CN112114154B - Kit for detecting nuclear factor-kappa B and application thereof - Google Patents
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Abstract
The invention provides a probe which can detect nuclear factor-kappa B with high sensitivity, good specificity and short time consumption; the probe is a DNA double-stranded probe; the DNA double-stranded probe is formed by reverse complementation of DNA-1 and DNA-2; the nucleotide sequence of the DNA-1 is shown as SEQ ID No.1, and the nucleotide sequence of the DNA-2 is shown as SEQ ID No. 2. The invention develops a simple, convenient and sensitive nuclear factor-kappa B detection method. The method combines DNA binding protein, exonuclease III (Exo-III) and isothermal exponential amplification technology, and adopts molecular beacon dependent amplification fluorescence analysis technology to successfully realize double amplification of signals. Compared with other methods, the method has higher specificity and lower detection limit, and can be directly used for detecting the nuclear factor-kappa B in the cancer cell nuclear extract.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a kit for detecting nuclear factor-kappa B and application thereof.
Background
At present, severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been worldwide-engineered and has serious harm and impact on human health. Anatomical results show that the pathological features of patients with new coronary pneumonia (covd-19) suggest the occurrence of Acute Respiratory Distress Syndrome (ARDS), the etiology of which is a cytokine storm. Upon viral entry into cells, release of nucleic acid RNAs activates Toll-like receptor 7 (TLR 7) and recruits multiple proteins to form complexes, thereby promoting entry of Transcription Factors (TFs) such as interferon regulatory factor 7 (IRF 7) and nuclear factor- κb (NF- κb) into the nucleus, and then activation of pro-inflammatory cytokine expression, resulting in overactivity of the immune system, thus causing cytokine storms, which are often dangerous and fatal. In this process, the transcription factor plays an extremely important role, and thus accurate measurement of the transcription factor is of great importance in biological research and clinical diagnosis.
Transcription factors comprise one or more DNA Binding Domains (DBDs) that bind to specific DNA sequences to regulate gene transcription. Can be used as a natural switch to convert physical and chemical signals such as temperature change, illumination, drug concentration, redox state and the like into transcriptional change. Thus, transcription factors play an important role in the pathways and networks of gene expression regulation. Imbalance in transcription factor signaling has been associated with cancer, developmental disorders, inflammation, and autoimmunity. Of these, NF-. Kappa.B is an important inducible transcription factor, which is present in almost all cells. NF- κB dimer can bind to NF- κB site in genome to regulate expression of target gene, and is involved in many important biological processes such as immunity, inflammation and cancer. For NF- κB, it has become a potential target for medical diagnosis and drug development.
Because of the importance of transcription factors, detection techniques are becoming more and more important. The current methods for detecting transcription factors mainly include electrochemical methods, radioactive gel migration Experiments (EMSA) and enzyme-linked immunosorbent assays (ELISA). However, these methods have some drawbacks. For electrochemical methods, the sample consumption is large, and complex electrode modification processes are often time-consuming and laborious. Although the radioactive gel migration experiments are simple and sensitive, the use of radioactive isotopes presents safety hazards to researchers and the surrounding environment. The ELISA has too narrow application range due to the addition of specific antibodies against transcription factors. Furthermore, in some typical fluorescent amplification strategies, transcription factors in solution can be detected directly. The presence of transcription factors can promote the formation of DNA double strands, producing intense Fluorescence Resonance Energy Transfer (FRET). However, binding of proteins to DNA may create steric hindrance, resulting in a low fluorescent signal. Therefore, there is also a need to develop new transcription factor detection methods.
