CN110144395A - A kind of reporter gene and preparation method thereof monitoring miRNA dynamic change - Google Patents
A kind of reporter gene and preparation method thereof monitoring miRNA dynamic change Download PDFInfo
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- CN110144395A CN110144395A CN201910372849.4A CN201910372849A CN110144395A CN 110144395 A CN110144395 A CN 110144395A CN 201910372849 A CN201910372849 A CN 201910372849A CN 110144395 A CN110144395 A CN 110144395A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of reporter genes and preparation method thereof for monitoring miRNA dynamic change, the reporter gene includes PUF gene, plasmid psiCHECK-2-HM471G, Fluc gene, six sections of NRE tandem repetitive sequences with four sections of tandem repetitive sequences of purpose miRNA complete complementary pairing and with the pairing of PUF complete complementary, the downstream terminator codon TGA for being located at PUF albumen with four sections of tandem repetitive sequences of purpose miRNA complete complementary pairing, described six sections of NRE tandem repetitive sequences with the pairing of PUF complete complementary are located at the upstream Fluc gene start codon ATG.This report gene is to carry out real-time, quantitative monitoring to the specific miRNA generated during Neural Differentiation or during muscle, Myocardium Differentiation.
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field, and in particular to a kind of monitoring miRNA dynamic change
Reporter gene and preparation method thereof.
Background technique
The small molecule single stranded RNA that MicroRNAs (miRNA, miR) is made of 20 to 22 non-coding nucleotides, can lead to
It crosses and translation or degradation mRNA is inhibited to carry out the expression of controlling gene.Mitogenetic, increasing is such as broken up in the activity of these molecules and many cells
Grow apoptosis close relation.Again because it can induce specific mRNA degradation or inhibit translation, so much clinically important
It also plays an important role in disease.Some miRNAs it is verified that with cardiac muscle and muscle atomization it is related, such as
MiR-1, miR-133, miR-181, miR-195, miR-206, and miR-26a.Also some miRNAs are found and nerve point
Change process close relation, such as miR-9, miR-124 etc..
Traditional means currently used for monitoring miRNAs expression mainly have Northern to hybridize (Northern blot), base
Because of chip (microarray), real-time quantitative PCR (real-time polymerase chain reaction) technology etc..It is logical
Traditional means are crossed, the expression variation of dynamic miRNA in cellular activity process can not be able to complete and repeatable earth's surface
Reveal and.And these traditional technologies in monitoring process mostly require destroy biological tissue, cause cellular level cracking or
Death had not only expended time energy, but also cannot have been detected in living body level.The main molecular imaging means that shine include at present
Two aspect of bioluminescence and autofluorescence.The means such as molecular beacon, labelled with radioisotope, Reporter System are used.
Molecular beacon problems faced is mainly whether institute's band colour developing group is sufficiently stable, the fluorescence quenching capability pole when not combining
The background fluorescence activity of itself institute's band is excessively high；And radioactive isotope can then cause a degree of damage to organism；In the presence of
Reporter System be switch-Off system, not can determine that be as time goes by caused by luciferase expression decline also
It is the effect of Reporter System.
Summary of the invention
The purpose of the present invention is to provide it is a kind of it is noninvasive, in real time physical examination survey miRNA expression reporter gene and its
Construction method is realized and is monitored to the dynamic of correlation miRNA during Neural Differentiation process or muscle differentiation.To further investigate to phase
It closes in neurological disease model or muscle-wasting diseases, developed by molecule level is to regulating and controlling to lay the foundation caused by it.
In order to realize that above-mentioned technical effect, the present invention are realized especially by following technical scheme:
A kind of reporter gene monitoring miRNA dynamic change, the reporter gene includes PUF gene, plasmid
PsiCHECK-2-HM471G, Fluc gene, with purpose miRNA complete complementary pairing four sections of tandem repetitive sequences and with PUF it is complete
Six sections of NRE tandem repetitive sequences of full complementary pairing, four sections of tandem sequence repeats sequences with the pairing of purpose miRNA complete complementary
Column are located at the downstream terminator codon TGA of PUF albumen, six sections of NRE tandem repetitive sequences with the pairing of PUF complete complementary
Positioned at the upstream Fluc gene start codon ATG.
