CN108548780A - The method of transcription factor chip agent box and high flux screening target gene transcription factor - Google Patents
The method of transcription factor chip agent box and high flux screening target gene transcription factor Download PDFInfo
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Abstract
The present invention relates to technical field of biomedical detection, a kind of method for carrying out high flux screening target gene transcription factor the present invention provides transcription factor chip agent box and using above-mentioned transcription factor chip agent box, in the screening technique, using target gene promoters area DNA fragmentation as probe, applied to transcription factor activity chip, effectively compensate for the insufficient disadvantage of transcription factor activity chip specificity, the problem of making it possible fast and efficiently to filter out target gene transcription factor, while avoiding subjective selectivity and omitting potential transcription factor.
Description
Technical field
The present invention relates to technical field of biomedical detection, it is related to a kind of transcription factor chip agent box and using this turn
Record the method that factor chip agent box carries out high flux screening target gene transcription factor.
Background technology
Transcription factor is a kind of albumen that can be combined with target gene DNA sequence dna and start target gene transcription.Transcription factor with
The combination of target gene DNA is dependent on the Binding site for transcription factor (transcription in target gene promoters area
Factorbinding site, TFBS), promoter region is normally at the transcription initiation site upstream 1000bp ranges of the gene
It is interior.Transcription factor controlling gene by with TFBS is combined in gene DNA sequence is expressed.
The study found that in a variety of diseases, especially in cancer, the gene expression profile of pathological tissues is markedly different from normal group
It knits, gene expression regulation is prompted to change.Changes in gene expression plays key effect in the progress of a variety of diseases, at present
There are the disease treatment means intervened for specific gene.Since transcriptional control is the key link of gene expression regulation, because
This understands the mechanism of changes in gene expression, that is, the transcription factor of controlling gene expression variation is positioned, to understanding base in lysis
Because the reason of expression changes is of great significance, while can be to find gene therapy intervention target spot to give a clue.
Currently, the method for finding target gene transcription factor mainly may by transcription factor binding sequence database analysis
The transcription factor combined with target gene promoters area, as JASPAR databases (Khan A, Fornes O, Stigliani A,
Gheorghe M,Castro-Mondragon JA,van der Lee R,Bessy A,Cheneby J,Kulkarni SR,
Tan G et al: JASPAR 2018:update ofthe open-access database oftranscription
factor binding profiles and its web framework.Nucleic acids research 2018,46
(D1):) or UniPROBE databases (Hume MA, Barrera LA, Gisselbrecht SS, Bulyk D1284.
ML:UniPROBE,update 2015:new tools and content for the online database
ofprotein-binding microarray data on protein-DNA interactions. Nucleic acids
research 2015,43(Database issue):D117-122.), then tested by gel shift
(electrophoretic mobility shift assay, EMSA), chromatin immune co-precipitation experiment (chromatin
Immunoprecipitation assay, ChIP) validation database analysis prediction transcription factor whether with target gene promoters
Area has in conjunction with activity.
It is clear that the method for conventional identification target gene transcription factor takes time and effort, can only be predicted according to database analysis
Transcription factor list attempt one by one or there is subjectivity to selectively attempt to, and very likely in the case where attempting out positive findings
Miss other transcription factors that can equally combine.
Recently there is a kind of high-throughput chip technology of screening activity change transcription factor, be used for living under the conditions of observation experiment
The changed transcription factor of property.This method screens range although reducing target gene transcription factor to a certain extent,
After the transcription factor for filtering out activity change, the transcription factor of regulation and control target gene can not be still focused on, it can only be according to database point
The transcription factor that can be wherein combined with target gene is analysed, is then verified again by EMSA, ChIP, still time and effort consuming.
Invention content
The purpose of the present invention is to provide a kind of transcription factor chip agent box, it is a kind of that the purpose of the present invention lies also in offer
The method of high flux screening target gene transcription factor.
The first aspect of the present invention provides a kind of transcription factor chip agent box.Include that nucleoprotein is taken out in the kit
Extract, two groups of TranSignal probe groups, target gene promoters area to be studied DNA fragmentation, probe-transcription factor complex separation
Purification componentry, probe separates collection liquid, transcription factor chip and development camera system.
Preferably, nucleoprotein extract includes BufferA mixed liquors, Buffer B mixed liquors and PBS cell flushing liquors.
When extracting nucleoprotein from tissue sample, BufferA mixed liquors and Buffer B mixed liquors are only used, when being carried from cell sample
When taking nucleoprotein, also need to use PBS cell flushing liquors.
Preferably, every group of TranSignal probe groups respectively include 345 kinds of TranSignal probes, turn respectively with 345 kinds
Factor-specific is recorded to combine.
Preferably, the site combined with 345 kinds of TranSignal probes complementaries containing 345 on transcription factor chip.This
Commercialization transcription factor chip agent box can be used in invention, such as Panomics company models are TranSignalTM
The kit (Cat.MA1215) of Protein/DNAArray, including 345 kinds of TranSignal probes, can specificity with 345 kinds
Transcription factor specific binding (specific 345 kinds of probes, transcription factor referring to Panomics companies website-
www.panomics.com).Also it is purchased from Bo Ao biotech firms modelHuman transcription factor activity profile chip agent box
(Cat.No.230010 contains 270 kinds of transcription factors).