Molecular Beacon (MB) is a novel neck-ring structure probe, which is a fluorescent-labeled oligonucleotide chain composed of a loop region, a dry region, a fluorescent group and a quencher. Molecular beacons are increasingly being used to detect and analyze specific nucleic acid sequences or proteins in solution due to their high sensitivity, non-toxicity and high specificity. When the molecular beacon alone is present, the fluorescent group and the quencher on the stem are in close proximity to each other, and the fluorescence of the fluorescent group is quenched. When the target nucleic acid is present in solution, conformational changes of the molecular beacon may be induced during hybridization. The fluorescent group and the quencher are separated from each other, thereby recovering fluorescence. Thus, a change in fluorescence intensity can determine whether the target nucleic acid is present in the solution. In protein detection, the target nucleic acid may be released by reaction of the target protein with the DNA probe and the tool enzyme. The target protein is detected and analyzed by detecting nucleic acid using molecular beacons. The existing signal amplification strategies based on molecular beacon detection proteins have the defects of low sensitivity, long time consumption, excessively complex design and the like. Therefore, it is necessary to establish an economical, sensitive, rapid and highly specific transcription factor detection method.
Disclosure of Invention
Therefore, the current signal amplification strategies based on molecular beacon detection proteins have the defects of low sensitivity, long time consumption, excessively complex design and the like. The invention provides a probe for detecting nuclear factor-kappa B, which has high sensitivity, good specificity and short time consumption, and a related product and application thereof.
The invention provides a probe for detecting nuclear factor-kappa B, which is a DNA double-stranded probe; the DNA double-stranded probe is formed by reverse complementation of DNA-1 and DNA-2; the nucleotide sequence of the DNA-1 is shown as SEQ ID No.1, and the nucleotide sequence of the DNA-2 is shown as SEQ ID No. 2.
A kit for detecting nuclear factor- κb, comprising the above-described probe for detecting nuclear factor- κb.
Optionally, the kit further comprises two hairpin molecular beacons, independently packaged, MB-1 and MB-2, respectively; the nucleotide sequence of MB-1 is shown as SEQ ID No. 3; the MB-2 is obtained by marking a fluorescent group at the 5' -end of SEQ ID No.4 and marking a quencher at the 20 th position.
Optionally, the fluorescent group is cy3 and the quencher is BHQ-2; or the fluorescent group is cy5, and the quencher is BHQ-2; the fluorescent group is 6-FAM, and the quencher is BHQ-1.
Optionally, the kit further comprises an individually packaged exonuclease III. Alternatively, the enzyme may be used in an amount of 0.25U per uL assay.
Optionally, the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1:1:1:2.
the method for detecting the nuclear factor-kappa B comprises the steps of incubating the probe and a sample to be detected for more than 30 minutes at room temperature, adding exonuclease III, MB-1 and MB-2, incubating at 37 ℃ for more than 1 hour, and if the fluorescence intensity of the sample to be detected is increased after incubation compared with a negative control, indicating that the sample to be detected contains the nuclear factor-kappa B; the negative control is a sample without nuclear factor- κb. The temperature at which the probe and the sample to be tested are incubated together is 25.+ -. 2 ℃.
Alternatively, the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1:1:1:2.
the application of the probe or the kit in detecting the nuclear factor-kappa B also belongs to the protection scope of the invention.
Alternatively, the nuclear factor- κB is specifically NF- κBp65 or NF- κBp50.
The technical scheme of the invention has the following advantages:
1. the probe of the invention is a DNA double-stranded probe; the DNA double-stranded probe consists of DNA-1 and DNA-2; the DNA-1 and the DNA-2 are reversely complementary; the nucleotide sequence of the DNA-1 is shown as SEQ ID No.1, the nucleotide sequence of the DNA-2 is shown as SEQ ID No.2, and the probe can detect NF- κB with good specificity and high sensitivity.
2. The invention develops a simple, convenient and sensitive nuclear factor-kappa B detection method. The method combines DNA binding protein, exonuclease III (Exo-III) and isothermal exponential amplification technology, and adopts molecular beacon dependent amplification fluorescence analysis technology to successfully realize double amplification of signals. Compared with other methods, the method has higher specificity and lower detection limit, and can be directly used for detecting the nuclear factor-kappa B in the cancer cell nuclear extract.
