Optical fiber sensor, preparation method of optical fiber sensor and application of optical fiber sensor in detection of in-vitro ubiquitination and high-throughput drug screening

Abstract

The invention relates to an optical fiber sensor, a preparation method of the optical fiber sensor and application of the optical fiber sensor in detection of in-vitro ubiquitination and high-throughput drug screening. The optical fiber sensor comprises an optical fiber probe and ubiquitination substrate protein modified on the optical fiber probe, and the substrate protein can be ubiquitinated by HECT type E3 ubiquitin ligase WWP2. The in-vitro ubiquitin system can verify phenomena in cells, is not influenced by complex environments in the cells, and can more clearly observe the ubiquitination mechanism of E3 ubiquitin ligase, and the optical fiber sensing can monitor the dynamic process of ubiquitination of a substrate, improve the rate of in-vitro ubiquitin detection, reduce factors influencing the ubiquitin reaction, reduce the experiment cost, and improve the detection efficiency of the E3 ubiquitin ligase. The defects of the existing experimental technology in in-vitro ubiquitination research are overcome, and the effects of an inhibitor and PROTAC on E3 ubiquitin ligase and a substrate can be quantified. The method can also be applied to detection of endogenous E3 activity and substrate ubiquitination conditions in the cell lysis buffer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a fiber-optic sensor, a preparation method thereof and applications thereof in detecting in vitro ubiquitination and high-throughput drug screening. BACKGROUND

[0002] The ubiquitin-proteasome system (UPS) is a proteolytic system involved in the degradation of proteins in cells, which is involved in the regulation of many cellular processes in eukaryotic cells, such as DNA repair, stress response and cell proliferation, and plays an important role in maintaining cellular homeostasis. Ubiquitin is a highly conserved protein composed of 76 amino acids, and its main function is to mark proteins to be decomposed so that they are degraded by 26S proteasome. The ubiquitination modification of substrate proteins is a cascade reaction catalyzed by E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme and E3 ubiquitin ligase. The human genome contains genes encoding two E1 (UBA1, UBA6), about 50 E2 and nearly 700 E3 ubiquitin ligases. E3 ubiquitin ligase can directly interact with the substrate or interact with the substrate through an adapter protein, which determines the specificity of the substrate bound by the ubiquitin-proteasome system. According to the structural characteristics and action mechanism, the E3 ubiquitin ligases found in mammalian cells can be divided into the following three categories: HECT-containing E3 ubiquitin ligases, RING domain-containing E3 ubiquitin ligases and RBR protein family E3 ubiquitin ligases.

[0003] UPS dysregulation is closely related to many diseases such as cancer, diabetes, cardiovascular disease, etc. Bortezomib, the first drug targeting 26S proteasome, is used for the treatment of multiple myeloma and has been approved for marketing by the US Food and Drug Administration. However, due to the indiscriminate inhibition of all protein degradation pathways dependent on 26S proteasome, this type of inhibitor can cause damage to normal cells and has high toxicity and side effects. Due to the limitation of the types of E1 ubiquitin activating enzyme and E2 ubiquitin binding enzyme, the potential for developing targeted drugs is small. E3 ubiquitin ligase has the characteristics of specific domain, targeted substrate and low normal expression level, which is of great significance in reducing the toxicity and side effects of drugs in the research of small molecule inhibitors and targeted antibodies. And inhibition of E3 ubiquitin ligase-substrate targets has less impact on the growth and function of normal cells. Therefore, the development of targeted drugs for E3 ubiquitin ligase has become one of the research hotspots in recent years. At present, most of the small molecule drugs targeting E3 in the development stage are directed against the RING family. Small molecule inhibitors targeting E3 have off-targeting situations, which may cause toxicity and side effects. Therefore, the development of new small molecule inhibitors targeting E3 and drugs targeting "undruggable" targets of PROTACs are of great significance in providing new treatment strategies for cancer, neurodegenerative diseases, etc.

