CN1296491C - Active transcription factor detecting method and kit - Google Patents
Active transcription factor detecting method and kit Download PDFInfo
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- CN1296491C CN1296491C CNB2004100601387A CN200410060138A CN1296491C CN 1296491 C CN1296491 C CN 1296491C CN B2004100601387 A CNB2004100601387 A CN B2004100601387A CN 200410060138 A CN200410060138 A CN 200410060138A CN 1296491 C CN1296491 C CN 1296491C
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Abstract
The present invention relates to an active transcription factor detecting method and a kit thereof. The active transcription factor detecting method is an improvement based on an ELISA detecting method. The active transcription factor detecting method has the difference that a new double-strand oligonucleotide probe is used for overcoming the disadvantage of a double-strand oligonucleotide probe of the ELISA detecting method caused by designing construction. By experimental verification, the active transcription factor detecting method reaches high sensitivity and detecting specificity, which shows that the new double-strand oligonucleotide probe completely can be used for the active detection of transcription factors. The detecting effect of a probe with a copy and the detecting effect of a probe with a two-copy combining sequence are identical. The present invention also relates to the kit for the active transcription factor detecting method.
Description
Technical field
The invention belongs to biochemical and molecular biosciences subject field, more particularly it is a kind of transcription factor activity detection method, the invention still further relates to the used test kit of this transcription factor activity detection method.
Background technology
The nucleotide sequence that plays the regulate gene expression effect on same nucleic acid chains is called cis-acting elements, comprises promotor, enhanser, attenuator etc.; Transcription factor also claims trans-acting factor, thereby is meant and acts on the protein that cis-acting elements can play regulating and controlling effect to the genetic expression on the different nucleic acid chains.Transcription factor will play a role, and must have certain activity, this depend on transcription factor quantity and with cis-acting elements bonded avidity.Detect the activity of transcription factor, significant for illustrating certain expression of gene regulatory mechanism, thereby be widely used in biology and the medical research.
At present, the common method that is used to detect transcription factor activity mainly contains four kinds: one, and the gel electrophoresis Mobility Shift Assay, (electrophoretic mobility shift assay, EMSA), claim gel retardation analysis (gel retarding assay, gel shift assay) again.In this experiment, cell extract and the radiolabeled double chain oligonucleotide probe that contains certain transcription factor particular combination sequence are hatched altogether, the activatory transcription factor can with corresponding probe after external combination, last sample electrophoresis in the non-denaturing polyacrylamide gel carries out the radioautograph colour developing at last.Two bands can appear in the result, and one is moved soon, and corresponding to free probe, one is to be detained band, corresponding to the complex body of transcriptional factors and probe formation.Certainly, the specificity of its reaction needs to verify by the competitive assay of antibody and mutant oligonucleotide probe in addition; Though this method is responsive, is unsuitable for extensive detection, and need the contact radioelement; Two, reporter gene is analyzed (reporter gene assay), reporter gene (generally being luciferase or beta-galactosidase enzymes) is structured under the specific transcription factor bonded promotor, reflects the activity of transcription factor by the expression level of reporter gene indirectly.As long as the efficient height of cell transfecting, this method still compare responsive,, also can detect on a large scale simultaneously by means of fluorescence microplate reader; But in this case, other transcription factors are possibility Impact Report expression of gene level also, and the specificity of reaction is affected; And, owing to be the activity that reflects transcription factor by the activity of the luciferase of expressing indirectly, will certainly be subjected to downstream process as transcribe, the influence of translation etc.Three, use the nuclear translocation of antibody in situ detection transcription factor, but this is a kind of method qualitatively, is unsuitable for extensive detection.Four, to some transcription factor, as NF-κ B, also available western blot detects the degraded situation of its endochylema arrestin (I κ B), reflects the activity of transcription factor indirectly, also is unsuitable for extensive detection.
Though above method has been brought into play crucial effect in the research of gene transcription regulation, owing to do not possess quick, easy, high-throughout characteristics, it is very limited that the research of transcriptional control is still carried out.
