CN112114154A - Kit for detecting nuclear factor-kappa B and application thereof - Google Patents

Abstract

The probe provided by the invention can be used for detecting the nuclear factor-kappa B with high sensitivity, good specificity and short time consumption; the probe is a DNA double-strand 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-kB detection method. The method combines DNA binding protein, exonuclease III (Exo-III) and isothermal index 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-kB in the cancer cell nucleus extracting solution.

Description

Kit for detecting nuclear factor-kappa B and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting a nuclear factor-kappa B and application thereof.

Background

At present, severe acute respiratory syndrome coronavirus (SARS-CoV-2) has rolled the world, causing serious harm and influence to human health. Anatomical results show that the pathological features of patients with new coronary pneumonia (COVID-19) suggest the development of Acute Respiratory Distress Syndrome (ARDS), the etiology of ARDS is cytokine storm. When a virus invades a cell, the release of nucleic acid RNA activates Toll-like receptor 7(TLR7) and recruits multiple proteins to form complexes, thereby facilitating the entry of Transcription Factors (TFs), such as interferon regulatory factor 7(IRF7) and nuclear factor- κ B (NF- κ B), into the nucleus, and then activating the expression of pro-inflammatory cytokines, leading to overactivity of the immune system, which in turn causes cytokine storms, which are often dangerous and fatal. In this process, transcription factors play an extremely important role, and therefore accurate measurement of transcription factors is of great significance 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, oxidation-reduction state and the like into transcription change. Therefore, transcription factors play an important role in pathways and networks for regulation of gene expression. An imbalance in transcription factor signaling is associated with cancer, developmental disorders, inflammation, and autoimmunity. NF-. kappa.B is an important inducible transcription factor and is present in almost all cells. The NF-kB dimer can be combined with NF-kB sites in a genome, regulate the expression of target genes and relate to a plurality of important biological processes such as immunity, inflammation, cancer and the like. For NF- κ B, it has become a potential target for medical diagnosis and drug development.

Due to the importance of transcription factors, detection techniques are increasingly gaining attention. The current methods for detecting transcription factors mainly comprise an electrochemical method, a radioactive gel migration assay (EMSA) and an enzyme-linked immunosorbent assay (ELISA). However, these methods have some disadvantages. For electrochemical methods, sample consumption is large, and the complex electrode modification process is often time consuming and laborious. Although the radioactive gel migration experiment is simple and sensitive, the use of radioactive isotopes poses safety risks to researchers and the surrounding environment. The enzyme-linked immunosorbent assay has a narrow application range due to the addition of a specific antibody for resisting the transcription factor. Furthermore, in some typical fluorescence amplification strategies, transcription factors in solution can be detected directly. The presence of transcription factors can promote the formation of DNA double strands, resulting in strong Fluorescence Resonance Energy Transfer (FRET). However, binding of proteins to DNA may create steric hindrance, resulting in a low fluorescence signal. Therefore, development of a novel transcription factor detection method is also required.

The Molecular Beacon (MB) is a novel neck ring structure probe, and is a fluorescence labeling oligonucleotide chain which is composed of a ring region, a dry region, a fluorescent group and a quencher. Due to their high sensitivity, non-toxicity and high specificity, molecular beacons are increasingly being used for the detection and analysis of specific nucleic acid sequences or proteins in solutions. When the molecular beacon is present alone, the fluorophore and the quencher on the stem are in proximity to each other and the fluorescence of the fluorophore is quenched. When the target nucleic acid is present in solution, it causes a conformational change in the molecular beacon during hybridization. The fluorophore and the quencher are separated from each other, thereby recovering fluorescence. Thus, the change in fluorescence intensity can determine whether the target nucleic acid is present in the solution. In protein detection, target nucleic acid can be released by reaction of the target protein with a DNA probe and a tool enzyme. The detection and analysis of target proteins is achieved by using molecular beacons to detect nucleic acids. However, some current signal amplification strategies based on molecular beacons for detecting proteins have the disadvantages of low sensitivity, long time consumption, excessively complex design and the like. Therefore, there is a need to establish an economical, sensitive, rapid and specific method for detecting transcription factors.

Disclosure of Invention

Therefore, some current signal amplification strategies based on molecular beacons for detecting proteins have the disadvantages of low sensitivity, long time consumption, excessively complex design and the like. The invention provides a probe for detecting nuclear factor-kB, 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-kB, which is a DNA double-strand 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 the nuclear factor-kB comprises the probe for detecting the nuclear factor-kB.