[0004] Optical fiber sensor is a new type of sensor that detects and analyzes physical, chemical or biological quantities by modulating, demodulating and processing optical signals in optical fibers. In the field of biology, optical fiber sensors can be used to detect biological macromolecules such as proteins, DNA and lipids due to their high sensitivity, fast response and easy integration. The main principle of optical fiber biosensor based on surface plasmon resonance (SPR) is to study the changes of multi-dimensional information such as light intensity, wavelength and polarization state caused by the interaction of biological molecules to realize biological sensing and detection. As an auxiliary tool, it can improve the accuracy and scientificity of biological research. In the aspect of target drug screening, optical fiber sensing technology has higher sensitivity and selectivity, can realize the screening and evaluation of a large number of compounds in a short time, and can improve the efficiency and economy of drug research and development. SUMMARY

[0005] In view of the above-mentioned disadvantages of the prior art, the present application provides an optical fiber sensor, a preparation method thereof and applications in detecting in vitro ubiquitination and high-throughput drug screening.

[0006] To achieve the above-mentioned purpose, the scheme of the present application is as follows:

[0007] In a first aspect, the present application provides an optical fiber sensor, which comprises an optical fiber probe and a ubiquitination substrate protein modified on the optical fiber probe, wherein the substrate protein is ubiquitinated and modified by mouse ubiquitin-like modifier enzyme 1 (UBA1), WW domain-containing E3 ubiquitin-protein ligase 2 (WWP2), recombinant human ubiquitin-conjugating enzyme (UBCH5b) and WW domain-binding protein 2 (WBP2)-Avitag fusion protein through several enzyme-linked reactions.

[0008] Optionally, the molar ratio of the mouse ubiquitin-like modifier enzyme 1, the WW domain-containing E3 ubiquitin-protein ligase 2, the recombinant human ubiquitin-conjugating enzyme and the WW domain-binding protein 2-Avitag fusion protein is 0.5-1.5:0.5-1.5:1.5-2.5:1.5-2.5.

[0009] Optionally, the WW domain-binding protein 2-Avitag fusion protein is connected with biotin.

[0010] Optionally, the biotin comprises D-biotin.

[0011] In a second aspect, the present application further provides a preparation method of the optical fiber sensor as described above, comprising the following steps:

[0012] S1. mixing the mouse ubiquitin-like modifier enzyme 1, the WW domain-containing E3 ubiquitin-protein ligase 2, the recombinant human ubiquitin conjugating enzyme and the WW domain-binding protein 2-Avitag fusion protein, standing, adding adenosine triphosphate, performing enzyme-linked reaction to obtain the substrate protein;

[0013] S2. modifying the substrate protein on the optical fiber probe to obtain the optical fiber sensor.

[0014] Optionally, in step S1, the molar ratio of the mouse ubiquitin-like modifier enzyme 1 to the adenosine triphosphate is 0.5-1.5:4-6.

[0015] Optionally, in step S1, the standing time is 15-25 min.

[0016] Optionally, in step S2, the modification of the substrate protein on the optical fiber probe comprises:

[0017] S2.1 immersing the cleaned gold film in an alcohol solution of 11-mercaptoundecanoic acid and incubating;

[0018] S2.2 co-incubating the incubation product obtained in step S2.1 with streptavidin (SA);

[0019] S2.3 co-incubating bovine serum albumin, the incubation product obtained in step S2.2 with the optical fiber probe;

[0020] S2.4 co-incubating the substrate protein with the incubation product obtained in step S2.3.

[0021] Optionally, in step S2.1, the incubation temperature is 20-30℃.

[0022] Optionally, in step S2.2, the co-incubation temperature is 20-30℃.

[0023] Optionally, in step S2.3, the co-incubation temperature is 20-30℃.

[0024] Optionally, in step S2.4, the co-incubation temperature is 20-30℃.

