CN102128828B - Method for detecting N-acylated homoserine lactone - Google Patents
Method for detecting N-acylated homoserine lactone Download PDFInfo
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- CN102128828B CN102128828B CN 201010578919 CN201010578919A CN102128828B CN 102128828 B CN102128828 B CN 102128828B CN 201010578919 CN201010578919 CN 201010578919 CN 201010578919 A CN201010578919 A CN 201010578919A CN 102128828 B CN102128828 B CN 102128828B
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Abstract
The invention discloses a method for detecting N-acylated homoserine lactone. The method for detecting whether N-acylated homoserine lactone exists in an auxiliary detection sample comprises the following steps of: (1) preparing experimental liquid, namely mixing a sample to be tested, magnetic nanoparticles coupled with an N-acylated homoserine lactone receptor and a buffer solution and incubating the mixture to obtain the experimental liquid, wherein the pH value of the buffer solution is 7.4; and (2) detecting whether the experimental liquid becomes blue after meeting agrobacteriumtumefaciens KYC55, and if so, determining that the sample to be detected contains the N-acylated homoserine lactone as a candidate. The novel application of the method disclosed by the invention is application of the N-acylated homoserine lactone receptor in detecting the N-acylated homoserine lactone in the sample. The method for detecting the N-acylated homoserine lactone by using N-acylated homoserine lactone receptor molecules has high sensitivity, good specificity and wide application prospect in the fields of detecting N-acylated homoserine lactone single molecules and pathogenic bacteria toxicity.
Description
Technical field
The present invention relates to a kind of method of the N-of detection acylated homoserine lactone.
Background technology
Aeromonas hydrophila has widely pathogenicity at occurring in nature, is Amphixenosis's important pathogeny bacterium.Naturally be present in the water sample environment, aquatic products industry in serious threat.This bacterium pathogenic relevant with the multiple virulence factor of its generation, virulence factor mainly contains outer enterotoxin, endotoxin, hemolysin, cytotoxin, proteinase etc.
Quorum Sensing (QS) system refers to that bacterium can detect in the surrounding environment number change of self or other bacteriums according to the concentration of signal specific molecule, when signaling molecule reaches certain concentration threshold, can start the phenomenon of the variation during the expression of related gene conforms in the thalline.Many gramnegative bacteriums produce acyl homoserine lactones compounds (acyl-homoserine lactones; be called for short AHL) signaling molecule; AHL is synthetic by class LuxI albumen; and the class LuxR receptor protein in born of the same parents is combined; form acceptor-auto-inducer complex; be combined with the genes of interest promoter when this complex, just can activate transcribing of this gene.This class auto-inducer can freely pass through cell membrane when high-cell density, accumulate in the extracellular.The LuxR albuminoid contains two domains, and its aminoterminal contains a specific region to be responsible in conjunction with the AHLs molecule and to regulate the oligomerization of albumen, thereby and the shuttle cardinal extremity contains the helix turn helix structure of a high conservative can be combined with DNA transcribing of controlling gene.The poisonous effect of most aquatic products bacterial pathogens expresses all to be regulated and control by AHLs concentration, and the performance positive feedback effect.The aquatic products bacterial pathogen is of a great variety; by sick fish sign; pathogen changes; water-quality guideline etc. come the outburst of early warning bacterial disease to lack foresight and accuracy; but in the total quorum sensing mechanism of this class pathogen; all (the G-bacterium: active concentration N-acylated homoserine lactone AHLs) is regulated and control " the toxicity switch " of pathogen by the signal specific molecule; various pathogenic bacteria may there are differences at poisonous effect; but because AHLs exists chemical constitution general character and receptor-specific, by the AHLs active concentration being implemented the then accurately outburst of early warning bacteriosis of monitoring.
Summary of the invention
An object of the present invention is to provide a kind of and the nanometer magnetic bead coupling of N-acylated homoserine lactone acceptor.
The nanometer magnetic bead of provided by the present invention and the coupling of N-acylated homoserine lactone acceptor, prepare according to the method that comprises the steps: suspending with coupling buffer activates magnetic bead, add again N-acylated homoserine lactone acceptor, at 25 ℃ of reaction 3 h, obtain the magnetic bead of N-acylated homoserine lactone acceptor coupling; Described coupling buffer is prepared as follows and obtained: be that 7.4,1 x PBS damping fluid and Tween-20 mix with the pH value, obtain coupling buffer, the pH value is that the volume ratio of 7.4,1 x PBS damping fluid and Tween-20 is 100ml: 0.05ml; The described pH value of every 100ml is that 7.4,1 x PBS damping fluid is prepared as follows and obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, and obtaining described pH value is 7.4,1 x PBS damping fluid.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor; the rate of charge of described N-acylated homoserine lactone acceptor and described activation magnetic bead is 5 μ g～10 μ gN-acylated homoserine lactone acceptors: the 2mg magnetic bead is specially 10 μ gN-acylated homoserine lactone acceptors: the 2mg magnetic bead.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, described activation nanometer magnetic bead prepares according to the method that comprises the steps: EDC solution, NHS solution and nanometer magnetic bead are mixed, hatch the nanometer magnetic bead that obtains activating.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, the rate of charge of described EDC, NHS and nanometer magnetic bead is 1mg: 1mg: 2mg.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, described temperature of hatching is 37 ℃, and the time is 30min.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, the solute of described EDC solution is the EDC solid, and solvent is that concentration is that 10mM, pH value are 6.0 MES damping fluid, and the proportioning of solute and solvent is 5mg: 1ml.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, the solute of described NHS solution is the NHS solid, and solvent is that concentration is that 10mM, pH value are 6.0 MES damping fluid, and the proportioning of solute and solvent is 5mg: 1ml.
