WO2020215844A1 - Product for screening lrpprc regulators and method for identifying lrpprc regulators - Google Patents

Abstract

Disclosed are a product for screening LRPPRC regulators and a method for identifying LRPPRC regulators, which product comprises a kit and a device, wherein the kit comprises a recombinant LRPPRC protein, GST protein, fluorescein-labeled aptamer, R14 and fluorescein-labeled control nucleic acid; and the device comprises a multi-hole plate and a fluorescence polarization signal detector. A short aptamer is used, which aptamer, in comparison with naturally occurring RNA and DNA sequences, has a shorter sequence and smaller molecular weight, which ensures that a polarization signal detection window is sufficiently large in a screening process, and a detection signal-to-noise ratio is successfully ensured; fine crystal structure analysis of the target protein is not needed in advance, which can greatly reduce a drug screening threshold; and the screening method has good universality for nucleic acid binding proteins and is easy to popularize.

Description

Products for screening LRPPRC regulators and methods for identifying LRPPRC regulators Technical field

The embodiment of the present invention relates to the field of biomedicine, in particular to a product for screening LRPPRC modulators and a method for identifying LRPPRC modulators.

Background technique

Molecular targeted therapy is the theoretical basis for the treatment of diseases at this stage, including tumors, cardiovascular system, and nervous system diseases, which urgently need clinically available specific molecular inhibitors. At this stage, drug design and development for protein targets are mainly limited to the following two aspects: First, drug development is mainly for organisms with kinase activity, and targeted drugs developed are mainly to inhibit protein enzyme activity, such as cancer. In treatment, gefitinib inhibits EGFR kinase activity, dasatinib inhibits SRC kinase activity, cardiovascular statins inhibit HMG-CoA reductase activity and so on. However, the types of proteins with enzymatic activity in cells account for only a small percentage of the total number of proteins, and there are fewer proteases that can be used as drug targets. Second, the design and screening of enzyme activity inhibitors for proteases is extremely dependent on the understanding of protein structure. In the absence of protein crystal structure, the screening of protein inhibitors will face great challenges. Therefore, there is an urgent need to develop effective drug screening systems for more protein targets, especially non-enzymes, to improve the efficiency of molecular targeted therapy.

Nucleic acid binding proteins are a family of proteins that can specifically bind to nucleic acids, and can be divided into RNA binding proteins and DNA binding proteins according to the type of nucleic acid that is bound. This type of protein generally has a conserved nucleic acid binding domain, which can selectively bind to RNA or DNA with a specific base sequence. It has now been confirmed that nucleic acid binding proteins play a vital role in almost all life activities such as DNA replication, DNA damage repair, gene transcription, protein translation, etc., such as malignant tumors, cardiovascular and cerebrovascular diseases, infectious diseases, etc. It plays an indispensable role in diseases related to human life and health. Take the RNA-binding protein LRPPRC as an example. If you want to screen for small molecule regulators that can block the binding of LRPPRC to RNA, you need to know the nucleic acid characteristics of LRPPRC binding, that is, the conserved binding domain (motif), but it is often impossible to know the absolute Most LRPPRC bind RNA motifs, even if LRPPRC is known to bind mRNA motifs, there are complicated procedures for in vitro transcription and recovery of RNA, and RNA is easily degraded, which can easily lead to system instability. Therefore, there is an urgent need to provide a more stable, versatile, and sensitive method for identifying LRPPRC modulators.

Summary of the invention

The purpose of the embodiments of the present invention is to provide a product for screening LRPPRC modulators and a method for identifying LRPPRC modulators, and to provide a more stable, versatile and sensitive nucleic acid binding protein as a target to identify LRPPRC modulators. Agent method.

In order to solve the above technical problems, the present invention provides a product for screening LRPPRC modulators, including a kit and a device. The kit includes recombinant LRPPRC protein, GST protein, fluorescein-labeled nucleic acid aptamer R14, and Fluorescein-labeled control nucleic acid; the device includes a multi-well plate and a fluorescence polarization signal detector.

