E3 ubiquitin enzyme mutant, modification and protein targeted degradation system thereof and application of E3 ubiquitin enzyme mutant

Abstract

The invention discloses an E3 ubiquitin enzyme mutant as well as modification and a protein targeted degradation system and application thereof, and particularly relates to a series of targeted protein degradation enhanced E3 ubiquitin enzymes (Trim21-N) and a modification method thereof, the E3 ubiquitin enzymes (Trim21-N) are obtained by modifying a wild type Trim21 (wtTrim21) protein structural domain, and the E3 ubiquitin enzymes comprise Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e and Trim21-f. The efficiency of degrading the target protein through the ubiquitin proteasome is remarkably improved, and the method has the characteristics of strong endogenous and exogenous universality, high specificity, ubiquitin proteasome dependence, low cytotoxicity, strong cell compatibility and the like on the degradation of the target protein. The invention provides E3 ubiquitin enzyme (Trim21-N) based on a targeted protein degradation enhancement version and an exogenous fluorescence report degradation system of the E3 ubiquitin enzyme (Trim21-N), preferably Trim21-e with the optimal degradation effect, and the E3 ubiquitin enzyme (Trim21-N) is named as delta Trim21. The invention further provides an endogenous protein degradation system based on the delta Trim21. The method has the characteristics of high specificity, rapidness and small module, so that the method can be used for researching various biological and disease mechanisms, including the fields of cell targeted therapy, cell mechanism exploration, genetic engineering and the like.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology, gene and protein engineering, and in particular to an E3 ubiquitinase mutant and its transformation, as well as a protein targeted degradation system and application. Background Art

[0002] In the emerging field of synthetic biology, the study of realizing the functions of biological molecules through design, modification and recombination has become one of the increasingly popular biomedical research directions. In recent years, with the rapid development of synthetic biology, the modification of biological molecules is no longer limited to some small molecules (DNA and RNA), and more and more research is moving towards biological macromolecules (peptides and proteins). In mammalian synthetic biology, the modification of the functions of these biological molecules can be achieved through the redesign and assembly of these biological molecules, bringing new research value and direction in basic biology and disease treatment.

[0003] In current biology and disease research, exploring the physiological functions of proteins and new methods for disease treatment by degrading target proteins through the ubiquitin proteasome pathway has become a hot research field. E3 ubiquitin ligases have high specificity in the ubiquitination proteasome pathway, so some scientists try to start with E3 enzymes, fuse the targeting module that specifically recognizes the target protein with the E3 enzyme to transform the E3 enzyme, thereby redirecting the ubiquitin proteasome pathway, such as fusing the targeting module to transform the E3 enzyme, and targeting protein degradation technology PROTACs, etc. However, they have certain limitations, such as pre-modification of the target protein, poor specificity, high molecular weight resulting in poor water solubility and membrane permeability, off-target effects, etc., which makes biology and disease research complicated and difficult, limiting their subsequent applications.

[0004] Recently, a new protein degradation strategy, Trim-Away, was reported in the journal Cell. By introducing antibodies to the target protein into the cell, highly specific recognition of the target protein is achieved, and the E3 ligase Trim21 is further used to recognize the Fc region of the antibody and mediate ubiquitination, followed by degradation via the proteasome, thereby achieving specific recognition and degradation of the target protein. This technology not only does not require pre-modification of the target protein, but also avoids the limitations of previous strategies by utilizing antigen-antibody specific hybridization reactions as the system's specific recognition function for the target protein. However, the degradation effect of the Trim-Away technology is not good enough, and the degradation of the target protein in the population of cells can only reach about 30%, which greatly hinders in-depth development and clinical application research. Summary of the invention

[0005] In view of the limitation of the existing Trim-Away technology that has a poor effect on the degradation of group target proteins, the present invention innovatively proposes for the first time a new E3 ubiquitinase mutant, namely a series of E3 ubiquitinases Trim21 (Trim21-N) with enhanced targeted protein degradation, including Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, and Trim21-f; wherein the amino acid sequence of Trim21-a is shown in SEQ ID NO.5; the amino acid sequence of Trim21-b is shown in SEQ ID NO.6; the amino acid sequence of Trim21-c is shown in SEQ ID NO.7; the amino acid sequence of Trim21-d is shown in SEQ ID NO.8; the amino acid sequence of Trim21-e is shown in SEQ ID NO.9; the amino acid sequence of Trim21-f is shown in SEQ ID NO.10. Preferably, Trim21-e is also named ΔTrim21.

[0006] The novel E3 ubiquitinase mutant proposed for the first time in the present invention, namely the E3 ubiquitinase Trim21 (Trim21-N), includes but is not limited to the six modified versions of the enhanced version of Trim21, Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, and Trim21-f. The schematic diagram of its protein structure is shown in FIG. Figure 1 shown.

[0007] The present invention also provides a method for modifying the E3 ubiquitinase Trim21 (Trim21-N) with enhanced targeted protein degradation. In a specific embodiment, the E3 ubiquitinase Trim21 (Trim21-N) is based on the modification of the wild-type Trim21 (wtTrim21) protein domain. The modified E3 ubiquitinase Trim21 with enhanced targeted protein degradation, i.e., enhanced Trim21-N, includes Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, Trim21-f, etc. The wild-type Trim21 (wtTrim21) protein domain includes four domains: RING, B-BOX, Coiled-coil, and PRYSPRY. The amino acid sequences of the four domains are shown in SEQ ID NO.1-4, respectively.

[0008] The structural domains of the wtTrim21 protein or the enhanced version of the Trim21 protein of the present invention include several or all of the RING domain, the B-BOX domain, the Coiled-coil domain and the PRYSPRY domain.

