A programmable regulated protein targeting degradation system, construction and application thereof

Abstract

The application discloses a target protein degradation control system based on a modified version of Trim21 (Delta Trim21), which is based on the modified protein Delta Trim21 degradation system, uses blue light, abscisic acid, Danoprevir and / or MK-5172 as an inducer, and controls the combination of target proteins and Delta Trim21 by introducing and modifying a controllable protein dimerization system based on the modified protein Delta Trim21 degradation system, so as to realize modularly controllable protein targeted degradation. The two control modules, namely a target protein recognition module and a degradation module based on an E3 ubiquitinase, are formed by splitting and recombining the controllable protein dimerization system and the Delta Trim21 domain. The application further discloses a degradation device comprising the system. The application further discloses a method for precisely controlling endogenous proteins (degradation of tumor occurrence related proteins) by using a programmable controllable modular protein degradation device, so as to achieve the effect of tumor specific killing. The application further provides a eukaryotic expression vector and a control method of the programmable controllable protein targeted degradation device.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of synthetic biology, protein engineering, tumor targeted therapy, and the like, and particularly relates to a programmable and controllable protein targeted degradation system and a target protein degradation control device, and construction and application thereof. BACKGROUND

[0002] Proteins, as the main participants of life activities, their expression and degradation are the basis for maintaining cell function and supporting physiological function, and are precisely regulated by the cell's own regulatory network. Abnormal expression of proteins can lead to the occurrence and development of related diseases, such as some malignant tumors. Therefore, interfering with the expression and function of proteins is not only a major strategy for studying protein function in basic research, but also an effective means for preventing and treating diseases caused by abnormal proteins.

[0003] In the field of synthetic biology, the research on changing the function of biomolecules through design, modification and recombination has become one of the increasingly popular directions. In recent years, with the rapid development of synthetic biology, more and more research focuses on proteins, a kind of biological macromolecules. In mammalian synthetic biology, through the redesign of these biomolecules, the modification of cell function can be achieved, 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 have some limitations due to their direct action on the DNA or RNA level, such as off-target effects, non-universality and inability to act on long-lived proteins.

[0005] There are some natural high-efficiency protein degradation pathways in mammalian cells to realize the accurate regulation of the levels of various proteins in cytoplasm and nucleus, including ubiquitin-proteasome pathway, lysosomal degradation, autophagic degradation and other processes. Among them, the ubiquitin-proteasome pathway is an extremely important protein selective degradation pathway in eukaryotic cells, which is responsible for about 80% of endogenous protein degradation in cells. At present, through the ubiquitin-proteasome pathway to degrade target proteins to explore the influence of proteins on physiological functions and diseases has become a hot research field, and technologies such as PROTACs, deGradFP degradation technology, AID and Trim-Away have brought new directions for disease treatment. However, these protein degradation technologies based on ubiquitin-proteasome cannot 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 population cells is not good enough, and these problems directly limit the subsequent disease treatment. In order to overcome these limitations, it is urgent to invent a controllable, specific and efficient high-speed targeted degradation system, which can be used for basic research and clinical application research. SUMMARY

[0006] In view of the deficiencies of the above existing protein interference technology, the present application innovatively proposes a mammalian cell modularly controllable protein targeted degradation method and system and device, taking blue light, abscisic acid, Danoprevir and / or MK-5172 as inducers, based on the modified protein ΔTrim21, and taking the modified protein ΔTrim21 degradation system as the basis, by introducing and modifying a controllable protein dimerization system to control the binding of the target protein and the modified protein ΔTrim21, the mammalian cell modularly controllable protein targeted degradation is carried out.

[0007] In specific embodiments, taking blue light, abscisic acid and two clinical small molecules Danoprevir and MK-5172 as inducers, and taking ΔTrim21 degradation system as the chassis, different modularly controllable protein targeted degradation methods are carried out.

[0008] The present application first proposes a mammalian cell different modularly controllable protein targeted degradation system / device, which is based on the ΔTrim21 degradation system and constructs a modularly controllable degradation system, including but 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 application, the "modular regulation" refers to the combination of the ΔTrim21 degradation system and four different inducer-responsive proteins, resulting in four different regulatory degradation systems. Although each of the four systems is independently regulated, they have 70% common parts (ΔTrim21 degradation system). By replacing different responsive proteins, the common parts remain unchanged, and different inducers can be responded to trigger regulatory degradation. Therefore, it is called "modular". Preferably, the present application includes four modular regulation methods.