[0025] In a third aspect, the present application also provides the optical fiber sensor as described above or prepared according to the method as described above for quantifying the effect of inhibitors and PROTACs on E3 ubiquitin ligases and substrates, applying to detecting endogenous E3 activity and substrate ubiquitination in cell lysates and drug screening or drug monitoring.

[0026] The beneficial effects of the present application are:

[0027] The in vitro ubiquitin system can verify the phenomenon in the cell, is not affected by the complex environment in the cell, and more clearly observes the ubiquitination mechanism of the E3 ubiquitin ligase. The optical fiber sensing of the application can monitor the dynamic process of substrate ubiquitination, improve the rate of in vitro ubiquitin detection, reduce factors affecting the ubiquitin reaction, reduce experimental costs, make up for the shortcomings of existing experimental techniques in in vitro ubiquitination research, and quantify the effect of inhibitors and PROTACs on E3 ubiquitin ligase and substrates.

[0028] The optical fiber sensing of the application is used for in vitro research (such as high-throughput drug screening, monitoring, etc.) of small molecule drugs in the ubiquitination system, which helps to improve the specificity of the drug, reduce off-target effects, and more accurately control experimental conditions and exclude the interference of other factors.

[0029] The application also simulates the situation in the cell, detects endogenous E3 activity and substrate ubiquitination in the cell lysate, and explores the substrate ubiquitination spectrum in the cell lysate, which significantly improves its application value.

[0030] The optical fiber sensing of the application can quantify the effect of inhibitors and PROTACs on E3 ubiquitin ligase and substrates. Through the design of multiple channels, it can also be applied to the screening of small molecule drugs of E3 ubiquitin ligase activators and inhibitors. The fast response of the spectrum can be used to directly judge the activity information of the drug on the substrate of the E3 ubiquitin ligase. The small molecules that respond are further verified by cell experiments, which are more suitable for high-throughput screening of potential drug candidate molecules. It is expected to further improve the efficiency and accuracy of small molecule drug screening on this basis, and to realize the screening and evaluation of a large number of compounds in a relatively short period of time, and to improve the efficiency and economy of drug research and development.

[0031] The application is the first exploration and application of the SPR-based optical fiber sensor technology in in vitro ubiquitination detection. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings from the structures shown in the drawings without creative labor.

[0033] Figure 1 The schematic diagram of the principle of the application;

[0034] Figure 2 The result diagram of WB verification;

[0035] Figure 3The figure is the process flow chart of the preparation of the optical fiber sensor. The sensing probe is the optical fiber sensor. Protein represents protein, and blotin represents blotting.

[0036] Figure 4 The figure is the gel electrophoresis detection result chart of the in vitro ubiquitination reaction monitoring experiment of the substrate protein. Streptavidin represents streptavidin, and time represents time.

[0037] Figure 5 The figure is the curve chart of wavelength shift and time in the in vitro ubiquitination reaction monitoring experiment of the substrate protein. Wavelength represents wavelength.

[0038] Figure 6 The figure is the scanning electron microscope image before adding USP21 deubiquitinase. With substrate indicates that WBP2 is modified on the optical fiber probe by biotin. With substrate-polyUB indicates that the optical fiber probe is polyubiquitinated.

[0039] Figure 7 The figure is the surface chemiluminescence verification of the optical fiber probe. The reaction conditions are as follows: E3 is wild type WWP2, 100 μM UB; wild type WWP2, 25 μM UB; no ATP is added, and other conditions are the same.

[0040] Figure 8 The figure is the ubiquitination of WBP2 under the action of deubiquitinase USP21 detected by the optical fiber. The left figure is the WB result, and the right figure is the wavelength shift obtained by the optical fiber detection. Wavelength shift represents wavelength shift.

[0041] Figure 9 The figure is the response graph of the optical fiber under different UB concentrations. The substrate attached to the surface of the optical fiber is WBP2, and E3 is WWP2 Y369E , and the UB concentration is 5, 50, and 100 μM.

