Protein targeted degradation system capable of being regulated and controlled in programmable mode, construction and application of protein targeted degradation system

Abstract

The invention discloses a modified Trim 21 (delta Trim 21)-based target protein degradation control system, which takes a modified protein delta Trim 21 degradation system as a basis, takes blue light, abscisic acid, Danoprevir and / or MK-5172 as inductors, controls the combination of target protein and delta Trim 21 by introducing and modifying a controllable protein dimerization system on the basis of the modified protein delta Trim 21 degradation system, and improves the target protein degradation control system. The protein targeted degradation capable of being modularly regulated and controlled is carried out. Two regulation and control modules, namely a target protein recognition module and a degradation module based on E3 ubiquitinase, are constructed by splitting and recombining a controllable protein dimerization system and a delta Trim 21 structural domain. The invention also discloses a degradation device comprising the system. The invention also discloses a method for accurately regulating endogenous proteins (degradation of proteins related to tumorigenesis) by using the programmable regulation type modular protein degradation device, so as to achieve the effect of specifically killing tumors. The invention also provides a eukaryotic expression vector and a regulation method of the programmable regulation type protein targeted degradation device.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology, protein engineering, tumor targeted therapy, and in particular to a programmable and regulated protein targeted degradation system and a target protein degradation control device, as well as their construction and application. Background Art

[0002] As a major participant in life activities, protein expression and degradation are the basis for maintaining cell functions and supporting the physiological functions of the body, and are precisely regulated by the cell's own regulatory network. Abnormal protein expression can lead to the occurrence and development of related diseases. For example, the pathogenesis of some malignant tumors is closely related to abnormal protein expression. Therefore, interfering with protein expression and function is not only the main strategy for studying protein function in basic research, but also an effective means to prevent and treat related diseases caused by abnormal proteins.

[0003] In the field of synthetic biology, the study of changing the function of biological molecules by designing, modifying and reorganizing them has become one of the increasingly popular directions. In recent years, with the rapid development of synthetic biology, more and more research focuses have been placed on biological macromolecules such as proteins. In mammalian synthetic biology, the redesign of these biological molecules can achieve the transformation of cell functions, bringing new research value and direction in basic biology and disease treatment.

[0004] Current protein interference technologies can be divided into three aspects: DNA level, RNA level and post-translational level. Although methods such as CRISPR / Cas9 at the DNA level and RNAi at the RNA level are rapid, efficient and highly applicable, they directly act on the DNA or RNA level, have off-target effects, are non-universal and cannot act on longevity proteins, which brings considerable limitations to this type of protein interference technology.

[0005] There are some natural and efficient protein degradation pathways in mammalian cells to achieve precise regulation of the levels of various proteins in the cytoplasm and nucleus, including ubiquitin-proteasome pathway, lysosomal degradation, autophagic degradation and other processes. Among them, the ubiquitin-proteasome pathway is an extremely important pathway for selective protein degradation in eukaryotic cells, responsible for the degradation of about 80% of endogenous proteins in cells. At present, it is a hot research field to explore the effects of proteins on physiological functions and diseases by degrading target proteins through the ubiquitin-proteasome pathway. PROTACs, deGradFP degradation technology, AID, Trim-Away and other technologies have brought new directions to disease treatment. However, these protein degradation technologies based on ubiquitin-proteasomes are currently unable to accurately regulate the degradation speed and time, nor can they achieve specific and selective degradation of target proteins. At the same time, the degradation effect of these degradation technologies on group cells is not good enough, and these problems directly limit subsequent disease treatment. In order to overcome these limitations, it is urgent to invent a controllable, specific, efficient and high-speed targeted degradation system that can be used for basic research and clinical transformation application research. Summary of the invention

[0006] In view of the shortcomings of the above-mentioned existing protein interference technology, the present invention innovatively proposes a modularly regulated protein targeted degradation method, system and device in mammalian cells, which uses blue light, abscisic acid, Danoprevir, and / or grazoprevir (MK-5172) as inducers, is based on the modified protein ΔTrim21, and is based on the modified protein ΔTrim21 degradation system. By introducing and modifying a controllable protein dimerization system to control the binding of the target protein with the modified protein ΔTrim21, modularly regulated protein targeted degradation is performed in mammalian cells.

[0007] In a specific implementation scheme, blue light, abscisic acid and two clinical small molecules Danoprevir and Grazoprevir (MK-5172) are used as inducers, and the ΔTrim21 degradation system is used as the chassis to carry out a protein targeted degradation method with different modular regulation.

[0008] The present invention proposes for the first time a protein targeted degradation system / device with different modular regulation of mammalian cells. The protein targeted degradation system / device is based on the ΔTrim21 degradation system to construct a modularly regulated degradation system, which includes but is not limited to: a blue light modular degradation system, an abscisic acid modular degradation system, a DNCR2 modular degradation system regulated by the clinical small molecule Danoprevir, and / or a GNCR1 modular degradation system regulated by the clinical small molecule MK-5172.

[0009] In the present invention, the "modular regulation" refers to the combination of the ΔTrim21 degradation system and four different inducer response proteins to produce four different regulated degradation systems. Although these four systems are regulated separately, they have 70% common parts (ΔTrim21 degradation system). Keeping the common parts unchanged, different inducer regulations can be responded to by replacing different response proteins to trigger regulated degradation. Therefore, it is called "modular". Preferably, the present invention includes four modular regulation methods.

[0010] In the present invention, the protein degradation system / device is constructed by splitting and recombining the controllable protein dimerization system and the ΔTrim21 domain to form two regulatory modules, namely, a target protein recognition module and a degradation module based on E3 ubiquitinase.

[0011] In a specific embodiment, the controllable protein dimerization system includes any regulated protein dimerization system or device, for example, the pMag / nMag system regulated by blue light, the ABI / PYL1 system regulated by abscisic acid, the DNCR2 / NS3a system regulated by the clinical drug Danoprevir, and the GNCR1 / NS3a system regulated by the clinical drug MK-5172.

[0012] It should be noted that, in the present invention, "wild-type Trim2" refers to the natural E3 ubiquitinase Trim21 protein. In the present invention, "modified protein ΔTrim21" refers to a mutant recombinant protein with better E3 ubiquitination degradation effect, which is reasonably designed by the present invention and deletes the b-box domain on the basis of the natural E3 ubiquitinase Trim21 protein. In the present invention, the "modified protein ΔTrim21 degradation system" is an improved protein degradation system based on the modified protein ΔTrim21 and the reported technology Trim21-Away. The mechanism of action is that, 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. The trimer is specifically degraded through the ubiquitin proteasome pathway under the guidance of the RING domain of ΔTrim21. When ΔTrim21, the reporter protein Reporter or its Fc antibody are consumed, the degradation automatically stops.

[0013] In specific embodiments, two preferred target protein degradation control systems / devices are ChemΔ2Trim-TPD and OptoΔ2Trim-TPD, enabling chemical (ABA, GZV, DNV) and light-triggered programmable activation of targeted protein degradation in cells and animals.

[0014] The present invention provides a modularly controllable protein targeted degradation device, which comprises the modularly controllable protein targeted degradation system.

[0015] The present invention proposes for the first time a new modified protein Δ2Trim21. The "modified protein Δ2Trim21" refers to the ΔTrim21 protein of the ΔTrim21 degradation system that is further modified in order to achieve the effect of regulating protein degradation, and the PRYSPRY domain is deleted, and it is named Δ2Trim21.