[0010] In the present application, 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, i.e., a target protein recognition module and an E3 ubiquitinase-based degradation module.

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

[0012] It should be noted that in the present application, "wild-type Trim2" refers to the natural E3 ubiquitinase Trim21 protein. In the present application, "engineered protein ΔTrim21" refers to a mutant recombinant protein with better E3 ubiquitination degradation effect, which is obtained by deleting the b-box domain from the natural E3 ubiquitinase Trim21 protein based on the reasonable design of the present application. In the present application, the "engineered protein ΔTrim21 degradation system" is an improved protein degradation system based on the engineered protein ΔTrim21 and the reported technology Trim21-Away. The mechanism of action is that, for example, after the exogenous reporter protein Reporter binds with its Fc antibody, ΔTrim21 binds with the reporter protein-antibody complex through its PRYSPRY domain to form a trimer. The trimer is specifically degraded by the ubiquitin proteasome pathway under the guidance of the RING domain of ΔTrim21. When ΔTrim21, reporter protein Reporter or its Fc antibody is consumed, the degradation is automatically terminated.

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

[0014] The application provides a modularly regulated protein targeted degradation device, which comprises the modularly regulated protein targeted degradation system.

[0015] The application first provides a new modified protein Δ2Trim21, wherein the modified protein Δ2Trim21 refers to ΔTrim21 protein of a ΔTrim21 degradation system being further modified to delete a PRYSPRY domain so as to achieve the effect of regulating protein degradation, and is named as Δ2Trim21.

[0016] In an embodiment, a protein structure diagram of the modified protein Δ2Trim21 is shown in the figure, which comprises a RING and a coiled-coil two protein domain structure, and the modified protein Δ2Trim21 is reduced by about 50% compared with wild-type Trim21. Figure 1 In an embodiment, the Δ2Trim21 is formed by deleting a PRYSPRY domain on the basis of ΔTrim21. The PRYSPRY domain is a binding domain of an IgG-Fc fragment, and the protein is reduced by about 50% compared with wild-type Trim21.

[0017] The RING domain is a ubiquitin binding domain, and the Δ2Trim21 is connected with a 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 whole 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 general zinc finger structures, the coordination sites of the two zinc ions in the RING domain are staggered to form a rigid spherical scaffold to facilitate protein-protein interaction.

[0018] The coiled-coil domain is a coiled-coil dimerization domain structure, and the Δ2Trim21 exists in a homodimer form in a solution through the coiled-coil domain.

[0019] Each amino acid sequence in the application can be prepared by an artificial synthesis method.

[0020] In the present application, the target protein recognition module is assembled by the C-terminal of the target protein specific antibody (Ab) through a flexible amino acid linker (Linker) with one of the proteins of the protein dimerization system, including but not limited to: pMag-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 1; pMag-Linker-Ab (anti-HER2), the amino acid sequence of which is shown as SEQ ID NO. 2; ABI-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 3; NS3a-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 4; and / or, NS3a-Linker-Ab (anti-HER2), the amino acid sequence of which is shown as SEQ ID NO. 5; etc. Wherein, the target protein specific antibody (Ab) does not require IgG-Fc fragment, only the protein domain specifically combined with the target protein is required.

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

[0022] Wherein, the flexible amino acid linker (Linker) can select various lengths of amino acid sequences. Preferably, the amino acid linker can include (GS)n, the amino acid sequence of which is shown as SEQ ID NO. 11-15.

[0023] In specific embodiments, the engineered protein Delta Trim21 degradation system, i.e., the target protein degradation control device based on Delta Trim21, includes a blue light-regulated pMag / nMag system (OptoDelta2Trim-TPD) and a drug-regulated ChemDelta2Trim-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). Chemical (ABA, GZV, DNV) and light (blue) trigger programmable activation of targeted protein degradation in cells and animals.

[0024] In specific embodiments, the target protein degradation control device based on Delta Trim21 can precisely regulate the degradation of exogenous proteins (fluorescent proteins, enzymes, etc.) and endogenous proteins (such as HER2, PDL1, EGFR, etc.), the amino acid sequences of which are shown in SEQ ID NO. 16-20.

