CN108064300B - A kind of arsenite inhibiting factor reporter plasmid and its construction method and application - Google Patents

A kind of arsenite inhibiting factor reporter plasmid and its construction method and application Download PDF

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CN108064300B
CN108064300B CN201780001826.1A CN201780001826A CN108064300B CN 108064300 B CN108064300 B CN 108064300B CN 201780001826 A CN201780001826 A CN 201780001826A CN 108064300 B CN108064300 B CN 108064300B
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arsenite
inhibiting factor
plhpars9
arsr
reporter plasmid
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李先强
方云
姜昕
许玫英
郭俊
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St Grace Ltd
Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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Abstract

Disclose a kind of arsenite inhibiting factor reporter plasmid and its construction method and application.Arsenite inhibiting factor reporter plasmid is pLHPars9 and pLLPars9, and nucleotide sequence is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.Arsenite inhibiting factor reporter plasmid of the present invention is all very sensitive to arsenite, detection range is 0.04~50 μM, wide dynamic detection range is presented, therefore, arsenite inhibiting factor reporter plasmid of the present invention can be used as biosensor, the detection for arsenite.In addition, arsenite inhibiting factor reporter plasmid pLHPars9 of the present invention is in addition to can detect arsenite, it may also be used for detection antimonite.

Description

A kind of arsenite inhibiting factor reporter plasmid and its construction method and application
Technical field
The invention belongs to genetic engineerings and technical field of molecular biology, it is more particularly related to a kind of arsenious acid Salt inhibiting factor reporter plasmid and its construction method and application.
Background technique
Arsenic is widely distributed in entire environment as naturally occurring element, it exists with inorganic or organic form, inorganic Form has high toxicity.The guidelines for drinking water quality that the World Health Organization works out is 10 μ g/L, but in some countries, underground water is by arsenic Concentration is higher than the pollution of permissible level, this can constitute a threat to citizen's health.Exposed To Arsenic can draw from drinking water and food for a long time Play a variety of diseases, including cancer, cardiovascular disease, neurotoxicity and diabetes.Further Exposed To Arsenic in order to prevent quickly, passes through Effective local analysis technology help to monitor the arsenic in supplying water and necessitate.
Detection method based on bacterium is one kind when by arsenic pollution, monitors the emerging technology of arsenic inducible gene expression. With tradition based on fixed equipment, be not suitable for the method detected on the spot compared with, based on the detection method of bacterium for arsenic just Ground detection is relatively stable and cheap.Importantly, this can measure the bioavilability of arsenic, to illustrate contact and dosage Between difference.The report that the key component of measuring method based on bacterium is made of promoter/operon and reporter gene Gene.The synthetic biology occurred recently promotes the arsenic based on bacterium by the bacterium operon of redesign naturally occurring The development of biosensor, to realize the response more sensitive to the arsenic in environment.
Arsenic reactivity operon in Escherichia coli can adjust that (ArsR belongs to by arsenite inhibiting factor (ArsR) Smt/ArsR family), family's element can be used as transcription inhibitory factor, when arsenic compound is not present, RNA polymerase be blocked to enter It is combined afterwards with promoter/operon sequence of target gene.After arsenic, and isolation of promoter, and then activated gene is expressed. Plasmid R773 and escherichia coli chromosome ArsR is homodimer, is respectively had positioned at its DNA binding domain initial position Cys32-Val-Cys-Asp-Leu-Cys arsenic binding sequence.ArsR in Acidithiobacillus ferrooxidans strain GF does not have in the position Binding sequence, but its cysteine residues is located at 95,96 and 102.About ArsR albumen and its combination operon and to it The more information being adjusted will be helpful to rewrite biosensor basic molecular composition, including inhibiting factor, promoter and DNA binding site.
The reporter gene that people have developed various buildings carrys out the induction of gene expression.Ideally, good Reporter gene should show the wide dynamic range of hypersensitivity and specificity, low endogenous background and reaction.In addition, based on report The detection method of gene answer it is easy to use, it is relatively cheap and safe.Autofluorescence reporter gene green fluorescent protein (GFP) is one The ideal reporter gene of kind Noninvasive.Reality both can be provided without additional substrate without cell cracking in analytic process When detect.But since detection does not include enlarging function, sensibility is relatively low, to limit its application.Enzyme reports base Cause for example, luciferase and beta galactosidase have hypersensitivity, thus is widely applied.Beta galactosidase report The problem of accusing gene is, naturally occurring in bacterium to will lead to high background.In reported research, firefly is used Luciferase rather than bacterial luciferase are as reporter gene, because the quantum yield of firefly luciferase is close to 90%, And bacterial luciferase is only proximate to 5-10%.Measuring method based on luciferase quickly, conveniently, and has extensive linear Dynamic range.In addition, its sensibility and short-half-life become the ideal chose for measuring interim gene expression.
Summary of the invention
It is an object of the invention to: overcome deficiency present in existing arsenic detection, arsenite inhibiting factor report is provided Accuse gene plasmid pLHPars9 and pLLPars9 and its construction method and application.
