CN107290543A - It is a kind of to detect the intracellular method with p53 transcription activating domain interaction proteins - Google Patents
It is a kind of to detect the intracellular method with p53 transcription activating domain interaction proteins Download PDFInfo
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- CN107290543A CN107290543A CN201710300083.XA CN201710300083A CN107290543A CN 107290543 A CN107290543 A CN 107290543A CN 201710300083 A CN201710300083 A CN 201710300083A CN 107290543 A CN107290543 A CN 107290543A
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Abstract
The intracellular method with p53 transcription activating domain interaction proteins is detected the invention discloses a kind of, it is by testing sample and the cell pyrolysis liquid containing p53 His recombinant proteins, is coated with after the luminous microballoon of p53 monoclonal antibodies, biotinylated His tag antibodies and the coated photosensitive microballoon hybrid reaction of Streptavidin, the optical signal value of reacting hole is measured using light-induced chemiluminescent analyzer, is that can detect in sample with the presence or absence of the albumen interacted with p53 transcription activating domains according to optical signal value.The present invention is based on photo-induced chemiluminescence immunoassay detection technique, by building carrier for expression of eukaryon and being expressed in cell to obtain the cell pyrolysis liquid containing p53 His recombinant proteins, it can make to be coated with the luminous microballoon of p53 monoclonal antibodies and combine the distance between photosensitive microballoon of biotinylation His tag antibodies less than 200nm.The inventive method can be used for the albumen that selective mechanisms interact with p53 transcription activating domains, high with simple to operate, signal to noise ratio, be easy to the advantage of high flux screening.
Description
Technical field
The invention belongs to bioprotein detection field, it is more particularly related to which a kind of detection of high flux is intracellular
With the method for p53 transcription activating domain interaction proteins.
Background technology
P53 is the important tumor suppressor of human body, is referred to as " genome bodyguard ".It checks DNA damage in the G1 phases, leads to
Transcriptional activity regulating cell DNA reparations, growth cycle retardance, apoptosis etc. are crossed, to maintain cell normal function.Wild type p53 egg
It is made up of in vain 393 amino acid, molecular weight of albumen is 53KD, including following domain：N-terminal transcription activating domain, proline
Rich region, DNA binding structural domains, tetramerization domain and the structureless basic domain of C-terminal.It is used as discovery and the mankind so far
Cancer-related highest albumen, because by negative regulation albumen, such as MDM2 and MDMX in about 50% tumour, suppression and
Can not be brought into normal play function, wherein being combined with p53 transcription activating domains makes its inactivation be one of important mechanism.In expression wild type
In p53 tumour, the combination by suppressing negative regulation albumen and p53 transcription activating domains discharges activity p53 albumen has turned into anti-swollen
One of popular target spot of tumor medicine design.In order to which p53 transcription activating domains are lived so as to recover wild type p53 in release tumor cell
Property, find and p53 transcription activating domains interaction protein and develop corresponding inhibitor there is important clinical treatment meaning.
In the last few years, continuing to develop with proteomics research technology, the new method of Way for Studying Protein-Protein Interactions
Continue to bring out, except the technology that traditional everybody has been well known, such as co-immunoprecipitation, GST-Pull down, yeast two-hybrid
Some new technologies, such as tandem affinity purification technology, surface plasma resonance etc. Deng, current development.Make a general survey of existing
There is technology respectively there are advantage and disadvantage：Co-immunoprecipitation is answered as protein-protein interaction under classical analysis physiological condition is simple to operate
With extensive, but complex steps can not Large-scale Screening interaction protein；GST-Pull down are that Validation in vitro protein is mutual
The common technology of effect, the technology high specificity is still not suitable for Large-scale Screening；Yeast-two hybrid technique can be analyzed known
Interaction between protein and the gene that can also be used to screen agnoprotein, although this method is adapted to Large-scale Screening
Interaction protein but easily there is false positive etc.；Tandem affinity purification technology overcomes the vacation sun of yeast-two hybrid technique well
Property and can study the interactions between protein network of cellular level on a large scale, but the TAP labels that this technology is introduced may influence
Combination of target protein and affinity column etc.；Surface plasma resonance technology is modified without label, and detection sensitivity is high, Neng Goushi
Shi Gaoxiao determines the interaction between protein, although this technology has precision high detection speed fast etc. advantage but right
Sample requirement is high, and expensive, general applicability is low.In a word, although these methods can meet scientific research requirement, to a certain degree
On there is respective deficiency, detect the interaction between p53 transcription activating domains and protein with being unfavorable for economical and efficient.
