Cell indicator, and preparation method therefor and use thereof

Abstract

The present invention belongs the technical field of biomacromolecule detection. Provided are a cell indicator system, and a preparation method therefor and the use thereof. The cell of the cell indicator system comprises at least one of a vector A, a vector B and a vector C; the vector A comprises a gene encoding an TetR protein; the vector B comprises genes encoding a biotin acceptor peptide and ubiquitin; and the vector C comprises genes encoding a targeting E3 ligase and a biotin ligase. By means of constructing the cell line capable of expressing the TetR protein, biotin acceptor peptide, ubiquitin, targeting E3 ligase and biotin ligase, and inducing the expression by means of doxycycline, the targeting E3 ligase can bind to a substrate, thereby ubiquitinating and biotinylating the substrate, and thus a known ubiquitinated substrate can be rapidly detected and an unknown ubiquitinated substrate can be rapidly identified.

Description

A cell indicator and its preparation method and application Technical Field

[0001] The present application relates to the technical field of biomacromolecule detection, and in particular to a cell indicator system and a preparation method and application thereof. Background Art

[0002] Biotin labeling (bioID) is a proximity-dependent labeling technique that can be used to study interactions between proteins, proteins and RNA, and proteins and DNA. BioID methods are limited to interactions between two known proteins. For example, when protein A is linked to BirA (biotinylating enzyme), protein B will be biotinylated when the two proteins are in close proximity or bind to each other. However, their application to the ubiquitination of substrate proteins by E3 ligases remains significantly limited.

[0003] PROTAC (proteolysis-targeting chimeras) is a drug development technology that utilizes the ubiquitin-proteasome system (UPS) to degrade target proteins. With the rapid development of PROTAC technology, there is an urgent need to improve and innovate existing bioID technology to facilitate the application of bioID to PROTAC technology.

[0004] Summary of the Invention

[0005] The purpose of the present application is to overcome the deficiencies of the prior art and to provide a cell indicator system that can target E3 ubiquitin ligase to induce ubiquitination and biotinylation of substrate proteins, as well as a preparation method and application thereof.

[0006] To achieve the above objectives, the technical solutions adopted in this application are:

[0007] In a first aspect, the present application provides a cell indicator system, wherein the cell comprises at least one of a carrier A, a carrier B, and a carrier C;

[0008] The vector A contains a TetR protein encoding gene;

[0009] The vector B contains genes encoding biologically acceptable peptides and ubiquitin;

[0010] The vector C contains genes encoding targeting E3 ligase and biotin ligase.

[0011] The present application constructs a cell line capable of expressing TetR protein, biological acceptor peptide, ubiquitin, targeted E3 ligase and biotin ligase, and inducing expression through doxycycline, which can target E3 ligase to bind to the substrate, ubiquitinate and biotinylate the substrate, and can quickly detect known and identify unknown ubiquitinated substrates.

[0012] The cells in the cell indicator of the present application can be commonly used model cells in the laboratory, and tumor cells, pancreatic tissue cells, mouse liver cells and other cells can also be selected as carrier cells to construct the cell indicator according to actual conditions.

[0013] As a preferred embodiment of the cell indicator system described in the present application, it includes at least one of the following (I)-(II):

[0014] (I) The vector A is a lentiviral expression vector, and the TetR protein encoding gene sequence is shown in SEQ ID NO.1;

[0015] (II) the vector B further contains a TetO element and a FLAG tag;

[0016] The biotin acceptor peptide encoding gene sequence is shown in SEQ ID NO.2;

[0017] The ubiquitin encoding gene sequence is shown in SEQ ID NO.3;

[0018] The nucleotide sequence of the TetO element is shown in SEQ ID NO.4;

[0019] The nucleotide sequence of the FLAG tag is shown in SEQ ID NO.5.

[0020] The incorporation of the Tet-On system enables the controlled expression of a biotin acceptor peptide and ubiquitin using doxycycline, limiting the binding of substrate biotinylation and ubiquitination to a controlled time window. The biotin acceptor peptide (BAP) is a specific receptor for biotin that binds to it. Ubiquitin (UB) is a small protein that marks proteins for degradation. FLAG, a small peptide tag, facilitates subsequent determination of BAP-UB expression.

