of DdPrx.
Embodiment 3 recombinates DdPrx protein expressions
To embodiment 2 build expression vector in different temperatures (18-37 DEG C) and different IPTG concentration (1.4-0.2mmol/
L the optimization that expression condition is carried out under the conditions of), sees whether recombination DdPrx albumen expresses.As shown in Figure 2, with low molecular weight protein
Marker compares, it can be seen that recombination DdPrx albumen has an apparent thick band at 55kDa, consistent with expection.Illustrate,
18-37 DEG C, when a concentration of 1.4-0.2mmol/L of IPTG, it can induce the recombination protein stabilized expression of DdPrx.
BL21 (DE3) bacterium solution for importing recombination DdPrx plasmids is subjected to Fiber differentiation, is as follows：It goes bail for and deposits
The 2 μ l of BL21 (DE3) bacterium solution of the DdPrx plasmids containing recombination are added in LB liquid mediums of the 5ml containing 50 μ g/ml kanamycins 37 DEG C
It is incubated overnight.Next day takes the bacterium solution 1ml being incubated overnight to be added 37 in LB liquid mediums of the 100ml containing 50 μ g/ml kanamycins
DEG C culture is to OD600About 0.6, add the IPTG of final concentration of 0.2mmol/L, 25 DEG C, 200rpm cultures 18h.4 DEG C,
10000rmp collects thalline, and 20ml purifying equilibration buffer (300mM NaCl, 50mM NaH is added2PO4, 10mM
Imidazole, 10mM Tris base, pH8.0) wash bacterium 3 times after, add 20ml purifying equilibration buffer ultrasonic waves and break bacterium
(3s+3s 10min, power 10%, 3 cycles), 4 DEG C, 13000rpm collection supernatants.Through SDS-PAGE electrophoresis, purpose item is cut
Band send Sangon Biotech (Shanghai) Co., Ltd. to carry out Mass Spectrometer Method, and result is 2 peptide fragments
AFIGKPAPDFTADAVVDGDFK, QITINDLPVGR and Ditylenchus destructor Prx protein matches.Show to recombinate DdPrx
Protein expression success.It most preferably washs under imidazole concentration and 500mmol/L most preferably elute imidazole concentration, uses in 100mmol/L
ProteinIsoTM Ni-NTA Resin (the full formula gold in Beijing) purifying recombination DdPrx albumen, is usedUltra-15 from
Heart filter (Merck KGaA) replacement buffer solution is 1 × PBS.
The foundation of embodiment 42-Cys Prx inhibitor screening method in vitro
DdPrx recombinant protein activity is carried out after the DTT concentration, pH value and the temperature optimization that are related to screening method in vitro to survey
It is fixed.It measures and uses thiocyanate-ferric.It is slow that 85 μ l reactions are added in the recombination DdPrx albumen (about 3.5 μ g) that 5 μ l embodiments 3 are obtained
30 DEG C of incubation 3min in fliud flushing (10mmol/L DTT, 1 × PBS, pH=7.0), be then respectively adding 10 μ l it is a concentration of 0.01,
0.02, the H of 0.03,0.04 and 0.05mmol/L, 5 concentration2O2Reaction is set to start, the H of each concentration2O2Reaction solution is
4,6,8,10, the trichloroacetic acid that 26% is added when 12min terminates reaction.40 μ l iron ammonium sulfates (Fe (NH are added into system4)2
(SO4)2) and 20 μ l potassium rhodanides (KSCN), and red complex is formed after remaining hydroperoxidation, measure A475 (Tecan
Infinite M200 PRO microplate reader).To acquire recombination DdPrx albumen to H2O2Catalytic efficiency.
Embodiment 52-Cys Prx inhibitor screening method in vitro
In vitro inhibitory activity experiment is carried out to recombination DdPrx with bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3-, it is bis- with 2,3-
(bromomethyl) quinoline is control, specific as follows：
The recombination DdPrx albumen (about 3.5 μ g) that 5 μ l embodiments 3 obtain is separately added into 85 μ l and contains 10 μm of ol/L-40 μ
In bis- (bromomethyl) quinoline of 2,3- of mol/L and the reaction buffer of bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3-, in 30 DEG C of water
It is incubated 30min under the conditions of bath, adds the H of 10 a concentration of 0.02mmol/L of μ l2O2Start to react, other steps are the same as embodiment 4.
As a result, it has been found that suppressions of the bis- 10 μm of ol/L-40 μm of ol/L of (bromomethyl) quinoline of 2,3- of non-Prx inhibitor to recombination DdPrx albumen
Rate processed is 9%-20%, and bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3-, 10 μm of ol/L-40 μm of ol/L are to recombinating DdPrx albumen
Inhibiting rate reach 20%-40%, compared with comparison medicament 2, bis- (bromomethyl) the quinoline inhibitions of 3- are apparent, show Isosorbide-5-Nitrae-dioxy
Generation two bromomethyl quinolines of -2,3- have higher in vitro inhibitory activity to recombination DdPrx.