Optionally, the kit further comprises two hairpin molecular beacons, MB-1 and MB-2, packaged independently; the nucleotide sequence of the 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 fluorophore 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 exonuclease III in an individual package. Alternatively, the amount of the enzyme used per uL of the assay system is 0.25U.

Optionally, the molar ratio of the DNA-1, the DNA-2, the MB-1 and the MB-2 is 1: 1: 1: 2.

a method for detecting 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 for more than 1 hour at 37 ℃, and if the probe is compared with a negative control, increasing the fluorescence intensity of the sample to be detected after incubation, so that the sample to be detected contains the nuclear factor-kappa B; the negative control was a sample containing no nuclear factor- κ B. The temperature for co-incubation of the probe and the sample to be tested is 25 +/-2 ℃.

Alternatively, the molar ratio of the above 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-kB also belongs to the protection scope of the invention.

Optionally, the nuclear factor- κ B is specifically NF- κ Bp65 or NF- κ Bp 50.

The technical scheme of the invention has the following advantages:

1. the probe is a DNA double-strand probe; the DNA double-strand 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 be used for detecting NF-kappa B with good specificity and high sensitivity.

2. The invention develops a simple, convenient and sensitive nuclear factor-kB detection method. The method combines DNA binding protein, exonuclease III (Exo-III) and isothermal index 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-kB in the cancer cell nucleus extracting solution.

3. The sensitivity experiment result shows that the method of the invention incubates the probe or the reagent or the kit of the invention and the sample to be tested, if the fluorescence intensity after the incubation is increased compared with that before the incubation, the nuclear factor-kB is contained in the sample to be tested. The minimum detection limit of the method is 2.6 pm.

4. The feasibility study result shows that the fluorescence intensity is obviously enhanced only when the double-stranded probe of the nuclear factor-kappa B, DNA, MB-1, MB-2 and Exo-III exist simultaneously.

5. Reaction time experiment results show that the fluorescence intensity of the detection method is enhanced along with the increase of time, and the detection method enters a plateau period within about 45 minutes.

6. The results of the test of endogenous NF-kappa B p65 show that the method can overcome the interference of endogenous components in the nuclear extracting solution.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram illustrating the principle of the molecular beacon-dependent fluorescence amplification method for detecting nuclear factor- κ B according to the present invention;

FIG. 2 is a graph showing the fluorescence spectra of NF-. kappa. B p65 at various concentrations (0, 5, 10, 15, 20, 30, 60, 120, 250, 500, and 1000pM) in example 2 of the present invention; the curves correspond to the detection results of NF-kappa B p65 concentrations of 0, 5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000pM from bottom to top in sequence;

FIGS. 3(a) and (b) are graphs each showing the relationship between the concentration of NF-. kappa. B p65 and the fluorescence intensity; the abscissa is the concentration of NF- κ B p65, and the ordinate is the fluorescence intensity;

FIG. 4 is the results of a feasibility study experiment;

FIG. 5 is the results of a reaction time experiment;

FIG. 6 shows the results of a specificity test;

figure 7 comparison of experimental results with standard curves for example 5.

Detailed Description

The protein binding buffer was: 10mM Tris HCl (pH 8.0), 100mM KCl, 2mM MgCl₂0.25mM DTT, 10% (by volume) glycerol and 0.1mM EDTA;

the DNA reaction buffer was: 50mM Tris-HCl, 100mM NaCl, 1mM EDTA, pH 8.0.

Reagent and apparatus

PAGE-pure oligonucleotides were purchased from Genscript (oligonucleotide sequences are listed in Table 1 below),

threo-1, 4-dimercapto-2, 3-butadiene (DTT) and Exo III are available from Biotechnology engineering (Shanghai) Inc. (China, Shanghai); exo III has a good number B300061-0004.

Culture medium (DMEM), Fetal Bovine Serum (FBS) and antibiotics were purchased from Life Technologies (gland island, new york, usa);

the nuclear extraction kit is purchased from Active Motif (product number 40010, ca) and other chemicals from the national drug group chemical agents limited (china, shanghai);

the ultrapure water used in this study was prepared by a Milli-Q purification system (Bellca, Mass., USA) with a resistivity of 18M Ω cm.

All fluorescence spectra were obtained on a multifunctional microplate reader (SpetraMax M5; molecular devices, Sanneville, Calif., USA).

