CN104297220B - A kind of mercury ion detecting method and detection device - Google Patents
A kind of mercury ion detecting method and detection device Download PDFInfo
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- CN104297220B CN104297220B CN201410157445.0A CN201410157445A CN104297220B CN 104297220 B CN104297220 B CN 104297220B CN 201410157445 A CN201410157445 A CN 201410157445A CN 104297220 B CN104297220 B CN 104297220B
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Abstract
The invention discloses a kind of mercury ion detecting method and detection device, belong to field of environmental biotechnology.Mercury ion detecting method comprises the following steps: add mercurous solion in the Pmerr1 Omerr1 biotin double-stranded DNA system be combined with merR1 GFP fusion protein, take out solution after standing and carry out fluorescence intensity measurement, utilize standard curve to calculate mercury ion content in solution.The detection method of the present invention has the advantage that (1) can carry out sample detection at sample point, it is not necessary to by sample long-distance transportation to specific testing agency, it is not necessary to use the instrument and equipment of Large expensive；Detection required time is short, from starting to take sample to finally obtaining data, within 30 minutes, can complete, be particularly suited for the detection of Large scale field field soil sample mercury content, it is possible to human and material resources detection needed for are greatly reduced；(2) the mercury ion ultimate value of the method detection is 0.3 μ g/l, and with the ultimate value of cold atom spectral absorption analytic process closely, accuracy is high.
Description
Technical field
Present invention relates particularly to a kind of mercury ion detecting method, and the detection device of enforcement the method, belong to environment raw
Thing technical field.
Background technology
Hydrargyrum is the heavy metal that a kind of toxicity is extremely strong, it is possible to causing people's neurotoxic, visual organ defect etc., serious can cause
People is dead.The hydrargyrum manifold entering human body crosses food channel.Hydrargyrum in food is essentially from the soil for crop growth.Domestic many
Ground Mercury in Soil content has reached warning value.According to China's nineteen ninety-five formulate standard of soil environment quality (standard No.:
GB15618-1995), 1.0mg/kg is to ensure agricultural production, safeguards the soil limits value of health, and 1.5mg/kg is to ensure
Agricultural and forestry production and the soil phosphorus forms of plant normal growth.
Mercury content in soil is detected in real time for protection people are healthy, to take appropriate measures be to weigh very much
Want.Traditional mercury content analyze method include atomic absorption spectroscopy, atomic fluorescence spectrometry, naa,
Induction coupling mass-spectrometric technique, high pressure liquid chromatography (HPLC) etc..These detection methods are costly, and equipment volume is huge, to operator
Technology requires height, and sample needs remote transport and pre-treatment.Field, farmland detection then need easy to carry, operation cost
With cheap detecting instrument.
Summary of the invention
It is an object of the invention to provide a kind of mercury ion detecting method.
Meanwhile, the present invention also provides for a kind of detection device being exclusively used in and implementing said method.
In order to realize object above, the technical solution adopted in the present invention is:
A kind of mercury ion detecting method, comprises the following steps: at the Pmerr1-being combined with merR1-GFP fusion protein
Omerr1-biotin double-stranded DNA system adds mercurous solion, takes out solution after standing and carry out fluorescence intensity measurement, profit
Mercury ion content in solution is calculated with standard curve.
Nucleotide sequence coded by shown in SEQ ID NO:1 of described merR1-GFP fusion protein.
The preparation of described merR1-GFP fusion protein comprises the following steps:
(1) with bacillus megaterium genomic DNA as template, utilize primer P1F, P1R to expand merR1 gene, build matter
Grain P1；
(2) with plasmid P1 as template, primer P2F, P2R amplification two ends are utilized to carry BamH I and EcoR I restriction enzyme site
MerR1 gene, builds plasmid P2；
(3) with plasmid pEGFP as template, utilize primer P3F, P3R to expand GFP gene, with EcoR I enzyme action GFP gene and
Plasmid P2, builds plasmid P3 after connection；
(4) convert escherichia coli with plasmid P3, take positive colony and be enlarged cultivating, IPTG abduction delivering merR1-GFP
Fusion protein；
The nucleotide sequence of primer P1F as shown in SEQ ID NO:3, the nucleotide sequence of primer P1R such as SEQ ID NO:4
Shown in, the nucleotide sequence of primer P2F as shown in SEQ ID NO:5, the nucleotide sequence of primer P2R such as SEQ ID NO:6 institute
Showing, the nucleotide sequence of primer P3F is as shown in SEQ ID NO:7, and the nucleotide sequence of primer P3R is as shown in SEQ ID NO:8.
In described Pmerr1-Omerr1-biotin double-stranded DNA, 3 ' ends of a strand have biotin jag, and this is single
The oligonucleotide sequence of chain is as shown in SEQ ID NO:2.Pmerr1-Omerr1-biotin double-stranded DNA can by biotin with
Streptavidin combines.Use Streptavidin coated elisa plate, then be connected into Pmerr1-Omerr1-biotin double-stranded DNA successively
With merR1-GFP fusion protein, the Pmerr1-Omerr1-biotin double-strand being combined with merR1-GFP fusion protein can be formed
DNA system.
The preparation method of described Pmerr1-Omerr1-biotin double-stranded DNA comprises the following steps: by primer O/Pmerr1-
F-biotin, O/Pmerr1-R-biotin are dissolved in buffer solution respectively, and equal-volume mixes, and 94 DEG C of degeneration 2 minutes, is down to
Room temperature obtains Pmerr1-Omerr1 double-strand；The nucleotide sequence of primer O/Pmerr1-F-biotin such as SEQ ID NO:11 institute
Showing, the nucleotide sequence of primer O/Pmerr1-R-biotin is as shown in SEQ ID NO:12.
If detected sample is mercurous soil, need to filter by soil dispersion in water after mixing, filtrate is containing mercury ion
Solution.
Described standard curve is fluorescence intensity-ion concentration of mercury curve.
A kind of mercury ion detecting device, including the Pmerr1-Omerr1-biotin being combined with merR1-GFP fusion protein
Double-stranded DNA system.
Concrete, in system, Pmerr1-Omerr1-biotin double-stranded DNA is fixed on Streptavidin bag by biotin
In the ELISA Plate of quilt.(high-affinity black ELISA Plate as coated in Streptavidin, Thermo fisher scientific,
Yokohama, Japan) on.After adding the standing cultivation of mercurous solion in ELISA Plate, owing to mercury ion can be with merR1-
GFP fusion protein carries out specific binding, and by merR1-GFP fusion protein from Pmerr1-Omerr1-biotin double-stranded DNA
On disintegrate down, and the amount of the merR1-GFP fusion protein disintegrated down is proportional with the amount of the mercury ion of addition, after taking cultivation
Solution carries out fluorescence intensity measurement, utilizes fluorescence intensity-ion concentration of mercury standard curve can try to achieve mercury ion content in solution.
Cleaning Principle schematic diagram is as in figure 2 it is shown, I for add mercurous solion in ELISA Plate in Fig. 2, II is mercury ion and merR1-
GFP fusion protein combines, and merR1-GFP fusion protein disintegrates down from Pmerr1-Omerr1-biotin double-stranded DNA, and III is
Solution after cultivation is transferred out, detects its fluorescence intensity.
The preparation method of mercury ion detecting device comprises the following steps: put by Pmerr1-Omerr1-biotin double-stranded DNA
In the little indoor of the coated ELISA Plate of Streptavidin, under room temperature, concussion is cultivated；Again merR1-GFP fusion protein is placed in cell
In, under room temperature, concussion is cultivated, and to obtain final product.