The content of the invention
It is an object of the invention to：Overcome the deficiencies in the prior art there is provided a kind of high flux detection it is intracellular with
The method of p53 transcription activating domain interaction proteins.
In order to realize foregoing invention purpose, the invention provides one kind detection is intracellular with p53 transcription activating domain phase interactions
With the method for albumen, it comprises the following steps：
(1) people source p53 coded sequences are inserted in carrier for expression of eukaryon pENTER, obtains pENTER-p53 recombinant plasmids；
(2) by pENTER-p53 Transfected Recombinant Plasmid HEK 293T cells obtained by step (1)；
(3) cell pyrolysis liquid cleavage step (2) described cell is used, centrifuging and taking supernatant is obtained containing p53-His recombinant proteins
Cell pyrolysis liquid；
(4) the luminous microballoon and biotinylated His tag antibodies for being coated with p53 monoclonal antibodies are prepared；
(5) sample is added in reacting hole, step (3) is then sequentially added described containing the thin of p53-His recombinant proteins
Cellular lysate liquid, step (4) the luminous microballoon for being coated with p53 monoclonal antibodies and the mixing of biotinylated His tag antibodies
Reaction, adds the coated photosensitive microballoon hybrid reaction of Streptavidin；
(6) optical signal value of reacting hole is measured using light-induced chemiluminescent analyzer, is detectable sample according to optical signal value
It whether there is the albumen interacted with p53 transcription activating domains in product.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (2), it is described
The condition of centrifugation is 15000~20000g, and centrifugation time is 10~30min.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (3), it is described
The consumption of cell pyrolysis liquid is every 107Individual cell adds 0.5~1mL.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (4), it is described
It is coated with the luminous microballoon of p53 monoclonal antibodies, the mass ratio of p53 monoclonal antibodies and luminous microballoon is 1~2:10.
As the intracellular method with p53 transcription activating domain interaction proteins of present invention detection, the p53 monoclonals resist
The amino acid of Antigenic Peptide behaviour source p53N ends 1~100 of body.
As the intracellular method with p53 transcription activating domain interaction proteins of present invention detection, the luminous microballoon
A diameter of 100~400nm.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (4), it is described
In biotinylated His tag antibodies, the mass ratio of biotin and His tag antibodies is 10:1.
As the intracellular method with p53 transcription activating domain interaction proteins of present invention detection, step (5) is each reacted
Kong Zhong, adds the μ g of luminous microballoon 1~3 for being coated with p53 monoclonal antibodies, adds the biotinylated His labels anti-
100~150ng of body, adds the μ g of cell pyrolysis liquid 5~10 containing p53-His recombinant proteins.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (5), it is described
The addition of the coated photosensitive microballoon of Streptavidin is per the μ L of hole 120~180.
Detected as the present invention in the intracellular method with p53 transcription activating domain interaction proteins, step (5), it is described
Hybrid reaction is 15~20min of incubation at 37 DEG C.