3. The sensitivity experiment result shows that in the method, the probe or the reagent or the kit is incubated with the sample to be detected, and if the fluorescence intensity is increased after the incubation compared with that before the incubation, the sample to be detected contains the nuclear factor-kappa B. The minimum detection limit of the method is 2.6pm.
4. The feasibility study result shows that the fluorescence intensity is obviously enhanced only when the nuclear factor-kappa B, DNA double-stranded probe, MB-1, MB-2 and Exo-III exist simultaneously.
5. The experimental result of the reaction time shows that the fluorescence intensity of the detection method is enhanced along with the increase of time, and the detection method enters a plateau phase about 45 minutes.
6. The detection test result of the endogenous NF-kappa B p65 shows that the method can overcome the interference of endogenous components in the nuclear extract.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the detection of nuclear factor- κB by the molecular beacon-dependent fluorescent amplification method of the present invention;
FIG. 2 is a fluorescence spectrum of NF-. Kappa. B p65 at various concentrations (0, 5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000 pM) in example 2 of the present invention; the curves sequentially correspond to the detection results of NF-kappa B p concentration of 0,5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000pM from bottom to top;
FIGS. 3 (a) and (b) are both NF- κ B p65 concentration versus fluorescence intensity; the abscissa is the concentration of NF- κ B p65 and the ordinate is the fluorescence intensity;
FIG. 4 is a feasibility study experiment result;
FIG. 5 is the results of a reaction time experiment;
FIG. 6 is the result of a specificity experiment;
FIG. 7 comparison between the experimental results of example 5 and the standard curve.
Detailed Description
The protein binding buffer was: 10mM Tris HCl (pH 8.0), 100mM KCl, 2mM MgCl 2 0.25mM DTT, 10% (volume percent) glycerol and 0.1mM EDTA;
the DNA reaction buffer is: 50mM Tris-HCl,100mM NaCl,1mM EDTA,pH8.0.
Reagents and apparatus
PAGE-pure oligonucleotides were purchased from Genscript company (oligonucleotide sequences are listed in table 1 below),
threeo-1, 4-dimercapto-2, 3-butadiene (DTT) and Exo III were purchased from biological engineering (Shanghai) stock Co., ltd (China, shanghai); exo III is given the product number B300061-0004.
Media (DMEM), fetal Bovine Serum (FBS) and antibiotics were purchased from Life Technologies (glaland, new york, usa);
the nuclear extraction kit was purchased from Active Motif (us, california, cat No. 40010) and other chemicals purchased from national pharmaceutical group chemical company (china, shanghai);
ultrapure water used in this study was prepared by a Milli-Q purification system (Bellica, massachus, USA) having a resistivity of 18MΩ cm.
All fluorescence spectra were obtained on a multifunctional microplate reader (Spnetmax M5; molecular device, sanguis Viel, calif.).
Example 1
DNA-1 (SEQ ID No. 1) dissolved in the DNA reaction buffer and DNA-2 (SEQ ID No. 2) dissolved in the DNA reaction buffer were mixed to obtain a stock solution of double-stranded probe at a concentration of 5. Mu.M. Molecular beacons MB-1 (SEQ ID No. 3) and MB-2 (5' -end of SEQ ID No.4 labeled with Cy3 and 20 th site labeled with BHQ-2) were dissolved in DNA reaction buffer, respectively, to prepare stock solutions having MB-1 and MB-2 concentrations of 5. Mu.M. All solutions were heated at 95 ℃ for 5 minutes, then slowly cooled to room temperature and allowed to stand for at least 4 hours.
For detection of NF- κ B p65, NF- κ B p65 was added to 20uL of protein binding buffer system, and incubated with 40 uL of double-stranded DNA probe solution at room temperature for 45min. Then, 20. Mu.L of Exo III at a concentration of 2.5U/. Mu.L, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the total volume of the system was 200. Mu.L, the concentration of the double-stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and the mixture was incubated at 37℃for 1 hour, hereinafter abbreviated as a method for detecting NF-. Kappa. B p 65.