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, the amino acid sequence of described N-acylated homoserine lactone acceptor is shown in SEQ ID NO:1;
Among the nanometer magnetic bead preparation method of above-mentioned and the coupling of N-acylated homoserine lactone acceptor, described N-acylated homoserine lactone is C4-HSL.
Another object of the present invention provides the method that whether contains the N-acylated homoserine lactone in a kind of auxiliary detection sample.
Whether contain the method for N-acylated homoserine lactone in the auxiliary detection sample provided by the present invention, comprise the steps:
1) preparation experiment liquid as follows: testing sample, above-mentioned arbitrary described nanometer magnetic bead and damping fluid with the coupling of N-acylated homoserine lactone acceptor are mixed, potpourri is hatched, obtain testing liquid; The pH value of described damping fluid is pH 7.4;
2) detect whether become blue after described experiment liquid runs into bacterial strain (Agrobacterium tumefaciens) KYC55, if become blue, determine that then the candidate is contained the N-acylated homoserine lactone in the described testing sample.
Whether contain in the method for N-acylated homoserine lactone described step 1 in the above-mentioned auxiliary detection sample) in, described damping fluid is the PBS damping fluid, and described temperature of hatching is 30 ℃, and the described time of hatching is 30min;
Whether contain in the method for N-acylated homoserine lactone in the above-mentioned auxiliary detection sample; described step 2) in; the described experiment liquid of described detection runs into the method that whether becomes blue behind bacterial strain (Agrobacterium tumefaciens) KYC55 and comprises the steps: to prepare glucose agar medium; on the described nutrient culture media on straight line and the equidistant place bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution; the Oxford cup that experiment liquid is housed is positioned on the described nutrient culture media; Oxford cup and each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution is also in alignment; the central point of Oxford cup with from nearest some bacterial strains of Oxford cup (Agrobacterium tumefaciens) KYC55 nutrient solution distance is arranged; behind 30 ℃ of maintenance 48h, observe each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution and whether show blueness.
Whether contain in the method for N-acylated homoserine lactone described step 1 in the above-mentioned auxiliary detection sample) in, described testing sample, with the nanometer magnetic bead of N-acylated homoserine lactone acceptor coupling and the volume sum of damping fluid be 100uL; Or described testing sample, with the nanometer magnetic bead of N-acylated homoserine lactone acceptor coupling and the volume ratio of damping fluid be 5 μ l: 20 μ l: 75 μ l;
Whether contain in the method for N-acylated homoserine lactone in the above-mentioned auxiliary detection sample; described step 2) in; the method of bacterial strain on the described point (Agrobacterium tumefaciens) KYC55 nutrient solution is for being to dip bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution with toothpick, and then point is to described nutrient culture media.
Whether contain in the method for N-acylated homoserine lactone described step 1 in the above-mentioned auxiliary detection sample) in, the described PBS damping fluid of every 100ml is prepared as follows and is obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, obtains described PBS damping fluid.
Whether contain in the method for N-acylated homoserine lactone in the above-mentioned auxiliary detection sample; described step 2) in, the central point of described Oxford cup be 8mm from the distance of nearest some bacterial strains of Oxford cup (Agrobacterium tumefaciens) KYC55 nutrient solution.
Whether contain in the method for N-acylated homoserine lactone in the above-mentioned auxiliary detection sample, described step 2) in, the nutrient solution of described bacterial strain (Agrobacterium tumefaciens) KYC55 obtains as follows: described bacterial strain (Agrobacterium tumefaciens) KYC55 is inoculated in the nutrient culture media, cultivate 48h for 30 ℃, obtain the nutrient solution of described bacterial strain (Agrobacterium tumefaciens) KYC55; Described nutrient culture media consists of: LB nutrient culture media+2 μ g/mL tetracyclines+100 μ g/mL spectinomycins+100 μ g/mL gentamicins;
Whether contain in the method for N-acylated homoserine lactone described step 2 in the above-mentioned auxiliary detection sample) in, described each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution is at a distance of 4mm;
Whether contain in the method for N-acylated homoserine lactone described step 2 in the above-mentioned auxiliary detection sample) in, the amount of described experiment liquid is 100ul;
Whether contain in the method for N-acylated homoserine lactone in the above-mentioned auxiliary detection sample, the sample of described detection is water.