Further, the concentration of the recombinant LRPPRC is the same as the GST protein concentration, both 14-136 μg/mL, preferably 68-120 μg/mL.

Further, the sequence of the nucleic acid aptamer R14 is GGTGGGTGGGTTGGGTGG, the sequence of the control nucleic acid is AATTTTTTAATTATTTATATTA; the concentration of the fluorescently labeled nucleic acid aptamer R14 is the same as that of the fluorescently labeled control nucleic acid, and the concentration of the stock solution is both 100 nM, The liquid concentration is 10 ^-8 M.

Further, the fluorescence polarization signal detector is a microplate reader, preferably an Infinite M1000 PRO plate reader.

Further, the product is used for screening LRPPRC protein negative regulators or C-terminal binding agents.

The present invention also provides a method for identifying LRPPRC modulators based on fluorescence polarization signal detection, which includes:

1) Prokaryotic expression and purification of LRPPRC protein and GST protein;

2) Synthesis of fluorescein-labeled nucleic acid aptamer R14 and fluorescent-labeled control nucleic acid, the sequence of the control nucleic acid is AATTTTTTAATTATTTATATTA;

3) Mix LRPPRC protein with fluorescently labeled nucleic acid aptamer R14, mix LRPPRC protein with fluorescently labeled control nucleic acid, and mix fluorescently labeled nucleic acid aptamer R14 with GST, and add them to different wells in the multiwell plate;

4) Add candidate molecules to each well;

5) Detect the fluorescence polarization signal of each mixture in step 3) with a fluorescence polarization signal detector.

Further, the step 3) also includes a DMSO blank control.

Further, the reaction concentration of the LRPPRC and GST are both 120 μg/mL, the reaction concentration of the fluorescently labeled nucleic acid aptamer R14 and the fluorescently labeled control nucleic acid are both 40 nM; the working concentration of the candidate molecule is 24 μM.

Further, the method of the present invention also includes collecting the fluorescent signal and the polarized light signal of each hole of the orifice plate; normalizing the signal of the DMSO hole, and adding the small molecule compound, the polarized light signal of the hole The inhibition rate of LRPPRC is greater than 50%; and the small molecule compound whose fluorescence signal itself changes less than 1.5 times is a negative regulator of LRPPRC.

Furthermore, the method of the present invention is a high-throughput screening method.

Compared with the prior art, the embodiment of the present invention can obtain nucleic acid aptamers that specifically bind to the LRPPRC binding protein through SELEX screening without knowing the motif of RNA binding, and perform fluorescence on the obtained nucleic acid aptamers. The protein label interacts with the LRPPRC binding protein to form a nucleic acid-protein complex. At this time, it is in a state of high polarized light signal. Adding small molecule compounds to the nucleic acid-protein complex system can block the protein nucleic acid binding function. The labeled nucleic acid aptamer is dissociated from the protein and is in a state of low polarized light signal. Since the number of bases of the nucleic acid aptamer is smaller, the molecular weight is smaller. According to the principle of polarized light detection, the smaller the molecular weight, the polarization changes It will be more significant, according to the inhibition rate of the polarized light signal is greater than 50%; and the change of the fluorescence signal itself is less than 1.5 times the screening conditions to obtain qualified LRPPRC negative regulator.

Description of the drawings

One or more embodiments are exemplified by the pictures in the corresponding drawings. These exemplified descriptions do not constitute a limitation on the embodiments. Elements with the same reference numbers in the drawings are represented as similar elements. Unless otherwise stated, the figures in the attached drawings do not constitute a limitation of scale.