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 double-stranded probe stock solution with a concentration of 5. mu.M. Molecular beacons MB-1(SEQ ID No.3) and MB-2 (the 5' -end of SEQ ID No.4 is labeled with Cy3, and the 20 th position is labeled with BHQ-2) were dissolved in the DNA reaction buffer, respectively, to prepare stock solutions each having a MB-1 and MB-2 concentration of 5. mu.M. All solutions were heated at 95 ℃ for 5 minutes, then slowly cooled to room temperature and left for at least 4 hours.

When detecting NF-kappa B p65, 20uL of protein binding buffer solution system is added with NF-kappa B p65, and then 40 uL of double-stranded DNA probe solution is added and incubated 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 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 the mixture was incubated at 37 ℃ for 1 hour, hereinafter referred to as a method for detecting NF- κ B p 65.

When detecting the transcription factor NF-kB in the nuclear extract, mixing 20 mu L of the nuclear extract 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, and the system was incubated at 37 ℃ for 1 hour with the double-stranded DNA probe at 1. mu.M, MB-1 at 1. mu.M and MB-2 at 2. mu.M, hereinafter referred to as the nuclear extract detection method.

TABLE 1

In DNA-1 and DNA-2, the bold bases are the binding sites for NF-. kappa. B p 65. The underlined bases in DNA-2 and MB-1 are complementary sequences. In MB-1 and MB-2, the bold bases indicate the stem sequences that complement each other to form hairpin probes. The italic bases in MB-1 and MB-2 are complementary sequences.

Principle of experiment

FIG. 1 shows the principle of molecular beacon-dependent fluorescence amplification for the detection of nuclear factor- κ B. 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 p65 on the probe. According to the properties of Exo-III, it can act on the 3' end of the DNA double strand, gradually catalyzing the removal of mononucleotides in the 3' to 5 ' direction, but has no activity on single-stranded DNA. In addition, the invention also designs two types of hairpin type molecular beacons MB-1 and MB-2. MB-1 contains a base sequence complementary to DNA-2 and a base sequence complementary to MB-2. MB-2 contains a Cy3 fluorophore modified at the 5' end and a Black hole quencher (BHQ-2) modified at the corresponding complementary base

When NF-. kappa. 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 was present, DNA-2 was protected by the binding of NF-. kappa. B p65 to the DNA double-stranded probe. While DNA-1 was still digested by Exo-III, the final retained DNA-2 reflected the NF-. kappa. B p65 content. Then, MB-1 recognizes the specific complementary sequence located on DNA-2 and binds to it 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, an intact DNA-2 and a reporter DNA (a portion of MB-1) are released. DNA-2 will continue to bind to new MB-1 and the above steps are repeated to form a cycle, thereby generating more reporter DNA. On the other hand, the reporter DNA hybridizes with MB-2, the hairpin structure is opened, a new DNA double strand is formed by MB-2 and the reporter DNA, the digestion mechanism of Exo-III is triggered, the Cy3-DNA fragment is released after digestion, the fluorescent group (Cy3) breaks away from the quencher (BHQ-2) and restores fluorescence, and the released reporter DNA hybridizes with the new MB-2 and the steps are repeated to form a cycle. Finally, each DNA-2 and reporter DNA may undergo multiple cycles, resulting in more MBs being digested, producing a large number of cy3-DNA fragments. When the excitation wavelength was set at 546nm, the cy3-DNA fragment had a fluorescence emission at 566 nm.

Example 2 sensitivity test

When NF-. kappa. B p65 was detected according to the method described in example 1, 20uL of the protein binding buffer system was added with NF-. kappa. B p65, followed by 40. mu.L of the double-stranded DNA probe solution, and the mixture was incubated at room temperature for 45 min. 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, and the system was incubated at 37 ℃ for 1 hour with a double-stranded DNA probe concentration of 1. mu.M, an MB-1 concentration of 1. mu.M and an MB-2 concentration of 2. mu.M.

The fluorescence spectra of NF-. kappa. B p65 at various concentrations (0, 5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000pM) in the range of 556-660nm were measured with a multifunctional microplate reader, and the results are shown in FIG. 2. It can be seen from FIG. 2 that the fluorescence signal increases with increasing concentration of NF-. kappa. B p65 (the curves in FIG. 2 correspond, from bottom to top, to the detection results at concentrations of NF-. kappa. B p65 of 0, 5, 10, 15, 20, 30, 60, 120, 250, 500 and 1000pM, respectively). As shown in FIG. 3 (excitation wavelength of 546nm, emission wavelength of 566nm), 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- κ B p 65. The concentration of NF- κ B p65 below 60pm was linear with fluorescence intensity. The correlation equation is Y210.07 +10.92X (R2 0.9879), where X is the NF- κ B p65 concentration and Y is the fluorescence intensity. The minimum detection limit of the method was found to be 2.6pm by calculating the ratio of the 3-fold standard deviation to the slope of the standard curve (3 σ/slope).