[0021] As a preferred embodiment of the cell indicator system described in the present application, the vector C further contains an HA tag; the vector C is a lentiviral expression vector;

[0022] The targeted E3 ubiquitin ligase encoding gene sequence is shown in SEQ ID NO.6;

[0023] The biotin ligase encoding gene sequence is shown in SEQ ID NO.7;

[0024] The nucleotide sequence of the HA tag is shown in SEQ ID NO.8.

[0025] Targeting E3 ligase is an enzyme that can specifically ubiquitinate its substrate, recognizing and ubiquitinating target proteins. Biotin ligase (BirA) is an enzyme that catalyzes biotin activity and the binding of biotin to the biotin receptor.

[0026] As a preferred embodiment of the cell indicator system described in the present application, it includes at least one of the following (I)-(II):

[0027] (I) The biotin acceptor peptide in the carrier B is located at the N-terminus of ubiquitin;

[0028] (II) The biotin ligase in the vector C is located at the N-terminus or C-terminus of the targeting E3 ligase.

[0029] In a second aspect, the present application provides a method for preparing the above-mentioned cell indicator system, comprising the following steps:

[0030] S1. Performing lentiviral packaging of the above-mentioned vector A and vector C in a carrier cell to obtain lentivirus A containing vector A and lentivirus C containing vector C;

[0031] S2, infecting the vector cells with the lentivirus A obtained in step S1 to obtain cell A containing vector A;

[0032] S3, transfecting the above-mentioned vector B into the cell A obtained in step S2 to obtain the cell B containing the vector A and the vector B;

[0033] S4. Infect the cell B obtained in step S3 with the lentivirus C obtained in step S1 to obtain the cell C containing the vector A, vector B and vector C. The obtained cell C is the cell indicator system.

[0034] In a third aspect, the present application provides the use of the above-mentioned cell indicator system in detecting and / or identifying ubiquitinated substrates.

[0035] The present application detects whether the sample can bind to streptavidin agarose beads to form a precipitate during the immunoprecipitation experiment, and uses Western blotting or mass spectrometry to detect whether it is biotinylated to determine whether the substrate is a ubiquitinated substrate. This method is rapid and convenient, does not require complex instruments and equipment, and has high economic benefits.

[0036] In a fourth aspect, the present application provides the use of the above-mentioned cell indicator system in detecting the process of forming a ternary complex between a PROTAC molecule and a ligand and / or the degree of ubiquitination.

[0037] This application can evaluate the process of PROTAC molecules and ligands forming a ternary complex by detecting the degree of biotinylation, and monitor the ubiquitination degree of the ternary complex, which can quickly detect and monitor the ubiquitination process of the ternary complex.

[0038] In a fifth aspect, the present application provides a kit for detecting and / or identifying ubiquitinated substrates, comprising the above-mentioned cell indicator system, doxycycline, biotin and streptavidin agarose beads.

[0039] As a preferred embodiment of the kit described in the present application, the kit further comprises a PROTAC molecule.

[0040] In a sixth aspect, the present application provides a method for detecting and / or identifying a ubiquitinated substrate, comprising the following steps:

[0041] (1) co-culturing the above-mentioned cell indicator system and adding doxycycline for induction to obtain a biotin acceptor peptide-ubiquitin conjugate and an E3 ubiquitin ligase-biotin ligase conjugate;

[0042] (2) mixing the biotin acceptor peptide-ubiquitin conjugate and the E3 ubiquitin ligase-biotin ligase conjugate obtained in step (1) with the test sample, adding biotin and / or PROTAC molecules to obtain cells containing ubiquitinated and biotinylated test samples;

[0043] (3) Lysing the cells containing the ubiquitinated and biotinylated test sample obtained in step (2) to obtain a lysed supernatant, the obtained lysed supernatant is immunoprecipitated with streptavidin agarose beads, and then the ubiquitination and biotinylation of the test sample are detected and / or identified by immunoblotting and / or mass spectrometry.

[0044] The present application can confirm whether a substrate can be ubiquitinated by binding streptavidin agarose beads to biotin and using immunoprecipitation technology and WB or mass spectrometry technology, and can detect known ubiquitinated substrates and unknown ubiquitinated substrates.

[0045] Compared with the prior art, the present invention has the following advantages:

[0046] (1) The present application constructs a cell line capable of expressing TetR protein, bioreceptor peptide, ubiquitin, targeted E3 ligase and biotin ligase, and inducing expression by doxycycline, which can target E3 ligase to bind to substrates, ubiquitinate and biotinylate the substrates, and can quickly detect known and identify unknown ubiquitinated substrates.