To achieve the above object of the invention, the present invention provides a kind of arsenite inhibiting factor reporter plasmids PLHPars9 and pLLPars9, wherein the nucleotide sequence of pLHPars9 is as shown in SEQ ID NO:1, the nucleosides of pLLPars9 Acid sequence is as shown in SEQ ID NO:2.Arsenite inhibiting factor reporter plasmid of the present invention is with ArsR- luciferase Common original part, and be added before R773ArsR operon (Fig. 1) respectively from Escherichia coli and Acidithiobacillus ferrooxidans strain GF (A.ferrooxidans) the two basic change sequence of chromosome.And the metal specificity of the two is different: pLLPars9 is to arsenious acid Salt has specificity, and pLHPars9 has specificity to arsenite and antimonite.PLHPars9 and pLLPars9 it Between unique difference be the copy number of plasmid and the corresponding ratio of ArsR promoter/operon sequence in connection.
The construction method of arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 of the present invention include such as Lower step:
(1) high copy number plasmid from pGFPuv, containing pUC replication orgin is used, with Fluc GFPuv gene at the site gene substitution high copy number plasmid XbaI and EcoRI, and digested using HindIII and PvuII To eliminate lac promoter sequence；
(2) segment of the promoter region containing 773 arsenite inhibiting factor operon of R is synthesized, nucleotide sequence is such as Shown in SEQ ID NO:3, and the luciferase genes of upstream at HindIII and XbaI are cloned, obtains pLHPars4；
(3) by the segment of the promoter region containing 773 arsenite inhibiting factor operon of R described in step (3) together with volume The 1st~102, amino acid segment of code arsenite inhibiting factor is cloned together obtains pLHPars5；
(4) it is respectively synthesized containing arsenite inhibiting factor binding sequence (EC-BS) in escherichia coli chromosome, such as SEQ Shown in ID NO:4, and the segment (AF- containing arsenite inhibiting factor binding sequence in Acidithiobacillus ferrooxidans strain GF BS), as shown in SEQ ID NO:5, and it is inserted respectively between two sites HindIII and PvuII of pLHPars5, difference gram Grand acquisition pLHPars7 and pLHPars10；
(5) it is respectively synthesized Escherichia coli/Acidithiobacillus ferrooxidans strain GF chromosome arsenite inhibiting factor binding sequence (EC-BS/AF-BS), arsenite inhibiting factor binding sequence (EC-BS) and two parts of acidophilus oxygen in two parts of escherichia coli chromosomes Change arsenite inhibiting factor binding sequence (AF-BS) in ferrous Thiobacillus chromosome, and clones respectively into pLHPars5, point PLHPars9, pLHPars11 and pLHPars12 are not obtained；
(6) amplification contains EC-BS/AF-BS, arsenite inhibiting factor operon and arsenite inhibiting factor amino acid 1st~102 coding region is then inserted into pACYC184 matter together with the segment in the luciferase gene downstream of pLHPars9 Between the site XbaI and HindIII of grain, pLLPars9 is obtained；
Wherein, the pACYC184 plasmid is to obtain at New England Biolabs (Cat#E4152S), and contain Low copy number p15A replication orgin.
Since arsenite inhibiting factor reporter plasmid pLHPars9 and pLLPars9 of the present invention have arsenious acid Salt specificity, therefore, can be used for detecting arsenite.In addition, arsenite inhibiting factor reporter plasmid pLHPars9 is also There is specificity to antimonite, therefore, it may also be used for detection antimonite.
It in order to achieve the above-mentioned object of the invention, is by this hair the present invention also provides a kind of arsenite biosensor Bright arsenite inhibiting factor reporter plasmid pLHPars9 and/or pLLPars9 conversion bacillus coli DH 5 alpha obtains, the life Object sensor is made of high copy number plasmid or low-copy-number plasmid, very sensitive to arsenite.
In addition, the present invention also provides a kind of antimonite biosensor, be inhibited by arsenite of the present invention because Sub- reporter plasmid pLHPars9 conversion bacillus coli DH 5 alpha obtains, very sensitive to antimonite.
Compared with the existing technology, the invention has the following beneficial effects:
(1) it is a discovery of the invention that the sensibility of the biosensor based on arsenic reporter gene depends in the case where no arsenic The maximum suppression of reporter gene and its highest inductivity in the presence of arsenic.Present invention demonstrates that high by providing Copy number plasmid can be realized the high level expression of luciferase gene, but this method will lead to low-level induction, this show with Corresponding promoter/operon sequence is compared, and the amount of endogenous ArsR albumen is too low.Most of promoter/operons are without ArsR Albumen is for combining, so as to cause any inhibiting.In order to can be adjusted, the present invention be firstly introduced into a kind of Escherichia coli The expression of ArsR inhibiting factor-Luciferase fusion albumen, expressed fusion protein can apply its promoter/operon anti- Feedback inhibits.Secondly, introducing two ArsR binding sequences, one comes from Escherichia coli, another comes from acidophilus ferrous oxide sulphur Bacillus chromosome.Using highly expressed ArsR albumen and preferable ArsR binding sequence, can be carried out by high copy number plasmid high Horizontal luciferase expression, thus the detection of arsenite.