EXAMPLE 3 feasibility study

The method is divided into 4 groups:

a group: following the method of example 1, 20uL of protein binding buffer system, 40. mu.L of double-stranded DNA probe solution was added and incubated at room temperature for 45 min; then adding 40 mu L of MB-1 and 80 mu L of MB-2, supplementing the system to 200 mu L with a DNA reaction buffer solution, wherein the concentration of the double-stranded DNA probe in the system is 1 mu M, the concentration of the MB-1 is 1 mu M, the concentration of the MB-2 is 2 mu M, incubating the system for 1h at 37 ℃, and detecting the fluorescence spectrum of the system in the range of 556-660 nm; the results are shown in FIG. 4, curve a;

b group: NF- κ B p65 was added to 20uL of the protein binding buffer system, followed by 40 μ L of the double-stranded DNA probe solution and incubation at room temperature for 45min as in example 1. Add 40. mu.L MB-1 and 80. mu.L MB-2 and supplement to 200. mu.L with DNA reaction buffer, NF-. kappa. B p65 final concentration in the system was 250pM, double-stranded DNA probe concentration in the system was 1. mu.M, MB-1 concentration was 1. mu.M, MB-2 concentration was 2. mu.M, and incubate at 37 ℃ for 1 h; detecting the fluorescence spectrum of the system in the range of 556-660 nm; the results are shown in FIG. 4, curve b;

and c, group: according to the method of example 1, 20uL protein binding buffer system, adding 1. mu.M double-stranded DNA probe solution 40. mu.L, and incubating at room temperature for 45 min; 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 and the concentration of MB-2 was 2. mu.M, and the mixture was 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 FIG. 4, curve c;

and d, group: NF- κ B p65 was added to 20uL of the protein binding buffer system, followed by 40 μ L of the double-stranded DNA probe solution and incubation at room temperature for 45min as in example 1. 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, and the system was incubated at 37 ℃ for 1 hour with a total volume of 200. mu.L, a final concentration of 250pM of NF-. kappa. B p65, a concentration of 1. mu.M of the double-stranded DNA probe, a concentration of 1. mu.M of MB-1 and a concentration of 2. mu.M of MB-2. Detecting the fluorescence spectrum of the system in the range of 556-660 nm; the results are shown in FIG. 4, curve d. FIG. 4 shows that the fluorescence intensity of each of the groups a, b and c is at a low level. When the nuclear factor-kappa B and Exo-III are simultaneously present (group d), the fluorescence intensity is obviously enhanced and is 644.2% higher than that of group c without the addition of NF-kappa B p 65.

Example 4 reaction time experiment

NF- κ B p65 was added to 20uL of the protein binding buffer system, followed by 40 μ L of the double-stranded DNA probe solution and incubation at room temperature for 45min as in example 1. 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, and the system was incubated at 37 ℃ for 1 hour, in a total volume of 200. mu.L, at a final concentration of 250pM of NF-. kappa. B p65, at a concentration of 1. mu.M of double-stranded DNA probe, at a concentration of 1. mu.M of MB-1 and at a concentration of 2. mu.M of MB-2.

In parallel, 9 sets of experiments were performed, fluorescence was detected at 0, 5, 10, 15, 30, 45, 60, 75, and 90 minutes of incubation at 37 ℃, the above experiments were repeated 3 times, and the detected fluorescence intensities were averaged and plotted, as shown in fig. 5. It can be seen from FIG. 5 that the fluorescence intensity increased with time and entered the plateau at around 45 minutes.

Example 5 specificity test

Following the procedure of example 1, 20uL of protein binding buffer system was added to 40. mu.L of DNA probe solution and incubated at room temperature for 45 min. 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, and the system was made to have a total volume of 200. mu.L, a final concentration of NF-. kappa. B p65 in the system was 250pM, a concentration of the double-stranded DNA probe was 1. mu.M, a concentration of MB-1 was 1. mu.M and a concentration of MB-2 was 2. mu.M, and incubated at 37 ℃ for 1 hour.

The 250pM NF-. kappa. 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 measuring the fluorescence intensity (excitation wavelength: 546nm, emission wavelength: 566nm) of each of the above reaction systems, as shown in FIG. 6, the fluorescence intensity of human thrombin, alpha-fetoprotein, human IgG or bovine serum albumin was low even at a very high concentration (1mM) and was much weaker than that of NF- κ B p65 at 250 pM. The method is proved to have higher specificity to NF-kappa b p 65.