[0042] Figure 10 The figure is the response graph of the optical fiber under different temperatures. The UB is 10 μM, and the reaction temperature is room temperature and 37°C.

[0043] Figure 11 The figure is the response graph of the optical fiber under different E3 enzyme activities. The UB is 100 μM, E2 is 5 μM, and E3 is activated WWP2 Y369E , WWP2 WT .

[0044] Figure 12 The figure is the response graph of the optical fiber under different E3 enzyme activities. The left figure is the WB result, and the right figure is the wavelength shift obtained by the optical fiber detection. E3 is WWP2 WT , WWP2Y369E , WWP2 C838S . DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the protection scope of the present application.

[0046] As Figure 1 and Figure 3 shown, the principle of the present application is:

[0047] The ubiquitination substrate protein WBP2 is modified on the surface of the optical fiber probe by using the high affinity of biotin and streptavidin, when UB is combined on WBP2, the increase of molecular weight is in the form of red shift of light wavelength to quantify the activity degree of E3 ligase WWP2 on the ubiquitination of substrate WBP2.

[0048] In the following description, a large number of details are discussed to provide a more thorough explanation of the embodiments of the present application, however, it is obvious for a person of ordinary skill in the art to implement the embodiments of the present application without these specific details.

[0049] (I) Several enzymes and substrates required for in vitro ubiquitination are purified: UBA1 (mouse ubiquitin-like modifier enzyme 1), UBCH5b (recombinant human ubiquitin conjugating enzyme), WWP2 (WW domain-containing E3 ubiquitin protein ligase 2), WBP2 (WW domain-binding protein 2), and the specific purification steps are as follows:

[0050] 1. Transformation:

[0051] (1) 1 uL of pET28b-UBA1 (donated by others), pGEX4T-1-UBCH5b (donated by others), pGEX6P-1-WWP2 (commercially available), and pET28a-His-SUMO-WBP2 plasmids (commercially available) are added to an EP tube containing 100 uL of BL21 competent cells, the EP tube bottom is lightly shaken to uniformly distribute the plasmids and the competent cells, and incubated on ice for 30 min;

[0052] (2) The temperature is treated at 42°C for 45 s to introduce the plasmids into the competent cells by heat shock;

[0053] (3) The competent cells are placed back on ice for 2 min to shrink;

[0054] (4) Add 200uL sterilized LB liquid medium to the EP tube, and incubate at 37℃ and 220rpm for 1h to make E. coli proliferate;

[0055] (5) Transfer 200uL E. coli after step (4) to a plate with corresponding resistance to plasmid, and smear evenly with a swab, and incubate at 37℃ and 220rpm overnight.

[0056] 2. Small shake: pick single clone to 25mL liquid LB medium containing antibiotics (pET28b-UBA1, pGEX4T-1-UBCH5b, pGEX6P-1-WWP2 are ampicillin resistant, and pET28a-His-SUMO-WBP2 is kanamycin resistant) respectively, and incubate at 37℃ and 220rpm overnight.

[0057] 3. Large shake:

[0058] (1) Inoculate the above bacterial liquid to 1L sterilized LB medium at a ratio of 1:100, and add 1mL corresponding antibiotic, and treat at 37℃ and 220rpm for 4h;

[0059] (2) Detect the OD600 value of the system, and when the OD600 value of the system is 0.6-0.8, add IPTG (isopropyl thiogalactoside) to make the final concentration of IPTG 0.5mM;

[0060] (3) Incubate at 18℃ and 220rpm overnight;

[0061] (4) Centrifuge at 4℃ and 5000rpm for 10min, collect the bacteria, and store at -80℃.