Preferably, the length of target gene promoters area to be studied DNA fragmentation is 1000~2000bp.
The second aspect of the present invention, the method for providing high flux screening target gene transcription factor, this method steps flow chart
As shown in the flow chart in Fig. 1, mainly include the following steps that：
A. nucleoprotein extracts：Cell or tissue nucleoprotein extracting is carried out using the nucleoprotein extracting solution in kit；
B. target gene promoters area segment competitive binding transcription factor
It is divided to A, B two groups by the obtained nucleoprotein of step A, is separately added into TranSignal probe mixed liquors, has in nucleoprotein
Active transcription factor combines with corresponding probe and forms compound；Target gene promoters area DNA pieces to be studied are added in B groups simultaneously
Section, the segment and TranSignal probe competitive binding transcription factors, make TranSignal probes be combined with transcription factor and subtract
It is few；Show if several TranSignal probe signals of certain or certain weaken transcription corresponding to these TranSignal probes because
Son can be combined with target gene promoters area.
C. probe is collected
Probe-transcription factor is isolated and purified using adsorption column and incubation buffer (Column Incubation Buffer)
Compound then uses probe elution buffer (Column Elution Buffer) to elute probe-transcription factor complex,
And heating 5~10min at 95 DEG C makes transcription factor deform, and separates and collects two groups of probes of A, B.
D. probe and chip hybridization
Two groups of probes of A, B are hybridized with transcription factor chip respectively.Contain 345 and 345 on transcription factor chip
The site that kind TranSignal probes complementaries combine.
E. chemiluminescence detection
Developed using substrate luminescence method, being taken pictures using exposure system shows testing result, and measures hair using software
Luminous point gray value carries out quantitative analysis.
Wherein, in step A, from cell sample extract nucleoprotein the step of be：Take 1 × 105~1 × 107A cell, is adopted
Cell is cleaned with PBS cell flushing liquors, adds 0.1~1mLBufferA mixed liquor broken cell membranes after removing PBS cell flushing liquors, from
The heart collects nucleus, and then plus 15~150 μ LBuffer B mixed liquor lytic cell cores, extracting nucleoprotein are simultaneously quantitative；
The step of extracting nucleoprotein, is from tissue sample：100~500 μ g fresh tissue samples are taken, is cut into small pieces and is put into
Guan Zhong adds 0.2~1.5mLBufferA mixed liquors to be homogenized, nucleus is collected by centrifugation, adds 30~150 μ l Buffer B mixed liquors
Lytic cell core extracts nucleoprotein and quantifies.
In step E, after probe is by biotin labeling, with chip hybridization, the Streptavidin antibody and probe that add HRP to mark
In conjunction with, chemiluminescent substrate is then added on chip, carry out shine development, obtain development figure as shown in Figure 2.
Illustrate the probe if the luminous intensity of some probe of B groups is less than the intensity of A groups corresponding position probe by analysis
Corresponding transcription factor has combination with target gene.
The third aspect of the present invention provides the method for high flux screening target gene transcription factor in screening HNF4 alpha transcriptionals
Application in the factor.By the screening of the HNF4 alpha transcriptional factors, the screening technique in the present invention is verified.
The beneficial guarantee and effect of the present invention is as follows：
The problem of present invention is for transcription factor screening time and effort consuming in the prior art, uses target gene promoters area DNA
Segment is applied to transcription factor activity chip, it is insufficient effectively to compensate for transcription factor activity chip specificity as probe
Disadvantage makes it possible fast and efficiently to filter out target gene transcription factor, while avoiding subjective selectivity and omitting potential
The problem of transcription factor.
In order to achieve the purpose that rapidly and efficiently and specifically to screen target gene transcription factor, commodity in useization of the present invention turns
Factor chip agent box and target gene promoters area 1000bp DNA fragmentations to be studied are recorded, target gene promoters area DNA is used
Segment as probe application in transcription factor activity chip, it is effective for screening target gene transcription factor, and reflect with tradition
Targeting gene transcription factor method compare with it is high-throughput, rapidly and efficiently, without subjective selectivity, without potential transcription factor omit
Advantage.
Description of the drawings
Fig. 1 is the flow chart of the method for high flux screening target gene transcription factor in the present invention；
Fig. 2 is middle probe of the present invention and transcription factor chip hybridization results schematic diagram, and the brightness each put in figure is every
The luminous intensity of a probe represents corresponding transcription factor activity size.The brightness of some point increases compared with the control group
Then show that corresponding transcription factor activity increases, it is on the contrary then show corresponding transcription factor activity decline；
Fig. 3 is the mentality of designing figure of the high flux screening HNF4 alpha transcriptional factors in the embodiment of the present invention；
Fig. 4 be in the embodiment of the present invention ChIP experiment detection Iron overload under the conditions of transcription factor YY1 and FAST-1 with
HNF4 α promoter regions combine activity.Iron overload group L02 cells are collected using 30 μM of Holo-Tf reagents cultures after 24 hours thin
Born of the same parents, detection YY1 and FAST-1 are combined activity with HNF4 α promoter regions.Compared with Control groups, * * *：P ＜ 0.001, n=4.