Fig. 1 shows the protein levels of LRPPRC and Actin enriched in various nucleic acid aptamer sequences using western blot in Example 1 of the present invention;

Figure 2 shows five LRPPRC fragments in embodiment 2 of the present invention;

Fig. 3 shows the GST tags enriched in each nucleic acid aptamer sequence detected by western blot in Example 2 of the present invention;

4 is a response curve of polarization signal value and concentration of each reaction system in Example 3 of the present invention;

FIG. 5 is the S/N ratio and Z factor value of each LRPPRC protein concentration gradient in Example 3 of the present invention;

Fig. 6 is a graph showing the effect of 850 small molecule compounds in inhibiting the binding of LRPPRC to nucleic acid in Example 4 of the present invention;

Figure 7 is a schematic diagram of the polarization inhibition rate of 20 candidate small molecule compounds in Example 4 of the present invention;

Fig. 8 is a change curve of the fluorescence spectrum of LRPPRC protein under different GAA concentrations in Example 5 of the present invention;

9 is a temperature change curve of GAA and LRPPRC after mixing at different GAA concentrations in Example 5 of the present invention;

Figure 10 is a graph showing the change trend of LRPPRC protein at different GAA concentrations and at different times in Example 6 of the present invention;

Fig. 11 shows the enrichment of CDK6 mRNA in tumor cells after treatment with different compounds in Example 6 of the present invention.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following describes the embodiments of the present invention in detail with reference to the accompanying drawings. However, a person of ordinary skill in the art can understand that in each embodiment of the present invention, many technical details are proposed for the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solution claimed in this application can be realized.

Nucleic acid aptamers are selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to find oligonucleotide fragments that can specifically bind to a given specific target. Such nucleic acid aptamers have multiple advantages such as small molecular weight, strong specificity, and large binding capacity. In recent years, they have gradually shown obvious advantages in the fields of molecular analysis, biological characterization, and drug delivery.

Fluorescence polarization detection is a classic analysis method in the field of analytical chemistry, and it is a basic means to characterize the interaction between molecules. The basic principle of this method is that the fluorescent substance is irradiated with a single plane of polarized light, and absorbs light energy to jump into an excited state, then return to the ground state, and emit a single plane of polarized fluorescence. The intensity of polarized fluorescence is positively correlated with the size of fluorescent molecules, and is inversely correlated with the speed of rotation when excited. The polarization signal of small-molecule fluorescent molecules is weak. When the small-molecule fluorescent substance and the macromolecular substance are combined with each other, the overall molecular weight increases sharply, the molecular vibration becomes slower, and the polarization signal increases significantly. Theoretically, fluorescently labeled nucleic acid aptamers will change in polarized light after binding to large molecular weight target proteins. However, at this stage, there is still no combination of nucleic acid aptamer screening and polarized light detection for high-throughput drug screening. Report.

Example 1

Verify the specific binding of aptamer R14 to LRPPRC

Co-immunoprecipitation proved that LRPPRC protein specifically binds to aptamer R14

1) Collect the cells by centrifugation, and collect the cell lysate by centrifugation at 12000g after lysis;

2) Synthetic biotin-labeled AS1411 and R14, AS1411 is a reported specific binding protein nucleolin

Nucleic acid aptamer; R14 is a nucleic acid aptamer obtained through LRPPRC protein screening, where R14:

GGTGGGTGGGTTGGGTGG;

AS1411: GGTGGTGGTGGTTGTGGTGGTGGTGG), dissolve in water, and make a 100nM storage liquid;

3) Prepare a protein-nucleic acid aptamer mixture with PBS containing 5mM MgCl ₂ , protein 500ug, nucleic acid aptamer 200pm, final volume 1ml;

4) After the protein-nucleic acid aptamer mixture is exposed to room temperature for 30 minutes, add 50ul streptavidin-coated beads to recover the biotin-labeled nucleic acid aptamer-protein complex;

5) After washing the obtained aptamer-protein complex three times with PBS, add 30ul protein electrophoresis loading buffer, and boil it in a boiling water bath for 5 minutes;

6) Western blot was used to detect the protein levels of LRPPRC and Actin enriched by each nucleic acid aptamer sequence, as shown in Figure 1. From Figure 1, it can be seen that the nucleic acid aptamer R14 can bind to the LRPPRC protein well.