The application of N-acylated homoserine lactone acceptor in following (1) or (2) or (3) or (4) or (5) or (6) also belongs to protection scope of the present invention:
(1) whether contains the N-acylated homoserine lactone in the test sample;
(2) prepare the kit that whether contains the N-acylated homoserine lactone in the test sample;
(3) whether test sample contains dna fragmentation shown in the SEQ ID NO:7;
(4) prepare the kit that whether contains dna fragmentation shown in the SEQ ID NO:7 in the test sample;
(5) amount of N-acylated homoserine lactone in the test sample;
(6) kit of the amount of N-acylated homoserine lactone in the preparation test sample;
In the above-mentioned application, the sample of described detection is water;
In the above-mentioned application, the amino acid sequence of described N-acylated homoserine lactone acceptor is shown in SEQ ID NO:1; Described N-acylated homoserine lactone is C4-HSL.
N-acylated homoserine lactone acceptor molecule of the present invention is highly sensitive for detection of the method for N-acylated homoserine lactone, specificity good, has broad application prospects at detection N-acylated homoserine lactone molecule and bacterial detection pathogen toxicity field.
The characteristics that this paper mainly utilizes the AhyR receptor protein to be combined with signaling molecule, enrichment detects signaling molecule in the water body, improves the concentrations of signaling molecule, thereby quickly aquatic products Outbreak disease in the water body is done early warning.
Description of drawings
Signaling molecule receptor protein (AhyR) purifying of Figure 1A h ATCC 7966
The signaling molecule receptor protein (AhyR) of Fig. 2 Ah ATCC 7966 under 1.5% Ago-Gel and the mutual gel retardation assasy of doing of its identification DNA (inter) sequence
The signaling molecule receptor protein (AhyR) of Fig. 3 Ah ATCC 7966 under 12% non-denaturing polyacrylamide gel and the mutual gel retardation assasy of doing of its identification DNA (inter) sequence
Fig. 4 AhyR is to the diffusion blockade test of unlike signal Molecular Recognization
Fig. 5 magnetic bead-AhyR is to the diffusion blockade test of C4-HSL recognition reaction
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
1, bacterial strain and carrier: Aeromonas hydrophila (Aeromonas hydrophilia ATCC 7966, Ah ATCC 7966) is available from Chinese agriculture microorganism fungus kind preservation administrative center (ACCC), and this bacterium ACCC deposit number is 10482.
Host e. coli (Escherichia coli) BL21 (DE3), carrier pET28a are all available from Invitrogen company, and pGEM-T Easy carrier is available from Promega company.
Detect bacterial strain (Agrobacterium tumefaciens) KYC55 at document (Cho, K., C.Fuqua, and S.C.Winans.1997.Transcriptional regulation and locations of Agrobacterium tumefaciens genesrequired for complete catabolism of octopine.J.Bacteriol.179:1-8.) disclosed in, the public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.
2, kit, toolenzyme and biochemical reagents: bacterial genomes DNA extracts kit, agarose gel reclaims kit, and the DNAC purification kit is a day root biotech firm, and restriction enzyme is available from TaKaRa company, ligase is available from Invitrogen company, and other all is domestic reagent.N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-Octanoyl-L-homoserine lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL), N-dodecanoyl-L-homoserinelactone (C12-HSL), N-(3-Oxohexanoyl)-D-homoserine lactone (3-oxo-C6-HSL), N-(3-Oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), N-(3-Oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL), N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), N-(3-Oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone (3-hydroxy-C14-HSL) is all available from Sigma (St.Louis, Mo, USA).
3, nutrient culture media:.
Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
The preparation of embodiment 1, ahyr receptor protein
One, Aeromonas hydrophila ATCC 7966 total DNA extract, and the gene cloning of ATCC 7966 receptor protein AhyR
Aeromonas hydrophila ATCC 7966 total DNA extract with reference to DNA of bacteria and extract kit instructions (day root biochemical technology company limited, China, Beijing).Primer AhyR-EF (5 '-ccggaattca tgaccgttaa aaaactttac-3 ') (SEQ ID:3) and AhyR-ER (5 '-attgcggccg cttataaaaa ttcaggaaat g-3 ') (SEQ ID:4) have been synthesized according to the sequences Design of the gene of the ATCC 7966 receptor protein ahyr that deliver, introduce respectively restriction endonuclease EcoR I and Not I restriction enzyme site (underscore part) at the end of primer, carry out pcr amplification take Aeromonas hydrophila ATCC 7966 total DNA as template.The PCR response parameter is: be cooled to 4 ℃ behind 94 ℃ of sex change 5min; Then 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 rear 72 ℃ of insulation 10min of circulation.The PCR fragment that obtains is connected with pGEM-T Easy, and sequence verification, result obtain the recombinant vector of insetion sequence gene shown in SEQ ID NO:2, and is correct, is denoted as pGEM-T Easy/ahyR.The amino acid sequence of the ahyr receptor protein of gene code is shown in SEQ ID NO:1 shown in the SEQ ID NO:2.