Specific implementation mode
In conjunction with embodiment and attached drawing, the present invention is described in detail, but the implementation of the present invention is not limited only to this.
The reagents and materials used in the present invention are commercially available or can be prepared by literature method.Tool is not specified in the following example
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition or method described in document, or according to the normal condition proposed by manufacturer.
Unless otherwise stated, otherwise percentage and number are calculated by volume.
Embodiment 1：
Under to screen the transcription factor that Iron overload causes HNF4 alpha expressions to decline be used as practical application, to height of the invention
The method of flux screening transcription factor is described in detail and verifies, but the implementation of the present invention is not limited only to this.
One, about the HNF4 alpha transcriptional factors
4 α of liver cell nuclear transcription factor (Hepatocyte nuclear factor 4alpha, HNF4 α) is in liver spy
The transcription factor of opposite sex expression, expression variation can influence liver different physiological roles and pathologic process.The study found that liver
Dirty Iron overload leads to liver inflammation (LI M, TANGY, WU L, et al.The hepatocyte- by lowering HNF4 α
specific HNF4alpha/miR-122pathway contributes to iron overload-mediated
hepatic inflammation. Blood,2017,130(8):1041-51.), but Iron overload causes HNF4 alpha expressions to decline
Mechanism it is unclear.
Two, mentality of designing
Using L02 cells as experimental subjects, it is divided into Normal group, Iron overload group and HNF4 α promoter probe groups.Such as figure
Shown in 3, the transcription factor influenced by Iron overload can be determined by comparing control group and Iron overload group；By comparing control
Group can determine the transcription factor combined with HNF4 α promoter regions with HNF4 α promoter probes groups；Preceding two experimental results of association
It can determine that the transcription factor for not only being influenced but also being combined with HNF4 α promoter regions by Iron overload, i.e. Iron overload cause HNF4 α tables
Up to the intermediate link of decline.
Three, operating procedure
1, Nuclear extract is collected
1) L02 cell inoculations are set as 3 groups in 3 10cm culture dishes, and wherein Iron overload group adds 30 μm of ol/L
Holo-Tf reagents culture 24 hours, cell is washed after culture with cold PBS；
2) PBS is blotted, 1mLBufferA working solutions are added in each ware；
3) after 4 DEG C of shaking table 10min cell is collected with cell scraper；
4) cell being collected into is transferred to 1.5mL centrifuge tubes, 14000g, 4 DEG C of centrifugation 3min；
5) supernatant is removed, 150 μ LBuffer B working solutions, vortex 10s are added；
6) 60min is incubated in ice, per 20min jog pipes；
7) 4 DEG C, 14000g centrifuges 5min, takes supernatant, the Nuclear extract as needed；
8) albumen concentration is detected：Detergent can interfere Bradford or BCA to test in Buffer B, can only use Bio-Rad
Protein DC experiment detection albumen concentration.
2, chip detects transcription factor activity：
This experiment commodity in use transcription factor chip agent box (Panomics companies, model TranSignalTM
Protein/DNAArray)
2.1 prehybridization
1) 42 DEG C of water-bath preheatings of Hybridization Buffer are taken, until being completely dissolved.
2) every film is put into a hybridization bottle, pays attention to face-up (front cannot paste bottle wall), adds 1 × Pre- of 25mL
Treatment Buffer I, rotation hybridization 5min in 45 DEG C of hybrid heaters.
3) solution in bottle is outwelled, 25mL 1 × Pre-Treatment Buffer II, 45 DEG C of hybrid heater rotation hybridization are added
10min。
4) it rinses：Solution in bottle is outwelled, adds deionized water to half volume, unsuitable overfill, acutely rotation 30 seconds, wash 3
It is secondary.
5) prehybridization：The hybridization solution of 5mL preheatings is added, rotates 4h in 42 DEG C of hybrid heaters, can stay overnight.
2.2TranSignal probes-transcription factor combines
1) prepare TranSignal probes-transcription factor mixture：+ 10 μ L TranSignal of 3 μ g Nuclear extracts
Probe Mix +ddH2O (RNase, DNase free) to 20 μ L.Wherein HNF4 α promoter probes group is in this step addition etc.
HNF4 α promoter region 1000bp segments are measured, shown in sequence following (SEQ ID NO.1).
2) mixing, 15 DEG C of incubation 30min.
2.3 separation TranSignal probes-transcription factor complex
1) preincubate：Adsorption column is put into collecting pipe, and the cold Column Incubation of 500 μ L are added in adsorption column
Buffer, 4 DEG C, 10000g centrifuges 30s.
2) TranSignal probes-transcription factor complex binds adsorption column：Add in the TF-Probe mix being incubated
Enter 20 μ l Column Incubation Buffer, mixing is transferred in adsorption column, is incubated 30min on ice.
3) 4 DEG C, 7000g centrifuges 30s, abandons liquid.
4) adsorption column is put into new collecting pipe, the unbonded probe of washing removal：600 μ l Column are added in adsorption column
Wash Buffer are incubated 10 minutes on ice, and 4 DEG C, 7000g centrifuges 30s, abandons liquid, is repeated 3 times.
5) it is cleared Liquid Residue, 4 DEG C, 10000g centrifuges 30s.