[0025] In specific embodiments, the target protein degradation control device based on Delta Trim21 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 application, 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 light-sensitive protein pMag. The amino acid sequence of pMag-Linker-Ab (anti-EGFP) is shown in SEQ ID NO. 1.

[0027] The degradation element of the blue light modular degradation system includes Delta2Trim21 and a light-sensitive protein nMag. The Delta2Trim21 is an optimized version of the E3 ubiquitinase Trim21, and the amino acid sequence of the Delta2Trim21 is shown in SEQ ID NO. 6. The amino acid sequence of nMag-Linker-Delta2Trim21 is shown in SEQ ID NO. 7.

[0028] The mechanism of action of the blue light modular degradation system is shown in Figure 2As shown, the target protein specific antibody and the photosensitive protein pMag in the target protein recognition element are fusion expressed, and the Δ2Trim21 and the photosensitive protein nMag in the degradation element are fusion expressed, under the irradiation of blue light, the pMag specifically combines with the nMag, and then the antibody-pMag-nMag-Δ2Trim21 complex is formed, the complex is degraded through the ubiquitin proteasome pathway after the antibody binds to the target protein and the ubiquitin tag is given to the complex through the ubiquitin binding domain of Δ2Trim21.

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

[0030] The degradation element of the abscisic acid modular degradation system comprises Δ2Trim21 and a protein PYL1. The Δ2Trim21 is an optimized version of the E3 ubiquitinase Trim21, and the amino acid sequence of the Δ2Trim21 is shown in SEQ ID NO. 6. The PYL1-Linker-Δ2Trim21 amino acid sequence is shown in SEQ ID NO. 8.

[0031] The mechanism of the abscisic acid modular degradation system is shown in the present application. Figure 2 As shown, the target protein specific antibody and the protein ABI in the target protein recognition element are fusion expressed, and the Δ2Trim21 and the protein PYL1 in the degradation element are fusion expressed, under the induction of abscisic acid ABA, the ABI specifically combines with the PYL1, and then the antibody-ABI-PYL1-Δ2Trim21 complex is formed, the complex is degraded through the ubiquitin proteasome pathway after the antibody binds to the target protein and the ubiquitin tag is given to the complex through the ubiquitin binding domain of Δ2Trim21.

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

[0033] The degradation element of the DNCR2 modular degradation system comprises Delta2Trim21 and protein DNCR2. The Delta2Trim21 is an improved and optimized version of E3 ubiquitinase Trim21, and the amino acid sequence of the Delta2Trim21 is shown as SEQ ID NO. 6, and the amino acid sequence of the DNCR2-Linker-Delta2Trim21 is shown as SEQ ID NO. 9.

[0034] The mechanism of the GNCR1 modular degradation system is shown in the figure. Figure 2 The target protein specific antibody and protein NS3a in the target protein recognition element are fusion-expressed, and the Delta2Trim21 and protein GNCR1 in the degradation element are fusion-expressed. Under the induction of MK-5172, NS3a specifically binds to GNCR1, and then an antibody-NS3a-GNCR1-Delta2Trim21 complex is formed. The complex is bound to the target protein through the antibody, is given a ubiquitination label through the ubiquitin binding domain of Delta2Trim21, and then is degraded through the ubiquitin proteasome pathway.

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

[0036] The degradation element of the GNCR1 modular degradation system comprises Delta2Trim21 and protein GNCR1. The Delta2Trim21 is an improved and optimized version of E3 ubiquitinase Trim21, and the amino acid sequence of the Delta2Trim21 is shown as SEQ ID NO. 6, and the amino acid sequence of the GNCR1-Linker-Delta2Trim21 is shown as SEQ ID NO. 10.

[0037] The mechanism of the GNCR1 modular degradation system is shown in the figure. Figure 2 The target protein specific antibody and protein NS3a in the target protein recognition element are fusion-expressed, and the Delta2Trim21 and protein GNCR1 in the degradation element are fusion-expressed. Under the induction of MK-5172, NS3a specifically binds to GNCR1, and then an antibody-NS3a-GNCR1-Delta2Trim21 complex is formed. The complex is bound to the target protein through the antibody, is given a ubiquitination label through the ubiquitin binding domain of Delta2Trim21, and then is degraded through the ubiquitin proteasome pathway.