Beneficial effects of the present invention:
MerR is the metalloid modulin being widely present in antibacterial.The research being combined merR with DNA/dissociate is the biggest
Majority is all carried out in gram negative bacteria.In gram negative bacteria, when the promoter region of merR Yu mer operon
When territory combines, rna polymerase activity is suppressed, mer gene transcribe stopping.When in Hg (II) with merR-Pmer complex
MerR when combining, merR-Pmer conformation crimps downwards 30 degree, and merR-Pmer bivalent core is distorted deformation, and RNA is polymerized
Enzyme is combined with DNA, restarts and transcribes.But in vitro, after merR with DNA is combined, in merR-DNA complex solution
Adding Hg (II) can not make merR disintegrate down from DNA.Gram positive bacteria bacillus megaterium is intracellular three hydrargyrum behaviour
Vertical son/promoter region, they are O/Pmerb3, O/Pmerr1 and O/Pmerr2 respectively, and these three region is by two merR eggs
White matter regulates and controls, and the two merR albumen is respectively merR1 and merR2.Research finds, merR1 can be combined with O/Pmerr1,
After adding the Hg (II) of variable concentrations in merR-DNA complex solution, protein band moves down, and move down
The quantity of protein band is proportional with the amount adding mercury ion in O/Pmerr1.Experimental result explanation mercury ion can be by
MerR1 is from Pmerr1-Omerr1(O/Pmerr1) disintegrate down, and the Hg of the amount of the merR1 disintegrated down and interpolation
(II) amount is proportional.
Green fluorescent protein is the reporter molecules that a class function is extremely strong, it is possible to gene expression, protein positioning and albumen
Matter interaction detects.Trans factors and cis element in bacterial transcription regulatory can occur interaction in vitro.GFP is connected
At the C-terminal gene fusion construct of trans factors, by this process and bacillus megaterium merR1 and Pmerr1-Omerr1(O/
Pmerr1) combination/dissociation process combines, it is possible to develop a kind of device that mercury ion carries out in situ detection.
In the present invention, mercury ion detecting method has the advantage that
(1) sample detection can be carried out at sample point, it is not necessary to by sample long-distance transportation to specific testing agency, no
Need to use the instrument and equipment of Large expensive；Detection required time is short, and from starting to take sample to finally obtaining data, 30 minutes i.e.
Can complete, be particularly suited for the detection of Large scale field field soil sample mercury content；The professional skill of operator is not almost had
Requiring, chemical reagent used is normal conventional chemical reagent, this detection method can be greatly reduced the manpower needed for detection,
Material resources；
(2) the mercury ion ultimate value of the method detection is 0.3 μ g/l, non-with the ultimate value of cold atom spectral absorption analytic process
Very close to, accuracy is high.
In the present invention, mercury ion detecting device can carry out lyophilization after using, and the detection device after lyophilization preserves
Under the conditions of 4 DEG C, next time is directly added into testing sample when using, once prepare can repeatedly Reusability, be substantially reduced
Expense needed for preparation detection device.
Accompanying drawing explanation
Fig. 1 is the structural representation of mercury ion detecting device in the present invention；
Fig. 2 is mercury ion detecting principle schematic in the present invention；
Fig. 3 is the electrophoresis result in embodiment 1 after plasmid BamH I and Xho I double digestion；
Fig. 4 is to add after meR1-GFP fusion protein solution in Pmerr1-Omerr1 genetic fragment solution in embodiment 3
Electrophoresis result；
Fig. 5 is to add variable concentrations mercury ion in embodiment 3 in merR1-GFP/Pmerr1-Omerr1 articulated system
Electrophoresis result；
Fig. 6 is the fluorescence that in embodiment 3, fusion protein and Pmerr1-Omerr1-biotin double-stranded DNA react different time
Intensity；
Fig. 7 is to add HgCl in embodiment 3 in mercury ion detecting device2The fluorescence intensity of solution is shifted after solution；
Fig. 8 is to add other heavy metal ion in merR1-GFP/Pmerr1-Omerr1 articulated system in embodiment 4
Electrophoresis result；
Fig. 9 is the fluorescence intensity adding different heavy metal ion solution in embodiment 4 in mercury ion detecting device；
When Figure 10 is that in embodiment 5, merR1-GFP crude protein reacts different from Pmerr1-Omerr1-biotin double-stranded DNA
Between fluorescence intensity；
Figure 11 is to add HgCl in embodiment 5 in mercury ion detecting device prepared by crude protein2The fluorescence intensity of solution；
Figure 12 is the fluorescence adding different heavy metal ion in embodiment 5 in mercury ion detecting device prepared by crude protein
Intensity；
Figure 13 is sensitivity tests to mercury ion detecting after the freeze-dried process of mercury ion detecting device in embodiment 6
Result；
Figure 14 is fluorescence intensity in embodiment 8-ion concentration of mercury standard curve.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
In the present embodiment, the construction method of merR1-GFP fusion gene is as follows:
(1) plasmid p1 is built
Preparation LB fluid medium, incubated overnight bacillus megaterium (Bacillus megaterium) under the conditions of 37 DEG C
(purchased from Bei Nuo bio tech ltd, Shanghai).Extract bacillus megaterium genomic DNA, referring in particular to sky root bacterial gene
Group DNA extraction kit description (article No.: DP302-02).Design primer amplification merR1 gene, primer sequence is as follows:
P1F:5 '-ATGAAATTTCGTATCGGAGAACTGGCTGAC-3 ' (as shown in SEQ ID NO:3),
P1R:5 '-TTATTTCTTCATCAGTGTTTCAATAATGGG-3 ' (as shown in SEQ ID NO:4)；
PCR reaction system: 2 × Pfu MasterMix(Beijing CoWin Bioscience Co., Ltd., article No.:
CW0686A) 25 μ l, primer P1F1pmol, primer P1R1pmol, H2O22 μ l, DNA1 μ l；
PCR response procedures: 94 DEG C, 5min, (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min) 40 circulations, 72 DEG C of 10min.
Amplified fragments is purified recovery, with T4DNA ligase, amplified fragments after purification is connected to cloning vehicle
On pMD19-T, condition of contact is 4 DEG C, 24 hours.Competence escherichia coli DH10B converted (specifically grasp with connecting product
" the Molecular Cloning: A Laboratory guide third edition first volume " page 91 and page 92 is seen as method), containing 100 μ g/ml ammonia benzyl penicillium sp after conversion
Cultivating on the LB flat board of element, 4 positive colonies of picking are cultivated, and extract plasmid, carry out enzyme action with EcoRV, and enzyme action produces
Thing carries out electrophoresis in the agarose gel of 0.8%, checks order the plasmid containing an about 400bp fragment in digestion products,
If sequence is completely the same with the sequence (GenBank:AB066362.1) delivered, then named for this plasmid p1.
(2) plasmid p2 is built
With plasmid p1 as template, BamH I and the merR1 gene of EcoR I restriction enzyme site are carried in design primer amplification two ends,
Primer sequence is as follows:
P2F:5 '-CGCGGATCCATGAAATTTCGTATCGGAGAACTGGCTGAC-3 ' (as shown in SEQ ID NO:5,
Underscore is Bam HI restriction enzyme site),
P2R:5 '-CCGGAATTCTTTCTTCATCAGTGTTTCAATAATGGGGCA-3 ' (as shown in SEQ ID NO:6,
Underscore is EcoR I restriction enzyme site)；
PCR reaction system: 2 × Pfu MasterMix(Beijing CoWin Bioscience Co., Ltd., article No.:
CW0686A) 25 μ l, primer P2F1pmol, primer P2R1pmol, H2O22 μ l, DNA1 μ l；
PCR response procedures: 94 DEG C, 5min, (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min) 40 circulations, 72 DEG C of 10min.