The nucleotide sequence of reporter gene of the present invention is as shown in SEQ ID NO.1.
The present invention provides the preparation methods of the reporter gene of the monitoring miRNA dynamic change, comprising the following steps:
1) by PUF gene cloning to plasmid psiCHECK-2-HM471G multiple cloning sites, plasmid psiCHECK- is obtained
2-HM471G-PUF；
2) by Fluc gene cloning to psiCHECK2-HM471G, psiCHECK2-HM471G-Fluc is obtained；
3) it is interacted by way of base pair complementarity using miRNA and target, it will be with purpose miRNA complete complementary
Four sections of tandem repetitive sequences of pairing are cloned into, and obtain psiCHECK-2-HM471G-PUF-miR-Fluc；
4) it is interacted, will be matched with PUF complete complementary by way of base pair complementarity using PUF albumen and NRE
Six sections of NRE tandem repetitive sequences be cloned into, obtain reporter gene psiCHECK-2-HM471G-PUF-miR reporter.
Four sections of tandem repetitive sequences of the described and purpose miRNA complete complementary pairing are located at the end of PUF albumen
The only downstream codon TGA.
Described six sections of NRE tandem repetitive sequences with the pairing of PUF complete complementary are located at Fluc gene start codon ATG
Upstream.
In another aspect of this invention, reporter gene miRNA dynamic change in detection atomization is provided
Application.
The atomization includes Neural Differentiation and muscle differentiation.
The detailed process of the application are as follows: the reporter gene first passes around transcription in vivo, and translation becomes one
Linear peptide chain can express the two genes of PUF and Fluc, respectively by the regulation of different promoters simultaneously；When in cell not
There are when purpose miRNA, PUF can be integrated in 6xNRE sequence, thus inhibit the expression of Fluc, luciferase content in cell
It is low；When, there are when purpose miRNA, purpose miRNA can be integrated on 4x miR target sequence in cell, inhibit the expression of PUF, thus
The inhibition to Fluc is released, Fluc is expressed, generates the fluorescence signal that can detecte.
The invention has the benefit that
Reporter System of the invention can generate Neural Differentiation in the process or during muscle, Myocardium Differentiation
Specific miRNA carries out real-time, quantitative monitoring.The monitoring system positive as one, itself will not send out because of the variation of time
Life it is weak, it is possible to it is long-acting, non-invasively realize physical examination survey.This is to clinic further investigation related neural aspect disease or flesh
Meat atrophy disease is of great significance to.
Detailed description of the invention
Fig. 1 is the structure chart of reporter gene of the present invention；
Fig. 2 is in-vitro simulated induction measurement fluorescence intensity；
Fig. 3 is intracellular Neural Differentiation measurement fluorescence intensity；
Fig. 4 is intracellular muscle differentiation measurement fluorescence intensity.
Specific embodiment
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
The building of 1 reporter gene of embodiment
A kind of reporter gene for monitoring miRNA dynamic change is present embodiments provided, this report gene is by the following method
It obtains:
(1) luciferase expression can be inhibited in conjunction with target sequence using PUF and be applied in system as reporter gene, by PUF
In gene cloning to plasmid psiCHECK-2-HM471G multiple cloning sites, to obtain plasmid psiCHECK-2-HM471G-
PUF；
(2) characteristic that can be used as reporter gene bioluminescence using firefly luciferase, Fluc gene cloning is arrived
On psiCHECK2-HM471G, to obtain psiCHECK2-HM471G-Fluc；
(3) it is interacted by way of base pair complementarity using miRNA and target, it will be completely mutual with purpose miRNA
The four sections of tandem repetitive sequences recruited pair are cloned into, and are located at the downstream terminator codon TGA of PUF albumen, are obtained
psiCHECK-2-HM471G-PUF-miR-Fluc；
(4) it is interacted, will be matched with PUF complete complementary by way of base pair complementarity using PUF albumen and NRE
Six sections of NRE tandem repetitive sequences be cloned into, and be placed on the upstream Fluc gene start codon ATG.To obtain report base
Because of psiCHECK-2-HM471G-PUF-miR reporter (Fig. 1).