[0038] The four modular degradation system components provided by the application are constructed in a eukaryotic expression vector through genetic engineering technology, and then the degradation of the target protein is realized. The four modular degradation systems provided by the application can be used for degrading exogenous reporter proteins or endogenous proteins introduced into host cells. The host cells can be any type of mammalian cells, such as hMSC-TERT, HEK-293, Hela, etc.

[0039] The wavelength of the blue light is 430nm±10, the light intensity is 0-1mW / cm 2 , the irradiation time is 0-30min, and the irradiation method includes pulse irradiation and continuous irradiation. Different light irradiation times are generated by controlling the light source, so that different degrees of degradation of the target protein are realized. The blue light source can be an LED, a laser lamp, etc. The wavelength of the red light is 460nm±10nm, the light intensity is 0-1mW / cm 2 , the irradiation time is 0-12h, and the irradiation method includes pulse irradiation and continuous irradiation. Different light irradiation times are generated by controlling the light source, so that different degrees of degradation of the target protein are realized. The red light source can be an LED, a laser lamp, etc.

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

[0041] The application also provides a construction method of the four modular protein targeted degradation systems, comprising the following steps:

[0042] (1) Constructing a target protein recognition element

[0043] The fusion protein of the target protein specific antibody and the photosensitive protein pMag, the fusion protein of the target protein specific antibody and the protein ABI, the fusion protein of the target protein specific antibody and the protein NS3a, and the connecting peptide between the aforementioned fusion proteins are respectively constructed as the target protein recognition element.

[0044] The specific antibody of the target protein is a modified antibody, only the antigen binding domain is reserved; the specific modified antibody is fused and expressed with the sensing protein of different regulation 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 the amino acid sequences are shown in SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 4.

[0045] (2) Degradation element

[0046] The fusion proteins of Δ2Trim21 and photosensitive protein nMag, Δ2Trim21 and protein PYL1, Δ2Trim21 and protein DNCR2, Δ2Trim21 and protein GNCR1 and the connecting peptides between the foregoing fusion proteins are respectively constructed as degradation elements.

[0047] The Δ2Trim21 is a modified version of E3 ubiquitinase Trim21, and the effect of E ubiquitination is enhanced, and the Δ2Trim21 is obtained by deleting the B-BOX domain and PRYSPRY domain of Trim21; Δ2Trim21 is fused and expressed with the sensing protein of different regulation systems through a connecting peptide to form a degradation element, and the degradation element includes but is not limited to nMag-Linker-Δ2Trim21, PYL1-Linker-Δ2Trim21, DNCR2-Linker-Δ2Trim21 and GNCR1-Linker-Δ2Trim21, and the 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) Report system

[0049] The exogenous report protein of the degradation system is green fluorescent protein d2EGFP, and the amino acid sequence is shown in SEQ ID NO. 21; the degradation of the endogenous protein is the endogenous protein itself, including but not limited to HER2, PD-L1, EGFR and cMYC, and the amino acid sequences of the 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 kinds of modular regulation protein targeted degradation systems can respond to minute-level blue light stimulation or drug stimulation to start the degradation of the protein, and can also respond to micro-watt-level intensity blue light stimulation or micro-mole-level drug stimulation to start the degradation of the protein.

[0051] The four kinds of modular regulated protein targeted degradation systems described in the present application 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 four kinds of modular regulated protein targeted degradation systems described in the present application are small in protein module and can be packaged with lentivirus for efficient delivery in vivo. The four kinds of modular regulated protein targeted degradation systems can regulate protein degradation in different mammalian cell lines (for example, 293T cells, HeLa cells, B16-F10 cells).

[0053] The present application also provides a eukaryotic expression vector of a modular regulated protein targeted degradation system, wherein the host cell is transfected with the eukaryotic expression vector, and contains the four kinds of modular 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 an 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 containing the four kinds of modular regulated protein targeted degradation systems. The expression vector can be a vector containing only the target protein recognition element coding gene or a vector containing only the degradation element coding gene. Alternatively, the expression vector can be a vector containing both the target protein recognition element coding gene and the degradation element coding gene. The construction of all the aforementioned mammalian cell expression vectors is shown in Table 1.