[0062] 4. Protein purification:

[0063] (1) Add 35mL Buffer I (20mM Tris PH8.0, 200mM NaCl) and PMSF (final concentration 1mM) and DTT (final concentration 1mM) to the protein after large shake treatment, and resuspend the bacterial liquid on ice;

[0064] (2) Lysate E. coli 600ba, 1L bacterial liquid is lysed about 3-4 times until the effluent is liquid droplets, collect the lysate in a 50mL centrifuge tube and place on ice, and centrifuge at 4℃ and 20000rpm for 20min;

[0065] (3) Transfer the supernatant after centrifugation to a 50mL centrifuge tube (on ice) and take 10uL supernatant to add 3uL 5× protein loading buffer labeled as "L"; pick up the precipitate with a gun head, add 5uL 5× protein loading buffer and mix evenly, labeled as "P";

[0066] (4) Take 3 mL resin filler into a 20 mL volume purification column, UBA1, WBP2 are Ni-NTA Beads 6FF, UBCH5b, WWP2 are GST-glutathione resin, after equilibration, binding on the 4 °C carousel for 4 h;

[0067] (5) Collect the effluent, take 10 uL of the collected liquid on ice in a 50 mL centrifuge tube, add 3 uL of 5* protein loading buffer labeled as "ft";

[0068] (6) Add 60 mL of wash buffer (20 mM Tris PH8.0, 200 mM NaCl, 0.1% Triton X100) to wash the column, add 10 mL of buffer I to wash the column, and do not collect the effluent;

[0069] (7) Elution: UBCH5b, WWP2 elution method: add 5 mL of elution buffer (Buffer I + 25 mM reduced glutathione), collect; UBA1, WBP2 elution method: respectively flow 5 mL of 25 mM Tris solution containing 10, 50, 100, 250, 500 mM imidazole through nickel, respectively collect, a total of 4 tubes; mix 10 uL of the above sample with 3 uL of 5*SDS loading, run gel, and stain with coomassie brilliant blue (R250 2.5g, methanol 450 mL, glacial acetic acid 100 mL, pure water 450 mL) for 10 min, add decolorizing solution (00 ml glacial acetic acid, 53 mL ethanol, 850 ml water) to decolorize;

[0070] (9) Enzymatic cleavage: add the corresponding enzyme to cleave the tag in a 15 mL centrifuge tube, UBA1 and UBCH5b are TEV, WWP2 is thrombin, WBP2 is ULP1, mix well and stand at 4 °C overnight;

[0071] (10) Concentration: Concentrate the protein in the centrifuge tube to reduce the imidazole concentration to less than 10 mM and increase the protein concentration.

[0072] ① AKTA protein purification instrument further purifies the target protein: install a Superdex200 increase molecular sieve purification column (200 nm for large molecular weight, 75 nm for small molecular weight), confirm that the purification column and A1 tube are in water; if the purification column is in ethanol, first replace the ethanol in the column with water (wash with water pump, then wash the column at a flow rate of 0.5 mL / min, 25 mL, column pressure less than 3 Mpa);

[0073] 2. Put A1 tube into Buffer A (20 mM Tris, pH 8.0, 200 mM NACL, 0.1% BME), Pumpwash A;

[0074] 3. Equilibrate column with 0.5 mL / min, 30 mL, column pressure less than 3 Mpa;

[0075] 4. Add concentrated protein supernatant into protein purifier loading hole with 1 mL syringe, run Superdex200 purification program, and collect subsequent eluate in 30 1 mL centrifuge tubes;

[0076] 5. Select corresponding centrifuge tube according to uv peak of chromatogram;

[0077] 6. Take 5 uL of protein sample and add 2 ul of protein loading buffer, use coomassie brilliant blue to confirm the centrifuge tube where target protein is located, centrifuge to collect, add 10% glycerol, and store at -80℃.

[0078] 5. WBP2 biotin connection:

[0079] 1. Add WBP2-Avitag fusion protein (final concentration 100 uM) in 952 uL PBS buffer, add 5 uL of 1 M magnesium chloride solution, 20 uL of 100 mM ATP solution, 20 uL of 50 uM BirA enzyme solution, and 3 uL of 50 mM D-biotin solution;

[0080] 2. After mixing, incubate the sample in a metal bath at 30℃ for 1 h;

[0081] 3. Dialyze the reacted protein in PBS overnight with a 10 kD dialysis bag;

[0082] 4. Concentrate the next day with a 10 kD ultrafiltration column, add 10% glycerol, 30 uL / tube, and store at -80℃.

