CN105755106A - Method for detecting dioxin persistent organic pollutants - Google Patents
Method for detecting dioxin persistent organic pollutants Download PDFInfo
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- CN105755106A CN105755106A CN201610167440.5A CN201610167440A CN105755106A CN 105755106 A CN105755106 A CN 105755106A CN 201610167440 A CN201610167440 A CN 201610167440A CN 105755106 A CN105755106 A CN 105755106A
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Abstract
The invention discloses a method for detecting dioxin persistent organic pollutants and relates to gene engineering. A CYP1A promoter sequence is obtained on zebra fish chromosome DNA through PCR (polymerase chain reaction) amplification according to design, and PGL6-TA-pCYP1A plasmid containing luciferase genes and TAbox is constructed. The detection efficiency of the recombinant plasmid on TCDD (tetrachlorodibenzo-p-dioxin) is improved by modifying a CYP1A promoter, and a system for rapidly and efficiently detecting TCDD and poison which also is AHR (aryl hydrocarbon receptor) ligand is constructed. The zebra fish cyp1a promoter sequence is modified with an overlap pcr technology and linked to a PGL6-TA carrier, the sensitivity of the modified promoter sequence to the dioxin persistent organic pollutants is improved, the modified promoter sequence is transfected to HepG2 cells, and the dioxin persistent organic pollutants in the environment can be rapidly detected.
Description
Technical field
The present invention relates to a kind of genetic engineering, be specifically related to a kind of two English class persistence organic pollutant detection sides
Method.
Background technology
Persistence organic pollutant (Persistent Organic Pollutants is called for short POPs) refers to persistently deposit
It is in environment there is the longest half-life, and can be gathered by food web, and human health and environment are adversely affected
Organic chemicals.
Persistence organic pollutant (POPs) refer to by various surrounding mediums (big gas and water, organism etc.) can grow away from
In environment, there is extended residual, bioconcentration, half volatile and high toxicity, to human health from migrating also long-term existence
With the organic pollution materials that environment has the natural of serious harm or synthetic.Two English class persistence organic pollutants refer to
Can make with intracellular polycyclic aromatic compound receptor (Aryl Hydrocarbon Receptor, AHR) protein binding
With, the persistence organic pollutant of induction downstream gene expression.This pollutant generally comprise multiring aromatic hydrocarbon organic pollution,
Polychlorinated biphenyl organic pollution and some herbicides.Wherein included tetrachloro dibenzo-p-two evil by people's extensive concern
English (Tetrachlorodibenzo-p-dioxin TCDD), benzopyrene (Benzoapyrene, BaP), naphthalene, anthracene, phenanthrene etc..And
TCDD is as material the strongest with AHR binding ability in this pollutant, and the present invention, as reference material, is used for verifying transformation
Sensitivity.
AHR and core dystopy albumen (Ah Receptor Nuclear Translocator, ARNT) thereof are a kind of with multi-ring
Compound fragrant hydrocarbon is the transcription factor of part, and major part hydrophobic aromatic hydrocarbon environment compound etc. is all this receptoroid
Part.AhR participates in the induction to some drug metabolism enzymes (such as P450 family CYP1A1, CYP1A12, CYP1B1 etc.) of the external poisonous substance
Process, and participate in regulating the expression of exogenous compounds metabolic enzyme gene (such as Cytochrome P450 1A1), and cytochrome
P4501A1 relevant with external medicine, the metabolism of poisonous substance and inactivation (Nie Shuwei, Xuchang is safe. aryl hydrocarbon receptor and the danger to human body thereof
Evil present Research [J]. Medical review, 2011,17 (1): 24-25.) in the case of not being combined with part, major part AhR location
In Cytoplasm, with heatshock protein 90 (heat shock protein 90, HSP90) dimer and hepatitis B virus x protein
Associated proteins 2 (hepatitis B virus X protein-associated protein 2, XAP2) combines, and is formed compound
Thing.The combination of HSP90 makes AhR maintain a kind of inactive, the state that can be combined with part.After AhR is combined with part TCDD,
Dissociate with HSP90, transport in nucleus.This process and the nuclear export signal (nuclear of AhR N ' end
Localization signal, NLS) relevant.Owing to this NLS with bHLH overlaps, during so combining without part, quilt
HSP90 is sheltered.HSP90 dissociates, and makes the NLS of AhR come out, and is combined with entering core complex, transports into core, then with cell
ARNT in core forms dimer, is attached to the DNA motif xenobiotics response element (xenobiotic-of target gene
Responsive element, XRE, also referred to as DRE) on, start transcribing of downstream gene.And AHR activation CYP1A gene is logical
Cross and obtain 5 ' T-GCGTG3 ' combinations in its promoter, start the expression of CYP1A.(Denison M S,Fisher J M,
Whitlock J P.Inducible,Receptor-Dependent Protein-DNA Interactions at a
Dioxin-Responsive Transcriptional Enhancer[J].Proceedings of the National
Academy of Science,1988,85(8):2528-2532.)