Its specific construction method are as follows:
1) linearized vector psiCHECK-2-HM471G passes through 1 double digestion of restriction enzyme Nhe 1 and Xho, digestion
1% agarose gel electrophoresis of product, cuts target DNA fragment under ultraviolet light.Purpose band is recycled with plastic recovery kit
Purifying.
2) Insert Fragment: PUF sequence is obtained, 4x targets sequence, 6xNRE sequence, luc2 sequence is by Shanghai biochemistry
Company is synthesized using conventional chemical processes, and synthesis obtains PUF-miR in order.
3) Insert Fragment is recombinated with carrier: the segment that above-mentioned amplification obtains is mixed with carrier psiCHECK-2-HM471G
It is prepared into the DNA solution of 10ul, isometric ligase is added, mixes well reaction.
4) it converts: the recombining reaction liquid of 10ul being taken to mix with the competent escherichia coli cell of 0.1ml, 30 minutes on ice,
42 DEG C 5 minutes on ice rapidly after heat shock 90 seconds.800ulLB fluid nutrient medium is added, 37 DEG C of shaking tables are incubated for 1 hour.Take above-mentioned bacterium
Liquid is coated in LB solid medium tablets with ampicillin, and face up half an hour placement, is fallen after bacterium solution fully absorbs
Horizontalization plate, 37 DEG C incubator culture 16 hours.
5) positive clone identification: plasmid is extracted in picking single colonie culture.With Nhe 1 and 1 double digestion of Xho and carry out agar
Sugared gel electrophoresis.Digestion is accredited as positive clone and passes through sequencing further verifying.Its nucleotide sequence such as SEQ ID NO.1 institute
Show.
The in-vitro simulated induction of embodiment 2
293 cell of HEK is cultivated, 24 orifice plates is taken, makes it that can converge 80-90% after for 24 hours.After for 24 hours, psiCHECK-2- is transfected
HM471G-PUF-miR reporter Plasmid DNA 0.8ug, miR124a mimics, the NC control of various concentration gradient
mimics.After 48h, useless culture solution is sucked, PBS is washed 3 times, and 100ul 1x cell pyrolysis liquid is added, and shaking table shakes 30min, is measured glimmering
Luminous intensity.
As a result as shown in Fig. 2, with added mimics concentration increase, fluorescence intensity rise, and to NC compare it is reactionless.
The intracellular Neural Differentiation application of embodiment 3
P19 mouse teratocarcinoma cells are cultivated, the culture dish of 24 orifice plates is taken, 3x10 is added4P19 cell.Respectively at culture
2D, 4D, 6D be added inducer vitamin A, induce its differentiating into nerve cells；After inducing the 6D time, transfection
PsiCHECK-2-HM471G-PUF-miR reporter Plasmid DNA 0.8ug；After transfecting 48h, useless culture solution is sucked, PBS washes 3
It is secondary, 100ul 1x cell pyrolysis liquid is added, shaking table shakes 30min, measures fluorescence intensity.
As a result as shown in figure 3, with the increase for inducing number of days, p19 differentiating into nerve cells generates endogenic miR-
124a is incorporated into 4xmiR124a targets, and PUF is inhibited to be incorporated into 6xNRE, to release the inhibition to luc2, generates glimmering
Light.And the fluorescence enhancement with the increase of miR-124a.