[0083] 6. In vitro ubiquitination WB verification

[0084] 1. In vitro ubiquitination reaction

[0085] Mix 1x reaction buffer, final concentration 1 mM DTT, final concentration 1 uM UBA1 purified in step 4, final concentration 1 uM UBCH5b purified in step 4, final concentration 2 uM WWP2 purified in step 4, final concentration 2 uM WBP2 purified in step 4, and 100 uM UB in a 1.5 mL EP tube, mix well, stand at room temperature for 20 min, add ATP (final concentration 5 mM), and record as the start of the reaction.

[0086] 4.2 μL of reaction samples taken at 0, 1, 10, 30 min were taken for WB verification.

[0087] 6. WB verification

[0088] ① The reaction sample was treated in a 100℃ temperature metal bath for 6 min to cook the sample;

[0089] ② The loading order was 0, 1, 10, 30 min;

[0090] ③ Electrophoresis: 90V, 20 min, 160V, 45 min;

[0091] ④ Membrane transfer: 100V, 1h, membrane transfer on ice. Membrane transfer solution configuration: 200ml methanol + 100ml 10* membrane transfer solution + 700ml distilled water;

[0092] ⑤ Blocking: 5% skimmed milk (2.5g of skimmed milk powder to 50ml of TBS), room temperature for 1h;

[0093] ⑥ The primary antibody HRP-streptavidin was diluted at a ratio of 1:4000 with the primary antibody (specifically 1.2 μL of HRP-streptavidin primary antibody diluted in 5mL of primary antibody diluent), and was mixed and incubated at 4℃ and 220rpm overnight;

[0094] ⑦ The next day, the primary antibody was collected and washed with TBST buffer (Tris buffer saline containing Tween-20) for 5 min, repeated three times, and developed. The results are shown in Figure 4 .

[0095] As can be seen from Figure 4 , the WBP2 band moves up in seconds or even minutes. This result shows that the in vitro ubiquitination rate of WBP2 is fast.

[0096] 7. The substrate protein was modified on the surface of an optical fiber, and the specific steps were as follows:

[0097] 1. Gold film surface cleaning: immerse the gold film in piranha solution (volume ratio of concentrated sulfuric acid to hydrogen peroxide is 3:1) for 5 min, and rinse the gold film thoroughly with ultrapure water;

[0098] 2. Immerse the gold film in an ethanol solution containing 1mM MUA (11-mercapto-undecanoic acid) and incubate at room temperature for 12h;

[0099] 3. Incubate the incubation product obtained in step 2 with a solution of streptavidin at a final concentration of 0.4M at room temperature for 2h, and then incubate the incubation product with a solution of BSA at a concentration of 1% and an optical fiber probe at room temperature for 1h;

[0100] 4. The incubation product obtained in step 3 was incubated with biotin-labeled in vitro ubiquitination reaction product (30 min product) at room temperature for 2 h, and then washed with PBS buffer and ultrapure water in sequence to obtain the optical fiber sensor (as shown in Figure 3 ).

[0101] (ii) In vitro ubiquitination reaction monitoring experiment of substrate protein

[0102] The optical fiber sensor prepared in (i) was used to monitor the in vitro ubiquitination reaction of the substrate protein in real time, and the specific steps were as follows:

[0103] In a 1.5 mL EP tube, the reaction premix was added: 1x reaction buffer, final concentration 1 mM DTT, final concentration 1 mM UBA1 purified in step 4, final concentration 1 mM UBCH5b purified in step 4, final concentration 2 mM WWP2 purified in step 4, final concentration 2 mM WBP2 purified in step 4, and 100 mM UB were mixed in a 1.5 mL EP tube, and then mixed well and incubated at room temperature for 20 min. ATP (final concentration 5 mM) was added and mixed slightly. Immediately, the optical fiber probe of WBP2-biotin, i.e. the optical fiber sensor prepared in (i), was placed in the reaction system, and a wavelength value was collected every 10 s until the wavelength shift tended to be stable (i.e. the experimental group). Gel electrophoresis was performed, and the curve of wavelength shift versus time was drawn. A control group was set, and the solution of the control group did not contain ATP. The results are shown in Figure 4 and Figure 5 .