Example 2

Fragmentation of LRPPRC protein, proving that aptamer R14 binds to the C-terminus of LRPPRC protein

1) According to the sequence characteristics of the LPRRC protein, cut the LRPPRC design into 5 fragments, which are respectively marked as LRP1-LRP5, as shown in Figure 2;

2) Integrate all the LRPPRC fragments into the GST expression plasmid, perform prokaryotic expression and purification in E. coli, and dissolve the obtained LRPPRC prokaryotic expression protein in PBS;

3) Configure the LRPPRC fragmented protein-biotin-labeled R14 mixture, the protein fragment is 100ug, the biotin-labeled R14 is 200pmol, and the final volume is 1ml;

4) After the mixture of LRPPRC fragment protein-R14 acts at room temperature for 30 minutes, add 50ul streptavidin-coated beads to recover the biotin-labeled R14-LRPPRC fragment protein complex;

5) After washing the obtained R14-LRPPRC fragment protein complex with PBS for three times, add 30ul protein electrophoresis loading buffer, and bath in boiling water for 5 minutes;

6) Western blot was used to detect the GST tags enriched in each aptamer sequence. The experimental results are shown in Figure 3. The GST tag was detected in LRPPRC fragment protein 6 (LRP6), but not in other fragment proteins. The GST tag indicates that only the LRPPRC protein fragment LRP6 binds to R14, that is, the aptamer R14 binds to the C-terminus of LRPPRC.

Example 3

In order to verify whether the nucleic acid aptamer-LRPPRC protein polarization screening system is suitable for a high-throughput drug screening system, this example was verified through the following two aspects:

One): specific verification of screening system

After FITC labeling nucleic acid, the specificity of the system is verified

1) Prokaryotic expression and purification of LRPPRC protein, GST protein as control protein, and BCA method for quantification;

2) Synthesize fluorescein-labeled (including but not limited to HEX, CY3, CY5 and other fluorescent labeling methods) LRPPRC specific nucleic acid aptamer Aptamer (R14, GGTGGGTGGGTTGGGTGG) and irrelevant control sequence (control DNA, AATTTTTTAATTATTTATATTA), configured to 100nM Stock solution

2) Configure the complex of protein and nucleic acid sequence, the concentration of LRPPRC specific nucleic acid aptamer and irrelevant control sequence are both 10 ^-8 M, and the concentration gradient of GST empty protein or LRPPRC protein is (0,7,14,34,68 , 136ug/ml), placed at room temperature for 30min;

3) After 30 minutes, use Infinite M1000 PRO plate reader (Tecan, Switzerland) to detect the polarization signal value of each reaction system;

4) Draw a concentration response curve according to the polarization signal value of each reaction system, as shown in Figure 4.

It can be seen from Figure 4 that the polarized signal of the nucleic acid aptamer R14 combined with the LRPPRC protein can reach nearly 300mp, which is much higher than the polarized signal of other nucleic acid-protein binding reports. This advantage is due to the small fragment of the nucleic acid aptamer used. The molecular weight is small. At the same time, the polarization signals of the binding of R14 and GST control protein and the binding of control sequence RTT and LRPPRC were significantly lower than that of R14 and LRPPRC protein. Fully screen the specificity of nucleic acid and protein binding.