CYP450 enzyme system is one of biological catalytic enzyme with catalysis active function most in nature, and it has in organism
There is important physiological function.CYP450 can to various endogenous material (such as steroid, fatty acid etc.) and exogenous material (as
Herbicide, insecticide, carcinogen etc.) carry out I phase reaction (the Omura T.Forty years of of bioconversion
cytochrome P450[J].Biochemical and Biophysical Research Communications,1999,
266(3):690-698.).At present, due to the hypersensitivity of CYP450 enzyme system activity of isoenzyme exogenous material, can be made it
The important indicator polluted as earlier evaluations and detection aquatic ecological environmental system.In recent years, CYP450 is in environmental toxicology side
Face is of increased attention.
Summary of the invention
It is an object of the invention to provide a kind of two English class persistence organic pollutant detection methods.
The present invention specifically comprises the following steps that
1) with speckle known on National Center for Biotechnology Information (NCBI) website
Horse fish genome sequence is classified as stencil design pair of primers pCYP1A-F, pCYP1A-R, clones CYP1A (Chromosome 18-NC_
007129.6) promoter sequence, primer sequence is as follows:
PCYP1A-F:5 '-AAAGGTACCGAAACCCACGCCATGCAAAC-3 '
Kpn I
PCYP1A-R:5 '-AAAAAGCTTTCCTCCGCGTGACGTCACCG-3 '
Hind III
Wherein the forward primer at CYP1A promoter sequence is provided with KpnI restriction enzyme site, is provided with at downstream primer
HindIII restriction enzyme site, with Brachydanio rerio genome as template, with above-mentioned two primers, utilizes round pcr to amplify promoter sequence
Row:
This promoter sequence is positioned at 314, the upstream base of the expression start codon ATG of CYP1A gene.To amplify
The fragment come reclaims sequence fragment after Kpn I/Hind III double digestion, and with the experiment through Kpn I/Hind III double digestion
Room original plasmid vector PGL6-TA connects, and connects product Transformed E .coliDH5 α, with ampicillin (Amp) screening and cloning
Son, obtains recombiant plasmid PGL6-TA-pCYP1A, the recombiant plasmid PGL6-TA-pCYP1A obtained is delivered to Sheng Gong company with
Rvprimer3 universal primer checks order, and checking obtains the accuracy of plasmid sequence further；
2) it is template with the recombiant plasmid PGL6-TA-pCYP1A obtained, designs primer pCYP1A-2 × DRE-F,
PCYP1A-2 × DRE-R, carries out overlap PCR, transforms promoter sequence, and primer sequence is as follows:
2 × DRE-1F:5 '-TGTTACATACATCAATCTCC-3 '
2 × DRE-2F:5 '-ACTGCGAGCCCCCTCGCGTGTGTTACATACATCAATCTCC-3’
Aryl hydrocarbon receptor binding site
2 × DRE-1R:5 '-CACGCACACATACATGGTAC-3 '
2 × DRE-2R:5 '-CACGCGAGGGGGCTCGCAGTCACGCACACATACATGGTAC-3 '
Transformation postorder lists meaning:
With pCYP1A-F, 2 × DRE-1R is primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 1；2
× DRE-1F, pCYP1A-R are primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 2；PCYP1A-F, 2 ×
DRE-2R is primer, and sequence 1 is template, and PCR amplifies sequence 3；2 × DRE-2F, pCYP1A-R are primer, and sequence 2 is
Template, PCR amplifies sequence 4；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced sequence 3 and sequence 4, expands
Increase and aim sequence pCYP1A-2 × DRE, first aim sequence is cloned in carrier T, obtain correct transformation sequence through order-checking,
After Kpn I/Hind III double digestion, reclaim sequence fragment with the plasmid obtained, and with through Kpn I/Hind III double digestion
Laboratory original plasmid vector PGL6-TA connects, and connects product Transformed E .coliDH5 α, screens with ampicillin (Amp)
Clone, obtains recombiant plasmid PGL6-TA-pCYP1A-2 × DRE, the recombiant plasmid PGL6-TA-pCYP1A-2 × DRE that will obtain