[0047] (2) The present application determines whether the sample is a ubiquitinated substrate by detecting whether it can bind to streptavidin agarose beads to form a precipitate during the immunoprecipitation experiment and then using Western blotting or mass spectrometry to detect whether it is biotinylated. This method is fast and convenient, does not require complex instruments and equipment, and has high economic benefits.

[0048] (3) This application can evaluate the process of forming a ternary complex between the PROTAC molecule and the ligand by detecting the degree of biotinylation, and monitor the ubiquitination degree of the ternary complex, which can quickly detect and monitor the ubiquitination process of the ternary complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a plasmid map of vector A in Example 1 of the present application;

[0050] Figure 2 is a plasmid map of vector B in Example 1 of the present application;

[0051] FIG3 is a plasmid map of vector C in Example 1 of the present application;

[0052] FIG4 is the WB verification result of cell A in Example 1 of the present application;

[0053] FIG5 is the WB verification result of cell B in Example 1 of the present application;

[0054] Figure 6 is a plasmid map of vector C in Example 2 of the present application;

[0055] FIG7 is the WB verification result of cell C in Examples 1 and 2 of the present application, where enTEL represents endogenous TEL protein;

[0056] FIG8 is an operational flow for detecting and / or identifying ubiquitinated substrates in Example 4 of the present application;

[0057] FIG9 is a WB verification result of biotinylation and ubiquitination of the cell indicator system in Example 1 of the present application;

[0058] Figure 10 is the WB detection results of ubiquitination and biotinylation of POI B after adding PROTAC in Example 4 of the present application. DETAILED DESCRIPTION

[0059] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below in conjunction with specific embodiments.

[0060] Unless otherwise specified, the materials, reagents, etc. used in the Examples, Comparative Examples, and Experimental Examples can be obtained from commercial sources.

[0061] Lipofectamine TM 3000 transfection reagent was provided by Invitrogen, catalog number L3000075.

[0062] Doxycycline (DOX) was provided by MCE with the product number HY-N0565.

[0063] The PLV-EF1a-IRES-Hygro vector is available in catalog number 85134, the PLV-EF1a-IRES-Puro vector is available in catalog number 85132, the psPAX2 plasmid is available in catalog number 12260, and the pMD2.G plasmid is available in catalog number 12259, all provided by Addgene.

[0064] 293T cells were provided by Saiku Biotechnology with the catalog number CC4003.

[0065] The anti-TetR antibody product number is Abcam, ab302642, the anti-FLAG antibody product number is Proteintech, 20543-1-AP, the anti-TEL antibody product number is CST, 14866, and the anti-biotin antibody product number is Bethyl, A150-109A.

[0066] Biotin was provided by MCE with the product number HY-B0511.

[0067] Streptavidin agarose beads were provided by Millipore, catalog number 16-126.

[0068] Homologous recombination technology was performed using a kit, and the relevant operation steps were carried out according to the kit instructions. The kit was ClonExpress Ultra One Step Cloning Kit V2, provided by Nanjing Novozymes Biotech Co., Ltd., with the product number C116.

[0069] The PROTAC molecule can be a commercially available PROTAC molecule. The PROTAC molecules used in the following examples are designed and synthesized by the applicant.

[0070] The antibody against substrate A was provided by Proteintech, catalog number 10355-1-AP.

[0071] The cell lysis buffer was CHPAS lysis buffer.

[0072] Western blotting (WB) was performed according to the procedures described in the Molecular Biology Laboratory Manual. The secondary antibodies used were goat anti-mouse and goat anti-rabbit, provided by Transgen, with catalog numbers HS201-01 and HS101-01.

[0073] The plasmid synthesis and sequencing used in the following examples were completed by GenScript Biotech Co., Ltd.

[0074] In the following examples and effect examples, unless otherwise specified, the cell culture conditions used were 37° C. and 5% CO 2 , and the culture medium used was complete culture medium.

[0075] Example 1

[0076] This embodiment provides a cell indicator system and a preparation method thereof, wherein the preparation method comprises the following steps:

[0077] 1. Construction of vector

[0078] 1.1 Construction of vector A

[0079] The lentiviral expression vector PLV-EF1a-IRES-Hygro was used, and the tet-PLKO-sgRNA-pruo vector was used as a template to amplify the gene fragment of the TetR protein by PCR. The TetR fragment was inserted into PLV-EF1a-IRES-Hygro by homologous recombination. The restriction sites used were BamH1 and EcoR1. Sequencing confirmed that the vector contained the target fragment, and the vector TetR was obtained, namely vector A. The map of vector A is shown in Figure 1, and the TetR protein encoding gene is shown in SEQ ID NO.1.