[0104] As shown in Figure 4 , the optical fiber sensor can quickly respond in the in vitro ubiquitination solution, and the wavelength of the light is red-shifted. When there is sufficient UB, the degree of red shift gradually increases until the UB is exhausted. WBP2 is connected to UB for several hours, and the red shift curve is stable, indicating that the reaction has reached saturation. At this time, a sufficient amount of UB protein is modified on the substrate and recognized by the 26S proteasome for degradation. Ubiquitin chains are produced, and this phenomenon cannot occur in the solution without ATP.

[0105] (iii) Verification of whether the red shift of the light wavelength is caused by specific connection of UB on the surface of the optical fiber

[0106] The specific steps for verifying whether the red shift of the light wavelength is caused by specific connection of UB on the surface of the optical fiber were as follows: The optical fiber sensors of the experimental group and the control group of (ii) were subjected to UB protein immunoblotting, and the specific steps were as follows: WB verification was performed, and chemiluminescence verification was performed. The surface of the optical fiber probe was subjected to chemiluminescence verification, and the reaction conditions were as follows: E3 was wild-type WWP2, 100 mM UB; wild-type WWP2, 25 mM UB; no ATP was added, and the other conditions were the same. The results are shown in Figure 7 .

[0107] In (ii), when the wavelength shift of the light was stable, USP21 deubiquitinase was added to make the final concentration of USP21 deubiquitinase 1.4 μM, and the results are shown in Figure 8 ,

[0108] As shown in Figure 7 and Figure 8 , during the process of protein modification, substances gradually attached to the surface of the optical fiber probe; the optical fiber probe was subjected to UB immunofluorescence color development, and no fluorescence appeared on the surface of the optical fiber probe in the group without ATP, while obvious fluorescence appeared on the surface of the optical fiber probe in the group with ATP; when the wavelength shift of the light was stable, 1.4 μM USP21 deubiquitinase was added, and the spectrum gradually blue-shifted to a wavelength shift of 0 nm. The results prove that the red shift of the light wavelength is caused by the specific connection of UB on the surface of the optical fiber.

[0109] (iv) Comparison of the enzymatic reaction kinetics of the spectrum curve under different reaction conditions

[0110] In order to explore whether the red shift curve of the light wavelength can correctly respond under the change of factors affecting the enzymatic reaction kinetics, the present application compares the enzymatic reaction kinetics of the spectrum curve under different reaction conditions, and the specific steps are as follows:

[0111] The final concentration of UB was set to 25, 50 and 100 μM, and E3 was activated WWP2 Y369E , and the steps were the same as before, and the results are shown in Figure 9 .

[0112] The final concentration of UB was set to 10 μM, and the temperature was set to room temperature and 37°C, and the steps were the same as before, and the results are shown in Figure 10 .

[0113] The final concentration of UB was set to 100 μM, and the concentration of UBCH5b was set to 5 μM, and E3 was wild type WWP2 and activated WWP2 Y369E , respectively, and the steps were the same as before, and the results are shown in Figure 11 .

[0114] The final concentration of UB was set to 100 μM, and E3 was wild type WWP2, activated WWP2 Y369E and inactivated WWP2 C838S , respectively, and the steps were the same as before, and the results are shown in Figure 12 .