Delivering to Sheng Gong company check order with Rvprimer3 universal primer, checking obtains the accuracy of plasmid sequence further；
3) using the recombiant plasmid PGL6-TA-pCYP1A-2 × DRE obtained is template, design primer 3 × DRE-F, 3 ×
DRE-R, carries out overlap PCR, transformation further to promoter sequence；
Primer sequence is as follows:
3 × DRE-F:5 '-ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGTGTTACATACATCAAT
-3 '
3 × DRE-R:5 '-CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCACACATACATG
GTAC-3 '
Transformation postorder lists meaning:
With pCYP1A-F, 3 × DRE-R is primer, and sequence 3 is template, and PCR amplifies sequence 5；3 × DRE-F, pCYP1A-
R is primer, and sequence 4 is template, and PCR amplifies sequence 6；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced
Sequence 5 and sequence 6, amplify aim sequence pCYP1A-3 × DRE；First aim sequence is cloned in carrier T, obtains through order-checking
Correct transformation sequence, reclaims sequence fragment with the plasmid that obtains after Kpn I/Hind III double digestion, and with through Kpn I/
Laboratory original plasmid vector PGL6-TA of Hind III double digestion connects, and connects product Transformed E .coliDH5 α, with ammonia benzyl
Penicillin (Amp) screening and cloning, obtains recombiant plasmid PGL6-TA-pCYP1A-3 × DRE；The recombiant plasmid PGL6-that will obtain
TA-pCYP1A-3 × DRE delivers to Sheng Gong company and checks order with Rvprimer3 universal primer, and checking obtains plasmid sequence further
The accuracy of row.
4) using the recombiant plasmid PGL6-TA-pCYP1A-3 × DRE obtained is template, design primer 4 × DRE-F, 4 ×
DRE-R, carries out overlap PCR, transformation further to promoter sequence.
Primer sequence is as follows:
4 × DRE-F:ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGACTGCGAGCC CCCTCGCGTG
4 × DRE-R:CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCGAGGG GGCTCGCAGT
Transformation postorder lists meaning:
Primer sequence is as follows:
4 × DRE-F:ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGACTGCGAGCC CCCTCGCGTG
4 × DRE-R:CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCGAGGG GGCTCGCAGT
With pCYP1A-F, 4 × DRE-R is primer, and sequence 5 is template, and PCR amplifies sequence 7；3 × DRE-F, pCYP1A-
R is primer, and sequence 6 is template, and PCR amplifies sequence 8；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced
Sequence 7 and sequence 8, amplify aim sequence pCYP1A-4 × DRE；First aim sequence is cloned in carrier T, obtains through order-checking
Correct transformation sequence, reclaims sequence fragment with the plasmid that obtains after Kpn I/Hind III double digestion, and with through Kpn I/
Laboratory original plasmid vector PGL6-TA of Hind III double digestion connects, and connects product Transformed E .coliDH5 α, with ammonia benzyl
Penicillin (Amp) screening and cloning, obtains recombiant plasmid PGL6-TA-pCYP1A-4 × DRE, the recombiant plasmid PGL6-that will obtain
TA-pCYP1A-4 × DRE delivers to Sheng Gong company and checks order with Rvprimer3 universal primer, and checking obtains plasmid sequence further
The accuracy of row；
5) liposome transfection of the recombiant plasmid transformed and screening: utilize the ViaFect of Promega companyTM
Transfection Reagent by PGL6-TA-pCYP1A, PGL6-TA-pCYP1A-2 × DRE, PGL6-TA-pCYP1A-3 ×
DRE, PGL6-TA-pCYP1A-4 × DRE rotates in HepG2 cell, and processes 24h with 1nM TCDD, utilizes Promega companyReporter Assay System detection kit detection luciferase expression, it is determined that change
The recombiant plasmid the made size to poisonous substance detection efficiency.Show that PGL6-TA-pCYP1A-4 × DRE compares other recombiant plasmid, effect
Rate is the highest；Compare the PGL6-TA-pCYP1A not transformed and improve 37 times；And PGL6-TA-pCYP1A-2 × DRE only improves 12
Times, PGL6-TA-pCYP1A-3 × DRE improves 27 times.