Based on the transcription factor activity detection method of elisa technique, be a kind of brand-new transcription factor activity detection technique that grew up in recent years.This method is at first by Gubler, and foundation such as M.L are subsequently by improvement such as Patricia Renard.This method has been utilized the principle of traditional E LISA, and different with it is, detected protein is not by antibody capture, but the double chain oligonucleotide probe that comprises transcription factor particular combination sequence that is fixed on the enzyme plate is captured.Be used to detect the transcription factor that one of transcription factor resists the identification activatory to be connected with target DNA.By two anti-IgG coupling matter and color reactions, obtain quantitative results again.
In this technology, the design construction of double chain oligonucleotide probe is a key.This probe is normally biotin labeled, need be fixed on the enzyme plate of avidin bag quilt.In this probe sequence, (transcription factor is that double-stranded DNA is conjugated protein to the specific binding sequence of transcription factor, generally about 20 base pairs) be absolutely necessary naturally, but it is far from being enough that this binding sequence is only arranged, because stay enough spaces can not for the effect of transcription factor complex and probe, this can reduce the sensitivity of detection even cause and can not detect.Therefore (catenation sequence is necessary linker) to add the preceding paragraph sequence at the non-transcribed factor effect end of probe.But this that adds section double-stranded sequence very possible with original transcription factor particular combination sequence between the new transcription factor binding member of formation, increase some other unknown transcription factor in conjunction with chance, also can increase the purpose transcription factor that will detect and the non-specific binding between the catenation sequence, disturb specific detection, reduce the sensitivity that detects, even cause detecting failure; In order to address this problem, the treatment process of Patricia Renard etc. is that catenation sequence is designed to stochastic sequence, make the concentration and probe concentration of each particular sequence all very low,, also do not reach the level that influence detects even exist some other unknown transcription factor bonded to disturb.But at this time,, can't be directly take off fire and aggregate into two strands with the complementary strand of a certain particular sequence because the single-stranded probe sequence of synthetic is at random, therefore, also need to design a pair of primer, obtain double-chain probe by PCR reaction amplification, purified again, obtain detecting required double chain DNA probe.The preparation process more complicated of whole probe, (because the difficulty of short chain PCR reaction costs an arm and a leg, the length of stochastic sequence is generally about 100bp, the whole probe sequence length is about 140bp, synthesizing of long-chain oligonucleotide probe is much not only expensive, and domestic many DNA Synesis Company can not ensure the quality of products, the purifying of short chain dna probe also needs the special-purpose purification devices of some imports), test kit (as the Active Motif company) costliness of external exploitation has limited at home and has carried out, popularizes this technology and be used for scientific research.Nonetheless, this probe also can not effectively reduce the purpose transcription factor that will detect and the non-specific binding between the catenation sequence, and this background that can cause detecting is deepened.
Summary of the invention
The objective of the invention is to overcome the weak point of transcription factor activity detection method in the above-mentioned elisa technique and a kind of transcription factor activity detection method is provided.This method is based on the improvement of ELISA detection method, and its special feature is to adopt a kind of new double chain oligonucleotide probe to make up, to overcome the deficiency that design construction was brought of ELISA detection method double chain oligonucleotide probe.
Another object of the present invention provides a kind of test kit that contains above-mentioned double chain oligonucleotide probe in detecting.