Two): High-throughput screening adaptability verification of screening system

Determine the signal-to-noise ratio and the Z-factor variation interval of the entire reaction system to judge whether the screening system is suitable for high-throughput screening

1) Prokaryotic expression and purification of LRPPRC protein, and BCA method for quantification;

2) Synthetic fluorescein-labeled (including but not limited to HEX, CY3, CY5 and other fluorescent labeling methods) LRPPRC specific nucleic acid aptamer Aptamer (R14, GGTGGGTGGGTTGGGTGG) configured into a 100nM stock solution;

2) Configure the LRPPRC-R14 complex, the concentration of R14 is always 10 ^-8 M, the concentration gradient of LRPPRC protein is (0,7,14,34,68,136ug/ml), and it is placed at room temperature for 30 minutes. Use only the fluorescently labeled R14 without LRPPRC protein as the control background hole;

3) After 30 minutes, use the Infinite M1000 PRO plate reader (Tecan, Switzerland) microplate reader to calculate the S/N and Z factor values of each LRPPRC protein concentration gradient. Generally, the S/N value of the S/N ratio is greater than 8 , When the Z value is between 0.5-1.0, it indicates that the system is suitable for high-throughput screening. As shown in Figure 5, when the protein concentration is higher than 60ug/ml, the system can meet the high throughput requirements well.

Example 4

According to the above optimized protein and nucleic acid concentration, high-throughput screening is performed from a library containing 850 small molecule compounds, and small molecule compounds that can block the binding of aptamer R14 and LRPPRC are screened from the library, as shown in Figure 6. , A total of 20 compounds can hinder the binding of LRPPRC to nucleic acids. It can be seen from Figure 7 that gossypol acetate (GAA) shows a strong LRPPRC inhibitory function. The specific screening methods are as follows:

1) In vitro prokaryotic expression and purification of LRPPRC RNA binding region C-domain protein;

2) In vitro synthesis of fluorescein-labeled (including but not limited to HEX, CY3, CY5 and other fluorescent labeling methods) LRPPRC specific binding nucleic acid aptamer R14-flu;

3) Configure the complex system of LRPPRC and R14-flu, use PBS as the buffer, the concentration is LRPPRC (120ug/ml), R14-flu (40nM), in a black opaque 384-well plate, add 15ul to each well The complex of LRPPRC and R14-flu;

4) Dilute 850 small molecule compounds with PBS to 60uM stock solution, add 10ul to each well of a 384-well plate, use DMSO well as a control, and act at room temperature for 5 minutes;

5) The Infinite M1000 PRO plate microplate reader collects the fluorescence signal and polarized light signal of each well, and normalizes the signal with the signal of the DMSO well, and calculates the inhibition rate of polarized light after each small molecule is added. The factor of change of the fluorescence signal itself is obtained by taking the inhibition rate of polarized light as the abscissa and the factor of change of the fluorescence signal itself as the ordinate. As shown in Fig. 6, a total of 20 small molecule compounds can hinder the binding of LRPPRC to nucleic acid. , That is, a total of 20 negative regulators can hinder the binding of LRPPRC to nucleic acids;

6) The screening conditions are that the inhibition rate of the polarized light signal is greater than 50%, and the change of the fluorescence value itself is less than 1.5 times. The change in light is caused by the inhibition of protein and nucleic acid binding, rather than the addition of small molecules that reduce the fluorescence signal of fluorescent molecules.)

Statistics on the polarization inhibition rates of 20 candidate small molecule compounds are performed. The statistical results are shown in Figure 7. GAA shows the largest polarization inhibition ability, that is, GAA as a negative regulator can most effectively inhibit The binding of LRPPRC protein to nucleic acid.

Example 5

In this example, fluorescence titration and isothermal calorimetry were used to detect the direct binding of LRPPRC and GAA.

1. Fluorescence titration method to determine the direct binding of GAA and LRPPRC protein

1) In vitro prokaryotic expression and purification of the C-domain protein (60ug/ml) of the RNA binding region of LRPPRC;

2) The stock solution concentration of GAA is 50mM;

3) Add a concentration gradient of GAA to the LRPPRC protein solution, respectively 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, 32uM;

4) Use FLS980 fluorescence spectrometer (Edinburgh Instruments, Ltd.) to detect the change in fluorescence value of LRPPRC protein in the 300-380nm band. Plot the changes in the fluorescence spectrum of the LRPPRC protein at different GAA concentrations, as shown in Figure 8. As can be seen from Figure 8, as the GAA concentration increases, the autofluorescence of the LRPPRC protein gradually decreases.