Example 5 detection of endogenous NF-. kappa. B p65

Culturing A549 cells in the presence of 10% fetal bovine serum, penicillin (100. mu.g/mL) and streptomycin (100. mu.g/mL) in DMEM at 37 ℃ in a cell culture incubator containing 5% carbon dioxide. The cell number was measured using a Petroff-Haussercell counter (USA) and 5X 10⁷The cells were placed in 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 intended to activate the NF-. kappa.B signaling pathway, allowing NF-. kappa.B to be transferred into the nucleus). Collecting the cell nucleus extract with a cell nucleus extraction kit to obtain cell nucleus extract, and storing at-80 deg.C. As shown in Table 2, the recovery of NF-. kappa. B p65 in the nuclear extract ranged from 95.78 to 110.02%, within the acceptable maximum recovery range (80% -120%) [ Cordell RL, White IR, Monks PS. differentiation of an assay for the determination of levoglucosan and associated monomeric derivatives for the quantification of wood in organic microbiological analysis, analytical and biochemical chemistry 2014; 406, 5283-92.https:// doi.org/10.1007/s00216-014-]。

When detecting targets contained in complex samples such as nuclear extracts, the targets are usually interfered by a plurality of endogenous components, so that strong fluorescence interference is generated, and the efficiency of the conventional biosensor is greatly reduced. To test the ability of the method of example 1 to detect specific proteins in complex samples, dilute nuclear extracts containing varying concentrations of NF-. kappa. B p65 were tested.

Taking 7 parts of nuclear extracting solution, respectively marking as No. 1-7 samples, and adding NF-kappa B p65 with the concentration of 0pM into a detection system of the 1 st sample; adding NF-kappa B p65 with the concentration of 5pM into the 2 nd sample; … …, NF-. kappa. B p65 was added to each of the remaining samples, as shown in Table 2, column 2.

Each sample was tested as follows: mixing the cell nucleus extract 20 μ L added with NF-kappa B p65 with double-stranded DNA probe solution 40 μ L, and incubating at room temperature for 45 min; then, Exo III (2.5U/. mu.L, 20. mu.L), 40. mu.L of MB-1 and 80. mu.L of MB-2 were 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 concentration of MB-2 was 2. mu.M, and incubated at 37 ℃ for 1 hour.

A standard curve was prepared by the method for detecting NF-. kappa. B p65 in example 1 using pure NF-. kappa. B p65 at concentrations of 0, 5, 10, 15, 20, 30 and 60pM, respectively (FIG. 7 standard curve).

The fluorescence intensity of 7 test systems containing NF-. kappa. B p65 was measured and plotted (see FIG. 7 for experimental results). Fig. 7 shows a comparison between the experimental results and the standard curve. It is known that the slopes of the two curves should tend to coincide if there is no endogenous interference in the nuclear extract. The result shows that the method can overcome the interference of endogenous components in the nuclear extracting solution.

TABLE 2

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

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Claims (9)

1. A probe for detecting nuclear factor-kB is characterized in that 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- κ B, comprising the probe for detecting nuclear factor- κ B according to claim 1.

3. The kit for detecting nuclear factor- κ B according to claim 2, further comprising two hairpin molecular beacons, MB-1 and MB-2; the nucleotide sequence of the 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; preferably, 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.

4. A kit as claimed in any one of claims 1 to 3, wherein the kit further comprises separately packaged exonuclease III.

5. The kit according to any one of claims 2 to 4, wherein the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1: 1: 1: 2.

6. a method for detecting nuclear factor-kappa B, characterized in that, the probe of 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 ℃, if compared with a negative control, the fluorescence intensity of the sample to be detected after the incubation is increased, which indicates that the sample to be detected contains the nuclear factor-kappa B; the negative control was a sample containing no nuclear factor- κ B.

7. The method for detecting nuclear factor- κ B according to claim 6, wherein the molar ratio of DNA-1, DNA-2, MB-1 and MB-2 is 1: 1: 1: 2.

8. use of the probe of claim 1 or the kit of any one of claims 2 to 5 for the detection of nuclear factor- κ B.

9. The probe according to claim 1, the kit according to any one of claims 2 to 5, the method according to claim 6 or 7, or the use according to claim 8, wherein the nuclear factor- κ B is in particular NF- κ B p65 or NF- κ B p 50.

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