Compared with prior art, the present invention detects that the intracellular method with p53 transcription activating domain interaction proteins has
Following advantage：
Therefore, not enough to make up prior art, the present invention is provided using light-induced chemiluminescent technology combination immunological method
A kind of intracellular method with p53 transcription activating domain interaction proteins of high flux detection.Light-induced chemiluminescent technology is one
The technology based on nanoparticle of kind, including photosensitive microballoon and luminous microballoon.Wrapped up Photoactive compounds in photosensitive microballoon, when its by
Singlet oxygen can be produced after light irradiation to exciting for wavelength 680nm, because the half-life period of singlet oxygen can only propagate for 4 μ s
200nm or so around to photosensitive microballoon, when luminous microballoon can then receive singlet when within 200nm near photosensitive microballoon
Oxygen and then a series of chemical reaction of generation, the wavelength for launching high level is 615nm exciting light.The p53 that the present invention is selected
Monoclonal antibody is DO1 clones, and its Antigenic Peptide behaviour source p53 transcription activating domain can be with the negative regulation protein competition knot such as MDM2
Close p53 transcription activating domains.The detection method background is low, sensitivity is high, "dead" pollution, property stable and homogeneous, operation
Simplicity is easily achieved high flux, has a good application prospect.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, it is intracellular to present invention detection to be interacted with p53 transcription activating domains
The method and its advantage of albumen are described in detail.
Fig. 1 is photo-induced chemiluminescence immunoassay technology for detection p53-His schematic diagram：P53-His furthers luminous microballoon with feeling
Light microballoon, produces optical signal.
Fig. 2 is former for the interaction between photo-induced chemiluminescence immunoassay technology for detection target protein and p53 transcription activating domains
Reason figure：Target protein competition binding p53 transcription activating domains, cause luminous microballoon to be pulled open with photosensitive microballoon distance, singlet oxygen without
Method reaches luminous microballoon, is produced without optical signal.
Fig. 3 is the phase between photo-induced chemiluminescence immunoassay technology for detection MDM2 (murine double minute gene 2) and p53 transcription activating domains
Interaction：With MDM2 (murine double minute gene 2) concentration increase, increasing p53 transcription activating domains are from p53 monoclonal monomers
In discharge, the microballoon that causes more and more to light leaves photosensitive microballoon, and signal value is more and more lower.
Fig. 4 be photo-induced chemiluminescence immunoassay technology for detection MDM4 (MDM2 sample p53 associated proteins) and p53 transcription activating domains it
Between interaction：With MDM4 (MDM2 sample p53 associated proteins) concentration increase, increasing p53 transcription activating domains from
Discharged in p53 monoclonal monomers, the microballoon that causes more and more to light leaves photosensitive microballoon, and signal value is more and more lower.
Embodiment
In order that goal of the invention, technical scheme and the advantageous effects of the present invention become apparent from, with reference to embodiments,
The present invention will be described in further detail.It should be appreciated that the embodiment described in this specification is just for the sake of explanation
The present invention, is not intended to limit the present invention, formula, the ratio of embodiment etc. can suit measures to local conditions to make a choice and have no reality to result
Matter influences.
Embodiment
PENTER-p53 construction of recombinant plasmid：
Experiment material：
Carrier for expression of eukaryon pENTER is purchased from Wei Zhen bio tech ltd.
Competent cell TOP10 is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and article No. is CB104.
T4 ligases are purchased from the precious biotech firm in Dalian, and article No. is 2011A.
QIAquick Gel Extraction Kit of tapping rubber is purchased from Omega Bio-Tek companies of the U.S., and article No. is D2500-02.
Plasmid extraction kit is purchased from Omega Bio-Tek companies of the U.S., and article No. is D6948-01.