[0009] Among them, the RING domain is the ubiquitin-linking domain, through which Trim21 is connected to the ubiquitin-binding enzyme E2. The conserved cysteine ​​and histidine residues of the RING domain are embedded in the core of the domain, and the conformation of the entire domain is maintained by coordinating two zinc ions. Other semi-conserved amino acid residues in the domain mainly form a hydrophobic core or participate in the recruitment of other proteins. Compared with the general zinc finger structure, the coordination sites of the two zinc ions in the RING domain are intertwined to form a rigid spherical scaffold to facilitate protein-protein interactions.

[0010] Among them, the function of the B-BOX domain is not clear yet. Some literature speculates that it has the function of limiting the degradation rate of ubiquitinase, but the specific mechanism is unknown.

[0011] Among them, the Coiled-coil domain is a coiled-coil dimerization domain. Through the Coiled-coil domain, ΔTrim21 exists in the form of a homodimer in the solution.

[0012] Among them, the PRYSPRY domain is the antibody binding domain. The PRYSPRY domain is a globular fold containing two antiparallel β folds connected by flexible loops and can be subdivided into PRY and SPRY elements. The homologous dimeric ΔTrim21 forms a 1:1 complex with the IgG-FC antibody through the PRYSPRY domain, in which the two PRYSPRY domains bind symmetrically to the Fc.

[0013] Among them, the amino acid sequences of the RING domain, B-BOX domain, Coiled-coil domain and PRYSPRY domain are shown in SEQ ID NO.1-4 respectively.

[0014] In the present invention, the modification method for obtaining the Trim21-a is to add a RING domain on the basis of wtTrim21, and its amino acid sequence is shown in SEQ ID NO.5. The modification method for obtaining the Trim21-b is to add a RING domain on the basis of wtTrim21, and perform an S80E point mutation on it, and its amino acid sequence is shown in SEQ ID NO.6. The modification method for obtaining the Trim21-c is to add a RING domain on the basis of wtTrim21, delete the B-BOX domain and perform an S80E point mutation, and its amino acid sequence is shown in SEQ ID NO.7. The modification method for obtaining the Trim21-d is to add a RING domain on the basis of wtTrim21, delete the B-BOX domain, and perform an S80E point mutation, and its amino acid sequence is shown in SEQ ID NO.8. The modification method for obtaining the Trim21-e is to delete the B-BOX domain on the basis of wtTrim21, and its amino acid sequence is shown in SEQ ID NO.9; the Trim2-e is the version with the best targeted protein degradation effect, which is also named ΔTrim21. The modification method for obtaining the Trim21-f is to perform S80E point mutation on the basis of wtTrim21, and its amino acid sequence is shown in SEQ ID NO.10.

[0015] In a specific embodiment, the present invention proposes for the first time six modified versions of the enhanced version of Trim21, namely: Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, Trim21-f, and their protein structures are shown in the figure below: Figure 1 The modification method for obtaining Trim21-a is to add a RING domain; the modification method for Trim21-b is to add a RING domain and perform a S80E point mutation; the modification method for Trim21-c is to add a RING domain and delete the B-BOX domain and perform a S80E point mutation; the modification method for Trim21-d is to add a RING domain and delete the B-BOX domain; the modification method for Trim21-e is to delete the B-BOX domain; and the modification method for Trim21-f is to perform a S80E point mutation.

[0016] In a preferred embodiment, the version with the best targeted protein degradation effect is Trim21-e, also named ΔTrim21. Compared with wild-type Trim21 (wtTrim21), the effect of ΔTrim21 in degrading target proteins through ubiquitin proteasome is increased by about 10 times. In addition, ΔTrim21 has the characteristics of endogenous and exogenous universality, high specificity, ubiquitin proteasome dependence, low cytotoxicity and strong cell compatibility for target protein degradation.

[0017] The present invention also provides a target protein degradation system based on E3 ubiquitinase Trim21 (Trim21-N), which can be used for a variety of biological and disease mechanism studies, including cell targeted therapy, cell mechanism exploration, genetic engineering and other fields. In a specific embodiment, the E3 ubiquitinase Trim21 (Trim21-N) (also known as enhanced version of Trim21) with enhanced targeted protein degradation includes but is not limited to Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, Trim21-f, and their amino acid sequences are shown in SEQ ID NO.5-10, respectively. Preferably, it includes the optimal version of Trim21-e (also named ΔTrim21) for targeted protein degradation, and its protein size is reduced by about 20% compared with the wtTrim21 protein.

[0018] The present invention also proposes a target protein degradation system based on an enhanced version of the E3 ubiquitinase Trim21 (Trim21-N), wherein the degradation system includes a targeted degradation system for exogenous transferred proteins and a targeted degradation system for endogenous proteins. The exogenous protein targeted degradation system includes an enhanced version of Trim21 (i.e., the targeted protein degradation-enhanced E3 ubiquitinase Trim21 (Trim21-N)), an exogenous target protein and an antibody corresponding to the target protein. The antibody must contain an IgG-FC fragment. The endogenous protein targeted degradation system includes Trim21-e (ΔTrim21) and an antibody corresponding to the endogenous target protein. The antibody must contain an IgG-FC fragment.

[0019] Preferably, the antibody can be a conventional antibody with IgG-FC, or an antibody obtained by fusing the antigen recognition domain with the IgG-FC fragment. In a specific embodiment, for example, the antibodies such as EGFP, HER2, cMyc, PDL1, EGFR antibodies, etc., are obtained by fusing the C-terminus of the antigen recognition domain with the IgG-FC fragment, and their amino acid sequences are shown in SEQ ID NO.11-15.