6) probe-transcription factor complex is eluted：Adsorption column is put into new 1.5ml centrifuge tubes, takes 60 μ l Column
Adsorption column is added in Elution Buffer, is incubated at room temperature 5min.
7) room temperature, 10000g centrifuge 1min.
2.4 hybridize the label probe of elution with the film handled well
1) probe-transcription factor denaturation and hybridization：The 5 DEG C of 3min denaturation of probe-transcription factor 9, are added hybridization bottle, 42 DEG C miscellaneous
It hands in stove and rotates hybridized overnight.
2) hybridization solution is outwelled, the 50mLHybridizationWash I of 42 DEG C of preheatings is added, are rotated in 42 DEG C of hybrid heaters
20min is washed, is repeated twice.
3) hybridize the 50mL HybridizationWash II for adding 42 DEG C of preheatings in bottle, rotate washing in 42 DEG C of hybrid heaters
20min is repeated twice.
2.5 films detect, and notice whenever film is not competent
1) it closes：Prepare clean plate, tweezers move into film in disk (face-up), and 1 × Blocking of 20mL are added
Buffer。
2) it places and is vibrated at a slow speed on shaker, room temperature closes 30min.
3) antibody incubation：It takes in 1 × Blocking of 1mL Buffer to clean centrifuge tube, the chain of 20 μ LHRP labels is added
Mould Avidin, vortex mixing the HRP labelled streptavidins diluted are added in confining liquid, room temperature 30min.
4) liquid is abandoned, 20mL 1 × Wash Buffer are added, room temperature is washed 3 times, each 8min.
5) add 20mL1 × Detection Buffer, be incubated at room temperature 5min.
6) it detects：Prepare Working Substrate Solution：250 μ L Solution I add 250 μ L
Solution II, vortex mixing, then add 2ml Solution III, mixing.
7) chip is put on a plastic film, uniform fold 2.5mLWorking Substrate Solution on chip,
Another layer of plastic film is covered immediately.Ensure that substrate is uniformly distributed, without bubble, does not wrinkle.It is incubated at room temperature 5min.
8) top is rushed into extra substrate pressurization, paper handkerchief blots the extra substrate of frosting quiet, uses chemiluminescence
Imaging system (SynGene companies) exposes 5-15min.
9) GeneSnap softwares analysis spot gray value is used to carry out quantitative analysis.
3, interpretation of result
The present embodiment use target gene promoters area to be studied segment as probe application in existing commercialization transcription because
Sub- activity chip carries out high-throughput, specifically target gene transcription factor screening.
Compare control group and Iron overload group transcription factor activity result of variations is as shown in table 1：
Twice of activity change or more and difference has statistics meaning under the conditions of 1 transcription factor activity chip of table detection Iron overload
Transcription factor (n=3) statistical result of justice
It is found by comparing control group and the variation of Iron overload group transcription factor activity, in 345 kinds of transcription factors, iron mistake
Load leads to 15 kinds of transcription factor activities variation 2 times or more, wherein 11 transcription factor activities decline (variation multiple ＜ 0.5 and
P ＜ 0.05), 4 transcription factor activities increase (variation multiple ＞ 2 and p ＜ 0.05).
Control group and HNF4 α promoter probe group transcription factor activity result of variations are as shown in table 2：
Activity change weakens and poor twice or more after 2 transcription factor activity chip of table detection addition HNF4 α promoter probes
Different statistically significant transcription factor (n=3) statistical result
Find there are 3 transcription factors to exist by comparing control group and the variation of HNF4 α promoter probe group transcription factor activities
Reduced activity (variation multiple ＜ 0.5 and p ＜ 0.05), i.e. this 3 transcription factor energy and HNF4 after addition HNF4 α promoter probes
α promoter region 1000bp segments combine.
In summary two results are it can be found that the activity of transcription factor YY1 and FAST-1 are inhibited by Iron overload, again
It can be combined with HNF4 α promoter regions, it may be the original that Iron overload causes HNF4 alpha expressions to decline to prompt the two transcription factors
Cause.
As shown in figure 4, through ChIP it is experimentally confirmed that two kinds of transcription factors of YY1 and FAST-1 are opened with HNF4 α when Iron overload
Mover area declines in conjunction with activity, consistent with transcription factor activity Microarray results, prompts transcription factor YY1 and FAST-1
It has been involved in the process that Iron overload causes HNF4 alpha expressions to decline.
The above result shows that using target gene promoters area DNA fragmentation as probe application in transcription factor activity chip,
It is effective for screening target gene transcription factor, and have compared with conventional identification target gene transcription factor method it is high-throughput,
Rapidly and efficiently, without subjective selectivity, omitted without potential transcription factor the advantages of.
Comparative example：The method for predicting the HNF4 alpha transcriptional factors using database JASPAR
It, may be with HNF4 α using 2018 editions transcription factor database JASPAR predictions as the comparative example of above-described embodiment
The transcription factor combined within the scope of promoter region 1000bp has 177, and concrete outcome is as shown in table 3, the sequence of the transcription factor
Such as SEQ ID NO:2~SEQ ID NO:Shown in 178.Obviously with traditional transcription factor identification method in this 177 potential regulation and control
The transcription factor that really regulation and control HNF4 alpha expressions are filtered out in the transcription factor of HNF4 α is extremely difficult, is tested by gel shift
(electrophoretic mobility shift assay, EMSA), chromatin immune co-precipitation experiment (chromatin
Immunoprecipitation assay, ChIP) validation database analysis prediction transcription factor whether with target gene promoters
Area has in conjunction with activity, can expend a large amount of manpower and materials, and subjective bias caused by selectively attempting is larger and have very much can
Can be negative findings.