1. target gene, PCR system are obtained by PCR：1 μ of μ l, cDNA of Prime STAR Max 25
L, each 1 μ l of upstream and downstream primer, moisturizing to the μ l of final volume 50.PCR conditions：98 DEG C of 5min, 98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 15s, 35
72 DEG C of 10min after individual circulation, 4 DEG C of preservations.Product is after 2% agarose gel electrophoresis, and using tapping rubber, QIAquick Gel Extraction Kit is reclaimed
Purpose fragment；
2. carrier for expression of eukaryon pENTER and PCR recovery product carry out double digestion, digestion through the AsiS I and-HF of Mlu I respectively
Product is after 1% agarose gel electrophoresis, and using tapping rubber, QIAquick Gel Extraction Kit reclaims purpose fragment；
3. recovery product is in the presence of T4 ligases according to mol ratio 1：10 stay overnight in 16 DEG C of connections；
4. by 10 μ l connection product Transformed E .coli TOP10 competent cells, it is applied to the LB solids containing that antibiotic of card
Medium culture 13h, the single bacterium colony of picking is in the LB fluid nutrient medium cultures 8h containing that antibiotic of card afterwards；
5. extracting plasmid in a small amount using plasmid extraction kit to go forward side by side performing PCR identification, the clone for being accredited as the positive carries out double
Digestion is identified and send sequencing company to be sequenced, and checking insertion is correct and standby without plasmid extraction is carried out after mutation.
It is prepared by the cell pyrolysis liquid containing p53-His recombinant proteins：
Experiment material：
Hyclone is purchased from Biological Industries (BI) biotechnologies company of Israel, and article No. is 04-001-
1A。
DMEM culture mediums are purchased from silent winged scientific and technological (China) Co., Ltd of generation that of match, and article No. is 8116349.
JetPRIME transfection reagents are purchased from France Polyplus Transfection biotechnologies company, and article No. is 114-
07。
1. it is thin based on 293T is cultivated under the conditions of 37 DEG C, 5%CO2 in 10cm wares with the DMEM cultures containing 10% hyclone
Born of the same parents；
2. degree to be fused is transfected when reaching 80%, concrete operations are as follows：It is slow with 500 μ l jetPRIME first
Fliud flushing dilutes 5 μ g pENTER-p53 recombinant plasmids, flicks the mixing of EP tube walls；Then 10 μ l jetPRIME reagents are added, are flicked
Tube wall mixes and is stored at room temperature 10min；Finally mixed liquor is added dropwise in culture dish, after 4h change containing dual anti-culture medium after
Continuous culture；
3. after 24 hours, being washed with PBS 3 times, 1ml cell pyrolysis liquids are added per ware and are incubated on ice 20 minutes, 20000g centrifugations
20 minutes；
4. taking supernatant to dispense into EP pipes, freeze standby in -80 DEG C of refrigerators.
The preparation of testing sample MDM2-GST/MDM4-GST lysates and blank control GST lysates：
Experiment material：
Prokaryotic expression carrier pGEX-4T-3 is purchased from GE companies of the U.S., and article No. is 27-4583-01.
Competent cell BL21 is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and article No. is CB105.
BCA protein quantifications detection kit is purchased from Thermo Fischer Scient Inc., and article No. is 23227.
Ultrasonic cell disruptor is biological purchased from new sesame, model JY92-IIDN.
1. MDM2/MDM4 coded sequences are inserted into prokaryotic expression carrier pGEX-4T-3 acquisition recombinant plasmids by being subcloned,
Through the errorless transformed competence colibacillus cell BL21 afterwards of sequence verification；
2. picking monoclonal bacterium colony when OD600 reaches 0.8 with 5ml LB culture mediums, adding IPTG to 0.1mM, 37 DEG C
Induction 3 hours；
Sample is boiled 3. washing thalline with PBS 3 times and adding 120 μ 1 × Loading of l buffer afterwards, and passes through Coomassie brilliant blue
Dyeing identification expression；
4. after identification expression, expanding culture to 200ml, OD600 adds IPTG to 0.1Mm, 18 DEG C of inductions when reaching 0.8
Overnight；
5. thalline is washed after 3 times through PBS, simultaneously multigelation 3 times, the 20000g after carrying out ultrasonic bacteria breaking are resuspended with appropriate PBS
Centrifugation takes supernatant as testing sample for 20 minutes；
6. another be made blank control with same step operation empty carrier pGEX-4T-3；
7. measure the concentration of testing sample and blank control sample to specifications by BCA methods.