[0020] In a specific embodiment, the protein targeted degradation system can not only degrade exogenous transferred proteins such as fluorescent proteins, enzyme proteins and proteins with specific functions, but also degrade endogenous proteins such as HER2, PDL1, EGFR, etc. For example, the protein targeted degradation system can achieve the purpose of simultaneously degrading different target proteins by adding different target protein-specific antibodies at the same time, and the target proteins include but are not limited to endogenous proteins such as cMyc, PDL1, EGFR, etc. in A375 cells.

[0021] Among them, the mechanism of action of the targeted exogenous protein degradation system based on the E3 ubiquitin enzyme Trim21 (Trim21-N) (including but not limited to ΔTrim21) enhanced by targeted protein degradation described in the present invention is as follows: Figure 3 As shown, taking ΔTrim21 as an example, for example, after the exogenous reporter protein Reporter binds to its Fc antibody, ΔTrim21 binds to the reporter protein-antibody complex through its PRYSPRY domain to form a trimer. Under the guidance of the RING domain of ΔTrim21, the trimer is specifically degraded through the ubiquitin proteasome pathway. When ΔTrim21, the reporter protein Reporter or its Fc antibody are consumed, the degradation stops automatically.

[0022] The targeted protein degradation system can construct the required E3 ubiquitin enzyme Trim21 (Trim21-N) (including but not limited to ΔTrim21) exogenous protein degradation system components based on targeted protein degradation enhancement in a eukaryotic expression vector through genetic engineering technology, thereby achieving specific degradation of the targeted reporter protein.

[0023] The exogenous protein degradation system based on the E3 ubiquitin enzyme Trim21 (Trim21-N) with enhanced targeted protein degradation provided by the present invention degrades the target reporter protein through the ubiquitin proteasome pathway of the cell itself. The introduction of the system hardly damages the cell or the body itself, and the system has proteasome dependence and protein targeting specificity. The degradation system can work in any type of mammalian cells, such as MSC, HEK-293, Hela, etc., which shows that the system has universal applicability.

[0024] The eukaryotic expression vector is a mammalian cell expression vector containing the exogenous protein degradation system based on the targeted protein degradation enhancement E3 ubiquitinase Trim21 (Trim21-N) (including but not limited to ΔTrim21). The expression vector includes a eukaryotic expression vector constitutively expressing the gene of the E3 ubiquitinase Trim21 (Trim21-N) (including but not limited to ΔTrim21) for targeted protein degradation enhancement, or a eukaryotic expression vector constitutively expressing the target protein gene, or a eukaryotic expression vector constitutively expressing the Fc antibody gene corresponding to the target protein. Alternatively, the eukaryotic expression vector simultaneously includes a gene element encoding the E3 ubiquitinase Trim21 (Trim21-N) (including but not limited to ΔTrim21) for constitutively expressing the targeted protein degradation enhancement, a gene element constitutively expressing the target protein, and a gene element constitutively expressing the Fc antibody corresponding to the target protein. The construction method of the mammalian cell expression vector is detailed in Table 1.

[0025] The mechanism of action of the endogenous protein degradation system based on the E3 ubiquitin enzyme Trim21-e (ΔTrim21) with enhanced targeted protein degradation described in the present invention is as follows: Figure 4 As shown, after the endogenous target protein Fc antibody binds to the target protein, ΔTrim21 binds to the endogenous protein-antibody complex through its PRYSPRY domain to form a trimer. The trimer is specifically degraded through the ubiquitin proteasome pathway under the guidance of the RING domain of ΔTrim21. When ΔTrim21 or the Fc antibody of the target protein is consumed, the degradation automatically stops.

[0026] The endogenous protein degradation system based on the E3 ubiquitinase Trim21-e (ΔTrim21) with enhanced targeted protein degradation provided by the present invention can achieve degradation of one or more target proteins by introducing one or more endogenous protein-targeting Fc antibodies. For example, the endogenous target protein degradation system can achieve simultaneous degradation of endogenous proteins such as cMyc, PDL1, and EGFR in A375 cells.

[0027] The present invention also proposes a eukaryotic expression vector, which contains the targeted protein degradation enhanced E3 ubiquitinase Trim21 (Trim21-N) as claimed in claim 1. The present invention also proposes a method for preparing a eukaryotic expression vector containing the targeted protein degradation enhanced E3 ubiquitinase Trim21 (Trim21-N) exogenous protein degradation system and introducing it into cells. The targeted protein degradation enhanced E3 ubiquitinase Trim21 (Trim21-N) includes but is not limited to Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, Trim21-f, preferably including ΔTrim21 and the like.

[0028] The preparation method of the eukaryotic expression vector is shown in Table 1; the method of introducing the degradation system into the cell includes calcium phosphate transfection, PEI transfection, liposome transfection, electroporation transfection, or virus infection.

[0029] Furthermore, the present invention also proposes a protein degradation method based on a targeted degradation system of exogenously transferred proteins, comprising the following steps:

[0030] a) introducing a constitutively expressed enhanced version of Trim21 plasmid, a constitutively expressed target protein plasmid, and a constitutively expressed Fc antibody plasmid of the target protein into the host cell by PEI transfection to achieve the expression of the encoding gene in the host cell;

[0031] b) 24-48 hours after transfection, the target protein content is detected. If the target protein is a fluorescent protein, it can be observed by fluorescence microscopy. If the target protein is an enzyme protein, it can be detected by enzyme activity. Other types of proteins can also be detected by Western blotting. Finally, the degradation efficiency of the exogenous protein degradation system for the exogenous transferred protein is determined.

[0032] Furthermore, the present invention also proposes a protein degradation method based on a targeted degradation system of endogenous proteins, the method comprising the following steps:

[0033] a) introducing a plasmid constitutively expressing Trim21-e (ΔTrim21) and a plasmid constitutively expressing an Fc antibody of a target protein into the host cell by PEI transfection to achieve expression of the encoding gene in the host cell;

[0034] b) 24-48 hours after transfection, Western blotting was performed to determine the degradation efficiency of the endogenous protein degradation system for endogenous expressed proteins.