When detecting the transcription factor NF- κB in the nuclear extract, mixing 20 μL of the nuclear extract with 40 μL of double-stranded DNA probe solution, and incubating for 45min at room temperature; exo III (2.5U/. Mu.L, 20. Mu.L), 40. Mu.L MB-1 and 80. Mu.L MB-2 were then added, the concentration of the double-stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, and the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour, hereinafter referred to as the method for detecting nuclear extracts.
TABLE 1
In DNA-1 and DNA-2, the bold bases are the binding site for NF-. Kappa. B p 65. The underlined bases in DNA-2 and MB-1 are complementary sequences. In MB-1 and MB-2, bold bases represent the stem sequences of the complementary sequences to each other to form the hairpin probe. The italic bases in MB-1 and MB-2 are complementary sequences.
Principle of experiment
FIG. 1 shows the principle of detection of nuclear factor- κB by molecular beacon-dependent fluorescent amplification. The invention designs reverse complementary DNA-1 and DNA-2 to obtain a DNA double-stranded probe, and designs a site capable of combining with NF-kappa B p on the probe. According to Exo-III, it acts on the 3' -end of the double strand of DNA, gradually catalyzing the removal of single nucleotide in the 3' -5 ' direction, but is inactive to single strand DNA. In addition, the invention also designs a MB-1 and MB-2 hairpin molecular beacon. MB-1 contains a base sequence complementary to DNA-2 and a base sequence complementary to MB-2. MB-2 contains a 5' -end modified Cy3 fluorophore and a black hole quencher (BHQ-2) modified at the corresponding complementary base
When NF- κ B p65 was absent from the solution, the DNA duplex was simultaneously digested from the 3' end by Exo-III. When NF-. Kappa. B p65 is present, DNA-2 is protected by NF-. Kappa. B p65 binding to DNA double stranded probes. While DNA-1 can still be digested by Exo-III, the final retained DNA-2 can reflect the NF-. Kappa. B p65 content. MB-1 then recognizes and binds to a specific complementary sequence located on DNA-2 to form a new DNA duplex, which triggers the digestion mechanism of Exo-III and starts a new round of digestion. When MB-1 is partially digested, a complete DNA-2 and a reporter DNA (part of MB-1) are released. DNA-2 will continue to bind to the new MB-1 and repeat the above steps to form a loop, thereby generating more reporter DNA. On the other hand, the reporter DNA hybridizes with MB-2, the hairpin structure is opened to form a new DNA double strand between MB-2 and the reporter DNA, the digestion mechanism of Exo-III is triggered, cy3-DNA fragments are released after digestion, the fluorescent group (Cy 3) is free from the quencher (BHQ-2) and fluorescence is recovered, the released reporter DNA hybridizes with the new MB-2, and the steps are repeated to form a cycle. Finally, each of DNA-2 and reporter DNA can undergo multiple cycles, resulting in more MBs being digested, producing a large number of cy3-DNA fragments. When the excitation wavelength was set to 546nm, the cy3-DNA fragment had fluorescence emission at 566 nm.
Example 2 sensitivity experiment
When NF- κ B p65 was detected as in example 1, NF- κ B p65 was added to 20uL of the protein binding buffer system, 40 uL of the double-stranded DNA probe solution was added thereto, and incubated at room temperature for 45min. Then, 20. Mu.L of 2.5U/. Mu.L of Exo III, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the concentration of the double-stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour.