[0016] In a specific embodiment, the protein structure diagram of the modified protein Δ2Trim21 is as follows Figure 1 As shown, it includes two protein domains, RING and Coiled-coil. Compared with wild-type Trim21, the modified protein Δ2Trim21 is reduced by about 50%. In a specific embodiment, the Δ2Trim21 is formed by deleting the PRYSPRY domain on the basis of ΔTrim21. The PRYSPRY domain is the binding domain of the IgG-Fc fragment, and compared with the wild-type Trim21, the protein is reduced by about 50%.

[0017] Among them, the RING domain is the ubiquitin-linked domain, and Δ2Trim21 is connected to the ubiquitin-binding enzyme E2 through the RING domain. 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.

[0018] Among them, the Coiled-coil domain is a coiled-coil dimerization domain. Through the Coiled-coil domain, Δ2Trim21 exists in the form of a homodimer in the solution.

[0019] Each amino acid sequence described in the present invention can be prepared by artificial synthesis.

[0020] In the present invention, the target protein recognition module is formed by assembling the C-terminus of the target protein specific antibody (Ab) with one of the proteins of the protein dimerization system through a flexible amino acid linker peptide (Linker), including but not limited to: pMag-Linker-Ab (anti-EGFP), whose amino acid sequence is shown in SEQ ID NO.1; pMag-Linker-Ab (anti-HER2), whose amino acid sequence is shown in SEQ ID NO.2; ABI-Linker-Ab (anti-EGFP), whose amino acid sequence is shown in SEQ ID NO.3; NS3a-Linker-Ab (anti-EGFP), whose amino acid sequence is shown in SEQ ID NO.4; and / or, NS3a-Linker-Ab (anti-HER2), whose amino acid sequence is shown in SEQ ID NO.5; etc. Among them, the target protein specific antibody (Ab) does not require an IgG-Fc fragment, but only requires a protein domain that specifically binds to the target protein.

[0021] The E3 ubiquitinase-based degradation module is formed by assembling the C-terminus of another protein in the protein dimerization system with Δ2Trim21 through a flexible amino acid linker peptide (Linker). The amino acid sequence of Δ2Trim21 is shown in SEQ ID NO.6. It includes but is not limited to: nMag-Linker-Δ2Trim21, whose amino acid sequence is shown in SEQ ID NO.7; YL1-Linker-Δ2Trim21, whose amino acid sequence is shown in SEQ ID NO.8; NCR2-Linker-Δ2Trim21, whose amino acid sequence is shown in SEQ ID NO.9; and / or, NCR1-Linker-Δ2Trim21, whose amino acid sequence is shown in SEQ ID NO.10.

[0022] The flexible amino acid linker peptide (Linker) may be selected from amino acid sequences of various lengths. Preferably, the amino acid linker peptide may include (GS)n, and its amino acid sequence is shown in SEQ ID NO.11-15.

[0023] In a specific embodiment, the modified protein ΔTrim21 degradation system is a target protein degradation control device based on ΔTrim21, including a blue light-regulated pMag / nMag system (OptoΔ2Trim-TPD), and a drug-regulated ChemΔ2Trim-TPD system (including an abscisic acid-regulated ABI / PYL1 system, a clinical drug Danoprevir-regulated DNCR2 / NS3a system, and a clinical drug MK-5172-regulated GNCR1 / NS3a system). This enables chemical (ABA, GZV, DNV) and light (blue)-triggered programmable activation of targeted protein degradation in cells and animals.

[0024] In a specific embodiment, the ΔTrim21-based target protein degradation control device can accurately regulate the degradation of exogenous proteins (fluorescent proteins, enzymes and other proteins) and endogenous proteins (such as HER2, PDL1, EGFR, etc.), and its amino acid sequence is shown in SEQ ID NO.16-20.

[0025] In a specific embodiment, the ΔTrim21-based target protein degradation control device can regulate the degradation of target proteins in various mammalian cells. Preferably, the mammalian cells include HEK-293T, HEK-293A, HeLa, hMSC-TERT, etc.

[0026] In the present invention, the blue light modular degradation system uses blue light as an inducer, and the system includes a target protein recognition element and a degradation element. The target protein recognition element includes a target protein-specific antibody and a photosensitive protein pMag. The amino acid sequence of the pMag-Linker-Ab (anti-EGFP) is shown in SEQ ID NO.1.

[0027] The degradation elements of the blue light modular degradation system include Δ2Trim21 and the photosensitive protein nMag. The Δ2Trim21 is a modified and optimized version of the E3 ubiquitin enzyme Trim21, the amino acid sequence of the Δ2Trim21 is shown in SEQ ID NO.6, and the amino acid sequence of the nMag-Linker-Δ2Trim21 is shown in SEQ ID NO.7.

[0028] The mechanism of action of the blue light modular degradation system of the present invention is as follows Figure 2As shown, the target protein specific antibody and the photosensitive protein pMag are fused and expressed in the target protein recognition element, and the Δ2Trim21 and the photosensitive protein nMag are fused and expressed in the degradation element. Under blue light irradiation, pMag will specifically bind to nMag, and then form an antibody-pMag-nMag-Δ2Trim21 complex. The complex binds to the target protein through the antibody, and the complex is ubiquitinated through the ubiquitin linking domain of Δ2Trim21, and then the complex is degraded through the ubiquitin proteasome pathway.

[0029] In the present invention, the abscisic acid modular degradation system uses abscisic acid ABA as an inducer, and the system includes a target protein recognition element and a degradation element. Wherein, the target protein recognition element includes a target protein-specific antibody and a protein ABI. The amino acid sequence of the ABI-Linker-Ab (anti-EGFP) is shown in SEQ ID NO.3.

[0030] The degradation elements of the modular degradation system of abscisic acid include Δ2Trim21 and protein PYL1. The Δ2Trim21 is a modified and optimized version of the E3 ubiquitin enzyme Trim21, the amino acid sequence of the Δ2Trim21 is shown in SEQ ID NO.6, and the amino acid sequence of the PYL1-Linker-Δ2Trim21 is shown in SEQ ID NO.8.

[0031] The mechanism of action of the abscisic acid modular degradation system of the present invention is as follows Figure 2 As shown, the target protein specific antibody and protein ABI are fused and expressed in the target protein recognition element, and Δ2Trim21 and protein PYL1 are fused and expressed in the degradation element. Under the induction of abscisic acid ABA, ABI will specifically bind to PYL1, and then form an antibody-ABI-PYL1-Δ2Trim21 complex. The complex binds to the target protein through the antibody, and the complex is ubiquitinated through the ubiquitin linking domain of Δ2Trim21, and then the complex is degraded through the ubiquitin proteasome pathway.

[0032] In the present invention, the DNCR2 modular degradation system uses Danoprevir as an inducer, and the system includes a target protein recognition element and a degradation element. The target protein recognition element includes a target protein-specific antibody and protein NS3a. The amino acid sequence of the NS3a-Linker-Ab (anti-EGFP) is shown in SEQ ID NO.4.

[0033] The degradation elements of the DNCR2 modular degradation system include Δ2Trim21 and protein DNCR2. The Δ2Trim21 is a modified and optimized version of the E3 ubiquitin enzyme Trim21, the amino acid sequence of the Δ2Trim21 is shown in SEQ ID NO.6, and the amino acid sequence of the DNCR2-Linker-Δ2Trim21 is shown in SEQ ID NO.9.