(2) secondly, although the expression of luciferase gene is relatively low, present invention low-copy-number plasmid substitutes high copy Number plasmid still can express good inductivity.
(3) Monitoring lower-cut of biosensor of the present invention is 0.04 μM of arsenite (~5 μ g/L), with text so far The Monitoring lower-cut for offering the best biosensor of middle report is the same.In addition, by being copied to based on high copy number plasmid and based on low The biosensor of shellfish number plasmid is compared, it is found that it is different the reaction of arsenite and metal specificity, sub- Arsenate inhibiting factor reporter plasmid pLHPars9 has specificity to arsenite and antimonite.
Detailed description of the invention
With reference to the accompanying drawings and detailed description, the present invention and beneficial effect are described in detail.
Fig. 1 is that the arsenide of the reporter plasmid of all components of the present invention induces schematic diagram；Wherein, reporter plasmid After being transformed into bacillus coli DH 5 alpha, handled respectively 2 hours with and without 10 μM of sodium arsenites, according to processed cell and without The control cell of processing is compared, and is measured uciferase activity and is calculated fold induction.
Fig. 2 is that the EMSA of PLHPars11, pLHPars12 and pLHPars9 Reporter gene vector of the present invention compares.A is indicated It is handled e.colidh5αcell 2 hours with 10 μM of arsenites, prepares cell lysate and copied respectively with containing 2 copy AF-BS, 2 The probe mixing of shellfish EC-BS, the 1 EC-BS and AF-EC biotin labeling copied, carry out EMSA analysis, control is without arsenious acid Salt treatment e.colidh5αcell lysate is mixed with correspondent probe.B indicates to handle bacillus coli DH 5 with 10 μM of arsenites α cell 2 hours, cell lysate was prepared with the probe of the EC-BS biotin labeling of 1 copy and mixes progress EMSA analysis, control It is to be mixed without arsenite processing e.colidh5αcell lysate with 1 copy EC-BS probe.
Fig. 3 is the comparison of pLHPars9 and pLLPars9 transformed cells of the present invention.With range of concentrations be 0,0.1,1,10 to 100 μM of arsenite handles pLHPars9 and pLLPars9 transformed cells 2 and 4 hour.It collects cell and carries out luciferase survey It is fixed.
Fig. 4~8 are the dynamic range and detectable limit of pLHPars9 and pLLPars9 transformed cells of the present invention.With 0,50, 100,200,400,600 and 800 μM of arsenites (Fig. 4), 0,0.2,0.4,0.6,0.8 and 1.0 μM of arsenite (Fig. 5~6) PLHPars9 and pLLPars9 transformed cells are handled with 0,0.02,0.04,0.06,0.08 and 0.1 μM of arsenite (Fig. 7~8). After processing 1 hour, collects cell and carry out luciferase assay.
Fig. 9~10 are the metal specificity of pLHPars9 and pLLPars9 transformed cells of the present invention.Respectively with or without 1 μM K+、Na+、Mg2+、Zn2+、Ca2+、Hg2+、Sb3+、Mn2+、Ni2+、Cr3+、Co2+、Cd2+、Cr2+、Cu2+、As5+And As3+To pLHPars9 It is handled 1 hour with pLLPars9 transformed cells, and collects cell and carry out luciferase assay.
Specific embodiment
In order to be more clear the purpose of the present invention, technical solution and advantageous effects, with reference to embodiments, to this Invention is further elaborated.It should be understood that embodiment described in this specification is just for the sake of this hair of explanation It is bright, be not intended to limit the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substance had no to result It influences.
Embodiment 1
1. plasmid construction
From a series of high copy number plasmids of pGFPuv (Clontech), contain pUC replication orgin (ColE1).Make Substitute the GFPuv gene at the site XbaI and EcoRI with Fluc gene, and using HindIII and PvuII into Row digestion is to eliminate lac promoter sequence.The segment of the 91bp promoter region containing R 773ArsR operon is synthesized, and is cloned The luciferase genes of upstream at HindIII and XbaI obtain pLHPars4 carrier.Synthesize the segment containing 91bp promoter region Together with 1-102, the amino acid segments of coding ArsR, clone obtains pLHPars5.It is respectively synthesized and is dyed containing Escherichia coli ArsR binding sequence or the segment containing binding sequence in Acidithiobacillus ferrooxidans strain GF (AF-BS) in body (EC-BS), and respectively It is inserted between two sites HindIII and PvuII of pLHPars5, clone obtains pLHPars7 and pLHPars10 respectively.This Outside, it is respectively synthesized the segment of EC-BS/AF-BS, two parts of EC-BS and two part of AF-BS, and is cloned respectively into pLHPars5, respectively Obtain pLHPars9, pLHPars11 and pLHPars12.It is obtained at New England Biolabs (Cat#E4152S) PACYC184 plasmid contains low copy number p15A replication orgin.Amplification contains EC-BS/AF-BS, ArsR operon and ArsR amino The coding region of acid 1-102 is then inserted into pACYC184 together with the segment in the luciferase gene downstream of pLHPars9 The site XbaI and HindIII between, obtain pLLPars9.