There are binding sites in the prediction of table 3JASPAR transcription factor databases and HNF4 α promoter region 1000bp sequences
Transcription factor and binding sequence
Note：With transcription initiation site position for 0, upstream binding site position is with negative number representation.
The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent under the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Sequence table
<110>Second Military Medical University, PLA
<120>The method of transcription factor chip agent box and high flux screening target gene transcription factor
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gccata 6
<210> 35
<211> 15
<212> DNA
<213> HNF4A
<400> 35
ttggactctc acctc 15
<210> 36
<211> 8
<212> DNA
<213> PDX1
<400> 36
tccattag 8
<210> 37
<211> 11
<212> DNA
<213> BATF::JUN
<400> 37
gaaatgagca a 11
<210> 38
<211> 10
<212> DNA
<213> HOXB13
<400> 38
gccataaaac 10
<210> 39
<211> 8
<212> DNA
<213> FOXD1
<400> 39
gtaaccag 8
<210> 40
<211> 9
<212> DNA
<213> HIC2
<400> 40
atgccccca 9
<210> 41
<211> 10
<212> DNA
<213> OLIG1
<400> 41
aacatattta 10
<210> 42
<211> 11
<212> DNA
<213> ELF5
<400> 42
tgcaggaagt a 11
<210> 43
<211> 10
<212> DNA
<213> TFE3
<400> 43
gacacctgcc 10
<210> 44
<211> 8
<212> DNA
<213> LMX1B
<400> 44
taaattaa 8
<210> 45
<211> 12
<212> DNA
<213> ELF4
<400> 45
atgcaggaag ta 12
<210> 46
<211> 10
<212> DNA
<213> MIXL1
<400> 46
ttaaattaaa 10
<210> 47
<211> 11
<212> DNA
<213> NFIC
<400> 47
tccctggcat g 11
<210> 48
<211> 8
<212> DNA
<213> SHOX
<400> 48
ttaattta 8
<210> 49
<211> 11
<212> DNA
<213> FOS
<400> 49
ggtgagtgat a 11
<210> 50
<211> 13
<212> DNA
<213> ELF1
<400> 50
catgcaggaa gta 13
<210> 51
<211> 8
<212> DNA
<213> MSX2
<400> 51
ctaatcac 8
<210> 52
<211> 10
<212> DNA
<213> HOXA2
<400> 52
tctaatcacc 10
<210> 53
<211> 8
<212> DNA
<213> MEIS2
<400> 53
aggacagg 8
<210> 54
<211> 8
<212> DNA
<213> PRRX1
<400> 54
taaattaa 8
<210> 55
<211> 10
<212> DNA
<213> ETV3
<400> 55
gcaggaagta 10
<210> 56
<211> 10
<212> DNA
<213> MZF1
<400> 56
gaatggggaa 10
<210> 57
<211> 12
<212> DNA
<213> ZBTB7B
<400> 57
tgtacccccg aa 12
<210> 58
<211> 10
<212> DNA
<213> HOXC10
<400> 58
gccataaaac 10
<210> 59
<211> 10
<212> DNA
<213> TFCP2
<400> 59
aaactggtga 10
<210> 60
<211> 10
<212> DNA
<213> NOTO
<400> 60
ttaaattaaa 10
<210> 61
<211> 10
<212> DNA
<213> TFEB
<400> 61
gacacctgcc 10
<210> 62
<211> 7
<212> DNA
<213> FOXP3
<400> 62
ataaata 7
<210> 63
<211> 8
<212> DNA
<213> DLX6
<400> 63
ctatttat 8
<210> 64
<211> 10
<212> DNA
<213> HOXB2
<400> 64
ggtgattaga 10
<210> 65
<211> 10
<212> DNA
<213> FOSB::JUNB
<400> 65
ggtgagtgat 10
<210> 66
<211> 8
<212> DNA
<213> ISL2
<400> 66
tcacttag 8
<210> 67
<211> 10
<212> DNA
<213> MYB
<400> 67
agcaacagtc 10
<210> 68
<211> 11
<212> DNA
<213> GATA2
<400> 68
gtgttatctt c 11
<210> 69
<211> 10
<212> DNA
<213> GSX2
<400> 69
actatttata 10
<210> 70
<211> 7
<212> DNA
<213> FOXO4
<400> 70
ataaata 7
<210> 71
<211> 12
<212> DNA
<213> FOS::JUNB
<400> 71
ggtgtgtcac ca 12
<210> 72
<211> 10
<212> DNA
<213> HOXB3
<400> 72
ctccattagt 10
<210> 73
<211> 7
<212> DNA
<213> USF1
<400> 73
cacctgg 7
<210> 74
<211> 16
<212> DNA
<213> RFX3
<400> 74
cattgccatg gagaca 16
<210> 75
<211> 12
<212> DNA
<213> CREB1
<400> 75
catggtgaca ca 12
<210> 76
<211> 8
<212> DNA
<213> LHX9
<400> 76
tccattag 8
<210> 77
<211> 11
<212> DNA
<213> FOXK2
<400> 77
ttataaatag t 11
<210> 78
<211> 11
<212> DNA
<213> CEBPB
<400> 78
tgtttctcca c 11
<210> 79
<211> 8