[0034] The mechanism of action of the DNCR2 modular degradation system of the present invention is as follows Figure 2 As shown, the target protein specific antibody and protein NS3a are fused and expressed in the target protein recognition element, and Δ2Trim21 and protein DNCR2 are fused and expressed in the degradation element. Under the induction of Danoprevir, NS3a will specifically bind to DNCR2, and then form an antibody-NS3a-DNCR2-Δ2Trim21 complex. The complex binds to the target protein through the antibody, and the complex is ubiquitinated through the ubiquitin linking domain of Δ2Trim21, and then the complex is degraded through the ubiquitin proteasome pathway.

[0035] In the present invention, the GNCR1 modular degradation system uses Danoprevir as an inducer, and the system includes a target protein recognition element and a degradation element. The target protein recognition element includes a target protein-specific antibody and protein NS3a. The amino acid sequence of the NS3a-Linker-Ab (anti-EGFP) is shown in SEQ ID NO.4.

[0036] The degradation elements of the GNCR1 modular degradation system include Δ2Trim21 and protein GNCR1. The Δ2Trim21 is a modified and optimized version of the E3 ubiquitin enzyme Trim21, the amino acid sequence of the Δ2Trim21 is shown in SEQ ID NO.6, and the amino acid sequence of the GNCR1-Linker-Δ2Trim21 is shown in SEQ ID NO.10.

[0037] The mechanism of action of the GNCR1 modular degradation system of the present invention is as follows Figure 2 As shown, the target protein specific antibody and protein NS3a are fused and expressed in the target protein recognition element, and the Δ2Trim21 and protein GNCR1 are fused and expressed in the degradation element. Under the induction of MK-5172, NS3a will specifically bind to GNCR1, and then form an antibody-NS3a-GNCR1-Δ2Trim21 complex. The complex binds to the target protein through the antibody, and the complex is ubiquitinated through the ubiquitin linking domain of Δ2Trim21, and then the complex is degraded through the ubiquitin proteasome pathway.

[0038] The four modular degradation system components provided by the present invention are constructed in a eukaryotic expression vector through genetic engineering technology to achieve the degradation of the regulated target protein. The four modular degradation systems provided by the present invention can be used to degrade the reporter protein or endogenous protein introduced exogenously into the host cell. The host cell can be any type of mammalian cell, such as hMSC-TERT, HEK-293, Hela, etc.

[0039] The wavelength of the blue light is 430nm±10, and the light intensity is 0-1mW / cm 2 ; The irradiation time is 0-30min; The irradiation methods include pulsed irradiation and continuous irradiation. By controlling the light source to produce different irradiation times, different degrees of degradation of the target protein can be achieved. The blue light source can be LED, laser light, etc. The red light wavelength is 460nm±10nm, and the light intensity is 0-1mW / cm 2 ; The irradiation time is 0-12h; The irradiation method includes pulsed irradiation and continuous irradiation. By controlling the light source to produce different irradiation times, the degradation of the target protein to different degrees can be achieved. The red light source can be an LED, a laser lamp, etc.

[0040] The drug incubation concentration of abscisic acid ABA is 0-50 μM, and the incubation time is 0-30 min; the drug incubation concentration of Danoprevir and MK-5172 is 0-10 μM, and the incubation time is 0-30 min. By controlling the drug concentration and drug time, the degradation of the target protein can be achieved to different degrees.

[0041] The present invention also proposes a method for constructing the four modularly regulated protein targeted degradation systems, comprising the following steps:

[0042] (1) Construction of target protein recognition elements

[0043] Constructing a fusion protein of a target protein-specific antibody and a photosensitive protein pMag, a fusion protein of a target protein-specific antibody and a protein ABI, a fusion protein of a target protein-specific antibody and a protein NS3a, and a connecting peptide between the aforementioned fusion proteins as a target protein recognition element;

[0044] Among them, the specific antibody for the target protein is a modified antibody, and only the antigen binding domain is retained; the specific modified antibody is fused with the sensor protein of different regulatory systems through a connecting peptide to form a target protein recognition element, and the target protein recognition element includes but is not limited to pMag-Linker-Ab (anti-EGFP), ABI-Linker-Ab (anti-EGFP), NS3a-Linker-Ab (anti-EGFP), and its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.4.

[0045] (2) Degradation elements

[0046] Constructing fusion proteins of Δ2Trim21 and light-sensitive protein nMag, fusion proteins of Δ2Trim21 and protein PYL1, fusion proteins of Δ2Trim21 and protein DNCR2, fusion proteins of Δ2Trim21 and protein GNCR1, and connecting peptides between the aforementioned fusion proteins as degradation elements;

[0047] Among them, the Δ2Trim21 is a modified version of the E3 ubiquitin enzyme Trim21, which enhances its E ubiquitination effect. Δ2Trim21 is obtained by deleting the B-BOX domain and PRYSPRY domain of Trim21; Δ2Trim21 is fused with sensor proteins of different regulatory systems through connecting peptides to form degradation elements, and the degradation elements include but are not limited to nMag-Linker-Δ2Trim21, PYL1-Linker-Δ2Trim21, DNCR2-Linker-Δ2Trim21, and GNCR1-Linker-Δ2Trim21, and their amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.14, and SEQ ID NO.10.

[0048] (3) Reporting system

[0049] The exogenous reporter protein of the degradation system is the green fluorescent protein d2EGFP, and its amino acid sequence is as shown in SEQ ID NO.21; the degradation of endogenous proteins is the endogenous proteins themselves, including but not limited to HER2, PD-L1, EGFR, cMYC, and the amino acid sequences of their antibodies are shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively.

[0050] The four modularly regulated protein targeted degradation systems described in the present invention can respond to minute-level blue light stimulation or drug stimulation to initiate protein degradation; they can also respond to microwatt-level blue light stimulation or micromole-level drug stimulation to initiate protein degradation.

[0051] The four modularly regulated protein targeted degradation systems described in the present invention can achieve the effect of simultaneously degrading different target proteins by adding different target protein recognition elements (including but not limited to, for example, cMyc recognition elements, PD-L1 recognition elements, EGFR recognition elements and HER2 recognition elements).

[0052] The protein modules of the four modularly regulated protein targeted degradation systems of the present invention are small and can be packaged by lentivirus for efficient in vivo delivery. The four modularly regulated protein targeted degradation systems can regulate protein degradation in different mammalian cell lines (e.g., 293T cells, HeLa cells, B16-F10 cells).

[0053] The present invention also provides a eukaryotic expression vector of a modularly regulated protein targeted degradation system, wherein the host cell is transfected with the eukaryotic expression vector, comprising the four modularly regulated protein targeted degradation systems, including but not limited to: a eukaryotic expression vector of a blue light-regulated target protein degradation control device; a eukaryotic expression vector of a abscisic acid-regulated target protein degradation control device; a eukaryotic expression vector of a clinical drug Danoprevir-regulated target protein degradation control device; and / or a eukaryotic expression vector of a clinical drug MK-5172-regulated target protein degradation control device.

[0054] The eukaryotic expression vector is a mammalian cell expression vector comprising the four modularly regulated protein targeted degradation systems. The expression vector may be a vector containing a target protein recognition element encoding gene alone or a vector containing a degradation element encoding gene alone. Alternatively, the expression vector includes both a target protein recognition element encoding gene and a degradation element encoding gene. The construction methods of all the aforementioned mammalian cell expression vectors are detailed in Table 1.