[0080] 1.2 Construction of vector B

[0081] The base sequences of the TetO element, FLAG tag, BAP, and UB were integrated using gene editing software. BAP was located at the N-terminus of UB and was commissioned to be synthesized as fragment 1 by a biological company. Fragment 1 was ligated to the pCMV-Zeo vector by enzyme digestion and ligation. Sequencing confirmed that the vector contained the target fragment, resulting in the vector TetO-FLAG-BAP-UB, which is vector B. The map of vector B is shown in Figure 2. The coding gene sequence of BAP is shown in SEQ ID NO.2, the coding gene sequence of UB is shown in SEQ ID NO.3, the nucleotide sequence of the TetO element is shown in SEQ ID NO.4, and the nucleotide sequence of the FLAG tag is shown in SEQ ID NO.5.

[0082] 1.3 Construction of vector C

[0083] The HA-tagged TEL base sequence was integrated and commissioned to a biological company for synthesis to obtain a TEL fragment with an HA tag; the BirA fragment was obtained by PCR amplification, and the TEL fragment and the BirA fragment were ligated into the lentiviral expression vector PLV-EF1a-IRES-Puro using homologous recombination technology. The restriction sites were BamH1 and EcoR1, and the BirA fragment was located at the C-terminus of the TEL fragment. Sequencing confirmed that the vector contained the target fragment, and the vector TEL-C-BirA was obtained, which is vector C. The map of vector C is shown in Figure 3; the TEL encoding gene sequence is shown in SEQ ID NO.6, the BirA encoding gene sequence is shown in SEQ ID NO.7, and the HA tag nucleotide sequence is shown in SEQ ID NO.8.

[0084] 2. Construction of cell indicator system

[0085] 2.1 Use the helper plasmids psPAX2 and pMD2.G and the vector A obtained in step 1.1 or the vector C obtained in steps 1 and 3 to co-transfect 293T cells in the exponential growth phase, and concentrate the lentivirus using the PEG8000 concentration method to obtain lentivirus A or lentivirus C;

[0086] 2.2 Select 293T cells in the logarithmic growth phase, aspirate the culture medium, wash twice with PBS, replace with fresh culture medium, add lentivirus A obtained in step 2.1, the volume ratio of lentivirus to cells is lentivirus: cells = 1:4, mix and culture for 48 hours, aspirate the culture medium, add fresh culture medium containing hygromycin, and culture for 7 days. Collect some cells to extract protein for western blot, and use anti-TetR antibody to detect whether TetR can be stably expressed. The WB test results are shown in Figure 4. As shown in Figure 4, the anti-TetR antibody can bind to the cell protein, indicating that the obtained cells can stably express TetR, that is, cell A containing vector A is obtained;

[0087] 2.3 According to Lipofectamine TM 3000 transfection reagent instructions: The vector B obtained in step 1.2 was transiently transferred into the cell A obtained in step 2.2. After culturing for 24 hours, bleomycin was added for selection for 7 days. The cloned cells were picked and maintained in a bleomycin-resistant environment. 0, 10, 100, and 1000 ng / mL DOX was added for treatment for 24 hours. Some cells were collected to extract proteins for WB. Anti-FLAG antibody was used to detect the expression of BAP-UB. The WB results are shown in Figure 5. As shown in Figure 5, the anti-FLAG antibody can bind to FLAG, indicating that the obtained cells can express BAP-UB under DOX induction, that is, cell B containing vector A and vector B was obtained; in addition, 100 ng / mL DOX induced expression better, and 100 ng / mL DOX was used for subsequent induction of expression.

[0088] 2.4 Select cell B in the logarithmic growth phase obtained in step 2.3, aspirate the culture medium, wash twice with PBS, replace with fresh culture medium, add lentivirus C obtained in step 2.1, the volume ratio of lentivirus to cells is lentivirus:cell = 1:4, mix and culture for 48 hours, aspirate the culture medium, add fresh culture medium containing puromycin, and culture for 7 days. Pick clones and maintain culture in a puromycin-resistant environment, add 100 ng / mL DOX and treat for 24 hours, collect some cells to extract protein for western blot, and use anti-FLAG antibody and anti-TEL antibody to detect whether BAP-UB and TEL-C-BirA can be stably expressed. The western blot test results are shown in Figure 6. As shown in Figure 6, the anti-FLAG antibody and anti-TEL antibody can bind to the endogenous TEL protein in the cells, indicating that the obtained cells can express BAP-UB and TEL-C-BirA. In other words, cell C containing vector A, vector B and vector C is obtained, and the obtained cell C is the cell indicator system.