It is coated with the preparation of the luminous microballoon of p53 monoclonal antibodies：
Experiment material：
Anti- p53 monoclonal antibodies are purchased from Santa Cruz biotechnology, and article No. is sc-126.
Luminous microballoon is purchased from Bo Yang bio tech ltd.
Sodium cyanoborohydride (NaBH3CN sigma companies of the U.S.) are purchased from, article No. is 156159.
The hydrochloride (CMO) of O- (carboxymethyl) azanol half is purchased from sigma companies of the U.S., and article No. is C13408-1G.
Tween-20 is purchased from green skies Bioisystech Co., Ltd, and article No. is ST825.
Ultrasonic cell disruptor is biological purchased from new sesame, model JY92-IIDN.
1. the anti-p53 monoclonal antibodies of 150 μ g are taken into 100K retention posts, it is another to add 400 μ l MES buffer solutions 8000g centrifugations
6 minutes；
2. repeat step 1 is operated 2 times, finally antibody in 3 minutes collection retention posts is centrifuged with new collecting pipe 3000g standby
With；
3. taking 1mg to light microballoon, room temperature 14000g is centrifuged 15 minutes, abandons supernatant standby；
4. with MES buffers 25mg/ml NaBH3CN solution；
5. NaBH in antibody-solutions, step 4 is sequentially added in step 2 into luminous microballoon3CN solution and 1.25 μ l's
10%Tween-20, finally adds MES buffer solutions to the μ l of final volume 200,37 DEG C of incubation reactions；
6. add CMO solution of the 10 μ l with the 0.8mol/l NaOH 65mg/ml prepared, 37 in 48 hours backward reaction tubes
DEG C closing 1 hour；
7. 14000g is centrifuged 15 minutes, supernatant is abandoned, is washed with PBS 3 times, final adjustment lights microballoon concentration extremely
5mg/ml produces the luminous microballoon for being coated with p53 monoclonal antibodies.
The preparation of biotinylation His tag antibodies：
Experiment material：
(+)-Biotin N-hydroxysuccinimide ester (activated biotin) are purchased from sigma companies of the U.S., goods
Number be H1759-250MG.
Anti- His tag monoclonal antibodies are purchased from Ba Ao get bio tech ltd, and article No. is AP0032M.
1. the anti-His tag monoclonal antibodies of 100 μ g are taken into 100K retention posts, it is another to add 400 μ l biotin labelings buffering
Liquid (0.1M Na2CO3Buffer solution, pH9.5) 8000g centrifuge 6 minutes；
2. repeat step 1 is operated 2 times, finally antibody in 3 minutes collection retention posts is centrifuged with new collecting pipe 3000g standby
With；
3. the biotin solution for preparing 50mg/ml with DMSO is standby；
4. the antibody-solutions in step 2 are mixed with the biotin solution in step 3, and add biotin labeling buffer solution
To the μ l of final volume 200；
5. incubation at room temperature 2 hours；
6. washing reaction solution with PBS 5 times with step 1, unreacted biotin is removed；
7. produce biotinylated His tag antibodies with PBS adjustment antibody concentration to 0.5mg/ml.
Detect MDM2/MDM4 and p53 transcription activating domains interaction：
Experiment material：
Light-induced chemiluminescent analyzer is purchased from Bo Yang bio tech ltd.
The photosensitive microballoon of streptavidin is purchased from Bo Yang bio tech ltd.