[0115] As shown in Figures 8 to 12As shown, at room temperature, the reaction efficiency increases with the increase of substrate UB concentration; the wavelength shift increases in the same time with the increase of UBCH5b enzyme concentration; the slope of E3 ubiquitin ligase activity wavelength red shift curve is significantly enhanced, the wavelength shift in the same time is increased, which represents that more ubiquitin molecules are connected to WBP2 under the action of WWP2, and finally most of these ubiquitin molecules exist in the form of ubiquitin chain. In addition, the reaction rate can also be significantly increased and the reaction equilibrium time can be accelerated at 37℃. The results show that the strategy of using the optical fiber sensor of the application for high-throughput drug screening is feasible.

[0116] The above examples only illustrate the principles and effects of the present application, but are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. An optical fiber sensor, characterized in that, The fiber optic sensor includes a fiber optic probe and a ubiquitinated substrate protein modified on the fiber optic probe. The substrate protein is obtained by ubiquitination of a WW domain-binding protein 2-Avitag fusion protein with mouse ubiquitin-like modifying factor enzyme 1, WW domain-E3 ubiquitin protein ligase 2, and recombinant human ubiquitin coupling enzyme through several stages of enzyme-linked reactions.

2. The fiber optic sensor as described in claim 1, characterized in that, The molar ratio of the mouse ubiquitin-like modifying factor enzyme 1, the WW domain-containing E3 ubiquitin protein ligase 2, the recombinant human ubiquitin coupling enzyme, and the WW domain-binding protein 2-Avitag fusion protein is 0.5-1.5:0.5-1.5:1.5-2.5:1.5-2.

5.

3. The fiber optic sensor as described in claim 1, characterized in that, The WW domain-binding protein 2-Avitag fusion protein is linked to biotin.

4. The fiber optic sensor as described in claim 3, characterized in that, The biotin includes D-biotin.

5. The method for fabricating the fiber optic sensor as described in claims 1-4, characterized in that, Includes the following steps: S1. Mix the mouse ubiquitin-like modifying factor enzyme 1, the WW domain-containing E3 ubiquitin protein ligase 2, the recombinant human ubiquitin coupling enzyme, and the WW domain-binding protein 2-Avitag fusion protein, let stand, add adenine nucleoside triphosphate, and perform an enzyme-linked reaction to obtain the substrate protein. S2. Modify the substrate protein onto the optical fiber probe to obtain the optical fiber sensor.

6. The method for fabricating an optical fiber sensor as described in claim 5, characterized in that, In step S1, the molar ratio of mouse ubiquitin-like modifying factor enzyme 1 to adenine nucleoside triphosphate is 0.5-1.5:4-6.

7. The method for fabricating an optical fiber sensor as described in claim 5, characterized in that, In step S1, the settling time is 15-25 minutes.

8. The method for fabricating an optical fiber sensor as described in claim 5, characterized in that, In step S2, the substrate protein is modified onto the optical fiber probe, including: S2.1 The cleaned gold film is immersed in an alcoholic solution of 11-mercaptoundecanoic acid and incubated. S2.2 Incubate the incubation product obtained in step S2.1 with streptavidin; S2.3 Co-incubate bovine serum albumin, the incubation product obtained in step S2.2, and the fiber optic probe; S2.4 Co-incubate the substrate protein with the incubation product obtained in step S2.

3.

9. The method for fabricating an optical fiber sensor as described in claim 8, characterized in that, In step S2.1, the incubation temperature is 20-30℃; And / or, in step S2.2, the co-incubation temperature is 20-30°C; And / or, in step S2.3, the co-incubation temperature is 20-30℃; And / or, in step S2.4, the co-incubation temperature is 20-30°C.

10. The fiber optic sensor as described in claims 1-4 or the fiber optic sensor prepared according to the method described in any one of claims 5-9, is used in quantifying the effects of inhibitors and PROTAC on E3 ubiquitin ligases and substrates, in detecting endogenous E3 activity and substrate ubiquitination in cell lysates, and in drug screening or drug monitoring.

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