[0089] Example 2

[0090] This example provides a cell indicator system and a preparation method thereof. The preparation method is similar to that of Example 1, except that BirA in step 1.3 is located at the N-terminus of TEL, and the resulting vector is TEL-N-BirA. The vector map is shown in Figure 7 , and the remaining steps involving vector C are replaced with the TEL-N-BirA vector. In step 2.4, the WB test results are shown in Figure 6 . As shown in Figure 6 , the anti-FLAG antibody and the anti-TEL antibody can bind to the cell protein, indicating that the obtained cells can express BAP-UB and TEL-N-BirA, that is, cell C containing vector A, vector B, and vector C is obtained, and the obtained cell C is the cell indicator system; the remaining steps and their parameter conditions remain unchanged.

[0091] Comparative Example 1

[0092] This comparative example provides a cell indicator system and a preparation method thereof. The preparation method is similar to that of Example 1, except that step 2.3 is not performed. In step 2.4, lentivirus C is directly transferred into cell A obtained in step 2.2, and the remaining steps and parameter conditions remain unchanged.

[0093] Comparative Example 2

[0094] This comparative example provides a cell indicator system and a preparation method thereof. The preparation method is similar to that of Example 2, except that step 2.3 is not performed. In step 2.4, lentivirus C is directly transferred into cell A obtained in step 2.2, and the remaining steps and parameter conditions remain unchanged.

[0095] Effect Example 1

[0096] In order to test whether the obtained cell indicator system can biotinylate and ubiquitinate the sample to be tested, the cells C obtained in Example 1 and Example 2 were tested by Western blotting for verification. The specific scheme is as follows:

[0097] Cell C induced with 100 ng / mL DOX was mixed with 1 μg / mL biotin and incubated at 37°C for 6 h. The cells were washed, and cell proteins were extracted for Western blotting. Anti-biotin and anti-FLAG antibodies were used for Western blotting. The results are shown in Figure 7.

[0098] As shown in FIG7 , the cell indicator systems of Examples 1 and 2 can both bind to anti-biotin antibodies and anti-FLAG antibodies, indicating that the cell indicator system of the present application can ubiquitinate the sample to be tested and bind to biotin to achieve biotinylation.

[0099] Example 3

[0100] This embodiment provides a kit for detecting and / or identifying ubiquitinated substrates, comprising the cell indicator system of embodiment 1 or embodiment 2, doxycycline, biotin, and streptavidin agarose beads.

[0101] Example 4

[0102] This embodiment provides a method for detecting and / or identifying ubiquitinated substrates, wherein substrate A (known to be a TEL substrate) is used as a test sample for detection. The detection process is shown in FIG8 , and the steps are as follows:

[0103] 1. 100 ng / mL of DOX was added to the cell indicator systems of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, as well as the cell B obtained in Example 1, and induced for 12 h to express BAP-UB and TEL-C-BirA / TEL-N-BirA, thereby obtaining BAP-UB and TEL-C-BirA / EL-N-BirA;

[0104] 2. To detect ubiquitination of substrate A, add 1 μg / mL biotin and incubate at 37°C for 6 h to obtain cells containing ubiquitinated and biotinylated substrate A;

[0105] 3. Following the instructions for the lysis buffer, add the lysis buffer to lyse the cells containing the ubiquitinated and biotinylated test sample obtained in Step 2. Centrifuge to obtain the lysis supernatant. Immunoprecipitate the resulting lysis supernatant with streptavidin agarose beads according to the instructions for the streptavidin agarose beads. Then, use Western blotting (WB) to detect ubiquitination and biotinylation of Substrate A using an anti-biotin antibody. To verify the expression of TEL and Substrate A, antibodies against TEL and Substrate A were added during the WB process. The WB results are shown in Figure 9.

[0106] As shown in FIG9 , only the ubiquitinated and biotinylated substrate A can bind to the streptavidin agarose beads to achieve immunoprecipitation and thus be detected by WB. This shows that the cell indicator system of the present application can simultaneously achieve ubiquitination and biotinylation of the sample to be tested.