[0055] The present invention also proposes a control method and application of a target protein degradation control device based on a protein dimerization system, including application in the control and degradation of exogenous proteins and endogenous proteins.

[0056] The present invention also proposes the application and method of using the modularly regulated protein targeted degradation system / device to regulate the degradation of exogenous proteins in host cells (mammalian cells), including the steps of: (1) constructing the four modularly regulated protein targeted degradation systems in a host cell eukaryotic plasmid expression vector; (2) introducing the expression vector into the host cell, wherein the target protein recognition element is the recognition element of d2EGFP; (3) inducing or regulating the specific degradation of the target protein in the host cell by blue light, abscisic acid ABA, the drug Danoprevir, and MK-5172. Further, the degradation of the exogenous fluorescent protein is observed by fluorescence microscopy. In a specific embodiment, preferably, by controlling the light source to produce different illumination times and intensities; by controlling the drug concentration and incubation time, different degrees of degradation of the exogenous reporter protein d2EGFP can be achieved.

[0057] The present invention also proposes the application and method of the modularly regulated protein targeted degradation system in degrading endogenous proteins. Specifically, the present invention proposes a method for simultaneously degrading endogenous proteins HER2, PD-L1, EGFP, and cMYC in HeLa cells mediated by the four modularly regulated protein targeted degradation systems, the method comprising: (1) constructing the four modularly regulated protein targeted degradation systems in a host cell eukaryotic plasmid expression vector; (2) introducing the expression vector into the host cell, wherein there are four target protein recognition elements, including recognition elements of four endogenous proteins. (3) inducing or regulating the specific degradation of the target protein in the host cell by blue light, abscisic acid ABA, the drug Danoprevir, and MK-5172. Further, the degradation of endogenous proteins is detected by Western-blot experiments. Among them, by controlling the light source to produce different illumination times and intensities; by controlling the drug concentration and incubation time, different degrees of degradation of endogenous proteins can be achieved.

[0058] The present invention also proposes the use of the system / device in degrading tumor-related proteins in melanoma cell-transplanted mice and inhibiting tumor growth. In a specific embodiment, for example, the system / device is used in B16F10 Luc Degradation of tumor-associated proteins inhibited tumor growth in melanoma cell-transplanted C57BL / 6JGpt mice.

[0059] The present invention also proposes a method for utilizing the blue light modular protein targeted degradation system in B16F10 Luc A method for degrading tumor-related proteins in melanoma cell-transplanted C57BL / 6JGpt mice to inhibit tumor growth, the method comprising:

[0060] (1) constructing a blue light module-regulated protein targeted degradation system; preferably, the specific antibody is a PD-L1 antibody or a c-Myc antibody;

[0061] (2) preparing a lentiviral vector expressing a blue light module-regulated protein targeted degradation system, wherein the preparation method of the lentiviral vector comprises:

[0062] Preparing a lentiviral packaging plasmid containing the blue light module-regulated protein targeted degradation system;

[0063] Using the lentiviral packaging plasmid, preparing a lentivirus containing the blue light module-regulated protein targeted degradation system;

[0064] (3) Construction of melanoma cell transplanted mice; preferably B16F10 Luc Tumor cells were transplanted into C57BL / 6JGpt mice; wherein the method for making the mice bear tumors includes:

[0065] B16F10 Luc Melanoma cell culture and proliferation;

[0066] The tumor cells were collected and subcutaneously implanted to construct B16F10 Luc Tumor cells were transplanted into C57BL / 6JGpt mice.

[0067] (4) Using lentivirus as a vector to deliver blue light module to regulate protein targeted degradation system into the subcutaneous B16F10 of C57BL / 6JGpt mice Luc In tumors.

[0068] By regulating the degradation of endogenous proteins of tumor cells (preferably PD-L1 and c-Myc proteins) through blue light, the effect of killing tumor cells and inhibiting tumor growth is achieved.

[0069] The present invention also proposes a modularly regulated protein targeted degradation system / device, the modularly regulated protein targeted degradation method, the modified protein Δ2Trim21, the eukaryotic expression vector, and the control method of the target protein degradation control system / device based on the protein dimerization system for degrading tumor-related proteins and inhibiting tumor growth. In a specific embodiment, it is applied to mammalian cells, such as melanoma cell-transplanted mice, such as B16F10 Luc Melanoma cells were transplanted into C57BL / 6JGpt mice.

[0070] The beneficial effects of the present invention include: for the first time, an innovative protein targeted degradation system with different modular regulation of mammalian cells is proposed, and the modular degradation system includes four modules with different regulation modes, namely, a blue light module, an abscisic acid module, a DNCR2 module and a GNCR1 module. The light module has the advantages of no need to take, remote traceless regulation, and no toxic side effects; the abscisic acid module has the advantages of being widely used in human and animal cells, having an auxiliary effect on disease treatment, and having synergistic efficacy; the two clinical small molecule drug modules have the advantages of being clinically listed, safe and reliable, having synergistic efficacy, easy to accept, and accurate and efficient combined treatment. The degradation system has a simple structure, a variety of regulation modes, high sensitivity and a fast degradation rate. The degradation system can not only degrade exogenous reporter proteins, but also degrade endogenous proteins efficiently and quickly. The system can also degrade different target proteins at the same time by designing different target protein recognition elements. In addition, the degradation system can achieve the effect of tumor killing by accurately regulating the degradation of endogenous proteins in vivo in the future. The present invention provides a protein targeted degradation system with different modular regulation of mammalian cells, which can be used for a variety of diseases and biological research, and has research value in the fields of cell targeted therapy, mammalian genetic engineering, and cell mechanism exploration.

[0071] The beneficial effects and advantages of the present invention also include that light has the advantages of no need to take, remote and traceless regulation, and no toxic side effects; abscisic acid has the advantages of being widely present in human and animal cells, having an auxiliary effect on disease treatment, and having synergistic therapeutic effects; two clinical small molecule drugs Danoprevir and MK-5172 have passed clinical marketing, are safe and reliable, have synergistic therapeutic effects, are easy to accept, and have the advantages of precise and efficient combined treatment. The four modularly regulated protein targeted degradation methods and systems provided by the present invention can quickly and specifically degrade target proteins within 30 minutes, are easy to operate, and have strong practicality. The present invention overcomes the shortcomings of existing protein degradation technologies such as slow degradation speed, uncontrollable specificity, and selective degradation. The present invention also proposes an application of a blue light module to regulate a protein targeted degradation system. The application uses a blue light module to regulate a protein targeted degradation system in B16F10 Luc Specifically degrade tumor-related proteins in melanoma cells and inhibit tumor development in mice. In summary, the present invention has great potential value in the field of mammalian protein engineering and biomedicine, and in the research of specifically and efficiently regulating and degrading target proteins.

[0072] The present invention proposes a target protein degradation control system and / or control device based on a modified version of Trim21 (ΔTrim21), wherein the design principle of the target protein degradation control device is to control the binding of the target protein with ΔTrim21 by introducing and modifying a controllable protein dimerization system. The protein degradation device based on regulating the binding of the target protein with ΔTrim21 is constructed by splitting and recombining the controllable protein dimerization system and the ΔTrim21 domain to form two regulatory modules, namely, a target protein recognition module and a degradation module based on E3 ubiquitinase. Preferably, the two target protein degradation control devices are ChemΔ2Trim-TPD and OptoΔ2Trim-TPD, which enable chemical (ABA, GZV, DNV) and light-triggered programmable activation of targeted protein degradation in cells and animals. The programmable regulated modular protein degradation device proposed in the present invention has the characteristics of simple structure, diverse regulation methods, high sensitivity and fast degradation speed. The programmable regulated modular protein degradation device proposed in the present invention is used to accurately regulate endogenous proteins (degradation of proteins related to tumor occurrence) to achieve the effect of tumor-specific killing. The present invention also provides a eukaryotic expression vector and a regulation method for a programmable regulated protein targeted degradation device, providing new research methods and strategies for future applications in various diseases and biological research, as well as cell targeted therapy, mammalian genetic engineering, cell mechanism exploration and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Schematic diagram of the protein structure of the modified protein Δ2Trim21.