The objective of the invention is to reach by following measure: a kind of transcription factor activity detection method which comprises at least following steps:
1. the extraction of cell cultures, total protein of cell;
2. double chain oligonucleotide probe 1 or 2 is fixed on the enzyme plate of avidin bag quilt: 2pmol double chain oligonucleotide probe 1 or 2 is dissolved among the 50 μ lPBS, join in the enzyme plate aperture of avidin bag quilt, 37 ℃, hatch 1 hour after, PBS+0.1%Tween-20 washing 2 times, PBS washes 1 time;
3. the total protein that extracts is combined with double chain oligonucleotide probe 1 or 2: every hole adds 20 μ l total proteins and 30 μ l binding buffer[4mM HEPES pH7.5,100mM KCl, 8%glycerol, 5mM DTT, 0.2%BSA, 40 μ g/ml salmon sperm DNAs], room temperature vibration 80rpm, after hatching 1 hour, PBS+0.1%Tween-20 washing 3 times;
4. with transcription factor specificity first antibody, be attached on transcription factor-probe complex: every hole adds transcription factor specificity the one 100 μ l that does dilution in 1: 1000 with PBS+0.1%BSA, after the incubated at room 1 hour, PBS+0.1%Tween-20 washing 3 times;
5. two anti-IgG with the HRP mark are attached on transcription factor specificity first antibody-transcription factor-dna probe mixture: every hole adds the two anti-Ig-G100 μ l that make the HRP mark of dilution in 1: 10000 with PBS+0.1%BSA, after the incubated at room 1 hour, PBS+0.1%Tween-20 washing 4 times;
6. color reaction: every hole adds tetramethyl benzidine, and (tetramethylbenzidine TMB) uses liquid 100 μ l, adds stop buffer 2MH after 10 minutes
2SO
450 μ l, 450nm place reading, 655nm does reference;
7. blank setting: the 3. in the step, every hole does not add 20 μ l total proteins, but adds 20 μ l lysis buffer Lysis Buffer, and all the other processes are identical.All last results expression are the difference that reading on the microplate reader (OD value) deducts the blank gained;
The structure that it is characterized in that described double chain oligonucleotide probe 1 is:
By 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With 3 '-transcription factor particular combination sequence antisense strand-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The structure of described double chain oligonucleotide probe 2 is:
By 5 '-transcription factor particular combination sequence--transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With two 3 '-transcription factor particular combination sequence antisense strands-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The single stranded DNA of described double chain oligonucleotide probe 1 or probe 2 long-chains partly is a kind of among single base A, T, G, the C.
Described double chain oligonucleotide probe 1 or 2 prepares by the following method:
First chain: contain 2 copies of transcription factor particular combination sequence, 5 '-transcription factor particular combination sequence-transcription factor particular combination sequence-C-(C) n-C-3 ', this chain 3 ' end is made biotin labeling, and n is a positive integer;
Second chain: contain 1 copy of transcription factor particular combination sequence, 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', this chain 3 ' end is made biotin labeling, and n is a positive integer;
The 3rd chain: transcription factor particular combination sequence antisense strand, 5 '-transcription factor particular combination sequence antisense strand-3 ';
More than in three chains, second chain mixes by 1: 1,1: 2 mole ratio respectively with the 3rd chain with the 3rd chain, first chain, puts 94 ℃ of water-bath sex change and takes out after 10 minutes, makes it take off fire and spends the night, and aggregates into two strands and is probe 1, probe 2.
In technique scheme, described first chain, second chain are 58 bases, and the 3rd chain is 22 bases;
In technique scheme, the described preparation method who detects NF-κ B double chain oligonucleotide probe 1 or 2 is:
First chain: contain 2 copies of NF-κ B binding sequence, totally 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
12-C-3 ', this chain 3 ' end is made biotin labeling;
Second chain: contain 1 copy of NF-κ B binding sequence, 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
34-C-3 ', this chain 3 ' end is made biotin labeling;
The 3rd chain: NF-κ B binding sequence antisense strand, 22 bases, 5 '-GCCTGGGAAAGT
CCCCTCAACT-3 ';
In above-mentioned three chains, second chain mixes by 1: 1,1: 2 mole ratio respectively with the 3rd chain with the 3rd chain, first chain, putting 94 ℃ of water-bath sex change took out after 10 minutes, making it take off fire spends the night, aggregate into two strands and be probe 1 (D1W, contain 1 the copy double-chain probe), probe 2 (D2W, contain 2 the copy double-chain probes).
The used test kit of above-mentioned transcription factor activity detection method, it comprises: two anti-, the developer of the enzyme dash board of avidin bag quilt, biotin labeled probe 1 or 2, phosphate buffered saline buffer (PPS), binding buffer liquid (bindingbuffer), one anti-, HPR mark, stop buffer, lotion damping fluid, described probe is an aforesaid method synthetic probe 1 or 2.
The structure of described double chain oligonucleotide probe 1 is:
By 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With 3 '-transcription factor particular combination sequence antisense strand-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The structure of described double chain oligonucleotide probe 2 is:
By 5 '-transcription factor particular combination sequence--transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With two 3 '-transcription factor particular combination sequence antisense strands-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The single stranded DNA of described double chain oligonucleotide probe 1 or probe 2 long-chains partly is a kind of among single base A, T, G, the C.