The nucleotide sequence of gained pLHPars9 is as shown in SEQ ID NO:1, the nucleotide sequence of pLLPars9 such as SEQ Shown in ID NO:2.
In above-mentioned steps, the nucleotides sequence of the segment of the promoter region containing 773 arsenite inhibiting factor operon of R Column contain arsenite inhibiting factor binding sequence such as SEQ in escherichia coli chromosome (EC-BS) as shown in SEQ ID NO:3 Shown in ID NO:4, the segment containing binding sequence in Acidithiobacillus ferrooxidans strain GF (AF-BS) as shown in SEQ ID NO:5, The nucleotide sequence of the coding region of 1-102, ArsR amino acid is as shown in SEQ ID NO:6, and luciferase gene sequence is such as Shown in SEQ ID NO:7.
2. processing and measurement
Plasmid is converted into bacillus coli DH 5 alpha competent cell.Picking single bacterium colony is aggressively shaken inoculation 12- at 37 DEG C 16 hours.According to OD value, it is diluted overnight culture with the culture medium containing antibiotic, it is dilute that high copy number plasmid is about pressed to 1:500 It releases, low-copy-number plasmid is diluted by 1:10.Diluted cell is further cultured for 3-4 hours in 37 DEG C of incubators, until O.D. value Reach 0.6.The cell liquid for taking 400 μ L is handled with the sodium arsenite (Sigma) of various concentration.By 50 μ L luciferase substrates and The DH5 α cell of 20 μ L and the mixing of 5 μ L cell pyrolysis liquids, are measured with the machine (Veritas) that luciferase detects, PLHPars9 shows high-caliber luciferase gene expression.
3. electrophoretic mobility measures (EMSA)
1mL overnight culture is centrifuged 1 minute with 10,000rpm to prepare cell lysate.Sediment is suspended from again 300 μ L lysis buffers (10mM Tris-HCl, pH8.0,0.1M NaCl, 1mM EDTA and 0.5% [w/v] Triton X- 100) in, 30 are cultivated at room temperature with the 25 freshly prepd lysozyme solns of μ L (10mg/mL, pH8.0 in 10mM Tris-HCl) Minute.After the completion of centrifugation, gel shift measurement is carried out using supernatant.1-3 μ g cell lysate, 5 times of 2 μ L are combined into buffering Liquid and 1 μ L poly- (I-C) mixing, are incubated for 5 minutes on ice.1 μ L biotinylated probes are added in mixture, are cultivated at 22 DEG C 30 minutes；Using 6.5% non-denaturing polyacrylamide gel, by each reaction mixture in 0.5 times of TBE at 4 DEG C of 100V Separation about 50 to 60 minutes.After gel is transferred on NB film, 15mL Block buffer is added at room temperature and continues 20 minutes It is closed, then with Streptavidin-HRP and by biotin on luminol (Pierce) enhanced chemiluminescence substrate detection trace The probe of label.Image is obtained using imager.
Experimental example 1 realizes high-caliber luciferase gene expression using high copy number plasmid
It is with 10 μM of sodium arsenites that transformed cells processing 2 is small in order to assess the arsenite inductivity of luciferase gene When；As shown in Figure 1, not observing the induction that the arsenite of apparent uciferase activity mediates.Escherichia coli chromosome ArsR can be used as trans- regulatory factor, for combining with escherichia coli chromosome and plasmid R773 operon.Therefore, no induction A simplicity of explanation be, with ArsR combine promoter/operon sequence compared with, free ArsR occupies the majority, and reason is to provide There is high copy number plasmid in the cell of high level expression luciferase.Arsenite is added, it can be from promoter/operon Except ArsR, compared with non-binding form, do not make uciferase activity that significant change occur, this is because the starting in conjunction with ArsR Son/operon quantity is very limited.
The coexpression of experimental example 2ArsR reduces the basal expression of reporter gene
By the feedback control of reporter gene expression, usually dropped using the coexpression of external source ArsR and luciferase gene The foundation level of low Poison element enzymatic activity.ArsR can carry out the expression of polycistron formula by reporter gene or be combined into fusion protein To express.Missing functional analysis is carried out to the ArsR by being co-expressed with beta-lactamase and shows to adjust activity without big The C-terminal from amino acid 93 to 117 of enterobacteria ArsR；In order to introduce the feedback regulation to luciferase gene expression, A short ArsR is constructed in the 1-102 amino acid of luciferase N-terminal, to obtain Reporter gene vector pLHPars5 (Fig. 1).This with ArsR 1-102 amino acids short ArsR under R773ArsR promoter/operon with fluorescein Enzyme is expressed as fusion protein together.Cell 2 hours for having converted pLHPars5 are handled with 10 μM of sodium arsenites.Luciferase is surveyed Surely 1.5 times of inductivities (Fig. 1) of uciferase activity are shown.The inductivity of uciferase activity is not as expected.