Amplification fragment out is carried out agarose gel purification recovery, then carries out double enzyme with BamH I and EcoR I
Cut, digestion products is carried out agarose gel purification recovery.With BamH I and EcoR I, plasmid pET28a carried out double enzyme simultaneously
Cut, digestion products is carried out agarose gel purification recovery.With T4DNA ligase to the merR1 gene after enzyme action and enzyme action after
PET28a carry out under the conditions of 4 DEG C 24 hours connect.With connecting product, escherichia coli DH10B competent cell is converted
(concrete operation method see " the Molecular Cloning: A Laboratory guide third edition first volume " page 91 and page 92).Containing 50 μ g/ml cards after conversion
Cultivating on the LB flat board of that mycin, 4 positive colonies of picking carry out liquid culture, with the little extraction reagent kit of ordinary plasmids (in
Ke Ruitai, Cat No:RTP2102) extract plasmid；With BamH I and EcoR I, plasmid being carried out double digestion, enzyme action condition is 37
DEG C, 2 hours；Digestion products carries out electrophoresis in the agarose gel of 0.8%, to containing an about 400bp fragment in digestion products
Plasmid check order, sequence and forecasting sequence (see SEQ ID NO:14) the named p2 of on all four plasmid after order-checking.
(3) plasmid p3 is built
Under the conditions of 37 DEG C, incubated overnight contains the escherichia coli of plasmid pEGFP (purchased from north, the Shanghai promise limited public affairs of biotechnology
Department), extract plasmid, design primer amplification GFP gene, primer sequence is as follows:
P3F:5’-CCGGAATTCATGTCTAAAGGTGAAGAATTATTCACTGGT-3 ' (as shown in SEQ ID NO:7,
Underscore is EcoR I restriction enzyme site),
P3R:5’-CCGGAATTCTTATTTGTACAATTCATCCATACCATGGGT-3 ' (as shown in SEQ ID NO:8,
Underscore is EcoR I restriction enzyme site)；
PCR reaction system: 2 × Pfu MasterMix(Beijing CoWin Bioscience Co., Ltd., article No.:
CW0686A) 25 μ l, primer P3F1pmol, primer P3R1pmol, H2O22 μ l, DNA1 μ l；
PCR response procedures: 94 DEG C, 5min, (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min) 40 circulations, 72 DEG C of 10min.
To the 700bp fragment amplified with quick agarose gel DNA reclaim test kit (Beijing health is century, Cat No:
CW2302) being purified recovery, then carry out enzyme action with EcoR I, enzyme action condition is 37 DEG C, 2 hours.To plasmid p2 EcoR
I carries out enzyme action, and digestion products is carried out agarose gel recovery.With calf intestine alkaline phosphatase (TAKARA) to the matter after reclaiming
Grain p2 carries out dephosphorylation process.The p2 T4DNA ligase of the GFP after enzyme action and phosphatizing treatment is carried out 4 DEG C 24 hours
Connect.With connecting product escherichia coli DH10B competent cell converted that (concrete operation method is shown in " Molecular Cloning: A Laboratory
The guide third edition first volume " page 91 and page 92), cultivate on the LB flat board containing 50 μ g/ml kanamycin after conversion.Picking
5 positive colonies carry out liquid culture, and condition of culture is 37 DEG C, 12 hours.With the little extraction reagent kit of ordinary plasmids (middle Ke Ruitai,
Cat No:RTP2102) extract plasmid, carry out enzyme action with EcoR I, enzyme action condition is 37 DEG C, 2 hours.Digestion products is 0.8%
Agarose gel carries out electrophoresis, the plasmid containing a 700bp fragment in digestion products is checked order, GFP base after order-checking
Because sequence (particular sequence is shown in SEQ ID NO:15) is completely the same with the sequence (GenBank:KF040393.1) delivered
And the named p3 of plasmid that GFP sequence transcriptional orientation is identical with upstream gene.
(4) escherichia coli containing plasmid p3 are built
Extract p3 plasmid, e. coli bl21 (DE3) competent cell is converted that (concrete operation method is shown in " molecule
The cloning experimentation guide third edition first volume " page 91 and page 92).Carry out on the LB flat board containing 50 μ g/ml kanamycin after conversion
Cultivate.5 positive colonies of picking carry out liquid culture, and condition of culture is 37 DEG C, 120rpm, 12 hours.Carry with ordinary plasmids is little
Test kit (middle Ke Ruitai, Cat No:RTP2102) extracts plasmid, carries out double digestion with BamH I and Xho I, and enzyme action condition is
37 DEG C, 2 hours.Digestion products carries out electrophoresis in the agarose gel of 0.8%, to containing a 1.1kb fragment in digestion products
Plasmid (see Fig. 3, in figure, L is DNA Marker, and S is endonuclease bamhi electrophoretic band, 5.4kb large fragment be shown that plasmid carry
Body pET28a, 1.1kb small fragment is shown that antigen-4 fusion protein gene merR1-GFP) check order, sequencing result and SEQ ID
The on all four plasmid of nucleotide sequence shown in NO:1 is defined as p3, and the e. coli bl21 (DE3) containing p3 plasmid is named
BL21-E。
Embodiment 2
In the present embodiment, the method for extraction and purification of merR1-GFP fusion protein is as follows:
The monoclonal of one BL21-E of picking from LB flat board, enters in the LB culture fluid containing 50 μ g/ml kanamycin
37 DEG C of incubated overnight of row.Then in 4 triangular flasks being respectively contained with 1 liter of LB culture fluid, training it is enlarged with the ratio of 1:100
Support.At culture fluid OD600When rising to 0.6, in culture fluid, add the IPTG of final concentration of 0.4mmol/l, continue to cultivate 4 little
Time.To culture fluid with 6000rpm, 4 DEG C of pelleted by centrifugation 10 minutes.Abandoning supernatant, precipitates flushing two by buffer A to cell
Secondary.The formula of buffer A is 50mmol/l Tris-HCl, pH7.5,2mmo/l2-mercaptoethanol, 5%(v/v)
Glycerol, 0.2mol/l (NH4)2SO4.By 50 milliliters of buffer A, cell precipitation is suspended.Cell suspending liquid is injected
M-110EH Microfluidizer (Microfluidics, Newton, MA, USA), to bacterial cell under conditions of 11000psi
Carry out cracking process.Homogeneous liquid after process, at 16000rpm, is centrifuged 1 hour under the conditions of 4 DEG C, supernatant is transferred to another
In individual clean triangular flask, filter with the filter membrane that aperture is 0.45 micron.By buffer B to chromatographic column heparin-
Sepharose column(Hiprep, Amersham Biosciences, Buckinghamshire, UK) it is balanced, then
The chromatographic column heparin-sepharose column that filtrate injection balance is crossed.The formula of buffer B is 50mmol/l
Tris-HCl, pH7.5,2mmo/l2-mercaptoethanol, 5%(v/v) glycerol.By the buffer B of 60 milliliters to layer
Analysis post washs.Then eluting is carried out with 140 milliliters of buffer C fusion protein to being combined in chromatographic column.Buffer C's
Formula is 50mmol/l Tris-HCl, pH7.5,2mmo/l2-mercaptoethanol, 5%(v/v) glycerol, (NH4)2SO4Concentraton gradient is 0.2mol/l to 1mol/l.Eluent is carried out Tricine-SDS-PAGE(18%) electrophoresis, detects purification
Quality.(the NH of the optimal eluting of fusion protein4)2SO4Concentration is between 0.7mol/l to 0.9mol/l.The egg of the most backward generation
White matter refined solution adds the glycerol of final concentration of 50%, is stored in-80 DEG C of refrigerators standby after mixing.