The present invention according to design from Brachydanio rerio chromosomal DNA PCR amplify CYP1A promoter sequence, construct containing
Luciferase gene, the PGL6-TA-pCYP1A plasmid of TA box.The present invention, by transformation CYP1A promoter, improves weight
The group plasmid detection efficiency to TCDD, establish one quickly, efficient detection TCDD and be the poisonous substance of AHR part equally
System.
The present invention utilizes Brachydanio rerio cyp1a gene promoter sequence, utilizes overlap pcr technology to transform it,
It is connected in PGL6-TA carrier, makes promoter sequence that transformation the completes sensitivity to two English class persistence organic pollutants
Strengthen, be transfected in HepG2 cell, it is possible to quickly two English class persistence organic pollutants in detection environment.
Accompanying drawing explanation
Fig. 1 is recombiant plasmid reporter gene testing result.
Fig. 2 is recombiant plasmid reporter gene expression multiple.
Fig. 3 is that TCDD induces luciferase expression.
Fig. 4 is that the luciferase of TCC induction expresses standard curve.
Detailed description of the invention
Following example will the invention will be further described in conjunction with accompanying drawing.
Step one: clone's Brachydanio rerio CYP1A (Chromosome 18-NC_007129.6) promoter sequence
1) arrange as stencil design pair of primers pCYP1A-F, pCYP1A-R with Brachydanio rerio genome sequence known on NCBI,
On pCYP1A-F, pCYP1A-R, wherein it is respectively provided with Kpn I and Hind III digestion site.
PCYP1A-F:5 '-AAAGGTACCGAAACCCACGCCATGCAAAC-3 '
Kpn I
PCYP1A-R:5 '-AAAAAGCTTTCCTCCGCGTGACGTCACCG-3 '
Hind III
0.2ml sterile centrifugation tube is sequentially added into: ddH2O 34.5 μ l, 10 × Taq enzyme buffer 5 μ l, 4 × dNTP
(2.5mmol/L) 4 μ l, pCYP1A-F primer 2 μ l, pCYP1A-R primer 2 μ l, zebrafish dna 1 μ l, Taq enzyme 0.5 μ l, totally
Long-pending 50 μ l.
PCR response parameter is arranged:
After reaction terminates, the agarose gel electrophoresis that 50 μ l carry out 2% identifies occur unique one on electrophoresis pattern
Bar meets the 314bp fragment of expection size, utilizes glue to reclaim test kit and reclaims this sequence, reclaims afterproduct Kpn I and Hind
III carries out double digestion, and glue reclaims again, simultaneously by original for laboratory plasmid vector PGL6-TA Kpn I/Hind III
Double digestion, and glue recovery, then carry out 1h even with T4DNA ligase by CYP1A promoter sequence and carrier segments at 16 DEG C
Connect, convert.
2) by 100 μ l competent cell (CaCl2Prepared by method) it is equipped with in the centrifuge tube that 10 μ l connect product, hun mixes
Even, ice bath 30min.
3) 42 DEG C of heat shock 90s, then ice bath 2min, adds 400 μ l fresh LB, puts 37 DEG C of shaking table recovery 40min,
Take 200 μ l and be coated with flat board (containing Amp100 μ g/ml).37 DEG C of overnight incubation.
4) in, amount extraction plasmid carries out enzyme action qualification, it is thus achieved that expection fragment.
Promoter sequence is transformed by step 2
As a example by obtaining recombiant plasmid PGL6-TA-pCYP1A-2 × DRE, PGL6-TA-pCYP1A-3 × DRE, PGL6-
The step of TA-pCYP1A-4 × DRE transformation is essentially identical.
1) it is template with the recombiant plasmid PGL6-TA-pCYP1A obtained, designs primer 2 × DRE-1F, 2 × DRE-1R, 2
× DRE-2F, 2 × DRE-2R.Wherein 2 × DRE-2F adds aryl hydrocarbon receptor binding site 5 '-GCGTG-3 '.2×
DRE-2R adds the sequence 5 '-CACGCG-3 ' of an aryl hydrocarbon receptor binding site reverse complemental.