Fluorescence spectra of NF- κ B p65 at various concentrations (0, 5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000 pM) in the range 556-660nm were measured using a multifunctional microplate reader, and the results are shown in FIG. 2. From FIG. 2, it can be seen that the fluorescence signal increases with increasing concentration of NF- κ B p65 (the curves in FIG. 2 correspond to the detection results of NF- κ B p65 concentrations of 0,5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000pM, in order from bottom to top). As shown in FIG. 3 (excitation wavelength: 546nm, emission wavelength: 566 nm), the fluorescence signal increases in a concentration-dependent manner, which means that the fluorescence intensity of the released Cy3-DNA fragment is proportional to the concentration of NF-. Kappa. B p 65. The NF- κ B p65 concentration was below 60pm and was linear with fluorescence intensity. The correlation equation is y=210.07+10.92x (r2= 0.9879), where X is NF- κ B p65 concentration and Y is fluorescence intensity. The lowest detection limit of this method was found to be 2.6pm by calculating the ratio of 3 standard deviations to the slope of the standard curve (3σ/slope).
EXAMPLE 3 feasibility study
The groups are 4:
group a: according to the method of example 1, 40. Mu.L of double-stranded DNA probe solution was added to 20. Mu.L of the protein binding buffer system, and incubated at room temperature for 45min; then 40. Mu.L MB-1 and 80. Mu.L MB-2 were added and the system was supplemented to 200. Mu.L with a DNA reaction buffer, the concentration of the double-stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour, and the fluorescence spectrum of the system in the range of 556-660nm was detected; the results are shown in curve a of FIG. 4;
group b: NF-. Kappa. B p65 was added to 20uL of the protein binding buffer system according to the method of example 1, and then 40. Mu.L of the double-stranded DNA probe solution was added thereto and incubated at room temperature for 45 minutes. Add 40. Mu.L MB-1 and 80. Mu.L MB-2 and supplement 200. Mu.L with DNA reaction buffer, the final NF- κ B p65 concentration in the system was 250pM, the concentration of double stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubate at 37℃for 1h; detecting the fluorescence spectrum of the system in the range of 556-660 nm; the results are shown in curve b of FIG. 4;
group c: according to the method of example 1, 40. Mu.L of a 1. Mu.M double-stranded DNA probe solution was added to 20. Mu.L of the protein binding buffer system, and incubated at room temperature for 45min; then, 20. Mu.L of Exo III at a concentration of 2.5U/. Mu.L, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the concentration of the double-stranded DNA probe in the system was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour; detecting the fluorescence spectrum of the system in the range of 556-660 nm; the results are shown in curve c of FIG. 4;
d group: NF-. Kappa. B p65 was added to 20uL of the protein binding buffer system according to the method of example 1, and then 40. Mu.L of the double-stranded DNA probe solution was added thereto and incubated at room temperature for 45 minutes. Then, 20. Mu.L of Exo III at a concentration of 2.5U/. Mu.L, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the total volume of the system was 200. Mu.L, the final concentration of NF- κ B p65 was 250pM, the concentration of double-stranded DNA probe was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour. Detecting the fluorescence spectrum of the system in the range of 556-660 nm; the result is shown in curve d of FIG. 4. FIG. 4 shows that the fluorescence intensities of groups a, b, and c are all at a low level. In contrast, when nuclear factor- κB and Exo-III were present simultaneously (group d), fluorescence intensity was significantly enhanced, 644.2% higher than that of group c without NF- κ B p 65.
Example 4 reaction time experiment
NF-. Kappa. B p65 was added to 20uL of the protein binding buffer system according to the method of example 1, and then 40. Mu.L of the double-stranded DNA probe solution was added thereto and incubated at room temperature for 45 minutes. Then, 20. Mu.L of Exo III at a concentration of 2.5U/. Mu.L, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the total volume of the system was 200. Mu.L, the final concentration of NF- κ B p65 was 250pM, the concentration of double-stranded DNA probe was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour.
9 sets of experiments were performed in parallel, fluorescence detection was performed at 37℃for 0,5, 10, 15, 30, 45, 60, 75, and 90 minutes, the above experiments were repeated 3 times, the average of the detected fluorescence intensities was taken, and a curve was drawn as shown in FIG. 5. From fig. 5, it can be seen that the fluorescence intensity increases with time, and enters the plateau at around 45 minutes.