Embodiment 3
The merR1-GFP fusion protein binding characteristic to O/Pmerry, the fusion protein binding characteristic to mercury ion, hydrargyrum from
The binding characteristic of merR1-GFP/Pmerr1-Omerr1 complex is studied by son:
(1) merR1-GFP fusion protein and the binding tests of Pmerr1-Omerr1
With bacillus megaterium genomic DNA as template, carrying out PCR amplification, synthetic primer is as follows:
O/Pmerr1-F:5 '-AGGGTAAGTAAAATCTCATGAATGAAGTAA-3 ' (as shown in SEQ ID NO:9),
O/Pmerr1-R:5 '-TTCATCGCGATCGACAACCCCTAGCAATTT-3 ' (as shown in SEQ ID NO:10)；
PCR reaction system: 2 × Pfu MasterMix(Beijing CoWin Bioscience Co., Ltd., article No.:
CW0686A) 25 μ l, primer O/Pmerr1-F1pmol, primer O/Pmerr1-R1pmol, H2O22 μ l, DNA1 μ l；
PCR response procedures: 94 DEG C, 5min, (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min) 40 circulations, 72 DEG C of 10min.
PCR amplification obtains the fragment of a 316bp, and this fragment is Pmerr1-Omerr1.By this fragment from DNA agarose
Gel takes down last time.After order-checking identical with nucleotide sequence shown in SEQ ID NO:13 after carry out following experiment again.
Preparation gel shift buffer, formula is: 20mmol/l Tris-HCl, pH7.5,50mmol/l KCl, 5%
Glycerol, 50 μ g/ml bovine serum albumin, 1mmol/l cysteine, 0.05%Nonidet P-40.?
DNA solution is separately added into the merR1-GFP fusion protein of final concentration of 1 μm ol/l, 5 μm ol/l, 10 μm ol/l, with 1:1's
Mixed liquor is dissolved in gel shift buffer by ratio, takes 5 microlitre mixed liquors, cultivates 60 minutes under the conditions of 4 DEG C.Preparation is solidifying
Glue sample-loading buffer, formula is: 0.05%bromophenol blue, 50%glycerol.Take 5 microlitre reaction mixtures and 1 micro-
Rise sample-loading buffer mixing.Carry out non-denaturing polyacrylamide gel (acrylamide: the methylene bisacrylamide=29:1) electricity of 6%
Swimming, electrophoretic buffer is Tris-acetate buffer (without EDTA), and its formula is: 0.025mol/l Tris, 0.25mol/l
Glycine, 0.1% (w/v) SDS, pH8.0.At 4 DEG C, electrophoresis 90 minutes under conditions of 200V.EB(ethidium is used after electrophoresis
Bromide) gel is dyeed.Result shows, adds the DNA band of DNA sample of merR1-GFP fusion protein at gel
On all occur on move phenomenon (see Fig. 4, in figure, 0 be shown that in Pmerr1-Omerr1 genetic fragment solution without any egg
White matter, 1,5,10 are shown that adding 1 μm ol/l merR1-GFP in Pmerr1-Omerr1 genetic fragment solution respectively merges
Albumen, 5 μm ol/l merR1-GFP fusion protein and 10 μm ol/l merR1-GFP fusion protein, co-culture 60 under the conditions of 4 DEG C
Electrophoresis result after minute), illustrate that merR1-GFP Yu Pmerr1-Omerr1 there occurs combination, after being combined with merR1-GFP
Pmerr1-Omerr1 molecule quantitative change is big, and migration velocity is slack-off, presents shifting phenomenon on gel.
(2) mercury ion makes the probability test that fused protein disintegrates down from DNA
With bacillus megaterium genomic DNA as template, carrying out PCR amplification, synthetic primer is as follows:
O/Pmerr1-F:5 '-AGGGTAAGTAAAATCTCATGAATGAAGTAA-3 ',
O/Pmerr1-R:5 '-TTCATCGCGATCGACAACCCCTAGCAATTT-3 '；
Ibid, amplification obtains the fragment of a 316bp, and this fragment is Pmerr1-Omerr1 for PCR reaction system and program,
This fragment is taken down from DNA agarose gel last time.
Preparation gel shift buffer, formula is: 20mmol/l Tris-HCl, pH7.5,50mmol/l KCl, 5%
Glycerol, 50 μ g/ml bovine serum albumin, 1mmol/l cysteine, 0.05%Nonidet P-40.?
DNA solution adds the merR1-GFP fusion protein of final concentration of 5 μm ol/l, mixed liquor is dissolved in solidifying with the volume ratio of 1:1
Glue migrates in buffer.Take 5 microlitre reaction mixtures to cultivate 60 minutes under the conditions of 4 DEG C.Then add respectively in reaction liquid
Enter final concentration of 1 μm ol/l, 5 μm ol/l, the HgCl of 10 μm ol/l2.Preparation Gel Loading buffer, formula is: 0.05%
Bromophenol blue, 50%glycerol.Take 5 microlitre reaction mixtures and the mixing of 1 microlitre sample-loading buffer.Carry out 6%
Non-denaturing polyacrylamide gel (acrylamide: methylene bisacrylamide=29:1) electrophoresis, electrophoretic buffer is Tris-
Acetate buffer (without EDTA), its formula is: 0.025mol/l Tris, 0.25mol/l Glycine, 0.1% (w/v)
SDS, pH8.0.At 4 DEG C, electrophoresis 90 minutes under conditions of 200V.EB(ethidium bromide is used after electrophoresis) gel is entered
Row dyeing.Result shows, adds HgCl2After carry out the reaction mixture of electrophoresis, its DNA pillar location be not bound with merR1-
The DNA pillar location of GFP is identical, and (Fig. 5, in figure, 1 is shown that without mercury ion in reaction mixture, and 2～6 show successively
In reaction mixture, add final concentration of 1 μm ol/l, 5 μm ol/l, 10 μm ol/l, 20 μm ol/l, 30 μm ol/l
HgCl2), illustrate to add HgCl2After, merR1-GFP fusion protein can be dissociated down from Pmerr1-Omerr1 by mercury ion
Coming, after merR1-GFP fusion protein disintegrates down from Pmerr1-Omerr1, the molecular weight of Pmerr1-Omerr1 returns to
Former normal condition, its migration velocity on gel returned to again before normal condition, after electrophoresis, its position is again
Return to normal position.
(3) fusion protein and the associativity being fixed on solid phase surface DNA double chain are tested
Entrusting TAKARA company, synthetic primer is as follows:
O/Pmerr1-F-biotin:5’-TATATTTACCCTGTACTAAGGTACGTGGTTTATGCTGTAAGTG AGG-
Biotin-3 ' (its oligonucleotide sequence is as shown in SEQ ID NO:11),
O/Pmerr1-R-biotin:5’-CCTCACTTACAGCATAAACCACGTACCTTAGTACAGGGTAA ATATA-
3 ' (as shown in SEQ ID NO:12)；
Oligonucleotide O/Pmerr1-F-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is the denseest
Degree is 100 μm ol/l, and oligonucleotide O/Pmerr1-R-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is
Final concentration of 100 μm ol/l.The mixing of both equal-volumes, 94 DEG C of degeneration 2 minutes, the most progressively it is down to room temperature, forms double-strand
Pmerr1-Omerr1-biotin, concentration is 50 μm ol/l.At this moment in the double-stranded DNA formed, 3 ' ends of a strand have a life
Thing element jag, this biotin jag is fixed for following.
Pmerr1-Omerr1-biotin can be fixed on micro-by the high-affinity between biotin and Streptavidin
In the little chamber interior walls of orifice plate.Use Streptavidin coated high-affinity black ELISA Plate (Thermo fisher
Scientific, Yokohama, Japan) fix oligonucleotide, with the Tris-HCl(pH7.4 of 25mmol/l) rinse enzyme mark
Plate cell three times, all washes the DNA in cell off removing.Pmerr1-Omerr1-biotin is dissolved in 25mmol/l's
Tris-HCl(pH7.4) in buffer, final concentration of 0.25 μm ol/l of Pmerr1-Omerr1-biotin.Take 100 microlitres molten
Liquid is placed in cell.Pmerr1-Omerr1-biotin solution is in inner surface is coated with the ELISA Plate cell of Streptavidin
Cultivating 2 hours, condition of culture is 26 DEG C, and 120rpm rocks.Then the solution of little indoor is removed, with 200 microlitre 25mmol/l
Tris-HCl(pH7.4) cell is flushed three times, to remove, cell there is no fixing DNA, obtains mercury ion detecting device,
Structural representation is as shown in Figure 1.