[0035] The beneficial effects of the present invention include: for the first time, it is innovatively proposed to transform the wild-type Trim21 to obtain a series of enhanced versions of Trim21 with degradation effects, wherein preferably the ΔTrim21 protein with the best degradation effect is taken as an example, and the degradation effect of the target protein is improved by about 10 times compared with the wild-type Trim21. Secondly, the ΔTrim21 protein has the same characteristics as the Trim21 protein, such as endogenous versatility, specificity, ubiquitin proteasome dependence, low cytotoxicity and cell compatibility. Finally, the degradation system developed based on the enhanced version of the E3 ubiquitinase (Trim21-N) (preferably ΔTrim21) protein for targeted protein degradation is simple in design and small in system module, and is easy to test for disease treatment in animals later. In general, the enhanced version of the E3 ubiquitinase (Trim21-N) for targeted protein degradation provided by the present invention, i.e., the enhanced version of Trim21 and its degradation system, can be used for a variety of biological and disease mechanism studies, and can realize the transformation of cell functions, bringing new research value and direction in basic biology and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the protein structure of wild-type Trim21 and enhanced Trim21-N.

[0037] Figure 2 This is the statistical result of d2EYFP fluorescence targeted degradation by wild-type Trim21 and enhanced version Trim21-N.

[0038] Figure 3 Schematic diagram of the action mechanism of the Trim21-N exogenous target protein degradation system.

[0039] Figure 4 Schematic diagram of the action mechanism of the ΔTrim21 endogenous target protein degradation system.

[0040] Figure 5 The fluorescence statistical results of wild-type Trim21 and ΔTrim21 targeting degradation of d2EYFP are shown in the figure.

[0041] Figure 6 A diagram showing the results of validating the degradation specificity of the ΔTrim21-based exogenous protein degradation system.

[0042] Figure 7 A diagram showing the results of validating the ubiquitin-proteasome dependence of the ΔTrim21-based exogenous protein degradation system.

[0043] Figure 8 This is a diagram to verify the results of Trim21 self-degradation based on the ΔTrim21 exogenous protein degradation system.

[0044] Fig. 9 This is the WB result of degrading the endogenous protein HER2 based on the ΔTrim21 endogenous protein degradation system.

[0045] Fig.10 This is a graph showing the results of a systemic toxicity test based on the ΔTrim21 exogenous protein degradation system.

[0046] Fig.11 Fluorescence statistics to verify the targeted degradation of d2EYFP based on the ΔTrim21 exogenous protein degradation system in different mammalian cell lines.

[0047] Fig.12 This is the WB result diagram to verify the simultaneous degradation of different target proteins based on the ΔTrim21 endogenous protein degradation system. DETAILED DESCRIPTION

[0048] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0049] Example 1, design and construction of different modified versions of Trim21.

[0050] In this example, six modified versions were designed by adding a RING domain or deleting the B-BOX domain or performing an S80E point mutation, and the modified E-Trim21 gene was cloned into a mammalian expression vector to complete the construction of E-Trim21. Figure 1 , Figure 2 The construction method comprises the following steps:

[0051] The first step is to synthesize the full-length Trim21 gene through gene synthesis.

[0052] In the second step, the modified front and back gene fragments are cloned into a mammalian expression vector through PCR and gene seamless assembly.

[0053] The third step is screening and sequencing to obtain plasmids of various E-Trim21 versions.

[0054] Example 2, verification of the ΔTrim21-based exogenous protein degradation system.

[0055] In this example, d2EGFP was used as a reporter gene to verify the degradation effect of the ΔTrim21 exogenous protein degradation system on the reporter gene. Figure 3 The specific steps are as follows:

[0056] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0057] The second step is to seed cells. One day before transfection, HEK-293T cells were plated at 5×10 4 The cells were seeded in a 24-well plate, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0058] The third step is plasmid transfection. The transfection system of this embodiment is divided into ΔTrim21 group, Trim21 group and d2EGFP group. The ΔTrim21 group transfects pMD54, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the Trim21 group transfects pWL235, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the d2EGFP group transfects pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the total amount of each group of plasmids above is 415ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. 6 hours after transfection, the cells were cultured in 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture.

[0059] Step 4: Fluorescence photography and analysis: 24 hours later, the cells were placed under a fluorescence microscope for observation and photography, with 5 fluorescence photos taken in each group. The average fluorescence value was then calculated and analyzed.

[0060] Statistical analysis of fluorescence data showed that the average fluorescence value of the d2EGFP group was the strongest, and the average fluorescence value of the ΔTrim21 group was the weakest. The average fluorescence value of the ΔTrim21 group was about 10 times weaker than that of the d2EGFP group and about 10 times weaker than that of the Trim21 group. Figure 5 .

[0061] Example 3, verification of the degradation specificity of the ΔTrim21-based exogenous protein degradation system.

[0062] In this example, two fluorescent proteins, d2EGFP and mCherry, were used as reporter genes to verify the specificity of the ΔTrim21 exogenous protein degradation system for the degradation of exogenous reporter proteins. The specific steps are as follows:

[0063] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0064] The second step is to inoculate cells (the specific steps are the same as those in Example 2 of the present invention).