2 × DRE-1F:5 '-TGTTACATACATCAATCTCC-3 '
2 × DRE-2F:5 '-ACTGCGAGCCCCCTCGCGTGTGTTACATACATCAATCTCC-3’
Aryl hydrocarbon receptor binding site
2 × DRE-1R:5 '-CACGCACACATACATGGTAC-3 '
2 × DRE-2R:5 '-CACGCGAGGGGGCTCGCAGTCACGCACACATACATGGTAC-3’
2) with pCYP1A-F, 2 × DRE-1R is primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 1,
2 × DRE-1F, pCYP1A-R are primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 2.This step is in order to protect
Card hi-fi, has changed Pfu enzyme and has expanded.Hereinafter it is all to use Pfu enzyme.
0.2ml sterile centrifugation tube is sequentially added into: ddH2O 29 μ l, 10 × Taq enzyme buffer 10 μ l, 4 × dNTP
(2.5mmol/L) 5 μ l, pCYP1A-F primer 2 μ l, pCYP1A-R primer 2 μ l, PGL6-TA-pCYP1A (500 μ g/ul) 1 μ l,
Pfu enzyme 1 μ l, cumulative volume 50 μ l.
PCR response parameter is arranged:
After reaction terminates, the agarose gel electrophoresis that 50 μ l carry out 2% identifies occur unique one on electrophoresis pattern
Bar meets expection size fragment, utilizes glue to reclaim test kit and is separately recovered sequence 1 and 2.
3) with pCYP1A-F, 2 × DRE-2R is primer, and sequence 1 is template, and PCR amplifies sequence 3,2 × DRE-
2F, pCYP1A-R are primer, and sequence 2 is template, and PCR amplifies sequence 4, utilize glue to reclaim test kit and are separately recovered sequence
3 and 4.
4) with pCYP1A-F, pCYP1A-R is primer, and template is simultaneously introduced sequence 3 and sequence 4, when PCR expands, and annealing
Temperature, every 5 cycle down 2 DEG C, drops to 53 DEG C from 65 DEG C.Amplifying aim sequence pCYP1A-2 × DRE, glue reclaims purpose sequence
Row, are attached aim sequence with carrier T, connect product Transformed E .coliDH5 α, with ampicillin (Amp) screening and cloning
Son, extracts plasmid in a small amount and identifies, the sequence obtained is connected in carrier T.Correct transformation sequence is obtained through order-checking, with
The plasmid arrived reclaims sequence fragment after Kpn I/Hind III double digestion, and with the experiment through Kpn I/Hind III double digestion
Room original plasmid vector PGL6-TA connects, and connects product Transformed E .coliDH5 α, with ampicillin (Amp) screening and cloning
Son, obtains recombiant plasmid PGL6-TA-pCYP1A-2 × DRE.Middle amount is extracted plasmid and is carried out enzyme action qualification.
Step 3 liposome transfection recombiant plasmid
1. day before transfection, trypsin digestion cell also counts, plating cells so that it is transfection day density be 90%.Cell
Bed board is without serum, without in the culture medium of the normal growth of antibiotic.
2., for every porocyte, use 50 μ l plasma-free DMEM medium to dilute 1.0 μ gDNA.Every hole adds 3 μ l
ViaFectTMTransfection Reagent reagent.At incubation at room temperature 20min.Now cell culture medium is changed into containing 1nM
The culture medium of TCDD, matched group uses 1nM DMSO.
3. directly complex is joined in every hole, wave and culture plate, mix gently.
4. at 37 DEG C, the CO of 5%2Cultivate 24h.After adding complex 24h in cell, analyze cell extract, utilizeReporter Assay System detects luciferase reporter gene activity.
Obtain a result such as Fig. 1, can significantly improve the sensitivity to TCDD plus DRE on the basis of original promoter, and
And the sensitivity of TCDD is greatly reinforced by the recombiant plasmid having added three DRE.
With 1nM DMSO process PGL6-TA-pCYP1A for 1, Fig. 2 illustrates the multiple often organizing raising.Four DRE are added
Recombiant plasmid compared to original promoter sequence, the Detection results of TCDD is strengthened 37 times.
We reduce the concentration that TCDD exposes on this basis, have drawn Fig. 3 result.
It will be seen that the method can detect 0.01nM TCDD.Utilize PGL6-TA-pCYP1A-4 × DRE plasmid, no
TCDD with concentration is the most dirty, obtains detecting the standard curve (see Fig. 4) of TCDD.The fit equation obtained is y=-3.5142x2+
85.112x+21.1, degree of fitting has reached 0.97899.In the case of relatively low concentration, have reasonable for detection TCDD
Detection results, but when concentration rate of exchange height, due to intracellular AHR protein quantity and the mortality of cell, just cannot reach
To preferable Detection results.