<212> DNA
<213> VAX1
<400> 79
ctatttat 8
<210> 80
<211> 8
<212> DNA
<213> RAX2
<400> 80
taaattaa 8
<210> 81
<211> 11
<212> DNA
<213> NFKB1
<400> 81
cggttttccc a 11
<210> 82
<211> 9
<212> DNA
<213> CDX1
<400> 82
gttataaat 9
<210> 83
<211> 10
<212> DNA
<213> FOSL2::JUNB
<400> 83
ggtgagtgat 10
<210> 84
<211> 8
<212> DNA
<213> LMX1A
<400> 84
taaattaa 8
<210> 85
<211> 13
<212> DNA
<213> SMAD2::SMAD3::SMAD4
<400> 85
gagtctagca caa 13
<210> 86
<211> 16
<212> DNA
<213> RFX4
<400> 86
cattgccatg gagaca 16
<210> 87
<211> 10
<212> DNA
<213> MAX
<400> 87
tagcacaagg 10
<210> 88
<211> 10
<212> DNA
<213> OLIG3
<400> 88
accatattta 10
<210> 89
<211> 10
<212> DNA
<213> GSX1
<400> 89
actatttata 10
<210> 90
<211> 10
<212> DNA
<213> ETV4
<400> 90
gcaggaagta 10
<210> 91
<211> 9
<212> DNA
<213> RUNX2
<400> 91
taaccccag 9
<210> 92
<211> 8
<212> DNA
<213> OTX1
<400> 92
ttaatcag 8
<210> 93
<211> 11
<212> DNA
<213> DUX4
<400> 93
aaattcaacc t 11
<210> 94
<211> 10
<212> DNA
<213> ETV5
<400> 94
gcaggaagta 10
<210> 95
<211> 10
<212> DNA
<213> SP1
<400> 95
taggcgcgga 10
<210> 96
<211> 10
<212> DNA
<213> FEV
<400> 96
gcaggaagta 10
<210> 97
<211> 12
<212> DNA
<213> ZBTB7C
<400> 97
aagcccaccc ag 12
<210> 98
<211> 11
<212> DNA
<213> KLF16
<400> 98
gacacaccca t 11
<210> 99
<211> 10
<212> DNA
<213> HOXD11
<400> 99
gccataaaac 10
<210> 100
<211> 8
<212> DNA
<213> OTX2
<400> 100
ttaatcag 8
<210> 101
<211> 11
<212> DNA
<213> HOXC12
<400> 101
agttataaat a 11
<210> 102
<211> 10
<212> DNA
<213> ERG
<400> 102
gcaggaagta 10
<210> 103
<211> 8
<212> DNA
<213> LBX1
<400> 103
ataaatag 8
<210> 104
<211> 10
<212> DNA
<213> NEUROD2
<400> 104
accatattta 10
<210> 105
<211> 13
<212> DNA
<213> ZBTB18
<400> 105
gagccaggtg tat 13
<210> 106
<211> 10
<212> DNA
<213> MEOX1
<400> 106
tctaatcacc 10
<210> 107
<211> 10
<212> DNA
<213> GCM2
<400> 107
gatgctggga 10
<210> 108
<211> 9
<212> DNA
<213> NKX3-2
<400> 108
gtcacttag 9
<210> 109
<211> 10
<212> DNA
<213> HOXA13
<400> 109
gttataaata 10
<210> 110
<211> 10
<212> DNA
<213> ETV1
<400> 110
gcaggaagta 10
<210> 111
<211> 10
<212> DNA
<213> NFIA
<400> 111
agtgcctact 10
<210> 112
<211> 11
<212> DNA
<213> PROP1
<400> 112
taatttaaat a 11
<210> 113
<211> 8
<212> DNA
<213> NKX6-1
<400> 113
ctatttat 8
<210> 114
<211> 10
<212> DNA
<213> TEAD1
<400> 114
cccattctaa 10
<210> 115
<211> 12
<212> DNA
<213> TGIF1
<400> 115
tggcaggtgt ct 12
<210> 116
<211> 11
<212> DNA
<213> TBX2
<400> 116
gaggtgttat c 11
<210> 117
<211> 15
<212> DNA
<213> MEF2A
<400> 117
ttccaaaaaa aaagc 15
<210> 118
<211> 14
<212> DNA
<213> SPIC
<400> 118
agaatgggga agga 14
<210> 119
<211> 14
<212> DNA
<213> KLF14
<400> 119
tgacacaccc atag 14
<210> 120
<211> 7
<212> DNA
<213> FOXI1
<400> 120
ataaata 7
<210> 121
<211> 12
<212> DNA
<213> PBX2
<400> 121
ctgaaggaca ga 12
<210> 122
<211> 10
<212> DNA
<213> TBX21
<400> 122
gaggtgttat 10
<210> 123
<211> 11
<212> DNA
<213> ETV2
<400> 123
tgcaggaagt a 11
<210> 124
<211> 7
<212> DNA
<213> FOXO6
<400> 124
ataaata 7
<210> 125
<211> 8
<212> DNA
<213> VAX2
<400> 125
ctatttat 8
<210> 126
<211> 10
<212> DNA
<213> BARHL2
<400> 126
ctttaacgta 10
<210> 127
<211> 8
<212> DNA
<213> FOXO3
<400> 127
tataaata 8
<210> 128
<211> 13
<212> DNA
<213> NEUROD1
<400> 128
tggcagctgg ccg 13
<210> 129
<211> 11
<212> DNA
<213> NR2F2
<400> 129
tgaaggacag a 11
<210> 130
<211> 10
<212> DNA
<213> LHX6
<400> 130
cttaatcaga 10
<210> 131
<211> 11
<212> DNA
<213> FOXP2
<400> 131
ttataaatag t 11
<210> 132
<211> 16
<212> DNA
<213> RFX2