2. Isothermal calorimetry (ITC) to determine the direct binding of GAA and LRPPRC protein

1) In vitro prokaryotic expression and purification of the C-domain protein (60ug/ml) of the RNA binding region of LRPPRC;

2) The stock solution concentration of GAA is 50mM;

3) Drop the GAA stock solution into the LRPPRC protein buffer step by step, and the iTC200 instrument (MicroCal, GE) isothermal calorimeter records the temperature change after each addition of GAA. The experimental results are shown in Figure 9, which can be seen from Figure 9. After GAA and LRPPRC are mixed, there is a significant exothermic phenomenon.

It can be seen from the above that fluorescence titration and isothermal calorimetry experiments have confirmed the direct binding of LRPPRC and GAA.

Example 6

Experiment to verify the molecular function of GAA inhibiting LRPPRC

This embodiment mainly verifies that GAA has an inhibitory effect on LRPPRC from two aspects, as follows:

1. GAA directly degrades LRPPRC protein

1) Lung adenocarcinoma cell line A549, breast cancer cell line MCF7, lung cancer cell line H1299, breast cancer cell line BT474, breast cancer cell line MCF7, lung cancer cell line H460 were cultured to the logarithmic growth phase, and a concentration gradient of GAA (0 , 1, 5, 10uM), after 48 hours of culturing, the cells were collected by centrifugation, and the whole cell protein was obtained after lysis. 40ug total protein is used for western blot to detect the change trend of LRPPRC protein;

2) Lung adenocarcinoma cell line A549, breast cancer cell line MCF7, lung cancer cell line H1299, breast cancer cell line BT474, breast cancer cell line MCF7, lung cancer cell line H460 were cultured to the logarithmic growth phase, using GAA with a final concentration of 10uM Carry out the time gradient experiment, treat separately (0, 8, 16, 24 hours). The cells were collected by centrifugation, and the whole cell protein was obtained after lysis. 40ug total protein is used in western blot to detect the change trend of LRPPRC protein. The change trend of LRPPRC protein is shown in Figure 10.

2. RIP experiment (RNA Immunoprecipitation) verifies that GAA affects the binding of CDK6 mRNA and LRPPRC protein:

1) Trypsin digestion to collect lung adenocarcinoma cell line A549, breast cancer cell line MCF7, lung cancer cell line H1299, breast cancer cell line BT474, breast cancer cell line MCF7, lung cancer cell line H460, lyse the cells to extract the cell lysate, and transfer to the cells The indicated compounds (GAA, 7F2, 1E5, 10C5, 11E10) with a final concentration of 10uM were added to the lysate, and the sample with DMSO added was used as the NC control.

2) Add 2ug of LRPPRC antibody (abcam company, catalog number ab97505) and 50ul protein A/G magnetic beads to each tube, overnight at four degrees, after incubation, wash with PBS, extract mRNA enriched on protein A/G magnetic beads, and reverse After recording, Realtime-PCR amplification was performed to detect the abundance of CDK6 mRNA. The enrichment amount of samples processed with DMSO was subjected to normalized statistical analysis. The statistical results are shown in Figure 11.

It can be seen from Fig. 10 that the protein level of LRPPRC decreased significantly after treating tumor cells with GAA.

It can be seen from Figure 11 that after GAA is used to treat tumor cells, the CDK6 mRNA that can specifically bind to LRPPRC captured by the LRPPRC specific antibody is significantly reduced, indicating that GAA can also block the binding of LRPPRC to mRNA.

In summary, GAA can inhibit the growth of tumor cells by reducing the expression of LRPPRC gene or reducing the activity of LRPPRC protein, so as to achieve the purpose of treating tumor diseases with high expression of LRPPRC.