A (is contained using buffer gradient dilution testing sample (cell pyrolysis liquid containing MDM2/MDM4) and blank control
GST cell pyrolysis liquid)；
B uses buffer solution 1 respectively:50 dilutions are coated with the luminous microballoon of p53 monoclonal antibodies, 1：100 dilution biotinylations
Anti- His tag antibodies, 1：32 cell pyrolysis liquids of the dilution containing p53-His recombinant proteins；
C sequentially adds the luminous microballoon after dilution, biotinylated antibody into reacting hole, containing p53-His recombinant proteins
Cell pyrolysis liquid and testing sample each 25 μ L, 37 DEG C of concussions are incubated 20 minutes；
D adds photosensitive microballoon, preferably 150 μ L into reacting hole again, and 37 DEG C of concussions are incubated 15 minutes；
E measures each reacting hole signal value using light-induced chemiluminescent analyzer.
As a result as shown in Figure 3 and Figure 4, experimental group is gradually reduced with the quality increase signal value of testing sample, and blank
Control group signal value then keeps constant.Show that this method is applicable to the albumen that detection interacts with p53 transcription activating domains.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.Although in addition, this specification
In used some specific terms, but these terms are merely for convenience of description, do not constitute any limitation to the present invention.
Claims (10)
1. a kind of detect the intracellular method with p53 transcription activating domain interaction proteins, it is characterised in that including following step
Suddenly：
(1) people source p53 coded sequences are inserted in carrier for expression of eukaryon pENTER, obtains pENTER-p53 recombinant plasmids；
(2) by pENTER-p53 Transfected Recombinant Plasmid HEK 293T cells obtained by step (1)；
(3) cell pyrolysis liquid cleavage step (2) described cell is used, centrifuging and taking supernatant is obtained containing the thin of p53-His recombinant proteins
Cellular lysate liquid；
(4) the luminous microballoon and biotinylated His tag antibodies for being coated with p53 monoclonal antibodies are prepared；
(5) sample is added in reacting hole, step (3) cell containing p53-His recombinant proteins is then sequentially added and splits
Solution liquid, step (4) the luminous microballoon and biotinylated His tag antibodies hybrid reaction for being coated with p53 monoclonal antibodies,
Add the coated photosensitive microballoon hybrid reaction of Streptavidin；
(6) optical signal value of reacting hole is measured using light-induced chemiluminescent analyzer, is that can detect in sample according to optical signal value
With the presence or absence of the albumen interacted with p53 transcription activating domains.
2. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In in step (2), the condition of the centrifugation is 15000~20000g, and centrifugation time is 10~30min.
3. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In in step (3), the consumption of the cell pyrolysis liquid is every 107Individual cell adds 0.5~1mL.
4. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In described to be coated with the luminous microballoon of p53 monoclonal antibodies in step (4), p53 monoclonal antibodies and the matter of luminous microballoon
Amount is than being 1~2:10.
5. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 4, its feature exists
In the amino acid of Antigenic Peptide behaviour source p53N ends 1~100 of the p53 monoclonal antibodies.
6. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 4, its feature exists
In a diameter of 100~400nm of the luminous microballoon.
7. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In in step (4), in the biotinylated His tag antibodies, the mass ratio of biotin and His tag antibodies is 10:1.
8. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In step (5) each in reacting hole, is coated with the μ g of luminous microballoon 1~3 of p53 monoclonal antibodies described in addition, add the life
His 100~150ng of tag antibody of thing elementization, add the μ g of cell pyrolysis liquid 5~10 containing p53-His recombinant proteins.
9. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature exists
In in step (5), the addition of the coated photosensitive microballoon of Streptavidin is per the μ L of hole 120~180.
10. the intracellular method with p53 transcription activating domain interaction proteins of detection according to claim 1, its feature
It is, in step (5), the hybrid reaction is 15~20min of incubation at 37 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471686A (en) * | 2020-04-18 | 2020-07-31 | 南开大学 | Oligonucleotide sequence of targeted p53 protein, derivative probe and application thereof |
| CN114107348A (en) * | 2021-11-24 | 2022-03-01 | 南京理工大学 | Chlamydomonas reinhardtii-based protein interaction analysis method |
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