The used test kit of above-mentioned transcription factor activity detection method, described binding buffer liquid (bindingbuffer) comprising: 4mM HEPES pH7.5,100mM KCl, 8%glycerol, 5mMDTT, 0.2%BSA, 40 μ g/ml salmon sperm DNAs.
In this probe design of the inventive method, do not take stochastic sequence, it all is known array, can be directly and complementary strand take off fire and generate double-chain probe, and do not need to obtain through the PCR reaction, taking off the double-chain probe that fire generates does not need purifying yet, can be directly used in to detect and use, transcription factor and probe combine and transcription factor connects together with the lotus root that combines of antibody, settle at one go, the specificity that does not need to carry out transcription factor is in addition identified; Whole process is easy, cheap, and what obtain is quantitative results, is suitable for large-scale promotion and application.
The strand partial design of long-chain of the present invention is single base " C ", and just a kind of in the strand in fact can be any single stranded sequence, but unity and stability in order to detect is set at poly " C " sequence.
The inventive method sensitivity, specificity height not only are suitable for the detection of NF-kB activity, are suitable for the mass detection of other transcription factor activities too, need not to contact radioactive substance.This method can directly apply to scientific research; Based on this method, can develop the transcription factor activity detection kit, as commodity.
Description of drawings
Fig. 1 is the mode chart of probe 1 of the present invention;
Fig. 2 is the mode chart of probe 2 of the present invention;
Fig. 3 is based on the transcription factor activity traditional detection method mode chart of elisa technique;
Fig. 4 is a transcription factor activity detection method mode chart of the present invention;
Fig. 5 is that wild-type (D1W) compares (n=3) with mutant double-chain probe (D1M) detected result;
Fig. 6 is the linear relationship chart of NF-kB activity (n=3) under the protein concentration gradient among Fig. 5;
Fig. 7 is a copy (D1W) and two copies (D2W) wild-type double-chain probe detected result and comparison (n=3).
Embodiment
Describe performance of the present invention in detail below in conjunction with accompanying drawing:
The present invention is the breakthrough point with NF-κ B, adopt a kind of new double chain oligonucleotide probe to make up, promptly synthetic one is long and the other is short two oligonucleotide segments, lengthy motion picture is disconnected except that the specific combination sequence that comprises transcription factor, also add enough single stranded DNA sequences in addition, short chain is exactly the complementary sequence of the specific combination sequence of transcription factor, with these one is long and the other is short two oligonucleotide segment balanced mix, after taking off fire, heating can be grouped to a kind of existing single stranded DNA part, the oligonucleotide probe of double-stranded DNA part is arranged again, can be directly used in detection.Wherein, double-stranded DNA partly is the specific combination sequence of transcription factor, and single stranded DNA partly is a catenation sequence; Transcription factor is that double-stranded DNA is conjugated protein in the known cell, and the strand catenation sequence can not increase the interference of other unknown transcription factors; The minute quantity single-stranded DNA binding protein that exists in the propagation phase nucleus only may combine with the single stranded DNA part, combining between the double-stranded part that can not have influence on probe and the purpose transcription factor that will detect; And, because catenation sequence is a single stranded DNA, effectively avoided the non-specific binding between purpose transcription factor and the catenation sequence, also greatly reduce other unknown transcription factor interferential chance and non-specific binding.Through facts have proved, the susceptibility of its detection and specificity are very high, and whole process is simple, and be much cheap, is suitable for carrying out on a large scale and promoting.