[0074] Figure 2 Schematic diagram of the action mechanism of four modular regulatory protein targeted degradation systems.

[0075] Figure 3 This is a graph showing the illumination time and intensity dependence of the degradation of exogenous d2EGFP by the blue light modular regulated protein targeted degradation system.

[0076] Figure 4 This is a graph showing the incubation time and concentration dependence of the degradation of exogenous d2EGFP by the abscisic acid (ABA) modular protein targeted degradation system.

[0077] Figure 5 This is a graph showing the incubation time and concentration dependence of the DNCR2 modular protein targeted degradation system in degrading exogenous d2EGFP.

[0078] Figure 6 This is a graph showing the incubation time and concentration dependence of the GNCR1 modular protein targeted degradation system in degrading exogenous d2EGFP.

[0079] Figure 7This is the result of four modular protein targeted degradation systems simultaneously degrading endogenous proteins HER2, PD-L1, EGFR, and cMYC in HeLa cells.

[0080] Figure 8 The blue light modular regulation protein targeted degradation system is used to study the light intensity dependence of the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells.

[0081] Fig. 9 The blue light modular regulation protein targeted degradation system is used to study the light time dependence of the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells.

[0082] Fig.10 Modular blue light-regulated protein targeted degradation system in B16F10 Luc Schematic diagram of the principle of inhibiting tumor growth by degrading tumor-associated proteins in melanoma cell-transplanted C57BL / 6JGpt mice.

[0083] Fig.11 Modular blue light-regulated protein targeted degradation system in B16F10 Luc The results of degradation of tumor-related proteins in melanoma cell-transplanted C57BL / 6JGpt mice inhibiting tumor growth. DETAILED DESCRIPTION

[0084] 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.

[0085] The present invention proposes a target protein degradation control system based on a modified version of Trim21 (ΔTrim21), which is based on the modified protein ΔTrim21 degradation system, with blue light, abscisic acid, Danoprevir and / or MK-5172 as inducers, and based on the modified protein ΔTrim21 degradation system, by introducing and modifying a controllable protein dimerization system to control the binding of the target protein with ΔTrim21, and performing modularly regulated protein targeted degradation. It is constructed by splitting and recombining the controllable protein dimerization system and the ΔTrim21 domain to form two regulatory modules, namely a target protein recognition module and a degradation module based on E3 ubiquitinase; it has the advantages of simple structure, diverse regulation methods, high sensitivity, and fast degradation speed. The present invention also proposes a degradation device comprising the above system. The present invention also proposes a method for using a programmable regulated modular protein degradation device to accurately regulate endogenous proteins (degradation of proteins related to tumor occurrence) to achieve the effect of tumor-specific killing. The present invention also provides a eukaryotic expression vector and a regulation method for a programmable regulated protein targeted degradation device, providing new research methods and strategies for future applications in various diseases and biological research, as well as cell targeted therapy, mammalian genetic engineering, cell mechanism exploration and other fields.

[0086] Example 1: Construction of different modular protein targeted degradation systems

[0087] In this example, a target protein recognition element is formed by constructing a fusion protein with a target protein-specific antibody and a regulatory protein; a degradation element is formed by constructing a fusion protein with Δ2Trim21 and another regulatory protein. Figure 1 The mechanism of action of the four modular regulatory protein targeted degradation systems is detailed in the appendix of the manual. Figure 2 The construction method comprises the following steps:

[0088] In the first step, the Δ2Trim21 gene, the target protein-specific antibody gene and four different regulatory protein genes are synthesized through gene synthesis.

[0089] In the second step, the target protein recognition element and degradation element fragments are cloned into a mammalian expression vector through PCR and gene seamless assembly.

[0090] The third step was screening and sequencing to identify plasmids with different modular protein targeted degradation systems.

[0091] Example 2: Verification of the illumination time and intensity dependence of the blue light modular regulated protein targeted degradation system in degrading exogenous d2EGFP.

[0092] This example uses d2EGFP as a reporter gene to verify the light time and intensity dependence of the degradation of exogenous d2EGFP by the blue light modular regulation protein targeted degradation system. Figure 2 The specific steps are as follows:

[0093] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0094] 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.

[0095] The third step is plasmid transfection. The transfection system of this embodiment is divided into an illumination group, a dark group and a d2EGFP group. The illumination group and the dark group are the same transfection group divided into two, transfecting pMD97, pMD99 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the above plasmids in each group are premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3) according to a total amount of 315ng, and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution was 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 was replaced for culture.

[0096] Step 4: Illumination. 14-18 hours after the solution was replaced, the dark group was placed in the dark, and the light group was placed in an environment with a wavelength of 460nm and an intensity of 1000μW / cm 2 The light group was placed in the dark, and the light group was placed in the light with a wavelength of 460nm and an intensity of 0-1000μW / cm 2 The samples were irradiated with LED for 30 min and then placed in the dark immediately after the light treatment.

[0097] Step 5: Fluorescence analysis: After the last group of irradiation, the cells were immediately extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0098] Western-blot data statistical analysis showed that the degradation of exogenous d2EGFP by the blue light modular regulation protein targeted degradation system is dependent on illumination time and intensity. Figure 3 .

[0099] Example 3: Verification of the incubation time and concentration dependence of the abscisic acid (ABA) modular protein targeted degradation system in degrading exogenous d2EGFP.

[0100] In this example, d2EGFP was used as a reporter gene to verify the light time and intensity dependence of the degradation of exogenous d2EGFP by the abscisic acid (ABA) modular protein targeted degradation system. Figure 2 The specific steps are as follows:

[0101] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0102] 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.

[0103] The third step is plasmid transfection. The transfection system of this embodiment is divided into a drug-added group, a non-drug-added group and a d2EGFP group. The drug-added group and the non-drug-added group are the same transfection group divided into two, transfecting pMD104, pMD105 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the above plasmids in each group are premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3) according to a total amount of 315ng, and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution was 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 was replaced for culture.

[0104] Step 4: Add drugs and incubate. After changing the medium for 14-18 hours, incubate for time dependence: add 50μM ABA to the drug group, incubate for 0-30min, remove the culture medium immediately after incubation, wash three times with PBS, and then add fresh culture medium for culture. Incubation concentration dependence: add 0-50μM ABA to the drug group, incubate for 30min, remove the culture medium immediately after incubation, wash three times with PBS, and then add fresh culture medium for culture.

[0105] Step 5: Fluorescence analysis: After the last group of incubations, the cells were immediately extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0106] Western-blot data statistical analysis showed that the degradation of exogenous d2EGFP by the modular regulation of abscisic acid ABA protein targeted degradation system is dependent on light time and intensity. Figure 4 .

[0107] Example 4: Verification of the incubation time and concentration dependence of the DNCR2 modular protein targeted degradation system in degrading exogenous d2EGFP.