[0065] The third step is plasmid transfection. The transfection system of this embodiment is divided into a ΔTrim21 group and a Control group. The ΔTrim21 group is transfected with pMD54, pWL115 and pMD72, and the plasmid amounts are 300ng, 100ng and 15ng; the Control group is transfected with pcDNA3.1, pWL115 and pMD72, and the plasmid amounts are 300ng, 100ng and 15ng; the total amount of each group of plasmids is 415ng, and it is premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. After 6h of transfection, 500μL of DMEM medium containing 10% FBS, 1% (volume / volume) of penicillin and streptomycin mixture is replaced for culture.

[0066] The fourth step is fluorescence photography and analysis (the specific steps are the same as those in Example 2 of the present invention).

[0067] Statistical analysis of fluorescence data showed that the reporter protein d2EGFP was degraded in the ΔTrim21 group, but the reporter protein mCherry was not degraded; in the Control group, the reporter protein d2EGFP was not degraded, and the reporter protein mCherry was not degraded either. This proves that the ΔTrim21-based exogenous protein degradation system is specific for the degradation of exogenous reporter proteins, and only selectively degrades the reporter proteins into which specific antibodies have been introduced. For details of the degradation specificity results of the ΔTrim21-based exogenous protein degradation system, please refer to the attached manual. Figure 6 .

[0068] Example 4: Verification of the ubiquitin-proteasome dependence of the ΔTrim21-based exogenous protein degradation system.

[0069] In this example, d2EGFP was used as a reporter gene, and the proteasome inhibitor MG-132 was added to verify the dependence of the ΔTrim21 exogenous protein degradation system on the ubiquitin proteasome. The specific steps are as follows:

[0070] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0071] The second step is to inoculate cells (the specific steps are the same as those in Example 2 of the present invention).

[0072] The third step is plasmid transfection. The transfection system of this embodiment is divided into ΔTrim21+MG-132 group and ΔTrim21+DMSO group. The ΔTrim21+MG-132 group transfects pMD54, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the ΔTrim21+DMSO group transfects pMD54, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the total amount of each group of plasmids is 415ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI is 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells.

[0073] Step 4: Add proteasome inhibitor MG-132. Replace with 500 μL DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture for culture, and add 10 μM proteasome inhibitor MG-132 to the ΔTrim21+MG-132 group and 10 μM DMSO to the ΔTrim21+DMSO group.

[0074] Statistical analysis of fluorescence data showed that compared with the ΔTrim21+DMSO group, the d2EGFP fluorescent reporter protein in the ΔTrim21+MG-132 group was not degraded, indicating that the ΔTrim21 exogenous protein degradation system relies on the cell's own ubiquitin proteasome pathway to degrade the reporter protein. Figure 7 .

[0075] Example 5, verification of Trim21 self-degradation based on ΔTrim21 exogenous protein degradation system.

[0076] In this example, d2EGFP fluorescent protein is used as a reporter gene, and the N-terminus of the protein ΔTrim21 is fused with mCherry tag protein to verify the degradation of ΔTrim21 itself based on the ΔTrim21 exogenous protein degradation system. The specific steps are as follows:

[0077] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0078] The second step is to inoculate cells (the specific steps are the same as those in Example 2 of the present invention).

[0079] The third step is plasmid transfection. The transfection system of this embodiment is divided into mCherry-ΔTrim21 group and Control group. The mCherry-ΔTrim21 group transfects pMD74, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the Control group transfects pMD74, pcDNA3.1 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the total amount of each group of plasmids is 415ng, premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. After 6h of transfection, 500μL of DMEM medium containing 10% FBS, 1% (volume / volume) of penicillin and streptomycin mixture is replaced for culture.

[0080] The fourth step is fluorescence photography and analysis (the specific steps are the same as those in Example 2 of the present invention).

[0081] Statistical analysis of fluorescence data showed that the reporter protein d2EGFP in the mCherry-ΔTrim21 group was degraded, and the mCherry-ΔTrim21 protein was also degraded; the reporter protein d2EGFP in the Control group was not degraded, and the mCherry-ΔTrim21 protein was not degraded. This proves that ΔTrim21 itself is degraded based on the ΔTrim21 exogenous protein degradation system, indicating that ΔTrim21 binds to the reporter protein-antibody complex through its PRYSPRY domain to form a trimer, and enters the proteasome for degradation. For detailed experimental data, please see the attached manual Figure 8 .

[0082] Example 6, verification of systemic toxicity based on ΔTrim21 exogenous protein degradation system.

[0083] In this example, SEAP was used as a reporter gene to verify the system toxicity of the ΔTrim21 exogenous protein degradation system. The specific steps are as follows:

[0084] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0085] The second step is to inoculate cells (the specific steps are the same as those in Example 2 of the present invention).

[0086] The third step is plasmid transfection. The transfection system of this embodiment is divided into Trim21 group, ΔTrim21 group and Control group. The Trim21 group transfects pWL235, pWL115, pMD44 and pSEAP with plasmid amounts of 300ng, 100ng, 15ng and 100ng; the ΔTrim21 group transfects pMD54, pWL115, pMD44 and pSEAP with plasmid amounts of 300ng, 100ng, 15ng and 100ng; the Control group transfects pcDNA3.1 and pSEAP with plasmid amounts of 415ng and 100ng; the total amount of each group of plasmids is 515ng, premixed with transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. 6 hours after transfection, replace with 500μL DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture for culture. Step 4: Secreted alkaline phosphatase reporter SEAP detection. The specific steps are: 1. Pipette the cell culture supernatant and put 200μL into a centrifuge tube (Note: Generally more than 150μL is required, because subsequent heating will consume part of the volume). 2. 65℃ water bath for 30min (Note: Heating is mainly to inactivate endogenous alkaline phosphatase, while SEAP is resistant to high temperature and will not be inactivated at this temperature). 3. Pipette 80μL (dilute according to the experimental situation) into a 96-well plate, and quickly add 100μl of preheated 2x buffer and 20μl of substrate solution. 4. Measure 10 times at 405nm with an interval of 1min each time (other conditions can be set according to the experimental situation). 5. Calculation of enzyme activity: The enzyme activity of alkaline phosphatase (SEAP) is defined as: at 37°C and pH 9.8, the alkaline phosphatase that reacts with the substrate disodium p-nitrophenyl phosphate (PNPP-Na2) within 1 minute to generate 1 mol / L p-nitrophenol is defined as 1 activity unit (1U). p-Nitrophenol itself has a bright yellow color. At a wavelength of 405nm, different concentrations of p-nitrophenol correspond to different absorbance values. The calculation method is: the slope of the curve of the OD values ​​measured at different time points during the reaction of the sample and the substrate * 256.8 is the enzyme activity, in units of U / L.