Concrete technical application scheme:
One, main agents and material:
Human umbilical vein endothelial cells strain (ECV304) is available from Wuhan University China typical culture collection center (CCTCC, China Center for Type Culture Collection);
BCA reagent (pierce company)
The people IL-1 β that recombinates, Calbiochem-Novabiochem Corporation La Jolla, CA92039-2087;
High speed freezing centrifuge, 3K18, Sigma Laborzentrifugen GmbH, Postfach1713-D-37507 Osterode am Harz;
The single stranded oligonucleotide fragment is given birth to worker's biotechnology company limited by Shanghai and is synthesized;
Mouse NF-κ B P65 monoclonal antibody, Santa Cruz;
The goat anti-mouse igg of HRP mark, Santa Cruz;
96 orifice plates of streptavidin bag quilt, streptavidin-coated 96-well plate, Roche Diagnostics GmbH, Manheim, Germany; Roche DiagnosticsCorporation, Indianapolis, IN, USA;
Tetramethyl benzidine (tetramethylbenzidine, TMB, the beautiful pearl east wind in Shanghai Bioisystech Co., Ltd);
Microplate reader (TECAN, Sunrise Remote/Touch Screen, Austria);
Two, experimental arrangement:
1. cell cultures: Human umbilical vein endothelial cells strain (ECV304) adopts the M199 substratum that contains 10%FBS to cultivate, and gets the cell that is in logarithmic phase and is used for experiment; Experimental group added the people IL-1 β that recombinates in preceding 30 minutes in stop cultivating, and final concentration is 5ng/ml, and control group does not add the people IL-1 β that recombinates.
2. the extraction of total protein of cell: with reference to (Patricia Renard, Isabelle Ernest, Andr é e Houbion, Muriel Art, Herv é Le Calvez, Martine Raes and Jos é Remacle, Development of a sensitive multi-well colorimetric assayfor active NF κ B, Nucleic Acids Research, 2001, Vol.29, No.4e21) method that waits is extracted total protein of cell.Be specially: IL-1 β handled after 30 minutes, discarded substratum, collecting cell after precooling PBS washes 2 times, centrifugal 10 minutes of 1000rpm, abandon supernatant, cell mass is resuspended in Lysis Buffer (20mM HEPES pH7.5,0.35M NaCl, 20%glycerol, 1%NP-40,1mM MgCl
26H
2O, 0.5mM EDTA, 0.1mM EGTA), face time spent adding proteinase inhibitor (cocktail); Put on ice after 10 minutes, 14000rpm, centrifugal 20 minutes, collect supernatant, take a morsel and carry out protein quantification, surplus putting-85 ℃ preservation with the BCA method.
3. the preparation of double chain oligonucleotide probe: it is synthetic that the single stranded oligonucleotide fragment is given birth to worker's biotechnology company limited by Shanghai, and totally 5 chains are according to this:
First chain: contain 2 copies of NF-κ B binding sequence, totally 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
12-C-3 ', this chain 3 ' end is made biotin labeling;
Second chain: contain 1 copy of NF-κ B binding sequence, 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
34-C-3 ', this chain 3 ' end is made biotin labeling;
The 3rd chain: NF-κ B binding sequence antisense strand, 22 bases, 5 '-GCCTGGGAAAGT
CCCCTCAACT-3 ';
The 4th chain: the sudden change chain sports " CTC " with " GGG " in the NF-κ B binding sequence, totally 58 bases, 5 '-AGTTGAG
CTCACTTTCCCAGGC-C-(C)
34-C-3 ', this chain 3 ' end is made biotin labeling;
The 5th chain: sudden change chain antisense strand, 22 bases, 5 '-GCCTGGGAAAGT
GAGCTCAACT-3 ';
More than in 5 chains, second chain mixes by 1: 1,1: 2,1: 1 mole ratio respectively with the 5th chain with the 3rd chain, the 4th chain with the 3rd chain, first chain, put 94 ℃ of water-bath sex change and take out after 10 minutes, make it take off fire and spend the night, aggregate into two strands, respectively as probe 1 (D1W, contain 1 the copy double-chain probe), probe 2 (D2W, contain 2 the copy double-chain probes), mutant probe (D1M), its corresponding single-stranded probe is designated as S1W (second chain) respectively, S2W (first chain), S1M (the 4th chain);
With probe stationary on the enzyme plate of avidin bag quilt: the 2pmol probe is dissolved among the 50ulPBS, joins in the enzyme plate aperture of avidin bag quilt, 37 ℃, hatch 1 hour after, PBS+0.1%Tween-20 washing 2 times, PBS washes 1 time;
5. the total protein that extracts is combined with probe: every hole adds 20 μ l total proteins and 30 μ lbinding buffer[4mM HEPES pH7.5,100mM KCl, 8%glycerol, 5mMDTT, 0.2%BSA, 40 μ g/ml salmon sperm DNAs], after room temperature vibration 80rpm is hatched 1 hour, PBS+0.1%Tween-20 washing 3 times;