The present invention, according to existing Brachydanio rerio CYP1A promoter sequence in ncbi database, utilizes Brachydanio rerio genomic DNA
For template, utilize round pcr to expand out by promoter sequence, and utilize overlap round pcr, transform this promoter sequence
Row, and be connected in PGL6-TA carrier, obtain a series of recombiant plasmid.Followed by TCDD, recombiant plasmid is carried out
Screening, finds that PGL6-TA-pCYP1A-4 × DRE is most sensitive to 1nM TCDD, and the TCDD of 0.01nM can be detected.TCDD
As the representative substances of POPs, and a lot of POPs is the part of AHR, so PGL6-TA-pCYP1A-4 × DRE can detect
It is much the pollutant of AHR part, and the shortest, and efficiency is high.
Claims (1)
1. an English class persistence organic pollutant detection method, it is characterised in that it specifically comprises the following steps that
1) with Brachydanio rerio genome known on National Center for Biotechnology Information website
Sequence is stencil design pair of primers pCYP1A-F, pCYP1A-R, clone CYP1A (Chromosome 18-NC_007129.6)
Promoter sequence, primer sequence is as follows:
PCYP1A-F:5 '-AAAGGTACCGAAACCCACGCCATGCAAAC-3 '
Kpn I
PCYP1A-R:5 '-AAAAAGCTTTCCTCCGCGTGACGTCACCG-3 '
Hind III
Wherein the forward primer at CYP1A promoter sequence is provided with Kpn I restriction enzyme site, is provided with Hind at downstream primer
III digestion site, with Brachydanio rerio genome as template, with above-mentioned two primers, utilizes round pcr to amplify promoter sequence:
This promoter sequence is positioned at 314, the upstream base of the expression start codon ATG of CYP1A gene；To expand out
Fragment reclaims sequence fragment after Kpn I/Hind III double digestion, and former with the laboratory through Kpn I/Hind III double digestion
Some plasmid vector PGL6-TA connect, and connect product Transformed E .coliDH5 α, with ampicillin (Amp) screening and cloning,
Recombiant plasmid PGL6-TA-pCYP1A, is carried out the recombiant plasmid PGL6-TA-pCYP1A obtained with Rvprimer3 universal primer
Order-checking, checking obtains the accuracy of plasmid sequence further；
2) it is template with the recombiant plasmid PGL6-TA-pCYP1A obtained, design primer pCYP1A-2 × DRE-F, pCYP1A-2 ×
DRE-R, carries out overlap PCR, transforms promoter sequence, and primer sequence is as follows:
2 × DRE-1F:5 '-TGTTACATACATCAATCTCC-3 '
2 × DRE-2F:5 '-ACTGCGAGCCCCCTCGCGTGTGTTACATACATCAATCTCC-3’
Aryl hydrocarbon receptor binding site
2 × DRE-1R:5 '-CACGCACACATACATGGTAC-3 '
2 × DRE-2R:5 '-CACGCGAGGGGGCTCGCAGTCACGCACACATACATGGTAC-3 '
Transformation postorder lists meaning:
With pCYP1A-F, 2 × DRE-1R is primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 1；2×
DRE-1F, pCYP1A-R are primer, and PGL6-TA-pCYP1A is template, and PCR amplifies sequence 2；PCYP1A-F, 2 ×
DRE-2R is primer, and sequence 1 is template, and PCR amplifies sequence 3；2 × DRE-2F, pCYP1A-R are primer, and sequence 2 is
Template, PCR amplifies sequence 4；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced sequence 3 and sequence 4, expands
Increase and aim sequence pCYP1A-2 × DRE, first aim sequence is cloned in carrier T, obtain correct transformation sequence through order-checking,
After Kpn I/Hind III double digestion, reclaim sequence fragment with the plasmid obtained, and with through Kpn I/Hind III double digestion
Laboratory original plasmid vector PGL6-TA connects, and connects product Transformed E .coliDH5 α, screens with ampicillin (Amp)
Clone, obtains recombiant plasmid PGL6-TA-pCYP1A-2 × DRE, the recombiant plasmid PGL6-TA-pCYP1A-2 × DRE that will obtain
Delivering to Sheng Gong company check order with Rvprimer3 universal primer, checking obtains the accuracy of plasmid sequence further；