Two, preparation contains the recombinant protein of the gene of ATCC 7966 receptor protein ahyr
The positive recombinant vector pGEM-T Easy/ahyR that step 1 is obtained carries out enzyme with restriction enzyme EcoR I and Not I and cuts, be connected with the plasmid pET28a that cuts through same enzyme, the recombinant vector thermal shock transforms e. coli bl21 (DH3) competent cell, resistance screening, the picking monoclonal, enlarge and cultivate, extract plasmid, order-checking is identified, the gene order that the result inserts between the EcoR of pET28a I and Not I restriction enzyme site is shown in SEQ ID NO:2, the recombinant expression carrier structure that shows structure is correct, is denoted as pET28a-ahyR.
Get BL21 (DE3) inoculation (kanamycins of LB+100 μ g/mL) in 50mL LB liquid medium that contains pET28a-ahyR, 37 ℃, shaken cultivation 12h activated spawn.Get the 4ml nutrient solution and add in the 200mL LB nutrient solution (LB+100 μ g/mL kanamycins) (2% inoculum concentration), 37 ℃ of shaken cultivation are 2-3h (OD approximately
600Reach 0.6-0.8) the rear derivant IPTG that adds final concentration 1mmol/L, 12h are cultivated in 20 ℃ of 180rpm concussions.Nutrient solution 12, the centrifugal 5min of 000rpm collects respectively cleer and peaceful precipitation on the nutrient culture media, and cell precipitation is resuspended with pH 7.41xPBS damping fluid, after the ultrasonication 12, the centrifugal 10min of 000rpm, the collection supernatant is cell pyrolysis liquid.Cell pyrolysis liquid carries out purifying through Ni-NTA post (Qiagen, German), obtains purifying protein.Albumen is carried out the SDS-PAGE electrophoresis, and the result as shown in Figure 1.Obtain electrophoretically pure single band (the 7th swimming lane), molecular weight is about 30kDa and predicted molecular weight is close.
With the Escherichia coli that change empty carrier pET28a in contrast, the result does not detect the albumen of molecules of interest amount size.
The application of embodiment 2, receptor protein
One, with the target DNA binding function
1, the gene of Ah ATCC 7966 receptor protein AhyR identification DNA (inter) sequence obtains
5) and AhyR-BR (5 '-CAGTTGGTCTTGTTTCATATGC-3 ') (SEQ ID:6) primer AhyR-BE (5 '-CTCGTTTTCTACCTCCCATC-3 ') (SEQ ID:, carry out pcr amplification take Aeromonas hydrophila ATCC 7966 total DNA as template has been synthesized in design according to the recognition sequence of the ATCC 7966 receptor protein AhyR that deliver.The PCR response parameter is: be cooled to 4 ℃ behind 94 ℃ of sex change 5min; Then 94 ℃ of sex change 30sec, 50 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 rear 72 ℃ of insulation 10min of circulation.Purification storage.The nucleotides sequence of the dna fragmentation that obtains is classified as shown in the SEQ ID NO:7.
2, the signaling molecule receptor protein (AhyR) of AhATCC 7966 and the mutual gel retardation assasy of doing of its identification DNA (inter) sequence
Receptor protein (AhyR) and its identification DNA (inter) are by 1: 1 (amount ratio) ratio at room temperature jointly behind the combination 30min, take target DNA (inter) and 1 at room temperature acting in conjunction of x PBS as CK, check with 1.5% agarose gel electrophoresis, electrophoretogram (Fig. 2,1 expression CK, 2 expression bonds) the band mobility of receptor protein (AhyR) and its identification DNA (inter) combination obviously lags behind the CK band in.
In kind carry out gel retardation assasy at 12% native polyacrylamide gel electrophoresis, obvious hysteresis phenomenon also appears in result's (Fig. 3,1 expression CK, 2 expression bonds) bond.The result shows that receptor protein can identify this segment DNA sequence, thereby causes it to be blocked in electrophoresis process.
3 repetitions are established in experiment, and the result is all consistent.