<400> 132
cattgccatg gagaca 16
<210> 133
<211> 10
<212> DNA
<213> RAX
<400> 133
tataaatagt 10
<210> 134
<211> 8
<212> DNA
<213> MSX1
<400> 134
tccattag 8
<210> 135
<211> 11
<212> DNA
<213> HOXD12
<400> 135
agttataaat a 11
<210> 136
<211> 15
<212> DNA
<213> RFX5
<400> 136
ctgcatggtg acaca 15
<210> 137
<211> 10
<212> DNA
<213> KLF5
<400> 137
actccgcgcc 10
<210> 138
<211> 10
<212> DNA
<213> MNX1
<400> 138
atcaataaga 10
<210> 139
<211> 10
<212> DNA
<213> ESX1
<400> 139
actatttata 10
<210> 140
<211> 8
<212> DNA
<213> ISX
<400> 140
ccaatttg 8
<210> 141
<211> 18
<212> DNA
<213> KLF13
<400> 141
gtgacacacc catagttt 18
<210> 142
<211> 10
<212> DNA
<213> FOS::JUN
<400> 142
gtgtgtcacc 10
<210> 143
<211> 12
<212> DNA
<213> GLI2
<400> 143
aagcccaccc ag 12
<210> 144
<211> 10
<212> DNA
<213> ERF
<400> 144
gcaggaagta 10
<210> 145
<211> 10
<212> DNA
<213> TFAP4
<400> 145
gccagctgcc 10
<210> 146
<211> 11
<212> DNA
<213> E2F6
<400> 146
tagtgggaaa a 11
<210> 147
<211> 13
<212> DNA
<213> HSF4
<400> 147
ctccagcccc ttc 13
<210> 148
<211> 10
<212> DNA
<213> MEOX2
<400> 148
tctgattaag 10
<210> 149
<211> 11
<212> DNA
<213> NFE2L2
<400> 149
gtgacacacc c 11
<210> 150
<211> 9
<212> DNA
<213> THAP1
<400> 150
ccgcccggt 9
<210> 151
<211> 6
<212> DNA
<213> ZNF354C
<400> 151
ctccgc 6
<210> 152
<211> 12
<212> DNA
<213> FOSL2::JUN
<400> 152
gggtgtgtca cc 12
<210> 153
<211> 8
<212> DNA
<213> BSX
<400> 153
ctatttat 8
<210> 154
<211> 10
<212> DNA
<213> NR4A1
<400> 154
ttaaggtccg 10
<210> 155
<211> 10
<212> DNA
<213> MYF6
<400> 155
agcaacagtc 10
<210> 156
<211> 10
<212> DNA
<213> LHX2
<400> 156
actaatggag 10
<210> 157
<211> 21
<212> DNA
<213> IRF1
<400> 157
cttttgtctc agttttggat c 21
<210> 158
<211> 8
<212> DNA
<213> VDR
<400> 158
agagtgca 8
<210> 159
<211> 8
<212> DNA
<213> VSX2
<400> 159
ctatttat 8
<210> 160
<211> 11
<212> DNA
<213> FOXH1
<400> 160
attagtccac a 11
<210> 161
<211> 8
<212> DNA
<213> VSX1
<400> 161
ctatttat 8
<210> 162
<211> 13
<212> DNA
<213> EOMES
<400> 162
gaggtgttat ctt 13
<210> 163
<211> 10
<212> DNA
<213> MSC
<400> 163
gacacctgcc 10
<210> 164
<211> 10
<212> DNA
<213> LBX2
<400> 164
tctgattaag 10
<210> 165
<211> 10
<212> DNA
<213> ALX3
<400> 165
actatttata 10
<210> 166
<211> 15
<212> DNA
<213> SP2
<400> 166
gcccctccct cggtt 15
<210> 167
<211> 14
<212> DNA
<213> EBF1
<400> 167
tatccccaga gtct 14
<210> 168
<211> 11
<212> DNA
<213> NFYA
<400> 168
ggccaatggg a 11
<210> 169
<211> 8
<212> DNA
<213> TBX4
<400> 169
aggcgcgg 8
<210> 170
<211> 10
<212> DNA
<213> HINFP
<400> 170
taaggtccgt 10
<210> 171
<211> 15
<212> DNA
<213> CENPB
<400> 171
cccgtccacc aggaa 15
<210> 172
<211> 12
<212> DNA
<213> TGIF2
<400> 172
tggcaggtgt ct 12
<210> 173
<211> 10
<212> DNA
<213> MLXIPL
<400> 173
ggcaggtgtc 10
<210> 174
<211> 11
<212> DNA
<213> FOXA1
<400> 174
tggttactct t 11
<210> 175
<211> 19
<212> DNA
<213> CTCF
<400> 175
cagccaccag gagcccgtc 19
<210> 176
<211> 8
<212> DNA
<213> E2F1
<400> 176
tttggagc 8
<210> 177
<211> 8
<212> DNA
<213> EN1
<400> 177
ataaatag 8
<210> 178
<211> 12
<212> DNA
<213> EHF
<400> 178
atgcaggaag ta 12
Claims (9)
1. a kind of transcription factor chip agent box, which is characterized in that including：
Nucleoprotein extract, two groups of TranSignal probe groups, target gene promoters area to be studied DNA fragmentation, probe-transcription because
Sub- compound isolates and purifies component, probe separates collection liquid, transcription factor chip and development camera system.