3) using the recombiant plasmid PGL6-TA-pCYP1A-2 × DRE obtained is template, designs primer 3 × DRE-F, 3 × DRE-R,
Carry out overlap PCR, transformation further to promoter sequence；
Primer sequence is as follows:
3 × DRE-F:5 '-ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGTGTTACATACATCAAT-3 '
3 × DRE-R:5 '-CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCACACATACATG GTAC
-3 '
Transformation postorder lists meaning:
With pCYP1A-F, 3 × DRE-R is primer, and sequence 3 is template, and PCR amplifies sequence 5；3 × DRE-F, pCYP1A-R are
Primer, sequence 4 is template, and PCR amplifies sequence 6；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced sequence
Row 5 and sequence 6, amplify aim sequence pCYP1A-3 × DRE；First aim sequence is cloned in carrier T, just obtains through order-checking
True transformation sequence, reclaims sequence fragment with the plasmid that obtains after Kpn I/Hind III double digestion, and with through Kpn I/
Laboratory original plasmid vector PGL6-TA of Hind III double digestion connects, and connects product Transformed E .coliDH5 α, with ammonia benzyl
Penicillin (Amp) screening and cloning, obtains recombiant plasmid PGL6-TA-pCYP1A-3 × DRE；The recombiant plasmid PGL6-that will obtain
TA-pCYP1A-3 × DRE delivers to Sheng Gong company and checks order with Rvprimer3 universal primer, and checking obtains plasmid sequence further
The accuracy of row；
4) using the recombiant plasmid PGL6-TA-pCYP1A-3 × DRE obtained is template, designs primer 4 × DRE-F, 4 × DRE-R,
Carry out overlap PCR, transformation further to promoter sequence；
Primer sequence is as follows:
4 × DRE-F:ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGACTGCGAGCC CCCTCGCGTG
4 × DRE-R:CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCGAGGG GGCTCGCAGT
Transformation postorder lists meaning:
Primer sequence is as follows:
4 × DRE-F:ACTGCGAGCCCCCTCGCGTGACTGCGAGCCCCCTCGCGTGACTGCGAGCC CCCTCGCGTG
4 × DRE-R:CACGCGAGGGGGCTCGCAGTCACGCGAGGGGGCTCGCAGTCACGCGAGGG GGCTCGCAGT
With pCYP1A-F, 4 × DRE-R is primer, and sequence 5 is template, and PCR amplifies sequence 7；3 × DRE-F, pCYP1A-R are
Primer, sequence 6 is template, and PCR amplifies sequence 8；With pCYP1A-F, pCYP1A-R for primer, template is simultaneously introduced sequence
Row 7 and sequence 8, amplify aim sequence pCYP1A-4 × DRE；First aim sequence is cloned in carrier T, just obtains through order-checking
True transformation sequence, reclaims sequence fragment with the plasmid that obtains after Kpn I/Hind III double digestion, and with through Kpn I/
Laboratory original plasmid vector PGL6-TA of Hind III double digestion connects, and connects product Transformed E .coliDH5 α, with ammonia benzyl
Penicillin (Amp) screening and cloning, obtains recombiant plasmid PGL6-TA-pCYP1A-4 × DRE, the recombiant plasmid PGL6-that will obtain
TA-pCYP1A-4 × DRE checks order with Rvprimer3 universal primer, and checking obtains the accuracy of plasmid sequence further；
5) liposome transfection of the recombiant plasmid transformed and screening: utilize the ViaFect of Promega companyTM Transfection
Reagent is by PGL6-TA-pCYP1A, PGL6-TA-pCYP1A-2 × DRE, PGL6-TA-pCYP1A-3 × DRE, PGL6-TA-
PCYP1A-4 × DRE rotates in HepG2 cell, and processes 24h with 1nM TCDD, utilizes Promega company Dual-Reporter Assay System detection kit detection luciferase expression, it is determined that the weight of transformation
The group plasmid size to poisonous substance detection efficiency.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995823A (en) * | 2017-04-13 | 2017-08-01 | 厦门大学 | A kind of preparation method of the blue or green Medaka of transgenosis stain |
| CN107058386A (en) * | 2017-04-13 | 2017-08-18 | 厦门大学 | A kind of preparation method of transgenic zebrafish |