Experimental example 3 adds ArsR binding sequence before Ars operon
Common methods in mammlian system are, by before promoter gene be inserted into additional cis-acting elements It copies preferably to measure the inductivity of reporter gene.It is additional big by being inserted into one between ArsR gene and reporter gene Enterobacteria ArsR binding sequence copy, people have been reduced using this method to report in arsenite biosensor building process The basic background of gene activity.It shows slightly lower compared with the control reporter gene for being not added with additional ArsR binding sequence Background.In the present invention, an Escherichia coli ArsR combination sequence is constructed before R773ArsR promoter/operon of pLHPars5 (EC-BS) or Acidithiobacillus ferrooxidans strain GF ArsR binding sequence (AF-BS) are arranged, Reporter gene vector pLHPars7 is respectively obtained With pLHPars10 (Fig. 1).Show within 2 hours that 2 times of uciferase activity lure with 10 μM of arsenite processing DH5 α transformed cells The property led (Fig. 1).Compared with the pLHPars5 for being not added with additional ArsR binding sequence, with pLHPars7 in the reacting of arsenite It is not improved with pLHPars10.Then, the EC-BS of 2 copies is added respectively before promoter/operon of pLHPars5 Or each 1 copy of AF-BS and EC-BS and AF-BS, respectively obtain pLHPars11, pLHPars12 and pLHPars9 (Fig. 1).Band There are the Reporter gene vector pLHPars11 of 2 copy EC-BS and the pLHPars12 with 2 copy AF-BS to induce in arsenite In do not show effect (Fig. 1).It is worth noting that, the Reporter gene vector with 1 copy EC-BS and 1 copy AF-BS PLHPars9 can show 5 times of luciferase fold induction (Fig. 1) after being handled 2 hours with 10 μM of arsenites, be better than 2 The EC-BS or AF-BS of copy.These are the result shows that the two binding sequences together can be complimentary to one another, so that it is better than big The EC-BS or AF-BS of 2 copies are individually added in enterobacteria ArsR combination.
Difference of the experimental example 4ArsR in its binding sequence
When interacting for Direct Test and Escherichia coli ArsR, whether individual EC-BS and AF-BS are different from 2 and copy The EC-BS or AF-BS of shellfish use EMSA, prepare cell lysate from bacillus coli DH 5 alpha-pLHPars5, with and without 10 μ M arsenite is handled 2 hours.The ArsR protein of purifying is replaced to carry out EMSA using cell lysate, to measure natural phase Interaction.According to the sequence of EC-BS and AF-BS, the end chemical synthesis 5' has the probe of biotin labeling.Synthesis is with 2 copies EC-BS, 2 copy AF-BS and AF-BS and EC-BS it is each 1 copy probe, to carry out EMSA analysis.Fig. 2A result is aobvious Show, the combination of AF-BS-EC-BS is better than the AF-BS of the copy of EC-BS and 2 of 2 copies, consistent with induction data.This shows ArsR It can be better than individually in conjunction with EC-BS or AF-BS spatially in conjunction with the AF-BS and EC-BS of 1 copy.It is looked for by prioritization scheme To better ArsR binding sequence, and several key positions in ArsR binding sequence are determined.The EC-BS and AF-BS of 1 copy The nucleotide of junction containing EC-BS and AF-BS helps preferably to be interacted with Escherichia coli ArsR.
In addition, band is clearly then shifted there are two meetings in EMSA result using the probe of the binding sequence with 2 copies, This shows there is two kinds, and ArsR dimer and the tetramer coexist or the DNA probe of two kinds of forms；One kind having one ArsR dimer, there are two ArsR dimers for another kind tool.In former possibility, dimer and the tetramer are coexisted, and another There is only dimers in kind possibility.If dimer and the tetramer coexist, even if a DNA binding sequence is used to arrange as probe Both should also detect.As shown in Figure 2 B, a displacement band is only observed, the relatively low strap being equivalent in Fig. 2A, this shows only to deposit It coexists in ArsR dimer rather than with the tetramer.Therefore, two displacement bands with probe are respectively by a dimer of ArsR It is formed with two dimers.
Addition arsenite can induce ArsR dissociated from binding sequence, it is consistent with above-mentioned conclusion, inhibiting factor need with Isolation of promoter.Different from MerR family inhibiting factor, without dissociation, but metal bonding can induce conformation from undesirable Promoter is changed into stronger promoter.