2. transcription factor chip agent box according to claim 1, it is characterised in that：
Wherein, the nucleoprotein extract includes Buffer A mixed liquors, Buffer B mixed liquors and PBS cell flushing liquors.
3. transcription factor chip agent box according to claim 1, it is characterised in that：
Wherein, every group of TranSignal probe groups respectively include 345 kinds of TranSignal probes, respectively with 345 kinds of transcription factors
Specific binding.
4. transcription factor chip agent box according to claim 3, it is characterised in that：
Wherein, the site combined with 345 kinds of TranSignal probes complementaries containing 345 on the transcription factor chip.
5. transcription factor chip agent box according to claim 1, it is characterised in that：
Wherein, the length of the target gene promoters area to be studied DNA fragmentation is 1000~2000bp.
6. carrying out high flux screening target gene transcription using Claims 1 to 5 any one of them transcription factor chip agent box
The method of the factor, which is characterized in that include the following steps：
A. nucleoprotein extracts：Cell or tissue nucleoprotein extracting is carried out using the nucleoprotein extracting solution in kit；
B. target gene promoters area segment competitive binding transcription factor
It is divided to A, B two groups by the obtained nucleoprotein of step A, is separately added into TranSignal probe mixed liquors, it is active in nucleoprotein
Transcription factor and corresponding probe combine and form compound；Target gene promoters area to be studied DNA fragmentation is added in B groups simultaneously,
The segment and TranSignal probe competitive binding transcription factors, make TranSignal probes be combined reduction with transcription factor；
Show the transcription factor corresponding to these TranSignal probes if certain or if certain several TranSignal probe signals weakens
It can be combined with target gene promoters area,
C. probe is collected
Probe-transcription factor complex is isolated and purified using adsorption column and incubation buffer, then uses probe separates collection liquid
Elution probe-transcription factor complex simultaneously makes transcription factor deform in a heated condition, separates and collects two groups of probes of A, B；
D. probe and chip hybridization
Two groups of probes of A, B are hybridized with transcription factor chip respectively；
E. chemiluminescence detection
Developed using substrate luminescence method, being taken pictures using exposure system shows testing result, and measures luminous point using software
Gray value carries out quantitative analysis.
7. the method for high flux screening target gene transcription factor according to claim 6, it is characterised in that：
Wherein, in step E, after probe is by biotin labeling, with chip hybridization, adds the Streptavidin antibody that HRP is marked and visit
Needle combines, and chemiluminescent substrate is then added on chip, carries out the development that shines.
8. the method for high flux screening target gene transcription factor according to claim 6, it is characterised in that：
Wherein, in step A, from cell sample extract nucleoprotein the step of be：Take 1 × 105~1 × 107A cell, using PBS
Cell flushing liquor cleans cell, adds 0.1~1mL Buffer A mixed liquor broken cell membranes, centrifugation after removing PBS cell flushing liquors
Nucleus is collected, then plus 15~150 μ L Buffer B mixed liquor lytic cell cores, extracting nucleoprotein are simultaneously quantitative；
The step of extracting nucleoprotein, is from tissue sample：100~500 μ g fresh tissue samples are taken, is cut into small pieces and is put into pipe,
Add 0.2~1.5mL Buffer A mixed liquors to be homogenized, nucleus is collected by centrifugation, 30~150 μ l Buffer B mixed liquors is added to crack
Nucleus extracts nucleoprotein and quantifies.
9. the method for the high flux screening target gene transcription factor described in claim 6 answering in screening the HNF4 alpha transcriptional factors
With.
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| CN111575365A (en) * | 2020-05-11 | 2020-08-25 | 深圳市人民医院 | ES marker and application thereof |
| CN114427000A (en) * | 2022-01-21 | 2022-05-03 | 信阳师范学院 | Identification method of specific gene transcription initiation site |
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| CN114427000A (en) * | 2022-01-21 | 2022-05-03 | 信阳师范学院 | Identification method of specific gene transcription initiation site |
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