Take 30 microlitres merR1-GFP protein purification solution (50 μm ol/l) to mix with 20 microliter of buffer liquid.Slow
Rushing formula of liquid is: 50mmol/L kaliumphosphate buffer, 50 μ g/ml salmon sperm dnas.Kaliumphosphate buffer formula is: 10mmol/l
Potassium phosphate (pH6.0), 0.05%Tween20.Stand 15 minutes at ambient temperature.Taking 300 microlitre mixed liquors, average mark is placed on 3
In individual cell.Under room temperature condition, 120rpm whirlpool concussion respectively is cultivated 15 minutes, 30 minutes, 2 hours.Solution by little indoor
Remove, clean cell once with 200 microlitre kaliumphosphate buffers (pH6.0), to remove the protein that little indoor are not bound with.With
The fluorescence intensity of little indoor is detected by hand-held green fluorescent protein detector, it was found that cultivate 15 minutes little indoor tables
The fluorescence intensity that face produces with cultivate 0.5 hour and the fluorescence intensity of 2 hours little chamber internal surfaces generation is identical (see Fig. 6, in figure 1
～3 be shown that reacting 15 minutes, 30 minutes, fluorescence intensity after 2 hours successively).
(4) the probability test that the fusion protein being fixed on solid phase surface is disintegrated down by mercury ion
Preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/l
NaCl, 0.1%Tween20.The HgCl of final concentration of 0.5 μ g/l is added in detection buffer2, take 150 microlitres and be placed on cell
In, cultivate 5 minutes.Transfer to the liquid of little indoor, in another common microwell plate cell, use hand-held green fluorescent protein
Detector (GFP-pen, GFP100, Photon systems instruments, Brno, Czech Republic) is to its fluorescence
Intensity detects.Result shows, the fluorescence intensity of the liquid adding mercuric chloride in ELISA Plate cell is significantly higher than and does not adds chlorination
(see Fig. 7, in figure, 1 is shown that in solution not containing any mercury ion to the fluorescence intensity of the liquid of hydrargyrum, and 2 are shown that containing 0.5
μg/lHgCl2Solution).Illustrating in the little indoor of ELISA Plate, the merR1-GFP being fixed on solid phase surface can be merged by mercury ion
Albumen disintegrates down from Pmerr1-Omerr1-biotin double-stranded DNA；By to the merR1-GFP fusion protein disintegrated down
Fluorescence intensity detect, it is possible to infer the content of mercuric chloride in liquid.
Embodiment 4
Other heavy metal ion binding characteristic to fusion protein:
(1) other heavy metal ion and fusion protein reaction characteristics in liquid phase environment
With bacillus megaterium genomic DNA as template, carrying out PCR amplification, synthetic primer is as follows:
O/Pmerr1-F:5 '-AGGGTAAGTAAAATCTCATGAATGAAGTAA-3 ',
O/Pmerr1-R:5 '-TTCATCGCGATCGACAACCCCTAGCAATTT-3 '；
Ibid, amplification obtains the fragment of a 316bp, and this fragment is Pmerr1-Omerr1 for PCR reaction system and program,
This fragment is taken down from DNA agarose gel last time.
Preparation gel shift buffer, formula is: 20mmol/l Tris-HCl, pH7.5,50mmol/l KCl, 5%
Glycerol, 50 μ g/ml bovine serum albumin, 1mmol/l cysteine, 0.05%Nonidet P-40.?
DNA solution adds the merR1-GFP fusion protein of final concentration of 10 μm ol/l, with the volume ratio of 1:1, mixed liquor is dissolved in
In gel shift buffer.Take 5 portions of mixed liquors, every part of 5 microlitres respectively, cultivate 60 minutes under the conditions of 4 DEG C.Then at reaction liquid
In be separately added into the CaCl of final concentration of 5 μm ol/l2、PbCl2、CdCl2、AsCl3、MnCl2.Preparation Gel Loading buffer, joins
Fang Wei: 0.05%bromophenol blue, 50%glycerol.Take 5 microlitre reaction mixtures and 1 microlitre sample-loading buffer mixes
Close.Carrying out non-denaturing polyacrylamide gel (acrylamide: the methene=29:1) electrophoresis of 6%, electrophoretic buffer is: Tris-
Acetate buffer (without EDTA), formula is: 0.025mol/l Tris, 0.25mol/l Glycine, 0.1% (w/v) SDS,
pH8.0.At 4 DEG C, electrophoresis 90 minutes under conditions of 200V.EB(ethidium bromide is used after electrophoresis) gel is contaminated
Color.Result shows, after adding these several heavy metal ion in reaction mixture, DNA band position on gel is not sent out
Changing (see Fig. 8, in figure, L is DNA Marker, CK be shown that in reactant mixture without any heavy metal element from
Son, 1～5 are shown that in reactant mixture adding CaCl successively2、PbCl2、CdCl2、AsCl3、MnCl2), illustrate that these are several
Heavy metal element ion can not be combined with merR1-GFP；These several heavy metal ion can not make to be combined in Pmerr1-
MerR1-GFP on Omerr1 disintegrates down from DNA.
(2) reaction experiment of other heavy metal elements and the fusion protein being fixed on solid phase surface
Entrusting TAKARA company, synthetic primer is as follows:
O/Pmerr1-F-biotin:5’-TATATTTACCCTGTACTAAGGTACGTGGTTTATGCTGTAAGTGAGG-
Biotin-3 ',
O/Pmerr1-R-biotin:5’-CCTCACTTACAGCATAAACCACGTACCTTAGTACAGGGTAAATATA-
3’；
Oligonucleotide O/PmerR1-F-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is the denseest
Degree is 100 μm ol/l.Oligonucleotide O/PmerR1-R-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is
Final concentration of 100 μm ol/l.The mixing of both equal-volumes, 94 DEG C of degeneration 2 minutes, progressively it is down to room temperature, forms double-strand Pmerr1-
Omerr1-biotin, concentration is 50 μm ol/l.At this moment the double-stranded DNA formed has a strand 3 ' end has a biotin
Jag, this biotin jag is fixed for following.
Pmerr1-Omerr1-biotin can be fixed on micro-by the high-affinity between biotin and Streptavidin
In the little chamber interior walls of orifice plate.Use high-affinity black ELISA Plate (the Thermo fisher of Streptavidin parcel
Scientific, Yokohama, Japan) fix oligonucleotide.Tris-HCl(pH6.0 with 150 microlitre 25mmol/l) slow
Rush liquid ELISA Plate cell is flushed three times, to remove the DNA in cell.Pmerr1-Omerr1-biotin is dissolved in
The Tris-HCl(pH7.4 of 25mmol/l) in buffer, final concentration of 0.25 μm ol/l of Pmerr1-Omerr1-biotin.Take
100 microlitre Pmerr1-Omerr1-biotin solution are placed in ELISA Plate cell.Pmerr1-Omerr1-biotin solution exists
Inner surface is enclosed with in the cell of Streptavidin cultivation 2 hours, and condition of culture is 25 DEG C, and 120rpm rocks.Then will
Pmerr1-Omerr1-biotin solution remove, with the Tris-HCl(pH6.0 of 200 microlitre 25mmol/l) buffer to cell punching
Wash three times, to remove the DNA being not bound with in cell.