Experimental example 5 is compared the biosensor with high copy number plasmid and low-copy-number plasmid
In order to check whether inductivity is different, introduces from plasmid when promoter/operon copy number is lower The low copy number p15A replication orgin of pACYC184 obtains pLLPars9 instead of high copy number pUC replication orgin.With 10 μM Arsenite is handled pLLPars9 transformed cells 2 hours, and compared with untreated control cell, which show > 10 times lure The property led is better than pLHPars9 (Fig. 1).It is dense with wider range in order to more specifically be compared pLHPars9 and pLLPars9 The arsenite of degree handles transformed cells, is handled 2 and 4 hours with 0,0.1,1,10 to 100 μM of arsenite.It is the same as expected, The foundation level of luciferase is far below pLHPars9, while the also significant lower (figure of maximum horizontal in pLLPars9 transformed cells 3).Using high copy number plasmid, 0.1 to 1 μM of change dramatically can occur in 2 hours for inductivity, and arsenite in 4 hours Change in the range more slowly.Using 100 μM of arsenites, the uciferase activity of pLHPars9 is observed in 4 hours Decline, but do not observed at 2 hours.Induction pattern with low-copy-number plasmid is different from the processed high copy of arsenite Number plasmid.In addition, not observing that luciferase activity declines under conditions of there is low-copy-number plasmid.These results indicate that high The arsenite inductivity of luciferase is different between copy number and low-copy-number plasmid.
The dynamic range and detectable limit of the DH5 α cell of experimental example 6pLHPars9 and pLLPars9 conversion
Application of the biosensor in the field is estimated can be completed in a short time analysis.In some researchs, with arsenic chemical combination The shortest time of object processing is 1 hour.With the arsenite of various concentration range (0,50,100,200,400,600 to 800 μM) Handle cell 1 hour that there is conversion to have pLHPars9 or pLLPars9.Luciferase assay show pLHPars9 and PLLPars9 biosensor reaches peak value in 50 μM and 100 μM of arsenite concentration respectively, and activity begins to decline (figure later 4).In addition, handling two kinds cell 1 hour with 0,0.2,0.4,0.8 to 1.0 μM of arsenite, linear response (Fig. 5 is all shown ~6).In order to study detectable limit, handled cell 1 hour with 0,0.02,0.04,0.06,0.08 and 0.16 μM of arsenite.If The detectable limit for specifying the arsenite concentration that can cause obviously to induce is that background adds 2 times of standard deviations (SD), then PLHPars9 and pLLPars9 biosensor can show 0.04 μM of arseniteDetectable limit (Fig. 7~ 8), it is lower than the 10 μ g/L World Health Organization (WHO) guides.Accordingly, it is shown that the two can be employed as arsenite biosensor.
Difference of experimental example 7pLHPars9 and the pLLPars9 biosensor to the selective reaction of metal
The specificity of both biosensors is tested, and the metal different from 17 kinds is compared.In general, such as Shown in Fig. 9~10, biosensor pLLPars9 ratio pLHPars9 can show more specificity.In surveyed metal, PLLPars9 is only by the induction of arsenite, though slightly inhibited by Hg (II), using arsenate and when other metals not Observe that it has apparent induction.Biosensor pLHPars9 shows have very big difference with pLLPars9.In addition to Arsenite As (III) reaction is outer, also has reaction to antimonite Sb (III), also has slight reaction to arsenate As (V). Difference between pLLPars9 and pLHPars9 is its promoter/operon copy number and its ratio corresponding to ArsR albumen Example.Therefore, the metal specificity of ArsR can be by ArsR albumen, ArsR combination promoter/operon concentration or ArsR to it It is adjusted in conjunction with promoter/operon ratio.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party Formula carries out change and modification appropriate.Therefore, the invention is not limited to the specific embodiments disclosed and described above, to this Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification In use some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Sequence table
<110>Guangdong Microbes Inst；Holy Gneuss Co., Ltd
<120>a kind of arsenite inhibiting factor reporter plasmid and its construction method and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2686
<212> DNA
<213> pLHPars9 (artificial sequence)
<400> 1
atggaaancg ccaagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 60
tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 120
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 180
agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 240
cagctgacac attcgttaag tcatatatgt ttttgacttt ttatccacga atatttcttg 300
cagtattgaa agctttgtga ttaatcatat gcgtttttgg ttatgtgttg tttgacttaa 360
tatcagagcc gagagatact tgttttctac aaaggagagg gaaatgttgc aactaacacc 420
acttcagtta tttaaaaacc tgtccgatga aacccgtttg ggtatcgtgt tgttgctcag 480
ggagatggga gagttgtgcg tgtgtgatct ttgcatggca ctggatcaat cacagcccaa 540
aatatcccgt catctggcga tgctacggga aagtggaatc cttctggatc gtaaacaggg 600
aaaatgggtt cactaccgct tatcaccgca tattccttca tgggctgccc agattattga 660
gcaggcctgg ttaagccaac aggacgacgt tcaggtcatc gcacgcaagc ctctagagga 720
tccccgggta ccggtagaaa aaatggaaga cgccaaaaac ataaagaaag gcccggcgcc 780
attctatccg ctggaagatg gaaccgctgg agagcaactg cataaggcta tgaagagata 840
cgccctggtt cctggaacaa ttgcttttac agatgcacat atcgaggtgg acatcactta 900
cgctgagtac ttcgaaatgt ccgttcggtt ggcagaagct atgaaacgat atgggctgaa 960
tacaaatcac agaatcgtcg tatgcagtga aaactctctt caattcttta tgccggtgtt 1020
gggcgcgtta tttatcggag ttgcagttgc gcccgcgaac gacatttata atgaacgtga 1080
attgctcaac agtatgggca tttcgcagcc taccgtggtg ttcgtttcca aaaaggggtt 1140
gcaaaaaatt ttgaacgtgc aaaaaaagct cccaatcatc caaaaaatta ttatcatgga 1200
ttctaaaacg gattaccagg gatttcagtc gatgtacacg ttcgtcacat ctcatctacc 1260
tcccggtttt aatgaatacg attttgtgcc agagtccttc gatagggaca agacaattgc 1320
actgatcatg aactcctctg gatctactgg tctgcctaaa ggtgtcgctc tgcctcatag 1380