Compared with the prior art, the technical solution of the present invention has the following advantages:

First of all, the embodiment of the present invention uses short nucleic acid aptamer R14, which has a shorter sequence and molecular weight compared with naturally occurring RNA and DNA sequences, which ensures that the polarization signal detection window is sufficient during the screening process. Large, so that the detection signal-to-noise ratio has a good guarantee;

Secondly, during the operation of this screening method, there is no need to perform detailed crystal structure analysis of the target protein in advance, which can greatly reduce the threshold of drug screening;

Finally, this screening method has good versatility for a wide range of nucleic acid binding proteins and is easy to popularize.

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in actual applications, various changes can be made in form and details without departing from the spirit and spirit of the present invention. range.

Claims (10)

1. A product for screening LRPPRC regulators, which is characterized by comprising a kit and a device, the kit comprising recombinant LRPPRC protein, GST protein, fluorescein-labeled nucleic acid aptamer R14, and fluorescein-labeled control nucleic acid ; The device includes a porous plate and a fluorescence polarization signal detector.
2. The product for screening LRPPRC regulators according to claim 1, wherein the concentration of the recombinant LRPPRC is the same as the GST protein concentration, both 14-136 μg/mL, preferably 68-120 μg/mL.
3. The product for screening LRPPRC regulators according to claim 1, wherein the sequence of the nucleic acid aptamer R14 is GGTGGGTGGGTTGGGTGG, and the sequence of the control nucleic acid is AATTTTTTAATTATTTATATTA; the fluorescently labeled nucleic acid aptamer R14 The concentration of the fluorescently labeled control nucleic acid is the same, the concentration of the stock solution is 100nM, and the concentration of the working solution is 10 ^-8 M.
4. The product for screening LRPPRC modulators according to claim 1, wherein the fluorescence polarization signal detector is a microplate reader, preferably an Infinite M1000 PRO plate reader.
5. The product for screening LRPPRC modulators according to any one of claims 1 to 4, wherein the product is used for screening LRPPRC protein negative modulators or C-terminal binding agents.
6. A method for identifying LRPPRC modulators based on fluorescence polarization signal detection is characterized in that it comprises the following steps:

   1) Prokaryotic expression and purification of LRPPRC protein and GST protein;

   2) Synthesis of fluorescein-labeled nucleic acid aptamer R14 and fluorescent-labeled control nucleic acid, the sequence of the control nucleic acid is AATTTTTTAATTATTTATATTA;

   3) Mix LRPPRC protein with fluorescently labeled aptamer R14, LRPPRC protein with fluorescently labeled control nucleic acid, and fluorescently labeled aptamer R14 with GST protein, and add them to different wells of the multiwell plate;

   4) Add candidate molecules to each well;

   5) Detect the fluorescence polarization signal of each mixture in step 3) with a fluorescence polarization signal detector.
7. The method for identifying LRPPRC modulators based on fluorescence polarization signal detection according to claim 6, wherein the step 3) further comprises a DMSO blank control.
8. The method for identifying LRPPRC modulators based on fluorescence polarization signal detection according to claim 6, wherein the reaction concentrations of the LRPPRC and GST proteins are both 120 μg/mL, and the fluorescently labeled nucleic acid aptamer R14, fluorescently labeled The reaction concentration of the control nucleic acid is 40 nM; the working concentration of the candidate molecule is 24 μM.
9. The method for identifying LRPPRC modulators based on fluorescence polarization signal detection according to any one of claims 6-8, wherein the fluorescence signal and the polarization signal of each hole of the orifice plate are collected; After normalization, after adding small molecule compounds, the inhibition rate of the polarized light signal of the pore is greater than 50%; and the small molecule compounds whose fluorescence signal itself changes less than 1.5 times are LRPPRC negative regulators.
10. The method for identifying LRPPRC modulators based on fluorescence polarization signal detection according to any one of claims 6-8, wherein the method is a high-throughput screening method.

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