Take 30 microlitres merR1-GFP protein purification solution (50 μm ol/l) to mix with 20 microliter of buffer liquid.Slow
Rushing formula of liquid is: 50mmol/L kaliumphosphate buffer, 50 μ g/ml salmon sperm dnas.15 minutes are stood under the conditions of 25 DEG C.Take 300
Microlitre mixed liquor, average mark is placed in 3 cells.Under the conditions of 25 DEG C, the concussion of 120rpm whirlpool is cultivated 15 minutes.By mixture from
ELISA Plate cell removes away.Clean cells once with 200 microlitre kaliumphosphate buffers (pH6.0), do not have removing little indoor
There is the protein of combination.
Preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/l
NaCl, 0.1%Tween20.Detection buffer is equally divided into 5 parts, reaction liquid is separately added into CaCl2、PbCl2、
CdCl2、AsCl3、MnCl2So that it is final concentration of 10 μ g/l.Take 150 microlitre detection buffer to be placed in ELISA Plate cell.25
DEG C cultivate 5 minutes, transfer to the liquid of little for ELISA Plate indoor, in another common microwell plate cell, use hand-held green fluorescence
The fluorescence intensity of Protein Detection instrument indoor liquid little to microwell plate detects.Result shows, these are several for the little indoor interpolation of ELISA Plate
The fluorescence intensity of the liquid of Heavy Metallic Elements ion is identical with the fluorescence intensity of the solution not adding any heavy metal element ion
(see Fig. 9, in figure, 1 is without any heavy metal element ion in solution, and 2～6 to be followed successively by containing final concentration be 10 μ g/l
CaCl2、PbCl2、CdCl2、AsCl3、MnCl2Solution)；Illustrate that these several heavy metal element ions can not be with merR1-
GFP combines；These several heavy metal ion can not make the merR1-being combined on Pmerr1-Omerr1-biotin double-stranded DNA
GFP disintegrates down from DNA, when in utilizing detection device to sample, mercury ion content detects, and this several heavy metal species unit
Element ion does not results in interference.
Embodiment 5
Fusion protein crude extract replaces the probability of purifying protein to test:
(1) synthetic dsdna, is fixed on the little chamber internal surface of ELISA Plate
Entrusting TAKARA company, synthetic primer is as follows:
O/Pmerr1-F-biotin:5’-TATATTTACCCTGTACTAAGGTACGTGGTTTATGCTGTAAGTGAGG-
Biotin-3 ',
O/Pmerr1-R-biotin:5’-CCTCACTTACAGCATAAACCACGTACCTTAGTACAGGGTAAATATA-
3’；
Oligonucleotide O/PmerR1-F-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is the denseest
Degree is 100 μm ol/l.Oligonucleotide O/PmerR1-R-biotin is dissolved in the Tris-HCl(pH7.4 of 25mmol/L) in so that it is
Final concentration of 100 μm ol/l.The mixing of both equal-volumes, 94 DEG C of degeneration 2 minutes, progressively it is down to room temperature, forms double-strand Pmerra-
Omerr1-biotin, concentration is 50 μm ol/l.At this moment the double-stranded DNA formed has a strand 3 ' end has a biotin
Jag, this biotin jag is fixed for following.
Pmerr1-Omerr1-biotin can be fixed on micro-by the high-affinity between biotin and Streptavidin
In the little chamber interior walls of orifice plate.Use Streptavidin coated high-affinity black ELISA Plate (Thermo fisher
Scientific, Yokohama, Japan) fix oligonucleotide.Tris-HCl(pH6.0 with 150 microlitre 25mmol/l) slow
Rush liquid ELISA Plate cell is flushed three times, to remove the DNA in cell.Pmerr1-Omerr1-biotin is dissolved in
The Tris-HCl(pH7.4 of 25mmol/l) in buffer, final concentration of 0.25 μm ol/l of Pmerr1-Omerr1-biotin, take
100 microlitres are placed in cell.Pmerr1-Omerr1-biotin solution is in inner surface is enclosed with the cell of Streptavidin
Cultivating 2 hours, condition of culture is 25 DEG C, and 120rpm rocks.Then Pmerr1-Omerr1-biotin solution is removed, with 200
The Tris-HCl(pH6.0 of microlitre 25mmol/l) cell flushes three times by buffer, do not fixes removing the little indoor of ELISA Plate
DNA.
(2) protein crude extract administration and the DNA double chain combination being fixed on little chamber internal surface are tested
The monoclonal of one BL21-E of picking from LB flat board, in the LB culture fluid containing 50 μ g/ml kanamycin 37
DEG C carry out incubated overnight.Then in 4 triangular flasks being respectively contained with 1 liter of LB culture fluid, training it is enlarged with the ratio of 1:100
Support.At OD600When rising to 0.6, in culture fluid, add the IPTG of final concentration of 0.4mmol/l, continue to cultivate 4 hours.Cell
Culture with 6000rpm, 4 DEG C of pelleted by centrifugation 10 minutes.With buffer (50mmol/l Tris-HCl, pH7.5,2mmo/l2-
Mercaptoethanol, 5%v/v glycerol, 0.2mol/l (NH4)2SO4) to cell precipitation flushing twice.Delay with 50 milliliters
Rush liquid (50mmol/l Tris-HCl, pH7.5,2mmo/l2-mercaptoethanol, 5%v/v glycerol, 0.2mol/l
(NH4)2SO4) cell precipitation is suspended.Cell suspending liquid is injected M-110EH Microfluidizer
(Microfluidics, Newton, MA), carries out cracking process to bacterial cell under conditions of 11000psi.Equal after process
Matter liquid, at 16000rpm, is centrifuged 1 hour under the conditions of 4 DEG C, transfers to supernatant, in another clean triangular flask, use aperture
It is that the filter membrane of 0.45 micron filters.Filtrate is fusion protein crude extract.
Take 30 microlitre merR1-GFP albumen runic thing solution and 20 microliter of buffer liquid mix.Buffer formulation is:
50mmol/L kaliumphosphate buffer, 50 μ g/ml salmon sperm dnas.Stand 15 minutes at ambient temperature.Take 300 microlitre mixed liquors,
Average mark is placed in 3 cells.Under room temperature condition, 120rpm whirlpool concussion respectively is cultivated 15 minutes, 30 minutes, 2 hours.By little
Liquid removal in room removes, and cleans cell once with 200 microlitre kaliumphosphate buffers (pH6.0), is not bound with removing
Runic thing protein.Detect by the fluorescence intensity of hand-held green fluorescent protein detector chamber internal surface little to ELISA Plate.
Result shows, cultivates the fluorescence intensity of ELISA Plate little chamber internal surface generation in 15 minutes and cultivates 0.5 hour or in 2 hours ELISA Plate
Fluorescence intensity that surface produces identical (see Figure 10, in figure 1～3 be shown that successively reacting 15 minutes, 30 minutes, after 2 hours
Fluorescence intensity).
(3) fusion protein being fixed on DNA double chain is disintegrated down by mercury ion
Preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/l
NaCl, 0.1%Tween20.The HgCl of final concentration of 10 μ g/l is added in detection buffer2.Take 150 microlitre detection buffer
It is placed in cell, cultivates 5 minutes.The liquid of little indoor is transferred in another common microwell plate cell, with hand-held green
Color fluorescin detector (GFP-pen, GFP100, Photon systems instruments, Brno, Czech
Republic) its fluorescence intensity is detected.Result shows, adds the fluorescence intensity of the liquid of mercuric chloride in ELISA Plate cell
Be significantly higher than the liquid not adding mercuric chloride fluorescence intensity (see Figure 11, in figure 1 be shown that in solution not containing any hydrargyrum from
Son, 2 are shown that containing 0.5 μ g/l HgCl2Solution).Illustrate that, in the little indoor of ELISA Plate, mercury ion can will be fixed on
The merR1-GFP fusion protein of phase surface disintegrates down from Pmerr1-Omerr1-biotin；By to disintegrating down
The fluorescence intensity of merR1-GFP fusion protein detects, it is possible to infer the content of mercuric chloride in liquid；By merR1-GFP
After fusion protein runic thing is placed in ELISA Plate cell, the merR1-GFP fusion protein in protein crude extract administration can be with solid
The oligonucleotide being scheduled on the little chamber internal surface of ELISA Plate combines, this combination and bond strength and the merR1-GFP of purification
The combination between oligonucleotide fixing on fusion protein and the little chamber internal surface of ELISA Plate and bond strength do not have anything
Different.