The intracellular muscle differentiation application of embodiment 4
C2C12 Skeletal Muscle Cells of Mouse is cultivated, the culture dish of 24 orifice plates is taken, 3x10 is added4C2C12 cell.Respectively at training
The differential medium for containing 1% fetal calf serum is added in feeding 2D, 4D, 6D, it is induced to break up to muscle cell；The induction 6D time terminates
Afterwards, psiCHECK-2-HM471G-PUF-miR reporter Plasmid DNA 0.8ug is transfected；After transfecting 48h, useless culture is sucked
Liquid, PBS are washed 3 times, and 100ul 1x cell pyrolysis liquid is added, and shaking table shakes 30min, measure fluorescence intensity.
As a result as shown in figure 4, with the increase for inducing number of days, C2C12 breaks up to muscle cell, generates endogenic miR-
133a is incorporated into 4xmiR133a targets, and PUF is inhibited to be incorporated into 6xNRE, to release the inhibition to luc2, generates glimmering
Light.And the fluorescence enhancement with the increase of miR-133a.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Sequence table
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<120>a kind of reporter gene and preparation method thereof for monitoring miRNA dynamic change
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gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 180
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 240
caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 300
attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 360
cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 420
agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca 480
ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct 540
tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc 600
actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga 660
gtacttaata cgactcacta taggctagcc accatgggcc gcagccgcct tttggaagat 720
tttcgaaaca accggtaccc caatttacaa ctgcgggaga ttgctggaca tataatggaa 780
ttttcccaag accagcatgg gtccagattc attcagctga aactggagcg tgccacacca 840
gctgagcgcc agcttgtctt caatgaaatc ctccaggctg cctaccaact catggtggat 900
gtgtttggta attacgtcat tcagaagttc tttgaatttg gcagtcttga acagaagctg 960
gctttggcag aacggattcg aggccacgtc ctgtcattgg cactacagat gtatggctgc 1020
cgtgttatcc agaaagctct tgagtttatt ccttcagacc agcagaatga gatggttcgg 1080
gaactagatg gccatgtctt gaagtgtgtg aaagatcaga atggcaatca cgtggttcag 1140
aaatgcattg aatgtgtaca gccccagtct ttgcaattta tcatcgatgc gtttaaggga 1200
caggtatttg ccttatccac acatccttat ggctgccgag tgattcagag aatcctggag 1260
cactgtctcc ctgaccagac actccctatt ttagaggagc ttcaccagca cacagagcag 1320
cttgtacagg atcaatatgg aaattatgta atccaacatg tactggagca cggtcgtcct 1380
gaggataaaa gcaaaattgt agcagaaatc cgaggcaatg tacttgtatt gagtcagcac 1440
aaatttgcaa gcaatgttgt ggagaagtgt gttactcacg cctcacgtac ggagcgcgct 1500
gtgctcatcg atgaggtgtg caccatgaac gacggtcccc acagtgcctt atacaccatg 1560
atgaaggacc agtatgccaa ctacgtggtc cagaagatga ttgacgtggc ggagccaggc 1620
cagcggaaga tcgtcatgca taagatccgg ccccacatcg caactcttcg taagtacacc 1680
tatggcaagc acattctggc caagctggag aagtactaca tgaagaacgg tgttgactta 1740
gggtgattct aggcgatcga ctagttggca ttcaccgcgt gccttaatag tatggcattc 1800
accgcgtgcc ttaatagtat ggcattcacc gcgtgcctta atagtatggc attcaccgcg 1860
tgccttaaca tatgctcgag cccgggaatt cgtttaaacc tagagcggcc gctggccgca 1920
ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgag gatctaaatg 1980
agtcttcgga cctcgcgggg gccgcttaag cggtggttag ggtttgtctg acgcgggggg 2040
agggggaagg aacgaaacac tctcattcgg aggcggctcg gggtttggtc ttggtggcca 2100
cgggcacgca gaagagcgcc gcgatcctct taagcacccc cccgccctcc gtggaggcgg 2160
gggtttggtc ggcgggtggt aactggcggg ccgctgactc gggcgggtcg cgcgccccag 2220
agtgtgacct tttcggtctg ctcgcagacc cccgggcggc gccgccgcgg cggcgacggg 2280
ctcgctgggt cctaggctcc atggggaccg tatacgtgga caggctctgg agcatccgca 2340