6. one anti-(anti-NF κ Bp65) is attached on the NF-κ b form dna mixture: every hole adds the anti-NF-κ Bp65100 μ l that does dilution in 1: 200 with PBS+0.1%BSA, and incubated at room is after 1 hour, and PBS+0.1%Tween-20 washs 3 times;
7. two anti-(Ig-G) with the HRP mark are attached on the anti-NF-κ Bp65-NF-κ b form dna mixture: every hole adds two anti-(Ig-G) the 100 μ l that make the HRP mark of dilution in 1: 10000 with PBS+0.1%BSA, after the incubated at room 1 hour, PBS+0.1%Tween-20 washing 4 times;
8. color reaction: every hole adds tetramethyl benzidine, and (tetramethylbenzidine TMB) uses liquid 100 μ l, adds stop buffer (2MH after 10 minutes
2SO
4) 50 μ l, 450nm place reading, 655nm does reference;
9. blank setting: in the 5th step, every hole does not add 20 μ l total proteins, but adds 20 μ l lysis buffers (Lysis Buffer), and all the other processes are identical.All last results expression are the difference that reading on the microplate reader (OD value) deducts the blank gained.
Experimental result:
1. wild-type (D1W) compares with mutant double-chain probe (D1M) detected result:
When detecting with wild-type double-chain probe D1W, compare with control group, under each concentration, (P＜0.01 n=3), and increases in gradient from the rising of the detected NF-kB activity of IL-1 β stimulating group significance; After core sequence " GGG " in the activation NF-κ B binding sequence was mutated into " CTC ", the OD value of control group and IL-1 β stimulating group was all an extremely low level, in each protein concentration level, do not have between the two significant difference (P＞0.05, n=3); (as Fig. 5)
Above result is made linear graph following (as Fig. 6): through linear correlation analysis, when protein concentration during at 2 μ g--10 μ g/well, no matter be control group, or IL-1 β stimulating group, measured NF-kB activity all shows good straight linear match relation, when protein concentration was higher than 10 μ g/well, measured NF-kB activity was tending towards state of saturation gradually, reached a platform.
2. a copy (D1W) and two copies (D2W) wild-type double-chain probe detected result and comparison:
Compare with control group, under each concentration, IL-1 β stimulating group detected NF-kB activity is significantly raise (P＜0.01 n=3), and increases in gradient; Probe D1W compares with the result that D2W detects, and there was no significant difference (P＞0.05, n=3); (see figure 7)
In detecting based on the transcription factor activity of elisa technique, this research improves it, with probe design one-tenth one is long and the other is short two complementary strands at one end, obtains through taking off fire first; When detecting, under each concentration, raise, and increase in gradient from the detected NF-kB activity of IL-1 β stimulating group significance with the wild-type double-chain probe; After core sequence " GGG " in the activation NF-κ B binding sequence is mutated into " CTC ", from control group and the detected NF-kB activity of IL-1 β stimulating group all an extremely low level, in each protein concentration level, there is not significant difference between the two, these show that all this detection has very high sensitivity and detection specificity, the catenation sequence that links to each other with enzyme plate keeps strand, effectively avoided the interference of non-specific binding, illustrated that this designed probe can be used for transcription factor activity and detect.Containing a copy is identical with two detection effects that copy the probe of binding sequences.Linear correlative analysis shows, when protein concentration during at 2 μ g--10 μ g/well, detected NF-kB activity shows good straight linear relation, explanation, detecting the NF-kB activity in this protein concentration scope can accomplish accurately quantitatively, the result has comparability, is that 5 μ g/well are the best with protein concentration wherein; When protein concentration was higher than 10 μ g/well, measured NF-kB activity was tending towards state of saturation gradually, reached a platform, illustrated, it is not accurate enough to detect the NF-kB activity in this protein concentration scope, should not carry out quantitative comparison.