6 anti-Plant nematode of embodiment infects the Vivo Studies on Screening verification of compound
By the two kinds of changes of bis- (bromomethyl) quinoline of the 2,3- used in embodiment 4 and two bromomethyl quinolines of 1,4- dioxos -2,3-
It closes object and carries out the Vivo Studies on Screening that anti-Plant nematode infects.The live body that rice root-knot nematode is infected as anti-Plant nematode is selected in experiment
Screening test material.Rice root-knot nematode can observe apparent root knot in 3 days after infecting rice root, transparentIt can directly be visually observed in F-127 matrix, be convenient for the activity of the anti-nematode infection of real-time tracking candidate compound
Variation.
The 25% of the precooling of 18ml nutrient solutions Han 1 × south China is poured into 25ml beakersF-127 (m/v, it is beautiful
SIGMA companies of state) matrix, then it is separately added into 300 rice root-knot nematodes and final concentration of 12.5,25,50,100,200 μm of ol/
Bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3- of L, moisturizing to 20ml often transplant the consistent rice seedlings of growing way (after germination in beaker
5 days rice seedlings) 3 plants.There is the beaker of rice seedlings to be placed in transparent plastic case kind, place mat about 5cm high, water content under plastic box
For 30%, the thin river sand of sterilizing, seal, put it into 25 ± 1 DEG C of temperature, illumination 16h/ dark 8h illumination box in
It cultivates (Fig. 3).It is respectively the carbofuran and 2 of 50 μm of ol/L with concentration, bis- (bromomethyl) quinoline of 3- are compareed as medicament, 50 μ
Mol/L acetone solns are as solvent control.4 repetitions of every group of medicament.Daily by transparentF-127 matrix
Observation counts the root knot in each beaker, observes 12 days altogether.Variance analysis is carried out with DPS v7.05.Observation result is shown in Fig. 4.
By the observation interpretation of result discovery to 6 days after medicine and 12 days, bis- bromomethyl quinoline of Isosorbide-5-Nitrae-dioxo -2,3- is to water
The inhibition of rice root-knot nematode shows to improve the increased trend of inhibition with concentration, as a result referring to the following table 1, after Chinese medicine
6 days, 50 μm of ol/L Isosorbide-5-Nitraes-bis- bromomethyl quinoline of dioxo -2,3- to the inhibition of rice root-knot nematode up to 64.8%, after medicine
29.0% then is dropped to the inhibiting rate of rice root-knot nematode within 12 days, comparison medicament 2, bis- (bromomethyl) quinoline of 3- are to rice root knot
The inhibition of nematode is only 33.0%, then falls to 4.7% within 12 days after medicine, hence it is evident that it is less than Isodose Isosorbide-5-Nitrae-dioxo -2,
The effect of bis- bromomethyl quinolines of 3-.Show that the screening technique that compound is infected using 2-Cys Prx as the anti-Plant nematode of target spot exists
Preventing in the screening of plant nematode drug has repeatability well and credibility, and with the method, we obtain one kind
Two bromomethyl quinolines of compound 1,4- dioxos -2,3- that anti-Plant nematode infects.
Inhibition of two bromomethyl quinolines of table 11,4- dioxos -2,3- to rice root-knot nematode
Claims (2)
1. recombinant expression plasmid pET41b-DdPrx is invaded carrying out anti-Plant nematode as target spot using 2-Cys types peroxiredoxin
The application in screening compound is contaminated, anti-Plant nematode infects screening compound and includes the following steps：
A, the recombinant expression plasmid pET41b-DdPrx of the Prx genes of 2-Cys containing Ditylenchus destructor is built；
B, recombinant expression plasmid is transferred to BL21 (DE3) competent cell, by IPTG Fiber differentiations to stablize expression recombination
DdPrx albumen；
C, by candidate compound and recombination DdPrx albumen be incubated altogether, according to thiocyanate-ferric detect candidate compound whether counterweight
Group DdPrx albumen has in vitro inhibitory activity；
IPTG inducing culturing conditions are in the step b：18-37 DEG C of temperature, a concentration of 0.2-1.4mmol/L of IPTG；
Total incubation in the step c refers to that will recombinate DdPrx albumen and be added to contain 10 μm of ol/L-40 μm of ol/L candidate compounds
In reaction buffer, 30min is incubated under 30 DEG C of water bath conditions, wherein the reaction buffer be the DTT containing 10mmol/L 1 ×
PBS buffer solution, pH 7.0.
2. application as described in claim 1, it is characterised in that：There is described pair of recombination DdPrx albumen higher in vitro inhibition to live
Property compound be two bromomethyl quinolines of 1,4- dioxos -2,3-.
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Citations (2)
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3560616A (en) * | 1968-06-17 | 1971-02-02 | Du Pont | Process of protecting vegetation from fungi using quinoxaline foliar fungicides |
| CN102046796A (en) * | 2008-02-10 | 2011-05-04 | 孟山都技术公司 | Methods and compositions for plant pest control |
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| Identification of conoidin A as a covalent inhibitor of peroxiredoxin II;Jeralyn D. Haraldsen等;《Organic & Biomolecular Chemistry》;20090807;第7卷(第15期);摘要和Introduction部分Fig.1 * |
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