| CN108642082A (en) * | 2018-03-29 | 2018-10-12 | 苏州木芮生物科技有限公司 | Construction method of transgenic zebra fish with overexpression of systemic glucocorticoid receptor gene |
| CN108642081A (en) * | 2018-03-29 | 2018-10-12 | 苏州木芮生物科技有限公司 | Construction method of luciferase transgenic zebra fish driven by glucocorticoid response original |
| CN109609469A (en) * | 2018-11-26 | 2019-04-12 | 浙江海洋大学 | A kind of novel sea biological pollution detection marker |
| KR20200105122A (en) * | 2019-02-28 | 2020-09-07 | 상명대학교산학협력단 | Detection method of marine pollutants using marine teleost CYP1A gene |
| CN114134197A (en) * | 2021-12-09 | 2022-03-04 | 南京大学 | Luciferase reporter gene-based cytotoxicity rapid detection method, cell strain construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749403A (en) * | 2004-09-15 | 2006-03-22 | 正修科技大学 | Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene |
| CN102851312A (en) * | 2012-03-07 | 2013-01-02 | 深圳华大基因研究院 | Carrier suitable for cell transformation, recombinant cell and application thereof |
| CN103820497A (en) * | 2014-03-18 | 2014-05-28 | 中国科学院生态环境研究中心 | Recombinant vector used for biologically detecting dioxin substances |
| CN104593417A (en) * | 2015-02-04 | 2015-05-06 | 江苏省农业科学院 | Xylanase intestinal directional expression vector and cell line thereof |
-
2016
- 2016-03-23 CN CN201610167440.5A patent/CN105755106A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749403A (en) * | 2004-09-15 | 2006-03-22 | 正修科技大学 | Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene |
| CN102851312A (en) * | 2012-03-07 | 2013-01-02 | 深圳华大基因研究院 | Carrier suitable for cell transformation, recombinant cell and application thereof |
| CN103820497A (en) * | 2014-03-18 | 2014-05-28 | 中国科学院生态环境研究中心 | Recombinant vector used for biologically detecting dioxin substances |
| CN104593417A (en) * | 2015-02-04 | 2015-05-06 | 江苏省农业科学院 | Xylanase intestinal directional expression vector and cell line thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张诺等: "荧光素酶表达基因法(CALUX)用于二噁英检测的研究进展", 《生态毒理学报》 * |
| 郑明辉主编: "《二恶英类生物检测技术》", 30 November 2013, 中国环境科学出版社 * |
| 黄留玉主编: "《PCR最新技术原理、方法及应用》", 31 January 2011, 化学工业出版社 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995823A (en) * | 2017-04-13 | 2017-08-01 | 厦门大学 | A kind of preparation method of the blue or green Medaka of transgenosis stain |
| CN107058386A (en) * | 2017-04-13 | 2017-08-18 | 厦门大学 | A kind of preparation method of transgenic zebrafish |
| CN108642082A (en) * | 2018-03-29 | 2018-10-12 | 苏州木芮生物科技有限公司 | Construction method of transgenic zebra fish with overexpression of systemic glucocorticoid receptor gene |
| CN108642081A (en) * | 2018-03-29 | 2018-10-12 | 苏州木芮生物科技有限公司 | Construction method of luciferase transgenic zebra fish driven by glucocorticoid response original |
| CN108642081B (en) * | 2018-03-29 | 2022-05-13 | 苏州木芮生物科技有限公司 | Construction method of luciferase transgenic zebra fish driven by glucocorticoid response original |
| CN108642082B (en) * | 2018-03-29 | 2022-05-13 | 苏州木芮生物科技有限公司 | Construction method of transgenic zebra fish with overexpression of systemic glucocorticoid receptor gene |
| CN109609469A (en) * | 2018-11-26 | 2019-04-12 | 浙江海洋大学 | A kind of novel sea biological pollution detection marker |
| KR20200105122A (en) * | 2019-02-28 | 2020-09-07 | 상명대학교산학협력단 | Detection method of marine pollutants using marine teleost CYP1A gene |
| KR102209087B1 (en) | 2019-02-28 | 2021-01-28 | 상명대학교 산학협력단 | Detection method of marine pollutants using marine teleost CYP1A gene |
| CN114134197A (en) * | 2021-12-09 | 2022-03-04 | 南京大学 | Luciferase reporter gene-based cytotoxicity rapid detection method, cell strain construction method and application thereof |
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