Two, the signaling molecule receptor protein (AhyR) of Ah ATCC 7966 is tested with the signaling molecule interaction of its identification
Detection method is identical with the described method of step 2 among the embodiment 3, and different is the composition difference of experiment liquid.Experiment liquid is prepared as follows in this experiment:
With signaling molecule C4-HSL solution 5 μ l (5 μ M), 1 x PBS damping fluid, the 25 μ l of ahyr receptor protein solution 20 μ l (5 μ g/ml) and pH7.4 mix, 30 ℃ of common water-bath 30min, (purpose that adds Ni-NTA post matter is to adsorb fixedly AhyR albumen to add Ni-NTA post matter 50 μ l again, make it can not free diffusing), obtain testing liquid.
CK is that the 1x PBS damping fluid 95 μ l of signaling molecule C4-HSL solution 5 μ l (5 μ M), pH 7.4 add in the cup of Oxford, puts 48h in 30 ℃ of incubators, obtains contrasting liquid.
Identical described in the step 2 among the preparation method of signaling molecule C4-HSL solution and the embodiment 3.
The Bluepoint number of ck group Duo 2 points (C4 organizes among Fig. 4) than experimental group as a result.Illustrate that receptor protein and C4-HSL can adsorb mutually.
Measure receptor protein and all the other signaling molecule effect (C6-HSL, C7-HSL, C8-HSL with same procedure, C10-HSL, C12-HSL, oxo-C6-HSL, oxo-C8-HSL, oxo-C10-HSL, oxo-C12-HSL, oxo-C14-HSL, 3-hydroxy-C8-HSL, 3-hydroxy-C14-HSL), result such as Fig. 4, the Bluepoint number of control group and experimental group does not have difference, illustrates that ahyr receptor protein and all the other signaling molecules do not have suction-operated.
3 repetitions are established in experiment, and the result is all consistent.
Signaling molecule receptor protein (AhyR) the nanometer magnetic bead finishing of embodiment 3, Ah ATCC 7966
Nanometer magnetic bead is available from Aorun Weina New Material Science and Technology Co., Ltd., Shanghai (Shanghai, China), AllMag
TMPM3-008.
The preparation of the nanometer magnetic bead of, modifying:
1, carbodiimide N-hydroxy-succinamide method (EDC/NHS method) activation magnetic bead
(1) get 2mg PM3-008 magnetic bead in the 1.5ml centrifuge tube, with 500 μ l MEST damping fluid washed twice, magnetic removes supernatant after separating.The MEST damping fluid is prepared as follows: be that the MES damping fluid (2-(N-morpholine) ethyl sulfonic acid) of the pH 6.0 of 10mM and 0.05ml Tween 20 mix with 100ml concentration, obtain the MEST damping fluid.
(2) EDC solution preparation: the concentration with 4 ℃ of storages is the MES damping fluid dissolving EDC solid (1ml: 5mg), obtain EDC solution (now with the current) of the pH 6.0 of 10mM;
The NHS solution preparation: the concentration with 4 ℃ of storages is the MES damping fluid dissolving NHS solid (1ml: 5mg), obtain NHS solution (now with the current) of the pH 6.0 of 10mM;
(3) the EDC solution of 200 μ l and 200 μ l NHS solution are joined (rate of charge of EDC, NHS and nanometer magnetic bead is 1mg: 1mg: 2mg in the centrifuge tube that the PM3-008 magnetic bead is housed; ), spiral mixing magnetic bead fully suspends, 37 ℃ of insulation 30min, during keep the suspended state of magnetic bead, the magnetic bead that obtains activating.
2, the AhyR albumen of magnetic bead coupling preparation
(1) relaunder activated magnetic bead 3 times with the coupling buffer of 500 μ l after, again with the coupling buffer washing magnetic bead of 500 μ l 2 times.
Described coupling buffer is prepared as follows and obtained: be that 7.4,1 x PBS damping fluid and Tween-20 mix with the pH value, obtain coupling buffer, the pH value is that the volume ratio of 7.4,1 x PBS damping fluid and Tween-20 is 100ml: 0.05ml; The described pH value of every 100ml is that 7.4,1 x PBS damping fluid is prepared as follows and obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, and obtaining described pH value is 7.4,1 x PBS damping fluid.
(2) add magnetic bead after the resuspended washing of 400 μ l coupling buffers, the AhyR albumen that adds again the purifying of 100 μ l, 100 μ g/ml, so that the carboxyl of magnetic bead surfaces activation in magnetic bead surfaces, obtains the magnetic bead of coupling with albumen coupling with amino room temperature (25 ℃) the reaction 3h of AhyR albumen.The rate of charge of AhyR albumen and nanometer magnetic bead is 10 μ g AhyR albumen: the 2mg magnetic bead.
(3) with magnetic bead after the 500 μ l coupling buffers washings coupling 2 times, add 25 ℃ of sealings of the coupling buffer magnetic bead 30min suspended state of magnetic bead (during keep) (BSA is with not binding site sealing of AhyR) that 500 μ l contain 1% bovine serum albumin(BSA) (BSA).