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

[0056] The application also provides a method for regulating degradation of an exogenous protein in a host cell (mammalian cell) by using the modular regulated protein targeted degradation system / device, comprising the following steps: (1) constructing the four modular regulated protein targeted degradation systems in a eukaryotic plasmid expression vector of the host cell; (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), drug Danoprevir and MK-5172. Further, the degradation of the exogenous fluorescent protein can be observed by fluorescence microscopy. In specific embodiments, preferably, different illumination times and intensities of the light source are controlled; and different degrees of degradation of the exogenous reporter protein d2EGFP can be achieved by controlling the drug concentration and incubation time.

[0057] The application also provides a method for degrading an endogenous protein by using the modular regulated protein targeted degradation system. Specifically, the application provides a method for simultaneously degrading endogenous proteins HER2, PD-L1, EGFP and cMYC in HeLa cells mediated by the four modular regulated protein targeted degradation systems, which comprises the following steps: (1) constructing the four modular regulated protein targeted degradation systems in a eukaryotic plasmid expression vector of the host cell; (2) introducing the expression vector into the host cell, wherein the target protein recognition element is four, including the recognition elements of the four endogenous proteins; (3) inducing or regulating the specific degradation of the target protein in the host cell by blue light, abscisic acid (ABA), drug Danoprevir and MK-5172. Further, the degradation of the endogenous protein can be detected by Western-blot experiment. In specific embodiments, different illumination times and intensities of the light source are controlled; and different degrees of degradation of the endogenous protein can be achieved by controlling the drug concentration and incubation time.

[0058] The application also provides an application of the system / device in degrading tumor-related proteins and inhibiting tumor growth in melanoma cell transplanted mice. In specific embodiments, the application is applied to degrading tumor-related proteins and inhibiting tumor growth in B16F10 Luc degrading tumor-related proteins and inhibiting tumor growth in melanoma cell transplanted C57BL / 6JGpt mice.

[0059] The application also provides a method for degrading tumor-related proteins and inhibiting tumor growth in melanoma cell transplanted C57BL / 6JGpt mice by using the blue light modular protein targeted degradation system, which comprises the following steps: Luc degrading tumor-related proteins and inhibiting tumor growth in melanoma cell transplanted C57BL / 6JGpt mice.

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

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

[0062] Preparation of a lentivirus packaging plasmid containing the blue light module regulated protein targeted degradation system;

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

[0064] (3) Constructing a melanoma cell transplanted mouse; preferably B16F10 Luc Transplanting tumor cells into C57BL / 6JGpt mice; wherein the tumor-bearing method of the mice comprises:

[0065] B16F10 Luc Culture and proliferation of melanoma cells;

[0066] Collecting the tumor cells subcutaneously, constructing B16F10 Luc Transplanting tumor cells into C57BL / 6JGpt mice.

[0067] (4) Delivering the blue light module regulated protein targeted degradation system to the subcutaneous B16F10 Luc tumor in C57BL / 6JGpt mice by lentivirus as a carrier.

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

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

[0070] The beneficial effects of the present application include: for the first time, a different modular regulation protein targeted degradation system for mammalian cells is proposed, which comprises 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 non-marking regulation, no toxic side effects, etc.; the abscisic acid module has the advantages of being widely used in human and animal cells, having an auxiliary effect on disease treatment, having a synergistic effect, etc.; the two clinical small molecule drug modules have the advantages of being safe and reliable, having a synergistic effect, being easy to accept, and being precise and efficient in combination therapy. The degradation system has the characteristics of simple structure, various regulation modes, high sensitivity and fast degradation speed. The degradation system not only can degrade exogenous reporter proteins, but also can efficiently and rapidly degrade endogenous proteins. By designing different target protein recognition elements, the system can also degrade different target proteins simultaneously. In addition, the degradation system can later be used to precisely regulate the degradation of endogenous proteins in vivo to achieve the effect of tumor killing. The present application provides a protein targeted degradation system with different modular regulation for mammalian cells, which can be used for various 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 application also include that light has the advantages of no need to take, remote non-marking regulation, no toxic side effects, etc.; abscisic acid has the advantages of being widely used in human and animal cells, having an auxiliary effect on disease treatment, having a synergistic effect, etc.; the two clinical small molecule drugs Danoprevir and MK-5172 have the advantages of being safe and reliable, having a synergistic effect, being easy to accept, and being precise and efficient in combination therapy. The four modular regulation protein targeted degradation methods and systems provided by the present application can rapidly and specifically degrade target proteins within 30 minutes, are convenient to operate, and have strong practicality. The present application overcomes the shortcomings of existing protein degradation techniques, such as slow degradation speed, uncontrollable specificity, and selective degradation. The present application also proposes an application of a blue light module regulated protein targeted degradation system. The application uses the blue light module regulated protein targeted degradation system to specifically degrade tumor-related proteins in B16F10 Luc melanoma cells and inhibit the development of tumors in mice. In summary, the present application has great potential value in the research of specific and efficient regulation and degradation of target proteins in the field of mammalian protein engineering and biomedicine.