[0087] SEAP data analysis showed that compared with the control group, there was no significant difference in SEAP expression between the experimental group Trim21 and the ΔTrim21 group, indicating that the ΔTrim21 exogenous protein degradation system has almost no toxicity to the cells themselves. For detailed experimental data, please refer to the attached manual Fig.10 .

[0088] Example 7, Verification of cell compatibility based on ΔTrim21 exogenous protein degradation system.

[0089] In this example, d2EGFP fluorescent protein was used as a reporter gene to verify the cell compatibility of the ΔTrim21 exogenous protein degradation system. The specific steps are as follows:

[0090] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0091] Step 2: Inoculate cells. HEK-293A cells, HEK-293T cells, hMSC cells, and HeLa cells that are in good growth state were digested with 0.25% trypsin and seeded in 24-well plates, with 3 wells for each cell type and 6×10 cells per well. 4 cells, and add 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin.

[0092] The third step is plasmid transfection. The transfection system of this embodiment is divided into ΔTrim21 group and d2EGFP group, and each group is divided into 4 subgroups (HEK-293A cell group, HEK-293T cell group, hMSC cell group, HeLa cell group, each group is transfected). The ΔTrim21 group is transfected with pMD54, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 300ng, 100ng and 15ng; the total amount of each group of plasmids is 415ng, and it is premixed with the transfection reagent PEI (mass ratio of plasmid to PEI is 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After standing for 15 minutes, the DNA-PEI premix solution is added dropwise to each well of cells. 6 hours after transfection, the cells were cultured in 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture.

[0093] The fourth step is fluorescence photography and analysis (the specific steps are the same as those in Example 2 of the present invention).

[0094] Statistical analysis of fluorescence data showed that the degradation effect of the HEK-293A cell group on the reporter protein d2EGFP reached 7 times; the degradation effect of the HEK-293T cell group on the reporter protein d2EGFP reached 6 times; the degradation effect of the HeLa cell group on the reporter protein d2EGFP reached 11 times; the degradation effect of the hMSC-TERT cell group on the reporter protein d2EGFP reached 10 times; the results show that the ΔTrim21-based exogenous protein degradation system can work well in a variety of different cell lines. For detailed experimental data, please refer to the attached manual Fig.11 .

[0095] Example 8, Validation of the ΔTrim21-based endogenous protein degradation system.

[0096] This example uses the endogenous protein Her2 as the research object to verify the degradation effect of the ΔTrim21 endogenous protein degradation system. The schematic diagram of the mechanism of action of the ΔTrim21 endogenous protein degradation system is shown in the appendix of the specification. Figure 4 The specific steps are as follows:

[0097] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0098] The second step is to seed cells. One day before transfection, HeLa cells were seeded at 5×10 4 The cells were seeded in a 24-well plate, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0099] The third step is plasmid transfection. The transfection system of this embodiment is divided into ΔTrim21 group, Trim21 group and pcDNA3.1 group. The ΔTrim21 group transfects pMD54 and pMD51, and the plasmid amount is 300ng and 100ng; the Trim21 group transfects pWL235 and pMD51, and the plasmid amount is 300ng and 100ng; the pcDNA3.1 group transfects pcDNA3.1, and the plasmid amount is 400ng; the total amount of each group of plasmids above is 400ng, and it is premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. After 6h of transfection, 500μL of DMEM medium containing 10% FBS, 1% (volume / volume) of penicillin and streptomycin mixture is replaced for culture.

[0100] Step 4: Western-blot analysis: After 24 hours, the cells were collected for Western-blot analysis, and then the band fluorescence was counted and analyzed.

[0101] Western-blot analysis showed that the pcDNA3.1 group had the most endogenous protein HER2, while the ΔTrim21 group had the least endogenous protein HER2, proving that the ΔTrim21 endogenous protein degradation system can degrade endogenous proteins. For detailed experimental data, please refer to the appendix of the manual Fig. 9 .

[0102] Example 9, verification of simultaneous degradation of different target proteins based on the ΔTrim21 endogenous protein degradation system.

[0103] This example uses endogenous proteins cMyc, PD-L1, and EGFR as research objects to verify the effect of simultaneously degrading different target proteins based on the ΔTrim21 endogenous protein degradation system. The specific steps are as follows:

[0104] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0105] The second step is to seed cells. One day before transfection, A375 cells were seeded at 5×10 4 The cells were seeded in a 24-well plate, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0106] The third step is plasmid transfection. The transfection system of this embodiment is divided into a ΔTrim21 group and a pcDNA3.1 group. The ΔTrim21 group transfects pMD54, pMD127, pMD136, and pMD144 plasmids in an amount of 200ng, 100ng, 100ng, and 100ng; the pcDNA3.1 group transfects pcDNA3.1, and the plasmid amount is 500ng; the total amount of each group of plasmids above is 500ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI 1:3), and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution is added dropwise to each well of cells. After 6h of transfection, 500μL of DMEM medium containing 10% FBS and 1% (volume / volume) of penicillin and streptomycin mixture is replaced for culture.