(4) with the magnetic bead after the coupling buffer of the 500 μ l washing sealing 2 times, with the resuspended magnetic bead of 100 μ l preservation liquid, be stored in 4 ℃ of refrigerators, stand-by.Preserve the preparation of liquid: will add 0.02%NaN in the PBST damping fluid
3Mix with 0.05%BSA, obtain preserving liquid.
Two, use the method for the nanometer magnetic bead detection signal molecule of coupling AhyR:
Principle: signaling molecule C4-HSL becomes blue after running into indicator bacteria KYC55, and this complex can not spread after signaling molecule C4-HSL and magnetic bead coupling AhyR protein combination, so the constant indigo plant of indicator bacteria KYC55.
Detection method:
1) the configuration glucose agar medium is cut to strip, places on the flat board that is decorated with the cross grid again;
2) dip indicator bacteria KYC55 nutrient solution with toothpick, indicator bacteria KYC55 nutrient solution on the right-angled intersection place point of the centre of each strip nutrient culture media, every at a distance of 4mm and each point on the same straight line;
3) place the Oxford cup in the forefront of nutrient culture media bar, Oxford cup and each point bacterial strain KYC55 nutrient solution are also in alignment, and 100 μ l experiment liquid is placed the Oxford cup, the central point of Oxford cup and from its nearest bacterial strain KYC55 nutrient solution point apart from 8mm;
4) will detect again flat board and Oxford cup and place together 30 ℃ of incubators to cultivate 48h, and then observe each point bacterial strain KYC55 nutrient solution and whether become blueness; If it is blue that the some bacterial strain KYC55 nutrient solution nearest apart from the Oxford cup shows, determine that then the candidate is contained the N-acylated homoserine lactone and reached the detectable order of magnitude in the experiment liquid.
5) add up again the number of Bluepoint.The number of Bluepoint the fewer of clear signal molecular diffusion of more saving your breath, the amount that contains C4-HSL in the bright experiment liquid of more saving your breath of signaling molecule diffusion is fewer, and combination has occured in fewer coupled to Nano magnetic bead and the C4-HSL of preparation of showing of amount that contains C4-HSL in the experiment liquid.
The nutrient solution of indicator bacteria (Agrobacterium tumefaciens) KYC55 obtains as follows: described bacterial strain (Agrobacterium tumefaciens) KYC55 is inoculated in the nutrient culture media, cultivate 48h for 30 ℃, obtain the nutrient solution of described bacterial strain (Agrobacterium tumefaciens) KYC55; Described nutrient culture media consists of: LB nutrient culture media+2 μ g/mL tetracyclines+100 μ g/mL spectinomycins+100 μ g/mL gentamicins.
The composition of glucose agar medium (mg/L): KH
2PO
4535, (NH
4)
2SO
410, MgSO
43.9, CaCl
20.38, FeSO
47H
2O 0.25, MnSO
4H
2O 0.11, glucose 5000, and agar 2%, all the other are water.
The preparation of experiment liquid: with signaling molecule C4-HSL solution 5 μ l (5 μ M), the AhyR protein 20 μ l of magnetic bead coupling, and pH 7.4,1 x PBS damping fluid, 75 μ l mixing, 30 ℃ of common water-bath 30min namely obtain testing liquid.
The preparation of CK: be that 7.4,1 x PBS damping fluid, 95 μ l mix 30 ℃ of water-bath 30min with signaling molecule C4-HSL solution 5 μ l (5 μ M) and pH value.
The compound method of signaling molecule C4-HSL solution: C4-HSL and ethanol are mixed, and the potpourri that obtains is signaling molecule C4-HSL solution; The concentration of C4-HSL is 5 μ M in the potpourri.-20 ℃ of preservations.
The described pH value of every 100ml is that 7.4,1 x PBS damping fluid is prepared as follows and obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, and obtaining described pH value is 7.4,1 x PBS damping fluid.
The result as shown in Figure 5.It is blue that first bacterial strain KYC55 nutrient solution of experimental group becomes, but color is obviously shallow than control group, and become the Bluepoint number and be less than the CK group, illustrates that the nanometer magnetic bead of coupling AhyR is successfully prepared, and can be effectively in conjunction with C4-HSL.
Three, the sensitivity of method detects
Experimental group: C4-HSL is dissolved in the PBS damping fluid of pH 7.4, final concentration is 5 μ M, obtains C4-HSL solution.(C4-HSL concentration is denoted as 10 to add the solution of 5 μ L C4-HSL in the PBS damping fluid of 45 μ L pH 7.4
-1), and to carry out successively 10 times gradient dilution to C4-HSL concentration be 10
-6, be prepared into the solution of variable concentrations C4-HSL.Detect according to method described in the experiment two.
Control group (Ni-NTA adsorption method): except the preparation of experiment liquid from experimental group is different, all the other conditions are all identical with experimental group.