[0072] The present application provides a target protein degradation control system and / or control device based on modified Trim21 (Delta Trim21), wherein the design principle of the target protein degradation control device is to control the binding of target protein and Delta Trim21 by introducing and modifying a controllable protein dimerization system. The protein degradation device based on the regulation of the binding of target protein and Delta Trim21 is constructed by splitting and recombining the controllable protein dimerization system and the domain of Delta Trim21 to form two regulation modules, i.e. a target protein recognition module and a degradation module based on E3 ubiquitinase. Preferably, the two target protein degradation control devices are ChemDelta2Trim-TPD and OptoDelta2Trim-TPD, which enable programmable activation of targeted protein degradation in cells and animals triggered by chemicals (ABA, GZV, DNV). The programmable regulation type modular protein degradation device provided by the present application has the characteristics of simple structure, various regulation modes, high sensitivity and fast degradation speed. The programmable regulation type modular protein degradation device provided by the present application is used for precise regulation of endogenous proteins (degradation of tumor occurrence related proteins) to achieve tumor specific killing effect. The present application also provides a eukaryotic expression vector and a regulation method of the programmable regulation type protein targeted degradation device, which provides a new research method and a new strategy for future application in various diseases and biological researches, cell targeted therapy, mammalian genetic engineering, cell mechanism exploration and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 The protein structure diagram of the modified protein Delta2Trim21.

[0074] Figure 2 The mechanism diagram of the four kinds of modular regulation protein targeted degradation systems.

[0075] Figure 3 The light irradiation time and intensity dependence result diagram of the blue light modular regulation protein targeted degradation system degrading exogenous d2EGFP.

[0076] Figure 4 The incubation time and concentration dependence result diagram of the abscisic acid ABA modular regulation protein targeted degradation system degrading exogenous d2EGFP.

[0077] Figure 5 The incubation time and concentration dependence result diagram of the DNCR2 modular regulation protein targeted degradation system degrading exogenous d2EGFP.

[0078] Figure 6 The incubation time and concentration dependence result diagram of the GNCR1 modular regulation protein targeted degradation system degrading exogenous d2EGFP.

[0079] Figure 7Figure 1 shows the results of four modular regulatory protein targeted degradation systems simultaneously degrading endogenous proteins HER2, PD-L1, EGFR, and cMYC in HeLa cells.

[0080] Figure 8 To investigate the light intensity dependence of a blue light modular regulation protein targeted degradation system on the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells.

[0081] Figure 9 To investigate the light-time dependence of a blue light modular regulation protein targeted degradation system on the degradation of endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells.

[0082] Figure 10 For blue light modular regulation of protein targeted degradation system in B16F10 Luc Schematic diagram of the mechanism by which tumor-associated proteins are degraded to inhibit tumor growth in C57BL / 6JGpt mice with melanoma cell transplantation.