EXAMPLE 5 specificity experiments
In accordance with the method of example 1, 20uL of protein binding buffer system was then incubated with 40 uL of added DNA probe solution for 45min at room temperature. Then, 20. Mu.L of Exo III at a concentration of 2.5U/. Mu.L, 40. Mu.L of MB-1 and 80. Mu.L of MB-2 were added, the total volume of the system was 200. Mu.L, the final concentration of NF-. Kappa. B p65 in the system was 250pM, the concentration of the double-stranded DNA probe was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, the concentration of MB-2 was 2. Mu.M, and incubated at 37℃for 1 hour.
250pM NF- κ B p65 in the above system was replaced with 1mM Human thrombin (Human thrombin), 1mM Alpha Fetoprotein (AFP), 1mM Human immunoglobulin G (Human IgG), 1mM Bovine Serum Albumin (BSA), respectively, and a set of blank controls (controls) was set.
As a result of examining the fluorescence intensities (excitation wavelength: 546nm and emission wavelength: 566 nm) of the respective reaction systems, the fluorescence intensities of human thrombin, alpha fetoprotein, human IgG or bovine serum albumin were low and far lower than those of NF-. Kappa. B p65 at 250pM even at a very high concentration (1 mM) as shown in FIG. 6. The method has higher specificity to NF-kappa b p 65.
Example 5 endogenous NF-. Kappa. B p65 detection
A549 cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin (100 μg/mL) and streptomycin (100 μg/mL) and in a cell incubator containing 5% carbon dioxide at 37 ℃. Cell number was measured by a Petroff-Haussercell counter (USA) and 5X 10 7 Individual cells were placed into EP tubes, washed 2 times with PBS (0.1M, pH7.4,4 ℃) and incubated with 20ng/mL tumor necrosis factor alpha (TNF-. Alpha.) for 35min (this step was aimed at activating the NF-. Kappa.B signaling pathway to allow NF-. Kappa.B to migrate into the nucleus). And (3) collecting the cell nucleus extract by using a nuclear extraction kit to obtain a cell nucleus extract, and preserving at-80 ℃ to be tested. As shown in Table 2, the recovery of NF-. Kappa. B p65 in the nuclear extracts was 95.78-110.02% within an acceptable maximum recovery range (80% -120%) [ Cordell RL, white IR, monks PS.validation of an assay for the determination of levoglucosan and associated monosaccharide anhydrides for the quantification of wood smoke in atmospheric ]aerosol.Analytical and bioanalytical chemistry2014；406:5283-92.https://doi.org/10.1007/s00216-014-7962-x]。
When detecting targets contained in complex samples such as nuclear extracts, the targets are often interfered by a plurality of endogenous components, so that strong fluorescence interference is generated, and the efficiency of a common biosensor is greatly reduced. To verify the ability of the method of example 1 to detect specific proteins in complex samples, diluted nuclear extracts containing different concentrations of NF- κ B p65 were tested.
Taking 7 parts of nuclear extract, respectively marking the nuclear extract as samples 1-7, and adding NF-kappa B p65 with the concentration of 0pM into a detection system of the sample 1; NF- κ B p65 was added to sample 2 at a concentration of 5pM; … … NF- κ B p65 was added to each of the remaining samples as shown in Table 2, column 2.
Each sample was tested as follows: mixing 20 mu L of the nuclear extract added with NF-kappa B p65 with 40 mu L of double-stranded DNA probe solution, and incubating for 45min at room temperature; exo III (2.5U/. Mu.L, 20. Mu.L), 40. Mu.L MB-1 and 80. Mu.L MB-2 were then added to form a detection system in which the concentration of the double-stranded DNA probe was 1. Mu.M, the concentration of MB-1 was 1. Mu.M, and the MB-2 was 2. Mu.M and incubated at 37℃for 1 hour.
A standard curve (FIG. 7 standard curve) was prepared by the method for detecting NF- κ B p65 in example 1 using NF- κ B p65 pure product with concentrations of 0,5, 10, 15, 20, 30, 60pM, respectively.