aactgcctgc gtgagattct cgcatgccag agatcctatt tttggcaatc aaatcattcc 1440
ggatactgcg attttaagtg ttgttccatt ccatcacggt tttggaatgt ttactacact 1500
cggatatttg atatgtggat ttcgagtcgt cttaatgtat agatttgaag aagagctgtt 1560
tctgaggagc cttcaggatt acaagattca aagtgcgctg ctggtgccaa ccctattctc 1620
cttcttcgcc aaaagcactc tgattgacaa atacgattta tctaatttac acgaaattgc 1680
ttctggtggc gctcccctct ctaaggaagt cggggaagcg gttgccaaga ggttccatct 1740
gccaggtatc aggcaaggat atgggctcac tgagactaca tcagctattc tgattacacc 1800
cgagggggat gataaaccgg gcgcggtcgg taaagttgtt ccattttttg aagcgaaggt 1860
tgtggatctg gataccggga aaacgctggg cgttaatcaa agaggcgaac tgtgtgtgag 1920
aggtcctatg attatgtccg gttatgtaaa caatccggaa gcgaccaacg ccttgattga 1980
caaggatgga tggctacatt ctggagacat agcttactgg gacgaagacg aacacttctt 2040
catcgttgac cgcctgaagt ctctgattaa gtacaaaggc tatcaggtgg ctcccgctga 2100
attggaatcc atcttgctcc aacaccccaa catcttcgac gcaggtgtcg caggtcttcc 2160
cgacgatgac gccggtgaac ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat 2220
gacggaaaaa gagatcgtgg attacgtcgc cagtcaagta acaaccgcga aaaagttgcg 2280
cggaggagtt gtgtttgtgg acgaagtacc gaaaggtctt accggaaaac tcgacgcaag 2340
aaaaatcaga gagatcctca taaaggccaa gaagggcgga aagatcgccg tgtaactggc 2400
ttcagttaac tgctccggta gcagtaaggc tgtctgcatc taaaaaattt gcctgaacat 2460
atatgtttta tcaaatgcga ggtatttaag atgaaaacgt taatggtatt tgacccggcg 2520
atgtgttgca gcaccggcgt ctgcggtaca gatgttgatc aggctctggt cgatttttct 2580
acagatgtgc aatggctcaa acaatgcggt gtacaaattg agcgtttcaa tcttgcgcaa 2640
caaccgatga gctttgtaca gaacgagaag gtcaaagcgt ttattg 2686
<210> 2
<211> 5654
<212> DNA
<213> pLLPars9 (artificial sequence)
<400> 2
gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60
gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120
ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180
tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240
aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300
ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360
ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420
ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480
gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540
acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600
ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660
aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720
actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780
ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840
agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900
agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960
tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020
gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080
tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140
gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200
agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260
tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320
cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380
aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga acacattcgt 1440
taagtcatat atgtttttga ctttttatcc acgaatattt cttgcagtat tgaaagcttt 1500
gtgattaatc atatgcgttt ttggttatgt gttgtttgac ttaatatcag agccgagaga 1560
tacttgtttt ctacaaagga gagggaaatg ttgcaactaa caccacttca gttatttaaa 1620
aacctgtccg atgaaacccg tttgggtatc gtgttgttgc tcagggagat gggagagttg 1680
tgcgtgtgtg atctttgcat ggcactggat caatcacagc ccaaaatatc ccgtcatctg 1740
gcgatgctac gggaaagtgg aatccttctg gatcgtaaac agggaaaatg ggttcactac 1800
cgcttatcac cgcatattcc ttcatgggct gcccagatta ttgagcaggc ctggttaagc 1860
caacaggacg acgttcaggt catcgcacgc aagccggatc ctggaagacg ccaaaaacat 1920
aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca 1980
taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat 2040
cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat 2100
gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca 2160
attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga 2220
catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt 2280
cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca 2340
aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt 2400
cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag agtccttcga 2460
tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg 2520
tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag atcctatttt 2580
tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt 2640
tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag 2700
atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa gtgcgctgct 2760
ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc 2820
taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg gggaagcggt 2880
tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg agactacatc 2940
agctattctg attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc 3000
attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcaaag 3060
aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc 3120
gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga 3180
cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta 3240
tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc 3300
aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt 3360
ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac 3420
aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac 3480
cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa 3540
gatcgccgtg taagtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg 3600
tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc 3660
gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc 3720
gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc 3780
ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg 3840
gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc 3900
cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg 3960
tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc 4020
agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc 4080
acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg 4140
ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg 4200
ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg 4260
cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc 4320
ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt 4380
tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac 4440
gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa 4500
tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcccct acgtgctgct 4560
gaagttgccc gcaacagaga gtggaaccaa ccggtgatac cacgatacta tgactgagag 4620
tcaacgccat gagcggcctc atttcttatt ctgagttaca acagtccgca ccgctgtccg 4680
gtagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 4740
ttatcatgca actcgtagga caggtgccgg cagcgcccaa cagtcccccg gccacggggc 4800
ctgccaccat acccacgccg aaacaagcgc cctgcaccat tatgttccgg atctgcatcg 4860
caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctaaccgt 4920
ttttatcagg ctctgggagg cagaataaat gatcatatcg tcaattatta cctccacggg 4980
gagagcctga gcaaactggc ctcaggcatt tgagaagcac acggtcacac tgcttccggt 5040
agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga 5100
accgacgacc gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt 5160
attcaggcgt agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc 5220
ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag 5280
ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc 5340
gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt 5400
aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata 5460
aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg 5520
tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt 5580
tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct 5640
ttcattgcca tacg 5654
<210> 3
<211> 91
<212> DNA
<213>promoter region (artificial sequence) of 773 arsenite inhibiting factor operon of R
<400> 3
tgtgattaat catatgcgtt tttggttatg tgttgtttga cttaatatca gagccgagag 60
atacttgttt tctacaaagg agagggaaat g 91
<210> 4
<211> 32
<212> DNA
<213>contain escherichia coli chromosome (arsenite inhibiting factor binding sequence artifitial sequence in EC-BS)
<400> 4
cacattcgtt aagtcatata tgtttttgac tt 32
<210> 5
<211> 20
<212> DNA
<213>contain A.ferrooxidans (binding sequence artificial sequence in AF-BS)
<400> 5
atccacgaat atttcttgca 20
<210> 6
<211> 301
<212> DNA
<213>nucleotide sequence (artificial sequence) of 1-102, ArsR amino acid coding regions
<400> 6
caactaacac cacttcagtt atttaaaaac ctgtccgatg aaacccgttt gggtatcgtg 60
ttgttgctca gggagatggg agagttgtgc gtgtgtgatc tttgcatggc actggatcaa 120
tcacagccca aaatatcccg tcatctggcg atgctacggg aaagtggaat ccttctggat 180
cgtaaacagg gaaaatgggt tcactaccgc ttatcaccgc atattccttc atgggctgcc 240
cagattattg agcaggcctg gttaagccaa caggacgacg ttcaggtcat cgcacgcaag 300
c 301
<210> 7
<211> 1643
<212> DNA
<213>luciferase gene sequence (artificial sequence)
<400> 7
acgccaaaaa cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg 60
gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta 120
cagatgcaca tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt 180
tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg 240
aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg 300
cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc 360
ctaccgtggt gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc 420
tcccaatcat ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt 480
cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc 540
cagagtcctt cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg 600
gtctgcctaa aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca 660
gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat 720
tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg 780
tcttaatgta tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc 840
aaagtgcgct gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca 900
aatacgattt atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag 960
tcggggaagc ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca 1020
ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg 1080
gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg 1140
gcgttaatca aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa 1200
acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca 1260
tagcttactg ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta 1320
agtacaaagg ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca 1380
acatcttcga cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg 1440
ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg 1500
ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac 1560
cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca 1620
agaagggcgg aaagatcgcc gtg 1643

Claims (7)

1. a kind of arsenite inhibiting factor reporter plasmid pLHPars9, which is characterized in that the nucleotides sequence of pLHPars9 Column are as shown in SEQ ID NO:1.
2. a kind of arsenite inhibiting factor reporter plasmid pLLPars9, which is characterized in that the nucleotides sequence of pLLPars9 Column are as shown in SEQ ID NO:2.
3. the answering in arsenite detection of arsenite inhibiting factor reporter plasmid pLHPars9 described in claim 1 With.
4. the answering in arsenite detection of arsenite inhibiting factor reporter plasmid pLLPars9 described in claim 2 With.
5. the application of arsenite inhibiting factor reporter plasmid pLHPars9 according to claim 3, which is characterized in that The arsenite inhibiting factor reporter plasmid pLHPars9 is also used to the detection of antimonite.
6. a kind of arsenite biosensor, which is characterized in that be by arsenite inhibiting factor report described in claim 1 Gene plasmid pLHPars9 and/or arsenite inhibiting factor reporter plasmid pLLPars9 as claimed in claim 2 is accused to turn Change bacillus coli DH 5 alpha to obtain.
7. a kind of antimonite biosensor, which is characterized in that be by arsenite inhibiting factor report described in claim 1 Gene plasmid pLHPars9 conversion bacillus coli DH 5 alpha is accused to obtain.
CN201780001826.1A 2017-09-30 2017-09-30 A kind of arsenite inhibiting factor reporter plasmid and its construction method and application Active CN108064300B (en)

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