[0107] Example 5

[0108] This example provides a method for detecting and / or identifying ubiquitinated substrates, wherein substrate A (known to be a TEL substrate) is used as a test sample for detection. The steps are similar to those of Example 4, except that in step 2, 5 μM PROTAC molecules are added after biotin is added. The remaining steps and parameters remain unchanged. The obtained WB results are shown in Figure 10.

[0109] As shown in Figure 10, the method of detecting ubiquitinated substrates of the present application can detect the degree of ubiquitination of the ternary complex formed by the PROTAC molecule and the ligand, indicating that the cell indicator of the present application can be used to monitor the ubiquitination process of the ternary complex.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A cell indicator system, characterized in that, The cells include at least one of vector A, vector B, and vector C; Vector A contains the TetR protein coding gene; Vector B contains the coding genes of a biotin acceptor peptide and ubiquitin; Vector C contains the coding genes of a targeting E3 ligase and a biotin ligase.

2. The cell indicator system according to claim 1, wherein It includes at least one of the following (Ⅰ)-(Ⅱ): (Ⅰ) Vector A is a lentiviral expression vector, and the TetR protein coding gene sequence is as shown in SEQ ID NO.1; (Ⅱ) Vector B also contains a TetO element and a FLAG tag; The biotin acceptor peptide coding gene sequence is as shown in SEQ ID NO.2; The ubiquitin coding gene sequence is as shown in SEQ ID NO.3; The nucleotide sequence of the TetO element is as shown in SEQ ID NO.4; The nucleotide sequence of the FLAG tag is as shown in SEQ ID NO.

5.

3. The cell indicator system according to claim 1, wherein Vector C also contains an HA tag; Vector C is a lentiviral expression vector; The targeting E3 ligase coding gene sequence is as shown in SEQ ID NO.6; The biotin ligase coding gene sequence is as shown in SEQ ID NO.7; The nucleotide sequence of the HA tag is as shown in SEQ ID NO.

8.

4. The indicator system according to claim 1, characterized in that, It includes at least one of the following (Ⅰ)-(Ⅱ): (Ⅰ) The biotin acceptor peptide in vector B is located at the N-terminus of ubiquitin; (Ⅱ) The biotin ligase in vector C is located at the N-terminus or C-terminus of the targeting E3 ligase.

5. The preparation method of the cell indicator system according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Perform lentiviral packaging of vector A and vector C as described in any one of claims 1-4 in vector cells to obtain lentivirus A containing vector A and lentivirus C containing vector C; S2. Infect the vector cells with lentivirus A obtained in step S1 to obtain cell A containing vector A; S3. Transfect vector B as described in any one of claims 1-4 into cell A obtained in step S2 to obtain cell B containing vector A and vector B; S4. Infect cell B obtained in step S3 with lentivirus C obtained in step S1 to obtain cell C containing vector A, vector B, and vector C, and the obtained cell C is the cell indicator system.

6. The application of the cell indicator system as described in any one of claims 1-4 in detecting and / or identifying ubiquitinated substrates.

7. The application of the cell indicator system as described in any one of claims 1-4 in detecting the process of the formation of a ternary complex between a PROTAC molecule and a ligand and / or the degree of ubiquitination.

8. A kit for detecting and / or identifying ubiquitinated substrates, characterized in that, It includes the cell indicator system as described in any one of claims 1-4, doxycycline, biotin, and streptavidin agarose beads.

9. The kit according to claim 8, characterized in that, The kit also includes a PROTAC molecule.

10. A method for detecting and / or identifying ubiquitinated substrates, characterized in that, It includes the following steps: (1) Add doxycycline to the cell indicator system as described in any one of claims 1-4 for induced expression to obtain a biotin acceptor peptide-ubiquitin conjugate and an E3 ubiquitin ligase-biotin ligase conjugate; (2) Mix the biotin acceptor peptide-ubiquitin conjugate and the E3 ubiquitin ligase-biotin ligase conjugate obtained in step (1) with the sample to be tested, and add biotin and / or PROTAC molecules to obtain cells containing the ubiquitinated and biotinylated sample to be tested. (3) Lyse the cells containing the ubiquitinated and biotinylated sample to be tested obtained in step (2) to obtain a lysate supernatant. Perform immunoprecipitation of the obtained lysate supernatant with streptavidin agarose beads, and then detect and / or identify the ubiquitination and biotinylation of the sample to be tested by immunoblotting and / or mass spectrometry techniques.

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