The fluorescence intensity of the above-mentioned detection system to which NF- κ B p65 was added in 7 parts was measured, and a curve was prepared (see FIG. 7 for the experimental result). Fig. 7 shows a comparison between the experimental results and the standard curve. It is known that the slopes of the two curves should be consistent if no endogenous interference is present in the nuclear extract. The results show that the method can overcome the interference of endogenous components in the nuclear extract.
TABLE 2
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
<110> Jiangsu province atomic medical institute
<120> kit for detecting nuclear factor-kappa B and application thereof
<130> SHA202000524
<160> 4
<170> PatentIn version 3.5
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Claims (10)
1. A probe for detecting nuclear factor- κb, wherein the probe is a DNA double-stranded probe; the DNA double-stranded probe is formed by reverse complementation of DNA-1 and DNA-2; the nucleotide sequence of the DNA-1 is shown as SEQ ID No.1, and the nucleotide sequence of the DNA-2 is shown as SEQ ID No. 2.
2. A kit for detecting nuclear factor- κb, comprising the probe of claim 1.
3. The kit for detecting nuclear factor- κb according to claim 2, further comprising two hairpin molecular beacons of MB-1 and MB-2, respectively, packaged separately; the nucleotide sequence of MB-1 is shown as SEQ ID No. 3; the MB-2 is obtained by marking a fluorescent group at the 5' -end of SEQ ID No.4 and marking a quencher at the 20 th position.
4. The kit for detecting nuclear factor- κb according to claim 3, wherein the fluorescent group is cy3 and the quencher is BHQ-2; or the fluorescent group is cy5, and the quencher is BHQ-2; the fluorescent group is 6-FAM, and the quencher is BHQ-1.
5. The kit of claim 2, further comprising an individually packaged exonuclease III.
6. The kit of any one of claims 2-5, wherein the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1:1:1:2.
7. a method for detecting nuclear factor-kappa B is characterized in that the probe according to claim 1 and a sample to be detected are incubated for more than 30 minutes at room temperature, then exonuclease III, MB-1 and MB-2 are added, and the incubation is carried out for more than 1 hour at 37 ℃, and if the fluorescence intensity of the sample to be detected is increased after the incubation compared with a negative control, the sample to be detected contains nuclear factor-kappa B; the negative control is a sample without nuclear factor- κb.
8. The method for detecting nuclear factor- κb according to claim 7, wherein the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1:1:1:2.
9. use of a probe according to claim 1 or a kit according to any one of claims 2 to 6 for the detection of nuclear factor- κb.
10. The probe of claim 1, or the kit of any one of claims 2 to 5, or the method of claim 7 or 8, or the use of claim 9, wherein the nuclear factor- κb is in particular NF- κ B p65 or NF- κ B p50.
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| WO2013062931A2 (en) * | 2011-10-24 | 2013-05-02 | Furchak Jennifer | A molecular beacon based assay for the detection of biomarkers of breast cancer metastasis |
| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
| CN109439735A (en) * | 2018-11-06 | 2019-03-08 | 江苏集萃分子工程研究院有限公司 | A kind of fluorescent detection probe, kit and the application of abasic endonuclease 1 |
| CN109706224A (en) * | 2018-12-14 | 2019-05-03 | 山东师范大学 | A kind of method that the Multiple Cycle amplification of exonuclease III auxiliary is used to delicately detect DNA |
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| WO2013062931A2 (en) * | 2011-10-24 | 2013-05-02 | Furchak Jennifer | A molecular beacon based assay for the detection of biomarkers of breast cancer metastasis |
| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
| CN109439735A (en) * | 2018-11-06 | 2019-03-08 | 江苏集萃分子工程研究院有限公司 | A kind of fluorescent detection probe, kit and the application of abasic endonuclease 1 |
| CN109706224A (en) * | 2018-12-14 | 2019-05-03 | 山东师范大学 | A kind of method that the Multiple Cycle amplification of exonuclease III auxiliary is used to delicately detect DNA |
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