(4) the probability test that other heavy metal elements and fusion protein runic thing-DNA complex react
Preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/l
NaCl, 0.1%Tween20.Detection buffer is equally divided into 5 parts, reaction liquid is separately added into CaCl2、PbCl2、
CdCl2、AsCl3、MnCl2So that it is final concentration of 10 μ g/l.Take 150 microlitre detection buffer to be placed in ELISA Plate cell.25
DEG C cultivate 5 minutes, transfer to the liquid of little for ELISA Plate indoor, in another common microwell plate cell, use hand-held green fluorescence
The fluorescence intensity of Protein Detection instrument indoor liquid little to microwell plate detects.Result shows, these are several for the little indoor interpolation of ELISA Plate
The fluorescence intensity of the liquid of Heavy Metallic Elements ion is identical with the fluorescence intensity of the solution not adding any heavy metal element ion
(see Figure 12, in figure, 1 is without any heavy metal element ion in solution, and 2～6 to be followed successively by containing final concentration be 10 μ g/l
CaCl2、PbCl2、CdCl2、AsCl3、MnCl2Solution)；Illustrate that these several heavy metal element ions can not be with merR1-
GFP combines；These several heavy metal ion can not make the merR1-GFP combining on Pmerr1-Omerr1-biotin from DNA
On disintegrate down, utilize detection device to sample in mercury ion content detect time, these several heavy metal element ions are not
Can interfere；The most raw materials used is the merR1-GFP albumen of purification, or engineering bacteria protein crude extract administration, at detection sample
During middle mercury ion, these several heavy metal element ions are all without interfering.
From above-described embodiment it can be seen that replace the merR1-GFP fusion protein of purification with crude protein extract, to enzyme mark
The DNA of the little indoor of plate makes fusion protein disintegrate down from DNA with protein bound, mercury ion with protein bound, mercury ion
The most do not impact Deng indices, protein crude extract administration therefore can be used to replace the merR1-GFP fusion protein of purification, from
And save the man power and material required for preparation detection device, testing cost is greatly reduced.
Embodiment 6
The lyophilization impact on fusion protein crude extract character:
Detection device after using in embodiment 5 is positioned over-80 DEG C of freezing processing 2 hours, then takes out and is placed in vacuum
Freezer dryer (FreeZone4.5, Labconco, USA) carries out lyophilization process, then is placed in 4 DEG C of refrigerators, after 30 days from
Taking out in 4 DEG C of refrigerators, preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/
LNaCl, 0.1%Tween20.It is separately added into final concentration of 0.1 μ g/l, 0.2 μ g/l, 0.3 μ g/l, 0.4 μ in detection buffer
G/l, the mercury ion of 0.5 μ g/l.Then take 150 Al of Solution and be respectively put into the different little indoor of detection device ELISA Plate, 25 degree
Cultivate 5 minutes, the liquid of little for ELISA Plate indoor is transferred in another common microwell plate cell, with hand-held green fluorescence egg
The fluorescence intensity of white detector indoor liquid little to microwell plate detects.Result shows, mercury ion content be 0.1 μ g/l and
The fluorescence intensity of the sample of 0.2 μ g/l does not has any difference with the fluorescence intensity of the sample without mercury ion, but mercury ion contains
Amount is that the fluorescence intensity of 0.3 μ g/l or the higher sample of concentration is then significantly higher than comparison, the inspection after lyophilization processes
The mercury ion detection minimum surveying device is that 0.3 μ g/l(is shown in Figure 13, and in figure, 1～6 are followed successively by interpolation final concentration in detection device
Be 0 μ g/l, 0.1 μ g/l, 0.2 μ g/l, 0.3 μ g/l, 0.4 μ g/l, the testing result of 0.5 μ g/l Hg (II) solution).And without
The mercury ion detection limit value crossing the detection device that lyophilization processes also is 0.3 μ g/l.This explanation lyophilization can't make
The detection function of mercury ion is impacted by detection device, and detection device can preserve at low temperatures for a long time, its mercury ion
Detection function can be maintained for a long time, and detection device, can repeatedly Reusability after making.
Embodiment 7
The preparation of soil extract sample:
Take 50 grams of dry soil to be suspended in 500 milliliters of ultra-pure waters, with Glass rod, soil is smashed to pieces so that it is in suspended
Liquid, stands 5 minutes, takes supernatant coarse filter paper and filter, then with the filter membrane that aperture is 0.45 micron, filtrate is carried out two
Secondary filtration, the liquid obtained by secondary filter is soil extract sample.
Embodiment 8
In the present embodiment, mercury ion detecting device includes the Pmerr1-Omerr1-being combined with merR1-GFP fusion protein
Biotin double-stranded DNA system, in system, Pmerr1-Omerr1-biotin double-strand is fixed on Streptavidin bag by biotin
In the high-affinity black ELISA Plate (Thermo fisher scientific, Yokohama, Japan) of quilt, merR1-GFP melts
The preparation method of hop protein is with embodiment 1,2, and the preparation method of Pmerr1-Omerr1-biotin double-strand system is with in embodiment 3
(3).
Taking out detection device, preparation detection buffer from 4 DEG C of refrigerators, detection buffer components is: 20mmol/l
Tris-HCl, pH7.9,1.0mol/l NaCl, 0.1%Tween20.It is separately added into a series of Concentraton gradient in detection buffer
HgCl2.The final concentration making mercury ion is respectively 0 μ g/l, 10 μ g/l, 20 μ g/l, 30 μ g/l, 40 μ g/l, 50 μ g/l, 60 μ g/
l、70μg/l、80μg/l、90μg/l、100μg/l、500μg/l、1000μg/l、2000μg/l、3000μg/l.Take 150 microlitre inspections
Survey buffer to be placed in detection device cell, cultivate 5 minutes, supernatant is transferred to another one common microwell plate cell
In.With hand-held green fluorescent protein detector (GFP-pen, GFP100, Photon systems instruments, Brno,
Czech Republic) its fluorescence intensity is measured.To drawing standard curve, knot between fluorescence intensity and mercury ion content
As shown in figure 14, in figure, 0～15 are shown that containing 0 μ g/l, 10 μ g/l, 20 μ g/l, 30 μ g/l, 40 μ g/l, 50 μ g/ fruit successively
l、60μg/l、70μg/l、80μg/l、90μg/l、100μg/l、500μg/l、1000μg/l、2000μg/l、3000μg/l Hg
(II) solution.By Figure 14 it will be seen that present one " S " type curve, mercury ion content between mercury ion content and fluorescence intensity
And present relation one to one between fluorescence intensity, the mercury ion content of its correspondence can be calculated according to fluorescence intensity level.
Experiment pedotheque picks up from certain truck garden, and this vegetable plot is owing to carrying out sewage irrigation and mercury-contaminated.This plot
The soil weight is 2.3 × 103kg/m3, water content is 18%, and pH value is 7.4.Take 50 grams of surface soils be suspended in 500 milliliters ultrapure
In water, with Glass rod, soil is smashed to pieces so that it is in suspension, stand 5 minutes, take supernatant coarse filter paper and filter, then use
Aperture is that the filter membrane of 0.45 micron carries out secondary filter to filtrate, and the liquid obtained by secondary filter is soil extract sample
Product.