[0107] Step 4: Western-blot analysis: After 24 hours, the cells were collected for Western-blot analysis, and then the band fluorescence was counted and analyzed.

[0108] Western-blot analysis showed that the pcDNA3.1 group had a lot of endogenous proteins cMyc, PD-L1, and EGFR, while the ΔTrim21 group had almost no endogenous proteins cMyc, PD-L1, and EGFR, proving that the ΔTrim21-based endogenous protein degradation system can degrade different target proteins at the same time and has a good effect. For detailed experimental data, please refer to the attached manual Fig.12 .

[0109] Table 1 Plasmid construction table

[0110]

[0111] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

[0112] The * in the following sequence represents a stop codon

[0113] SEQ ID NO.1: Amino acid sequence of RING domain

[0114] CPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVC

[0115] SEQ ID NO.2: Amino acid sequence of B-BOX domain

[0116] TQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVPLE

[0117] SEQ ID NO.3: Amino acid sequence of Coiled-coil domain

[0118] EAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAE

[0119] FVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDR

[0120] RCHSSALELLQEVIIVLERSESEQ ID NO.4: Amino acid sequence of the PRYSPRY domain

[0121] AVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFH

[0122] SGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWLWNKQKY

[0123] EAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPL

[0124] RPFFSPGFNDGGKNTAPLTLCPLSEQ ID NO.5: Amino acid sequence of Trim21-a

[0125] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0126] CCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQ

[0127] LANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRK

[0128] HRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKT

[0129] VETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQ

[0130] ALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGL

[0131] KKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMV

[0132] LGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWL

[0133] WNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0134] SEQ ID NO.6: Amino acid sequence of Trim21-b

[0135] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0136] CCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQ

[0137] LANMVNNLKEIEQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRK

[0138] HRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKT

[0139] VETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQ

[0140] ALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGL

[0141] KKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMV

[0142] LGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWL

[0143] WNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0144] SEQ ID NO.7: Amino acid sequence of Trim21-c

[0145] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0146] CCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQ

[0147] LANMVNNLKEIEQEAREGEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIK

[0148] RADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKE

[0149] AKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPEL

[0150] RSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNE

[0151] ERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLS

[0152] SKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0153] SEQ ID NO.8: Amino acid sequence of Trim21-d

[0154] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0155] CCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQ

[0156] LANMVNNLKEISQEAREGEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIK

[0157] RADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKE

[0158] AKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPEL

[0159] RSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNE

[0160] ERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLS

[0161] SKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0162] SEQ ID NO.9: Amino acid sequence of Trim21-e / ΔTrim21

[0163] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0164] CRQRFLLKNLRPNRQLANMVNNLKEISQEAREGEAAQEYQEKLQVALGELRR

[0165] KQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELE

[0166] KDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSES

[0167] WNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQV

[0168] RLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCR

[0169] DSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLD

[0170] YEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0171] SEQ ID NO.10: Amino acid sequence of Trim21-f

[0172] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0173] CRQRFLLKNLRPNRQLANMVNNLKEIEQEAREGEAAQEYQEKLQVALGELR

[0174] RKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQEL

[0175] EKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERS

[0176] ESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRR

[0177] QVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLG

[0178] VCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGI

[0179] FLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY*

[0180] SEQ ID NO.11: Amino acid sequence of the Fc antibody of HER2 protein

[0181] MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMG

[0182] LIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVG

[0183] YCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPS

[0184] VSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDR

[0185] FSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGDKT

[0186] HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

[0187] YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

[0188] LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW

[0189] ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0190] SEQ ID NO.12: Amino acid sequence of the Fc antibody of PD-L1 protein

[0191] MMAQVQLVETGGGLVQPGGSLRLSCTASGFTFSMHAMTWYRQAPGKQRELV

[0192] AVITSHGDRANYTDSVRGRFTISRDNTKNMVYLQMNSLKPEDTAVYYCNVPR

[0193] YDSWGQGTQVTVSSGGLPETGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0194] MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

[0195] VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

[0196] EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0197] SEQ ID NO.13: Amino acid sequence of the Fc antibody of EGFR protein

[0198] DLGKKLLEAARAGQDDEVRILMANGADVNADDTWGWTPLHLAAYQGHLEI

[0199] VEVLLKNGADVNAYDYIGWTPLHLAADGHLEIVEVLLKNGADVNASDYIGD

[0200] TPLHLAAHNGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQD

[0201] KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0202] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0203] KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

[0204] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0205] SEQ ID NO.14: Amino acid sequence of the Fc antibody of cMyc protein

[0206] QLQLVESGGGSVQPGGSLTLSCSASGFNISQYSVGWFREAPGEEREGISCLDID

[0207] GKITTFSDAIQGRFTIARDNAANMIYLHMDALNPLDTAVYRCVARWDCSRHD

[0208] FITQKTATGIWGPGTQVTVSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

[0209] RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0210] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM

[0211] TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

Claims

1. A targeted protein degradation enhanced version of E3 ubiquitinase (Trim21-N), characterized in that: It includes Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, and Trim21-f; wherein the amino acid sequence of Trim21-a is shown in SEQ ID NO.5; The amino acid sequence of Trim21-b is shown in SEQ ID NO.6; The amino acid sequence of Trim21-c is shown in SEQ ID NO.7; The amino acid sequence of Trim21-d is shown in SEQ ID NO.8; The amino acid sequence of Trim21-e is shown in SEQ ID NO.9; The amino acid sequence of Trim21-f is shown in SEQ ID NO.

10.