Because in the design of this probe, do not take stochastic sequence, all be known array, can directly take off fire and generate double-chain probe, and not need to obtain through the PCR reaction with complementary strand, the double-chain probe that takes off the fire generation does not need purifying yet, can be directly used in detection usefulness; Transcription factor and probe combine and transcription factor connects together with the lotus root that combines of antibody, settle at one go, do not resemble the specificity that EMSA need carry out transcription factor in addition and identify; Whole process is much easy, cheap, and what obtain is quantitative results, is suitable for large-scale promotion and application.
In this research, be single base " C " with the strand partial design of long-chain, just a kind of in the strand in fact can be any single stranded sequence, but unity and stability in order to detect is set at poly " C " sequence.
This research is subjected to the subsidy of project of national nature science fund project No.30200099.
Need to prove: to those of ordinary skill in the art, can also make some changes or distortion to the present invention under the prerequisite that does not change the principle of the invention, this belongs to the protection domain of invention equally.
Claims (5)
1, a kind of transcription factor activity detection method which comprises at least following steps:
1. the extraction of cell cultures, total protein of cell,
2. double chain oligonucleotide probe 1 or 2 is fixed on the enzyme plate of avidin bag quilt: 2pmol double chain oligonucleotide probe 1 or 2 is dissolved among the 50 μ l PBS, join in the enzyme plate aperture of avidin bag quilt, 37 ℃, after hatching 1 hour, PBS+0.1% Tween-20 washing 2 times, PBS washes 1 time;
3. the total protein that extracts is combined with double chain oligonucleotide probe 1 or 2: every hole adds 20 μ l total proteins and 30 μ l binding buffer liquid, room temperature vibration 80rpm, hatch 1 hour after, PBS+0.1% Tween-20 washs 3 times;
4. with an anti-transcription factor antibody, be attached on transcription factor-probe complex: every hole adds the transcription factor antibody 100 μ l that do dilution in 1: 200 with PBS+0.1%BSA, and incubated at room is after 1 hour, and PBS+0.1% Tween-20 washs 3 times;
5. two anti-Ig-G with the HRP mark are attached on transcription factor antibody-transcription factor-probe complex: every hole adds the two anti-Ig-G100 μ l that make the HRP mark of dilution in 1: 10000 with PBS+0.1%BSA, after the incubated at room 1 hour, PBS+0.1% Tween-20 washing 4 times;
6. color reaction: every hole adds tetramethyl benzidine and uses liquid 100 μ l, adds stop buffer 2MH after 10 minutes
2SO
450 μ l, 450nm place reading, 655nm does reference;
7. blank setting: the 3. in the step, every hole does not add 20 μ l total proteins, but adds 20 μ l lysis buffers, and all the other processes are identical; All last results expression are the difference that reading OD value deducts the blank gained on the microplate reader;
The structure that it is characterized in that described double chain oligonucleotide probe 1 is:
By 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With 3 '-transcription factor particular combination sequence antisense strand-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The structure of described double chain oligonucleotide probe 2 is:
By 5 '-transcription factor particular combination sequence--transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling; With two 3 '-transcription factor particular combination sequence antisense strands-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The single stranded DNA of described double chain oligonucleotide probe 1 or probe 2 long-chains partly is a kind of among single base A, T, G, the C.
2, a kind of transcription factor activity detection method according to claim 1 is characterized in that what described double chain oligonucleotide probe 1 or probe 2 prepared by the following method:
First chain: contain 2 copies of transcription factor particular combination sequence, 5 '-transcription factor particular combination sequence-transcription factor particular combination sequence-C-(C) n-C-3 ', this chain 3 ' end is made biotin labeling;
Second chain: contain 1 copy of transcription factor particular combination sequence, 58 bases, 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', this chain 3 ' end is made biotin labeling;
The 3rd chain: transcription factor particular combination sequence antisense strand, 22 bases, 5 '-transcription factor particular combination sequence antisense strand-3 ';
More than in three chains, second chain mixes by 1: 1,1: 2 mole ratio respectively with the 3rd chain with the 3rd chain, first chain, puts 94 ℃ of water-bath sex change and takes out after 10 minutes, makes it take off fire and spends the night, and aggregates into two strands and is probe 1, probe 2.