[0083] Figure 11 For blue light modular regulation of protein targeted degradation system in B16F10 Luc Figure showing the results of tumor growth inhibition by degradation of tumor-related proteins in C57BL / 6JGpt mice after melanoma cell transplantation. Detailed Implementation

[0084] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0085] The application provides a target protein degradation control system based on a modified version of Trim21 (ΔTrim21), which is based on a modified protein ΔTrim21 degradation system, and is induced by blue light, abscisic acid, Danoprevir and / or MK-5172. The target protein is combined with ΔTrim21 by introducing and modifying a controllable protein dimerization system based on the modified protein ΔTrim21 degradation system, and the protein target degradation can be modularly regulated. The two regulation modules, namely a target protein recognition module and a degradation module based on an E3 ubiquitinase, are formed by splitting and recombining the controllable protein dimerization system and the ΔTrim21 domain. The application has the advantages of simple structure, various regulation modes, high sensitivity and fast degradation speed. The application also provides a degradation device comprising the system. The application also provides a method for precisely regulating endogenous proteins (degradation of tumor-related proteins) by using a programmable and controllable modular protein degradation device, so that the tumor can be specifically killed. The application also provides a eukaryotic expression vector and a regulation method of the programmable and controllable protein target degradation device, which provides a new research method and a new strategy for future application in various diseases and biological research, cell targeted therapy, mammalian genetic engineering and cell mechanism exploration.

[0086] Example 1, construction of different modular protein target degradation systems

[0087] In this embodiment, a target protein specific antibody and a regulatory protein are fused to form a target protein recognition element, and Δ2Trim21 and another regulatory protein are fused to form a degradation element. The structure of Δ2Trim21 is shown in the attached Figure 1 The mechanism of the four modular protein target degradation systems is shown in the attached Figure 2 The construction method comprises the following steps:

[0088] First, Δ2Trim21 genes, target protein specific antibody genes and four different regulatory protein genes are synthesized by gene synthesis.

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

[0090] Third, plasmids with different modular protein target degradation systems are obtained by screening and sequencing.

[0091] Example 2, verification of the light irradiation time and intensity dependence of the blue light modular protein target degradation system on the degradation of exogenous d2EGFP.

[0092] The present embodiment takes d2EGFP as a reporter gene to verify the light time and intensity dependence of the degradation of exogenous d2EGFP based on the blue light modular regulation protein targeted degradation system. The schematic diagram of the mechanism of the blue light modular regulation protein targeted degradation system is shown in the description Figure 2 . The specific steps are as follows:

[0093] First, plasmid construction. The plasmid construction in the present embodiment is shown in Table 1.

[0094] Second, inoculate cells. One day before transfection, HEK-293T cells were inoculated in a 24-well plate at a cell amount of 5x10 4 cells per well, and 500ul of DMEM medium containing 10% FBS was added to each well.

[0095] Third, plasmid transfection. The transfection system of the present embodiment is divided into light group, dark group and d2EGFP group. The light group and the dark group are the same transfection group divided into two parts, and pMD97, pMD99 and pMD44 are transfected, with a plasmid amount of 150ng, 150ng and 15ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, with a plasmid amount of 150ng, 150ng and 15ng; the above plasmids in each group are premixed with a total amount of 315ng, and the transfection reagent PEI (plasmid to PEI mass ratio 1:3) is dissolved in 50ul of serum-free and antibiotic-free DMEM. After 15 minutes of standing, the DNA-PEI premix is added dropwise to each well of cells. After 6h of transfection, 500ul of DMEM medium containing 10% FBS, 1% (volume / volume) of penicillin, streptomycin mixture is added for culture.

[0096] Fourth, light. After 14-18 hours of medium change, the time-dependent exploration group: the dark group is placed in the dark, and the light group is placed under LED irradiation with a wavelength of 460nm and an illumination intensity of 1000ul / cm 2 . After light treatment, it is immediately placed in the dark. The intensity-dependent exploration group: the dark group is placed in the dark, and the light group is placed under LED irradiation with a wavelength of 460nm and an illumination intensity of 0-1000ul / cm 2 . After light treatment, it is immediately placed in the dark.

[0097] Fifth, fluorescence analysis. After the last group is irradiated, the cells are immediately extracted for protein, and the degradation of the reporter protein is detected by Western-blot, and then the average fluorescence value is calculated and analyzed.

[0098] Statistical analysis of western-blot data shows that the blue light modular regulated protein targeted degradation system has light time and intensity dependence for the degradation of exogenous d2EGFP. The experimental data are shown in the following table: Figure 3 .