[0108] In this example, d2EGFP was used as a reporter gene to verify the light time and intensity dependence of the degradation of exogenous d2EGFP by the DNCR2 modular protein targeted degradation system. Figure 2 The specific steps are as follows:

[0109] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0110] 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.

[0111] The third step is plasmid transfection. The transfection system of this embodiment is divided into a drug-added group, a non-drug-added group and a d2EGFP group. The drug-added group and the non-drug-added group are the same transfection group divided into two, transfecting pMD106, pMD107 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the above plasmids in each group are premixed with the transfection reagent PEI (plasmid to PEI mass ratio 1:3) according to a total amount of 315ng, and dissolved in 50ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution was 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 was replaced for culture.

[0112] Step 4: Add drugs and incubate. After changing the solution for 14-18 hours, incubate for time-dependent exploration group: add 5μM of the drug Danoprevir to the drug-added group, and the incubation time is 0-30min. After the incubation is completed, the culture medium is immediately removed, and the cells are washed three times with PBS and then fresh culture medium is added for cultivation. Incubation concentration-dependent exploration group: add 0-5μM of the drug Danoprevir to the drug-added group, and the incubation time is 30min. After the incubation is completed, the culture medium is immediately removed, and the cells are washed three times with PBS and then fresh culture medium is added for cultivation.

[0113] Step 5: Fluorescence analysis: After the last group of incubations, the cells were immediately extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0114] Western-blot data statistical analysis showed that the DNCR2 modular regulatory protein targeted degradation system has a light time and intensity dependence on the degradation of exogenous d2EGFP. Figure 5 .

[0115] Example 5, verification of the incubation time and concentration dependence of the GNCR1 modular protein targeted degradation system in degrading exogenous d2EGFP.

[0116] In this example, d2EGFP was used as a reporter gene to verify the light time and intensity dependence of the degradation of exogenous d2EGFP by the GNCR1 modular protein targeted degradation system. Figure 2 The specific steps are as follows:

[0117] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0118] 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.

[0119] The third step is plasmid transfection. The transfection system of this embodiment is divided into a drug-added group, a non-drug-added group and a d2EGFP group. The drug-added group and the non-drug-added group are the same transfection group divided into two, transfecting pMD106, pMD108 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amounts are 150ng, 150ng and 15ng; the total amount of each group of plasmids above is 315ng, 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 was 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 was replaced for culture.

[0120] Step 4: Add drugs and incubate. After changing the medium for 14-18 hours, incubation time dependence exploration group: 5μM of drug MK-5172 was added to the drug-added group, and the incubation time was 0-30min. After the incubation was completed, the culture medium was immediately removed, and fresh culture medium was added after washing three times with PBS for cultivation. Incubation concentration dependence exploration group: 0-5μM of drug MK-5172 was added to the drug-added group, and the incubation time was 30min. After the incubation was completed, the culture medium was immediately removed, and fresh culture medium was added after washing three times with PBS for cultivation.

[0121] Step 5: Fluorescence analysis: After the last group of incubations, the cells were immediately extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0122] Western-blot data statistical analysis showed that the GNCR1 modular regulatory protein targeted degradation system has a light time and intensity dependence on the degradation of exogenous d2EGFP. Figure 6 .

[0123] Example 6, verification of simultaneous degradation of endogenous proteins HER2, PD-L1, EGFR, and cMYC in HeLa cells based on four modular regulatory protein targeted degradation systems.

[0124] This example uses four endogenous proteins, HER2, PD-L1, EGFR, and cMYC, as target proteins to verify that four modular regulatory protein targeted degradation systems can simultaneously degrade different endogenous proteins in HeLa cells. The specific steps are as follows:

[0125] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0126] 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.

[0127] The third step is plasmid transfection. The transfection system of this embodiment is divided into four groups, namely, blue light group, ABA group, DNCR group and GNCR group, each group is further divided into a treatment group and a non-treatment group. The blue light group was transfected with pMD99, pWH62, pWH63, pWH64 and pMD145; the plasmid amount was 100 ng; the ABA group was transfected with pMD105, pMD301, pMD302, pMD303 and pMD304; the plasmid amount was 100 ng; the DNCR group was transfected with pMD107, pMD307, pMD308, pMD309, and pMD310; the plasmid amount was 100 ng; the GNCR group was transfected with pMD108, pMD307, pMD308, pMD309, and pMD310; the plasmid amount was 100 ng; the total amount of each group of plasmids was 500 ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI was 1:3), and dissolved in 50 ul serum-free and antibiotic-free DMEM. After 15 minutes of rest, the DNA-PEI premix solution was added dropwise to each well of cells. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0128] Step 4: Add drugs or treat with light. After changing the medium for 14-18 hours, the dark group in the blue light group was cultured in the dark, and the light group was irradiated with blue light for 30 minutes; the drug treatment group was treated with the corresponding drug for 30 minutes.

[0129] Step 5: Western-blot analysis: Immediately after the treatment, the cells were extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0130] Western-blot data statistical analysis showed that the four modular protein degradation systems can simultaneously degrade different endogenous proteins in HeLa cells. Figure 7 .

[0131] Example 7, verification of the light intensity dependence and light time dependence of the blue light modular regulation protein targeted degradation system for the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells.

[0132] This example uses four endogenous proteins HER2, PD-L1, EGFR, and cMYC as target proteins to verify the light intensity dependence of the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells based on the blue light modular regulation protein targeted degradation system. The specific steps are as follows:

[0133] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0134] 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.

[0135] The third step is plasmid transfection. The transfection system of this embodiment is divided into HER2 group, PD-L1 group, EGFR group and cMyc group. The HER2 group is transfected with pMD99 and pWH62, and the plasmid amount is 150ng each; the PD-L1 group is transfected with pMD99 and pWH63, and the plasmid amount is 150ng each; the EGFR group is transfected with pMD99 and pWH64, and the plasmid amount is 150ng each; the cMyc group is transfected with pMD99 and pMD145, and the plasmid amount is 150ng each; the total amount of each group of plasmids is 300ng, and it is 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 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.

[0136] Step 4: Illumination. 14-18 hours after the solution was replaced, the dark group was placed in the dark, and the light group was placed in an environment with a wavelength of 460nm and an intensity of 1000μW / cm 2 The light group was placed in the dark, and the light group was placed in the light with a wavelength of 460nm and an intensity of 0-1000μW / cm 2 The samples were irradiated with LED for 30 min and then placed in the dark immediately after the light treatment.

[0137] Step 5: Fluorescence analysis: After the last group of irradiation, the cells were immediately extracted for protein, and the degradation of the reporter protein was detected by Western-blot, and the average fluorescence value was calculated and analyzed.

[0138] Western-blot data statistical analysis shows that the blue light modular regulation protein targeted degradation system has light intensity dependence and light time dependence for the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc. For detailed experimental data, please refer to the attached manual. Figure 8 , 9 .

[0139] Example 8: Blue light modular regulation of protein targeted degradation system in B16F10 Luc Degradation of tumor-associated proteins inhibited tumor growth in melanoma cell-transplanted C57BL / 6JGpt mice.

[0140] In this example, three endogenous proteins, PD-L1 and c-Myc, were used as target proteins to verify the application of the blue light-based modular protein targeted degradation system in B16F10 Luc Melanoma cell transplantation in C57BL / 6JGpt mice degrades tumor-related proteins to inhibit tumor growth. See the appendix of the instruction manual for details. Fig.10 The specific steps are as follows:

[0141] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0142] The second step is cell culture. Luc The cells were cultured and expanded in DMEM complete medium to reach 5×10 per mouse. 5 Cell mass is the total amount of cells.