3, a kind of transcription factor activity detection method according to claim 2 is characterized in that the preparation method of described detection NF-κ B double chain oligonucleotide probe is:
First chain: contain 2 copies of NF-κ B binding sequence, totally 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
12-C-3 ', this chain 3 ' end is made biotin labeling;
Second chain: contain 1 copy of NF-κ B binding sequence, 58 bases, 5 '-AGTTGAG
GGGACTTTCCCAGGC-C-(C)
34-C-3 ', this chain 3 ' end is made biotin labeling;
The 3rd chain: NF-κ B binding sequence antisense strand, 22 bases, 5 '-GCCTGGGAAAGT
CCCCTCAACT-3 '.
4, the used test kit of transcription factor activity detection method, it comprises: two anti-, the developer of the enzyme plate of avidin bag quilt, double chain oligonucleotide probe 1 or double chain oligonucleotide probe 2, phosphate buffered saline buffer, binding buffer liquid, one anti-, HPR mark, stop buffer, lotion damping fluid;
The structure of described double chain oligonucleotide probe 1 is:
By 5 '-transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling and 3 '-transcription factor particular combination sequence antisense strand-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The structure of described double chain oligonucleotide probe 2 is:
By 5 '-transcription factor particular combination sequence--transcription factor particular combination sequence-C-(C) n-C-3 ', totally 58 bases, 3 ' end is made biotin labeling, with two 3 '-transcription factor particular combination sequence antisense strands-5 ', wherein transcription factor particular combination sequence antisense strand totally 22 bases are combined into two strands;
The single stranded DNA of described double chain oligonucleotide probe 1 or double chain oligonucleotide probe 2 long-chains partly is a kind of among single base A, T, G, the C.
5, according to the used test kit of the described transcription factor activity detection method of claim 4, it is characterized in that described binding buffer liquid comprises: 4mM HEPES pH 7.5,100mM KCl, 8% glycerine, 5mM DTT, 0.2% BSA, 40 μ g/ml salmon sperm DNAs.
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| CN101942513A (en) * | 2010-08-27 | 2011-01-12 | 深圳博尔美生物科技有限公司 | Biochip and production method thereof |
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| CN103048463A (en) * | 2012-05-02 | 2013-04-17 | 中国科学院广州生物医药与健康研究院 | Microwell plate nucleic acid hybridization ELISA (enzyme-linked immuno sorbent assay) method for detecting DNA-binding proteins based on different rigors |
| CN110172498B (en) * | 2019-04-28 | 2022-09-20 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for rapidly and efficiently analyzing interaction of transcription factor and target DNA binding sequence thereof |
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| WO2000012709A2 (en) * | 1998-08-31 | 2000-03-09 | Urogenesys, Inc. | Phelix: a testis-specific protein expressed in cancer |
| EP1136567A1 (en) * | 2000-03-24 | 2001-09-26 | Advanced Array Technologies S.A. | Method and kit for the screening, the detection and /or the quantification of transcriptional factors |
| US6379887B1 (en) * | 1998-09-10 | 2002-04-30 | The General Hospital Corporation | Gene for inhibiting melanin biosynthesis |
| JP2002306171A (en) * | 2001-04-09 | 2002-10-22 | Geneticlab Co Ltd | Method for identifying agent affecting activity of transcription factor |
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|---|---|---|---|---|
| WO2000012709A2 (en) * | 1998-08-31 | 2000-03-09 | Urogenesys, Inc. | Phelix: a testis-specific protein expressed in cancer |
| US6379887B1 (en) * | 1998-09-10 | 2002-04-30 | The General Hospital Corporation | Gene for inhibiting melanin biosynthesis |
| EP1136567A1 (en) * | 2000-03-24 | 2001-09-26 | Advanced Array Technologies S.A. | Method and kit for the screening, the detection and /or the quantification of transcriptional factors |
| JP2002306171A (en) * | 2001-04-09 | 2002-10-22 | Geneticlab Co Ltd | Method for identifying agent affecting activity of transcription factor |
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| CN101942513A (en) * | 2010-08-27 | 2011-01-12 | 深圳博尔美生物科技有限公司 | Biochip and production method thereof |
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