Preparation detection buffer, detection buffer components is: 20mmol/l Tris-HCl, pH7.9,1.0mol/l
NaCl, 0.1%Tween20.Equal-volume detection pedotheque it is separately added in detection buffer.Take 150 microlitres and contain detection sample
The buffer of product is placed in cell, cultivates 5 minutes, is transferred to by supernatant in another common microwell plate.Hand-held is green
Fluorescin measuring instrument (GFP-pen, GFP100, Photon systems instruments, Brno, Czech Republic)
Its fluorescence intensity fluorescence is measured.Measurement result is compared with standard curve, calculates this plot Mercury in Soil and contain
Amount is 2.1mg/kg.
With 200 microlitre detection buffer, detection device is rinsed 3 times, then device is placed in-80 DEG C of freezing processing 2 little
Time, it is put in after taking-up in freezer dryer (FreeZone4.5, Labconco, USA) and carries out lyophilization, further take out and be placed in 4 DEG C
Refrigerator preserves, in case next time uses.
Claims (7)
1. a mercury ion detecting method, it is characterised in that: comprise the following steps: be combined with merR1-GFP fusion protein
Pmerr1-Omerr1-biotin double-stranded DNA system adds mercurous solion, takes out solution after standing and carry out fluorescence intensity
Measure, utilize standard curve to calculate mercury ion content in solution；
The preparation method of described Pmerr1-Omerr1-biotin double-stranded DNA comprises the following steps: by primer O/Pmerr1-F-
Biotin, O/Pmerr1-R-biotin are dissolved in buffer solution respectively, and equal-volume mixes, and 94 DEG C of degeneration 2 minutes, is down to room
Temperature obtains Pmerr1-Omerr1-biotin double-strand；The nucleotide sequence of primer O/Pmerr1-F-biotin such as SEQ ID NO:
Shown in 11, the nucleotide sequence of primer O/Pmerr1-R-biotin is as shown in SEQ ID NO:12.
Mercury ion detecting method the most according to claim 1, it is characterised in that: described merR1-GFP fusion protein by
Nucleotide sequence coded shown in SEQ ID NO:1.
Mercury ion detecting method the most according to claim 2, it is characterised in that: the system of described merR1-GFP fusion protein
For comprising the following steps:
(1) with bacillus megaterium genomic DNA as template, utilize primer P1F, P1R to expand merR1 gene, build plasmid P1；
(2) with plasmid P1 as template, primer P2F, P2R amplification two ends are utilized to carry BamH I and EcoR I restriction enzyme site
MerR1 gene, builds plasmid P2；
(3) with plasmid pEGFP as template, primer P3F, P3R is utilized to expand GFP gene, with EcoR I enzyme action GFP gene and plasmid
P2, builds plasmid P3 after connection；
(4) converting escherichia coli with plasmid P3, take positive colony and be enlarged cultivating, IPTG abduction delivering merR1-GFP merges
Albumen；
The nucleotide sequence of primer P1F as shown in SEQ ID NO:3, the nucleotide sequence of primer P1R such as SEQ ID NO:4 institute
Show, the nucleotide sequence of primer P2F as shown in SEQ ID NO:5, the nucleotide sequence of primer P2R as shown in SEQ ID NO:6,
The nucleotide sequence of primer P3F is as shown in SEQ ID NO:7, and the nucleotide sequence of primer P3R is as shown in SEQ ID NO:8.
Mercury ion detecting method the most according to claim 1, it is characterised in that: described Pmerr1-Omerr1-biotin is double
In chain DNA, 3 ' ends of a strand have biotin jag, and the oligonucleotide sequence of this strand is as shown in SEQ ID NO:2.
5. a mercury ion detecting device, it is characterised in that: include the Pmerr1-being combined with merR1-GFP fusion protein
Omerr1-biotin double-stranded DNA system；The preparation method of described Pmerr1-Omerr1-biotin double-stranded DNA includes following step
Rapid: primer O/Pmerr1-F-biotin, O/Pmerr1-R-biotin to be dissolved in respectively in buffer solution, equal-volume mixes,
94 DEG C of degeneration 2 minutes, are down to room temperature and obtain Pmerr1-Omerr1-biotin double-strand；The core of primer O/Pmerr1-F-biotin
Nucleotide sequence as shown in SEQ ID NO:11, the nucleotide sequence of primer O/Pmerr1-R-biotin such as SEQ ID NO:12 institute
Show.
Mercury ion detecting device the most according to claim 5, it is characterised in that: Pmerr1-Omerr1-in described system
Biotin double-stranded DNA is fixed in the coated ELISA Plate of Streptavidin by biotin.
Mercury ion detecting device the most according to claim 6, it is characterised in that: its preparation method comprises the following steps: will
Pmerr1-Omerr1-biotin double-stranded DNA is placed in the coated ELISA Plate of Streptavidin, and under room temperature, concussion is cultivated；Again will
MerR1-GFP fusion protein is placed in ELISA Plate, and under room temperature, concussion is cultivated, and to obtain final product.
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| CN110283768A (en) * | 2019-07-05 | 2019-09-27 | 中国农业大学 | A kind of nucleic acid-protein compound allosteric type microbe whole-cell gas sensor for heavy metal Hg detection |
| CN110241064A (en) * | 2019-07-05 | 2019-09-17 | 中国农业大学 | A kind of building and its application of the nucleic acid-protein compound allosteric type microbial whole-cell sensor for mercury ion detecting |
| CN114152601B (en) * | 2021-12-28 | 2023-09-15 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
| CN114560950A (en) * | 2022-03-15 | 2022-05-31 | 甘肃民族师范学院 | Genetic-coded organic mercury fluorescent probe and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288967A (en) * | 2013-05-20 | 2013-09-11 | 广州柏纵生物科技有限公司 | Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation |
| CN103290132A (en) * | 2013-06-18 | 2013-09-11 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nano-gold biosensor for detecting mercury ions and kit |
| CN103667448A (en) * | 2013-11-05 | 2014-03-26 | 中国科学院深圳先进技术研究院 | Difunctional aptamer detection kit and detection method |
| KR20150042936A (en) * | 2013-10-14 | 2015-04-22 | 한국생명공학연구원 | Mannosylphosphorylation Reaction Using YlMpo1 Protein |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130004979A1 (en) * | 2010-06-11 | 2013-01-03 | Wisconsin Alumni Research Foundation | Glycosyltransferase reversibility for sugar nucleotide synthesis and microscale scanning |
-
2014
- 2014-04-18 CN CN201410157445.0A patent/CN104297220B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288967A (en) * | 2013-05-20 | 2013-09-11 | 广州柏纵生物科技有限公司 | Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation |
| CN103290132A (en) * | 2013-06-18 | 2013-09-11 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nano-gold biosensor for detecting mercury ions and kit |
| KR20150042936A (en) * | 2013-10-14 | 2015-04-22 | 한국생명공학연구원 | Mannosylphosphorylation Reaction Using YlMpo1 Protein |
| CN103667448A (en) * | 2013-11-05 | 2014-03-26 | 中国科学院深圳先进技术研究院 | Difunctional aptamer detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| Bioluminescent Sensors for Detection of Bioavailable Hg(Ⅱ)in the Environment;Olga Selifonova et al.;《Applied and Environmental Microbiology》;19930930;第59卷(第9期);3083-3090 * |
| Use of bioluminescent bacterial sensors as an alternative method for measuring heavy metals in soil extracts;Tiina Petanen,Martin Romantschuk;《Analytica Chimica Acta》;20021231;第456卷;55-61 * |
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