2. A method for modifying the enhanced version of E3 ubiquitinase (Trim21-N) targeted for protein degradation as claimed in claim 1, characterized in that: The method is based on the modification of the wild-type Trim21 (wtTrim21) protein domain, and the modified E3 ubiquitin enzyme Trim21 (Trim21-N) includes Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e, and Trim21-f; wherein the wild-type Trim21 (wtTrim21) protein domain includes four domains: RING, B-BOX, Coiled-coil, and PRYSPRY, and the amino acid sequences of the four domains are shown in SEQ ID NO.1-4, respectively.

3. The method according to claim 2, characterized in that In the method, The modification method for obtaining the Trim21-a is to add a RING domain to the wild-type Trim21 (wtTrim21); The modification method for obtaining the Trim21-b is to add a RING domain to the wild-type Trim21 (wtTrim21) and perform a S80E point mutation on it; The modification method for obtaining the Trim21-c is to add a RING domain, delete the B-BOX domain and perform an S80E point mutation on the basis of the wild-type Trim21 (wtTrim21); The modification method for obtaining the Trim21-d is to add a RING domain and delete the B-BOX domain on the basis of the wild-type Trim21 (wtTrim21); The modification method for obtaining the Trim21-e is to delete the B-BOX domain based on the wild-type Trim21 (wtTrim21); the Trim21-e is also named ΔTrim21; The modification method for obtaining the Trim21-f is to perform S80E point mutation on the basis of wild-type Trim21 (wtTrim21).

4. A protein targeted degradation system based on E3 ubiquitinase Trim21 (Trim21-N), characterized in that: The system includes a targeted degradation system for exogenous transferred proteins and / or a targeted degradation system for endogenous proteins; Wherein, the targeted degradation system of the exogenous transferred protein comprises the E3 ubiquitin enzyme Trim21 (Trim21-N) as claimed in claim 1, the exogenous target protein and the antibody corresponding to the target protein; The targeted degradation system of endogenous proteins comprises the E3 ubiquitinase Trim21 (Trim21-N) as claimed in claim 1, and an antibody corresponding to the endogenous target protein thereof; wherein the E3 ubiquitinase Trim21 (Trim21-N) is Trim21-e (ΔTrim21); The antibody is an antibody comprising IgG-FC, and / or the antibody is obtained by fusing an antigen recognition domain with an IgG-FC fragment.

5. A protein degradation method using the protein targeted degradation system as claimed in claim 4, characterized in that: The method comprises: The protein degradation method based on the targeted degradation system of exogenous transferred proteins comprises the steps 1-a): introducing a plasmid constitutively expressing the E3 ubiquitin enzyme Trim21 (Trim21-N) as claimed in claim 1, a plasmid constitutively expressing a target protein, and a plasmid constitutively expressing an Fc antibody of the target protein into the host cell by PEI transfection to achieve the expression of the encoding gene in the host cell; and / or, A protein degradation method based on a targeted degradation system of endogenous proteins, comprising step 2-a): introducing a plasmid constitutively expressing the E3 ubiquitin enzyme Trim21 (Trim21-N) as claimed in claim 1 and an Fc antibody plasmid constitutively expressing a target protein into the host cell by PEI transfection to achieve expression of the encoded gene in the host cell; wherein the E3 ubiquitin enzyme Trim21 (Trim21-N) is Trim21-e (ΔTrim21).

6. The protein degradation method according to claim 5, characterized in that The protein degradation method based on the targeted degradation system of exogenously transferred proteins comprises the following steps: 1-a) introducing a plasmid constitutively expressing the E3 ubiquitin enzyme Trim21 (Trim21-N) as claimed in claim 1, a plasmid constitutively expressing a target protein, and a plasmid constitutively expressing an Fc antibody of the target protein into the host cell by PEI transfection to achieve expression of the encoding gene in the host cell; 1-b) 24-48 hours after transfection, the degradation efficiency of the exogenous protein degradation system for the exogenous transferred protein is determined by detecting the target protein content; if the target protein is a fluorescent protein, it can be observed under a fluorescence microscope; if the target protein is an enzyme protein, it can be detected by enzyme activity; if the target protein is other types of proteins, it can be detected by protein blotting.

7. The protein degradation method according to claim 5, characterized in that: The protein degradation method based on the targeted degradation system of endogenous protein comprises the following steps: 2-a) introducing a plasmid constitutively expressing the E3 ubiquitinase Trim21 (Trim21-N) as claimed in claim 1 and a plasmid constitutively expressing an Fc antibody of a target protein into the host cell by PEI transfection to achieve the expression of the encoded gene in the host cell; wherein the E3 ubiquitinase Trim21 (Trim21-N) is Trim21-e (ΔTrim21); 2-b) 24-48 hours after transfection, Western blotting was performed to determine the degradation efficiency of the endogenous protein degradation system for the endogenous expressed protein.

8. The protein degradation method according to claim 5, characterized in that: The method can degrade exogenous transferred proteins including fluorescent proteins, enzyme proteins and proteins with specific functions, and can degrade endogenous proteins including HER2, PDL1, and EGFR; and / or, The method can achieve the purpose of simultaneously degrading different target proteins by simultaneously adding different target protein-specific antibodies; the target proteins include cMyc, PDL1, and EGFR endogenous proteins in A375 cells.

9. A eukaryotic expression vector, characterized in that: The invention contains the E3 ubiquitinase Trim21 (Trim21-N) with enhanced targeted protein degradation as claimed in claim 1.

10. Use of the E3 ubiquitinase Trim21 (Trim21-N) according to claim 1, the modification method according to claim 2, the protein targeted degradation system according to claim 4, the protein degradation method according to any one of claims 5 to 8, or the eukaryotic expression vector according to claim 9 in enhancing targeted protein degradation.

Images