[0143] The third step is to place the tumor subcutaneously in mice. After collecting the above cells by low-speed centrifugation, B16F10 Luc The cells were 5×10 per mouse. 5 The cells were injected subcutaneously on the back of mice.

[0144] Step 4: Grouping and delivery of degradation system. The mice in this example were divided into blue light group, dark group, PBS group, and empty virus group. The blue light group was injected with 5×10 6 TU was loaded with a virus that contained a degradation system and was treated with blue light for 30 min every day; the dark group was injected with 5×10 6 The virus with TU degradation system was protected from light; the PBS group was injected with an equal volume of PBS; the empty virus group was injected with 5×10 6 TU is a virus without a degradation system.

[0145] Step 5: Tumor fluorescence and volume monitoring. Tumor volume was measured every three days, and tumor size was monitored by in vivo imaging every five days. When the tumor volume of the mouse grew to 1500mm 3 The mice were then sacrificed for analysis.

[0146] Step 6: Analysis of treatment effects. After the experiment is completed, the tumor size is counted by Prism and the fluorescence intensity is analyzed by in vivo imaging. The tumor tissue is immediately extracted for protein, and the degradation of endogenous proteins is detected by Western-blot. The average fluorescence value is then counted and analyzed.

[0147] Western-blot detection, Prism and in vivo imaging fluorescence data statistical analysis showed that the blue light modular regulation protein targeted degradation system in B16F10 Luc Melanoma cells transplanted into C57BL / 6JGpt mice can simultaneously degrade endogenous proteins PD-L1 and c-Myc, achieving the effect of inhibiting tumor growth. For detailed experimental data, please refer to the attached manual Fig.11 .

[0148] Table 1 Plasmid construction table

[0149]

[0150] 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.

[0151] The * in the following sequence represents a stop codon

[0152] SEQ ID NO.1: Amino acid sequence of pMag-Linker-Ab (anti-EGFP) MHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETESDSAGSAGSAGSGSMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO.2: Amino acid sequence of pMag-Linker-Ab(anti-HER2) MHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETEAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGSEQ ID NO.3: Amino acid sequence of ABI-Linker-Ab(anti-EGFP) TRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKSDSAGSAGSAGSGSMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFF.

[0153] GSIPRTGTAFDYWGQGTQVTVSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0154] MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

[0155] VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

[0156] EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0157] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0158] SEQ ID NO.4: Amino acid sequence of NS3a-Linker-Ab(anti-EGFP)

[0159] KKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTA

[0160] TQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGS

[0161] RSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCP

[0162] AGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSPSDSAGSAGSAGSGSMAD

[0163] VQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRS

[0164] AGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSI

[0165] PRTGTAFDYWGQGTQVTVSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

[0166] RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0167] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM

[0168] TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0169] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0170] SEQ ID NO.5: Amino acid sequence of NS3a-Linker-Ab (anti-HER2)

[0171] KKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTA

[0172] TQTFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGS

[0173] RSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCP

[0174] AGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSPSDSAGSAGSAGSGSMAQ

[0175] VQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYP

[0176] GDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSS

[0177] SNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAA

[0178] PGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGS

[0179] KSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG

[0180] SEQ ID NO.6: Amino acid sequence of Δ2Trim21

[0181] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPV

[0182] CRQRFLLKNLRPNRQLANMVNNLKEISQEAREGEAAQEYQEKLQVALGELRR

[0183] KQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELE

[0184] KDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSES

[0185] WNLKDLDITSPELRSVCHVPGLKKMLRTCKKKGSVVIVGRINLSGDTAYAQQ

[0186] TRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWTVYHGAGTR

[0187] TIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIP

[0188] VRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDF

[0189] IPVESLETTMRSP

[0190] SEQ ID NO.7: Amino acid sequence of nMag-Linker-Δ2Trim21

[0191] HTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCALILCDLKQKDTPIVYAS

[0192] EAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDR

[0193] NAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETESDSAGSA

[0194] GSAGSGSMASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKG

[0195] GGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGEAAQEYQEKLQ

[0196] VALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEE

[0197] QRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQE

[0198] VIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTC

[0199] SEQ ID NO.8: Amino acid sequence of PYL1-Linker-Δ2Trim21

[0200] MGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDR。

[0201] PQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTG

[0202] FSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTR

[0203] LFADTVIRLNLQKLASITEAMNMASAARLTMMWEEVTCPICLDPFVEPVSIEC

[0204] GHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEA

[0205] REGEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRI

[0206] HAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISE

[0207] LDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTC

[0208] SEQ ID NO.9: Amino acid sequence of DNCR2-Linker-Δ2Trim21

[0209] MSSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEV

[0210] KRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAA

[0211] EEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVE

[0212] AAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVRE

[0213] AVERAEEVQRDPSGWLNHMASAARLTMMWEEVTCPICLDPFVEPVSIECGHS

[0214] FCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREG

[0215] EAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAE

[0216] FVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDR

[0217] RCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTC

[0218] SEQ ID NO.10: Amino acid sequence of GNCR1-Linker-Δ2Trim21

[0219] MDIEKLCKKAEEEAKEAQEKADELRQRHPDSQAAEDAEDLANLAVAAVLTAC

[0220] LLAQEHPNADIAKLCIKAASEAAEAASKAAELAQRHPDSQAARDAIKLASQA

[0221] ARAVILAIMLAAENPNADIAKLCIKAASEAAEAASKAAELAQRHPDSQAARD

[0222] AIKLASQAAEAVERAIWLAAENPNADIAKKCIKAASEAAEEASKAAEEAQRH

[0223] PDSQKARDEIKEASQKAEEVKERCKSMASAARLTMMWEEVTCPICLDPFVEP

[0224] VSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEI

[0225] SQEAREGEAAQEYQEKLQVALGELRRKQELAEKLEVIAIKRADWKKTVETQ

[0226] KSRIHAEFVQQKNFLVEEEEKRQLQELEKDEREQLRILGEKEAKLAQQSQALQE

[0227] LISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKM

[0228] LRTC

[0229] SEQ ID NO.11: Amino acid sequence of GS-1

[0230] GS

[0231] SEQ ID NO.12: Amino acid sequence of GS-2

[0232] GGGGS

[0233] SEQ ID NO.13: Amino acid sequence of GS-3

[0234] GGGGSGGGGS

[0235] SEQ ID NO.14: Amino acid sequence of GS-4

[0236] GGGGSGGGGSGGGGS

[0237] SEQ ID NO.15: Amino acid sequence of GS-6

[0238] GGGGSGGGGSGGGGR

[0239] SEQ ID NO.16: Amino acid sequence of d2EGFP

[0240] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL

[0241] PVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN

[0242] YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQ

[0243] KNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKD

[0244] PNEKRDHMVLLEFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQ

[0245] ESGMDRHPAACASARINV

[0246] SEQ ID NO.17: Amino acid sequence of HER2

[0247] MAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMG

[0248] LIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVG

[0249] YCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPS

[0250] VSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDR

[0251] FSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG

[0252] SEQ ID NO.18: Amino acid sequence of PD-L1

[0253] MMAQVQLVETGGGLVQPGGSLRLSCTASGFTFSMHAMTWYRQAPGKQRELV

[0254] AVITSHGDRANYTDSVRGRFTISRDNTKNMVYLQMNSLKPEDTAVYYCNVPR

[0255] YDSWGQGTQVTVSSGGLPETGG

[0256] SEQ ID NO.19: Amino acid sequence of EGFR antibody

[0257] DLGKKLLEAARAGQDDEVRILMANGADVNADDTWGWTPLHLAAYQGHLEI

[0258] VEVLLKNGADVNAYDYIGWTPLHLAADGHLEIVEVLLKNGADVNASDYIGD

[0259] TPLHLAAHNGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQ

[0260] SEQ ID NO.20: Amino acid sequence of cMYC antibody

[0261] QLQLVESGGGSVQPGGSLTLSCSASGFNISQYSVGWFREAPGEEREGISCLDID

[0262] GKITTFSDAIQGRFTIARDNAANMIYLHMDALNPLDTAVYRCVARWDCSRHD

[0263] FITQKTATGIWGPGTQVTVSA

Claims

1. A modularly regulated protein targeted degradation system, characterized in that: The system is based on a modified protein ΔTrim21 degradation system, with blue light, abscisic acid, Danoprevir and / or MK-5172 as inducers. Based on the modified protein ΔTrim21 degradation system, a controllable protein dimerization system is introduced and modified to control the binding of the target protein with the modified protein ΔTrim21, so as to perform modularly regulated targeted protein degradation in mammalian cells; the system includes a blue light modular degradation system, an abscisic acid modular degradation system, a DNCR2 modular degradation system and / or a GNCR1 modular degradation system.

2. The system according to claim 1, characterized in that It includes a pMag / nMag system regulated by blue light, an ABI / PYL1 system regulated by abscisic acid, a DNCR2 / NS3a system regulated by the clinical drug Danoprevir, and a GNCR1 / NS3a system regulated by the clinical drug MK-5172; and / or, it includes ChemΔ2Trim-TPD and OptoΔ2Trim-TPD.

3. The system according to claim 1, characterized in that It includes a target protein recognition module and a degradation module based on E3 ubiquitinase; the module is constructed by splitting and recombining a controllable protein dimerization system and a ΔTrim21 domain.

4. The system according to claim 3, characterized in that The target protein recognition module is formed by assembling the C-terminus of the target protein-specific antibody (Ab) with one of the proteins in the protein dimerization system through a flexible amino acid linker peptide (Linker), including: pMag-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown in SEQ ID NO. 1; pMag-Linker-Ab (anti-HER2), the amino acid sequence of which is shown in SEQ ID NO. 2; ABI-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown in SEQ ID NO. 3; NS3a-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown in SEQ ID NO.4; and / or, NS3a-Linker-Ab (anti-HER2), the amino acid sequence of which is shown in SEQ ID NO.5; The E3 ubiquitinase-based degradation module is formed by assembling the C-terminus of another protein in the protein dimerization system with Δ2Trim21 through a flexible amino acid linker peptide (Linker), and includes: nMag-Linker-Δ2Trim21, the amino acid sequence of which is shown in SEQ ID NO.7; PYL1-Linker-Δ2Trim21, the amino acid sequence of which is shown in SEQ ID NO.8; DNCR2-Linker-Δ2Trim21, the amino acid sequence of which is shown in SEQ ID NO.9; and / or, GNCR1-Linker-Δ2Trim21, the amino acid sequence of which is shown in SEQ ID NO.10; The amino acid sequence of Δ2Trim21 is shown in SEQ ID NO.6; Δ2Trim21 is formed by deleting the PRYSPRY domain on the basis of ΔTrim21; wherein the PRYSPRY domain is a binding domain of the IgG-Fc fragment; compared with the wild-type Trim21, the Δ2Trim21 protein is reduced by 50%; Wherein, the flexible amino acid linker peptide (Linker) includes (GS)n, and its amino acid sequence is shown in SEQ ID NO.11-15.

5. The system according to claim 1, wherein: The ΔTrim21-based target protein degradation can accurately regulate the degradation of exogenous proteins including fluorescent proteins and enzyme proteins, as well as endogenous proteins including HER2, PDL1, and EGFR, and its amino acid sequence is shown in SEQ ID NO.16-20.

6. A modularly controllable protein targeted degradation device, characterized in that: It comprises a system as claimed in any one of claims 1 to 5.

7. A modularly regulated protein targeted degradation method, characterized in that: In the method, the system as described in claim 1 and / or the device as described in claim 6 are used, blue light, abscisic acid, Danoprevir and / or MK-5172 are used as inducers, and based on the modified protein ΔTrim21 degradation system, a controllable protein dimerization system is introduced and modified to control the binding of the target protein with the modified protein ΔTrim21, so as to carry out modularly regulatable protein targeted degradation in mammalian cells.

8. A modified protein Δ2Trim21, characterized in that The Δ2Trim21 is formed by deleting the PRYSPRY domain on the basis of ΔTrim21; wherein the PRYSPRY domain is the binding domain of the IgG-Fc fragment; compared with the wild-type Trim21, the modified protein Δ2Trim21 is reduced by 50%.

9. A eukaryotic expression vector of a programmable and regulated protein targeted degradation system, characterized in that: The expression vector includes the system as described in claim 1 and / or the device as described in claim 6, which includes: a eukaryotic expression vector of a blue light-regulated target protein degradation control device; a eukaryotic expression vector of a abscisic acid-regulated target protein degradation control device; a eukaryotic expression vector of a clinical drug Danoprevir-regulated target protein degradation control device; and / or a eukaryotic expression vector of a clinical drug MK-5172-regulated target protein degradation control device.

10. A method for regulating a target protein degradation control system based on a protein dimerization system, characterized in that: The method comprises the following steps: (1) constructing the system according to claim 1 and / or the device according to claim 6 into a host cell eukaryotic plasmid expression vector; (2) introducing the expression vector into the host cell and expressing the encoded gene; (3) The blue light module is regulated by blue light; and / or, the abscisic acid module is regulated by adding the drug abscisic acid; and / or, the DNCR2 module is regulated by adding the drug Danoprevir; and / or, the GNCR1 module is regulated by adding the drug MK-5172.

11. A method for inhibiting tumor growth by degrading tumor-related proteins in melanoma cell-transplanted mice using the system according to claim 1 or the device according to claim 6, characterized in that: The method comprises: (1) constructing the system as claimed in claim 1 and / or the device as claimed in claim 6; wherein it is a protein targeted degradation system regulated by a blue light module; (2) preparing a lentiviral vector expressing the blue light module-regulated protein targeted degradation system; (3) Construction of melanoma cell transplanted mice; (4) By using lentivirus as a vector to deliver a blue light module to regulate the protein targeted degradation system, endogenous proteins can be specifically degraded in tumors to inhibit tumor proliferation.

12. The modularly controllable protein targeted degradation system according to any one of claims 1 to 5, the device according to claim 6, the modularly controllable protein targeted degradation method according to claim 7, the modified protein Δ2Trim21 according to claim 8, the eukaryotic expression vector according to claim 9, or the control system and / or control method of the device for controlling target protein degradation based on a protein dimerization system according to claim 10, in degrading tumor-related proteins and inhibiting tumor growth; characterized in that, It can be applied to regulate the degradation of target proteins in various mammalian cells; wherein the mammalian cells include HEK-293T, HEK-293A, HeLa, and hMSC-TERT.

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