Experiment liquid is prepared as follows in the control group: C4-HSL is dissolved in the PBS damping fluid of pH 7.4, final concentration is 5 μ M, obtains C4-HSL solution.(C4-HSL concentration is denoted as 10 to add the solution of 5 μ L C4-HSL in the PBS damping fluid of 45 μ L pH 7.4
-1), and to carry out successively 10 times gradient dilution to C4-HSL concentration be 10
-6, be prepared into the solution of variable concentrations C4-HSL.Detect according to method described in the experiment two.Be placed with Ni-NTA post matter in the cup of Oxford, purpose is to adsorb fixedly AhyR albumen, makes it can not free diffusing.
The testing result demonstration, it can detectable concentration be 25pmol (10 namely that the detection sensitivity of experimental group is about 25pmol[
-6) C4-HSL solution]; It can detectable concentration be 25pmol (10 namely that the detection sensitivity of control group is about 2.5nmol[
-3) C4-HSL solution]; The detection sensitivity of the AhyR protein ratio Ni-NTA absorption AhyR albumen of magnetic bead coupling improves approximately 100 times, reaches the pmol level.
3 repetitions are established in experiment, and the result is all consistent.
Claims (11)
1. nanometer magnetic bead with the coupling of N-acylated homoserine lactone acceptor, prepare according to the method that comprises the steps: with the nanometer magnetic bead after the coupling buffer suspension activation, add again N-acylated homoserine lactone acceptor, at 25 ℃ of reaction 3h, obtain the nanometer magnetic bead with the coupling of N-acylated homoserine lactone acceptor; Described coupling buffer is prepared as follows and is obtained: with the pH value be 7.4,1x PBS damping fluid and Tween-20 mix, and obtains coupling buffer, the pH value is 7.4, the volume ratio of 1x PBS damping fluid and Tween-20 is 100m1:0.05ml; The described pH value of every 100ml is 7.4,1x PBS damping fluid is prepared as follows and obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, obtains described pH value and be 7.4,1x PBS damping fluid.
2. nanometer magnetic bead according to claim 1, it is characterized in that: the rate of charge of the nanometer magnetic bead after described N-acylated homoserine lactone acceptor and the described activation is 5 μ g～10 μ g N-acylated homoserine lactone acceptors: the 2mg magnetic bead.
3. nanometer magnetic bead according to claim 1, it is characterized in that: the rate of charge of the nanometer magnetic bead after described N-acylated homoserine lactone acceptor and the described activation is 10 μ gN-acylated homoserine lactone acceptors: the 2mg magnetic bead.
4. according to claim 1 and 2 or 3 described nanometer magnetic beads, it is characterized in that: the nanometer magnetic bead after the described activation prepares according to the method that comprises the steps: EDC solution, NHS solution and nanometer magnetic bead are mixed, hatch the nanometer magnetic bead after obtaining activating;
The rate of charge of described EDC, NHS and nanometer magnetic bead is 1mg: 1mg: 2mg;
Described temperature of hatching is 37 ℃, and the time is 30min.
5. nanometer magnetic bead according to claim 4, it is characterized in that: the solute of described EDC solution is the EDC solid, and solvent is that concentration is that 10mM, pH value are 6.0 MES damping fluid, and the proportioning of solute and solvent is 5mg: 1ml;
The solute of described NHS solution is the NHS solid, and solvent is that concentration is that 10mM, pH value are 6.0 MES damping fluid, and the proportioning of solute and solvent is 5mg: 1ml;
The amino acid sequence of described N-acylated homoserine lactone acceptor is shown in SEQ ID NO:1;
Or described N-acylated homoserine lactone is C4-HSL.
6. whether contain the method for N-acylated homoserine lactone in the auxiliary detection sample, comprise the steps:
1) preparation experiment liquid as follows: arbitrary described nanometer magnetic bead and damping fluid with the coupling of N-acylated homoserine lactone acceptor among testing sample, the claim 1-5 mixed, potpourri is hatched, obtain testing liquid; The pH value of described damping fluid is 7.4;
2) detect whether become blue after described experiment liquid runs into bacterial strain (Agrobacterium tumefaciens) KYC55, if become blue, determine that then the candidate is contained the N-acylated homoserine lactone in the described testing sample.
7. method according to claim 6, it is characterized in that: described step 1), described damping fluid is the PBS damping fluid, and described temperature of hatching is 30 ℃, and the described time of hatching is 30min;
Or, described step 2) in, the described experiment liquid of described detection runs into the method that whether becomes blue behind bacterial strain (Agrobacterium tumefaciens) KYC55 and comprises the steps: to prepare glucose agar medium, on the described nutrient culture media on straight line and the equidistant place bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution, the Oxford cup that experiment liquid is housed is positioned on the described nutrient culture media, Oxford cup and each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution is also in alignment, the central point of Oxford cup with from nearest some bacterial strains of Oxford cup (Agrobacterium tumefaciens) KYC55 nutrient solution distance is arranged, behind 30 ℃ of maintenance 48h, observe each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution and whether show blueness.