cgactgcggt gatattaccg gagaccttct gcgggacgag ccgggtcacg cggctgacgc 2400
ggagcgtccg ttgggcgaca aacaccagga cggggcacag gtacactatc ttgtcacccg 2460
gaggcgcgag ggactgcagg agcttcaggg agtggcgcag ctgcttcatc cccgtggccc 2520
gttgctcgcg tttgctggcg gtgtccccgg aagaaatata tttgcatgtc tttagttcta 2580
tgatgacaca aaccccgccc agcgtcttgt cattggcgaa ttcgaacacg cagatgcagt 2640
cggggcggcg cggtcccagg tccacttcgc atattaaggt gacgcgtgtg gcctcgaaca 2700
ccgagcgacc ctgcagcgac ccgcttaaaa gcttggcatt tgtatatatg tatatatgta 2760
tatatgtata tatgtatata tgtatatacc ggtactgttg gtaaagccac catggccgat 2820
gctaagaaca ttaagaaggg ccctgctccc ttctaccctc tggaggatgg caccgctggc 2880
gagcagctgc acaaggccat gaagaggtat gccctggtgc ctggcaccat tgccttcacc 2940
gatgcccaca ttgaggtgga catcacctat gccgagtact tcgagatgtc tgtgcgcctg 3000
gccgaggcca tgaagaggta cggcctgaac accaaccacc gcatcgtggt gtgctctgag 3060
aactctctgc agttcttcat gccagtgctg ggcgccctgt tcatcggagt ggccgtggcc 3120
cctgctaacg acatttacaa cgagcgcgag ctgctgaaca gcatgggcat ttctcagcct 3180
accgtggtgt tcgtgtctaa gaagggcctg cagaagatcc tgaacgtgca gaagaagctg 3240
cctatcatcc agaagatcat catcatggac tctaagaccg actaccaggg cttccagagc 3300
atgtacacat tcgtgacatc tcatctgcct cctggcttca acgagtacga cttcgtgcca 3360
gagtctttcg acagggacaa aaccattgcc ctgatcatga acagctctgg gtctaccggc 3420
ctgcctaagg gcgtggccct gcctcatcgc accgcctgtg tgcgcttctc tcacgcccgc 3480
gaccctattt tcggcaacca gatcatcccc gacaccgcta ttctgagcgt ggtgccattc 3540
caccacggct tcggcatgtt caccaccctg ggctacctga tttgcggctt tcgggtggtg 3600
ctgatgtacc gcttcgagga ggagctgttc ctgcgcagcc tgcaagacta caaaattcag 3660
tctgccctgc tggtgccaac cctgttcagc ttcttcgcta agagcaccct gatcgacaag 3720
tacgacctgt ctaacctgca cgagattgcc tctggcggcg ccccactgtc taaggaggtg 3780
ggcgaagccg tggccaagcg ctttcatctg ccaggcatcc gccagggcta cggcctgacc 3840
gagacaacca gcgccattct gattacccca gagggcgacg acaagcctgg cgccgtgggc 3900
aaggtggtgc cattcttcga ggccaaggtg gtggacctgg acaccggcaa gaccctggga 3960
gtgaaccagc gcggcgagct gtgtgtgcgc ggccctatga ttatgtccgg ctacgtgaat 4020
aaccctgagg ccacaaacgc cctgatcgac aaggacggct ggctgcactc tggcgacatt 4080
gcctactggg acgaggacga gcacttcttc atcgtggacc gcctgaagtc tctgatcaag 4140
tacaagggct accaggtggc cccagccgag ctggagtcta tcctgctgca gcaccctaac 4200
attttcgacg ccggagtggc cggcctgccc gacgacgatg ccggcgagct gcctgccgcc 4260
gtcgtcgtgc tggaacacgg caagaccatg accgagaagg agatcgtgga ctatgtggcc 4320
agccaggtga caaccgccaa gaagctgcgc ggcggagtgg tgttcgtgga cgaggtgccc 4380
aagggcctga ccggcaagct ggacgcccgc aagatccgcg agatcctgat caaggctaag 4440
aaaggcggca agatcgccgt gtaataattc tagagtcggg gcggccggcc gcttcgagca 4500
gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 4560
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 4620
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 4680
gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccaggt 4740
ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 4800
aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4860
aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 4920
cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 4980
ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 5040
cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 5100
ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 5160
gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 5220
gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 5280
acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 5340
cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 5400
acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 5460
ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 5520
ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 5580
gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 5640
atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 5700
ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 5760
attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 5820
ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 5880
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 5940
aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6000
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 6060
tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 6120
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 6180
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 6240
agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 6300
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 6360
ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 6420
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 6480
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 6540
cacatggctc gac 6553
Claims (8)
1. a kind of reporter gene for monitoring miRNA dynamic change, which is characterized in that the reporter gene includes PUF gene, matter
Grain psiCHECK-2-HM471G, Fluc gene, four sections of tandem repetitive sequences and and PUF with the pairing of purpose miRNA complete complementary
Six sections of NRE tandem repetitive sequences of complete complementary pairing.
2. it is according to claim 1 it is a kind of monitor miRNA dynamic change reporter gene, which is characterized in that it is described with
Four sections of tandem repetitive sequences of purpose miRNA complete complementary pairing are located at the downstream terminator codon TGA of PUF albumen.
3. it is according to claim 1 it is a kind of monitor miRNA dynamic change reporter gene, which is characterized in that it is described with
Six sections of NRE tandem repetitive sequences of PUF complete complementary pairing are located at the upstream Fluc gene start codon ATG.
4. a kind of reporter gene for monitoring miRNA dynamic change described in any one of claim 1 to 3, feature exist
In the nucleotide sequence of the reporter gene is as shown in SEQ ID NO.1.
5. it is described in claim 1 monitoring miRNA dynamic change reporter gene preparation method, which is characterized in that including with
Lower step:
1) by PUF gene cloning to plasmid psiCHECK-2-HM471G multiple cloning sites, plasmid psiCHECK-2- is obtained
HM471G-PUF；
2) by Fluc gene cloning to psiCHECK2-HM471G, psiCHECK2-HM471G-Fluc is obtained；
3) it is interacted, will be matched with purpose miRNA complete complementary by way of base pair complementarity using miRNA and target
Four sections of tandem repetitive sequences be cloned into, obtain psiCHECK-2-HM471G-PUF-miR-Fluc；
4) it is interacted by way of base pair complementarity using PUF albumen and NRE, six will matched with PUF complete complementary
Section NRE tandem repetitive sequence is cloned into, and obtains reporter gene psiCHECK-2-HM471G-PUF-miR reporter.
6. application of the reporter gene described in claim 1 in detection atomization miRNA dynamic change.
7. application according to claim 6, which is characterized in that the atomization includes Neural Differentiation and muscle point
Change.
8. application according to claim 6, which is characterized in that when purpose miRNA is not present in cell, PUF can be combined
Onto 6xNRE sequence, to inhibit the expression of Fluc, luciferase content is low in cell；When there are purpose miRNA in cell
When, purpose miRNA can be integrated on 4x miR target sequence, inhibit the expression of PUF, to release the inhibition to Fluc, make Fluc
Expression, generates the fluorescence signal that can detecte.
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| CN110628815A (en) * | 2019-11-21 | 2019-12-31 | 西安电子科技大学 | Reporter gene probe for monitoring miRNA expression based on gene editing technology and construction method thereof |
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