8. according to claim 6 or 7 described methods, it is characterized in that: described step 1), described testing sample, with the nanometer magnetic bead of N-acylated homoserine lactone acceptor coupling and the volume sum of damping fluid be 100uL; Or described testing sample, with the nanometer magnetic bead of N-acylated homoserine lactone acceptor coupling and the volume ratio of damping fluid be 5 μ l: 20 μ l: 75 μ l;
Or, described step 2) in, the method of bacterial strain on the described point (Agrobacterium tumefaciens) KYC55 nutrient solution is for being to dip bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution with toothpick, and then point is to described nutrient culture media.
9. method according to claim 8, it is characterized in that: described step 1), the described PBS damping fluid of every 100ml is prepared as follows and is obtained: be the NaH of 0.2mol/L with 1.9ml concentration
2PO
4Aqueous solution and 8.1ml concentration are the Na of 0.2mol/L
2HPO
4Aqueous solution, water is settled to 100ml, obtains described PBS damping fluid;
Or, described step 2) in, the central point of described Oxford cup be 8mm from the distance of nearest some bacterial strains of Oxford cup (Agrobacterium tumefaciens) KYC55 nutrient solution.
10. method according to claim 9 is characterized in that:
Described step 2) in, the nutrient solution of described bacterial strain (Agrobacterium tumefaciens) KYC55 obtains as follows: described bacterial strain (Agrobacterium tumefaciens) KYC55 is inoculated in the nutrient culture media, cultivate 48h for 30 ℃, obtain the nutrient solution of described bacterial strain (Agrobacterium tumefaciens) KYC55; Described nutrient culture media obtains according to the method that comprises the steps: add tetracycline, spectinomycin and gentamicin in the LB nutrient culture media, obtain described nutrient culture media; The concentration of tetracycline in described nutrient culture media is 2 μ g/mL, and the concentration of spectinomycin in described nutrient culture media is 100 μ g/mL, and the concentration of gentamicin in described nutrient culture media is 100 μ g/mL;
Described step 2) in, described each point bacterial strain (Agrobacterium tumefaciens) KYC55 nutrient solution is at a distance of 4mm;
Described step 2) in, the amount of described experiment liquid is 100ul;
Described testing sample is water;
Or described N-acylated homoserine lactone is C4-HSL.
11.N-the application of acylated homoserine lactone acceptor in following (1) or (2) or (3) or (4) or (5) or (6):
(1) whether contains the N-acylated homoserine lactone in the test sample;
(2) prepare the kit that whether contains the N-acylated homoserine lactone in the test sample;
(3) whether test sample contains dna fragmentation shown in the SEQ ID NO:7;
(4) prepare the kit that whether contains dna fragmentation shown in the SEQ ID NO:7 in the test sample;
(5) amount of N-acylated homoserine lactone in the test sample;
(6) kit of the amount of N-acylated homoserine lactone in the preparation test sample;
The sample of described detection is water;
The amino acid sequence of described N-acylated homoserine lactone acceptor is shown in SEQ ID NO:1; Described N-acylated homoserine lactone is C4-HSL;
Wherein, whether contain the method for N-acylated homoserine lactone in the test sample, comprise the steps:
1) preparation experiment liquid as follows: arbitrary described nanometer magnetic bead and damping fluid with the coupling of N-acylated homoserine lactone acceptor among testing sample, the claim 1-5 mixed, potpourri is hatched, obtain testing liquid; The pH value of described damping fluid is 7.4;
2) detect whether become blue after described experiment liquid runs into bacterial strain (Agrobacterium tumefaciens) KYC55, if become blue, determine that then the candidate is contained the N-acylated homoserine lactone in the described testing sample.
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| CN105548058B (en) * | 2016-01-21 | 2018-04-27 | 广西大学 | A kind of method for detecting signaling molecule in pseudomonas aeruginosa |
| CN106086057A (en) * | 2016-06-22 | 2016-11-09 | 中国科学院生态环境研究中心 | A kind of method of Visual retrieval producing acyl homoserine lactones (AHL) bacterial strain and application thereof |
| CN106085939A (en) * | 2016-06-22 | 2016-11-09 | 中国科学院生态环境研究中心 | A kind of method of detection acyl homoserine lactones (AHL) |
| CN106191195B (en) * | 2016-07-11 | 2020-01-31 | 中国科学院生态环境研究中心 | method for determining total Acyl Homoserine Lactone (AHL) content and application thereof |
| CN109628562A (en) * | 2019-01-18 | 2019-04-16 | 内蒙古农业大学 | A method of detection ox muscle HSL gene expression amount |
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