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

[0100] In this example, d2EGFP was used as a reporter gene to verify the light time and intensity dependence of the abscisic acid ABA modular regulated protein targeted degradation system for degrading exogenous d2EGFP. The mechanism of action of the blue light modular regulated protein targeted degradation system is shown in the following figure: Figure 2 . The specific steps are as follows:

[0101] Step 1: Plasmid construction. The plasmid construction in this example is shown in Table 1.

[0102] Step 2: Cell inoculation. One day before transfection, HEK-293T cells were inoculated in a 24-well plate at a cell density of 5x10 4 cells per well, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0103] Step 3: Plasmid transfection. The transfection system in this example is divided into a drug-added group, a drug-free group, and a d2EGFP group. The drug-added group and the drug-free group are the same transfection group divided into two parts, and pMD104, pMD105, and pMD44 are transfected, with a plasmid amount of 150 ng, 150 ng, and 15 ng; the d2EGFP group is transfected with pcDNA3.1, pWL115, and pMD44, with a plasmid amount of 150 ng, 150 ng, and 15 ng; the above plasmids in each group are pre-mixed with a total amount of 315 ng with transfection reagent PEI (plasmid to PEI mass ratio 1:3) and dissolved in 50 ul of serum-free and antibiotic-free DMEM. After 15 minutes of standing, the DNA-PEI pre-mix was added to each well of cells. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (v / v) penicillin, and streptomycin mixture was added for culture.

[0104] Step 4: Drug incubation. After 14-18 hours of medium change, the incubation time dependence group: the drug-added group was added with 50 μM of drug ABA, and the incubation time was 0-30 min; after incubation, the medium was immediately removed, washed three times with PBS, and then fresh medium was added for culture. The incubation concentration dependence group: the drug-added group was added with 0-50 μM of drug ABA, and the incubation time was 30 min; after incubation, the medium was immediately removed, washed three times with PBS, and then fresh medium was added for culture.

[0105] Fifth step, fluorescence analysis. After the last group of incubation was completed, the cells were immediately extracted protein, and the degradation of the reporter protein was detected by Western-blot, and then the average fluorescence value was calculated and analyzed.

[0106] The statistical analysis of Western-blot data showed that the degradation of exogenous d2EGFP by the ABA modular regulated protein targeted degradation system was dependent on light time and intensity. The experimental data are shown in the attached Figure 4 .

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

[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 regulated protein targeted degradation system. The mechanism of action of the blue light modular regulated protein targeted degradation system is shown in the attached Figure 2 . The specific steps are as follows:

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

[0110] Second step, cell inoculation. One day before transfection, HEK-293T cells were inoculated in a 24-well plate at a cell density of 5 x 10 4 cells per well, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0111] Third step, plasmid transfection. The transfection system in this example was divided into a drug-added group, a drug-free group, and a d2EGFP group. The drug-added group and the drug-free group were the same transfection group divided into two parts, and pMD106, pMD107, and pMD44 were transfected, with a plasmid amount of 150 ng, 150 ng, and 15 ng; the d2EGFP group transfected pcDNA3.1, pWL115, and pMD44, with a plasmid amount of 150 ng, 150 ng, and 15 ng; the above plasmids in each group were pre-mixed with a total amount of 315 ng with transfection reagent PEI (plasmid to PEI mass ratio 1:3) and dissolved in 50 μl of serum-free and antibiotic-free DMEM. After 15 minutes of standing, the DNA-PEI pre-mix was added to each well of cells. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (v / v) penicillin, and streptomycin mixture was added for culture.

[0112] Fourth step, drug incubation. After 14-18 hours of medium change, incubation time-dependent group: drug group added 5 μM drug Danoprevir, incubation time was 0-30 min, after incubation, the medium was immediately removed, washed with PBS three times, and then fresh medium was added for culture. Incubation concentration-dependent group: drug group added 0-5 μM drug Danoprevir, incubation time was 30 min, after incubation, the medium was immediately removed, washed with PBS three times, and then fresh medium was added for culture.

[0113] Fifth step, fluorescence analysis. After the last group of incubation was completed, the cells were immediately protein extracted, and the degradation of the reporter protein was detected by Western-blot, and then the average fluorescence value was calculated and analyzed.

[0114] The Western-blot data statistical analysis showed that the DNCR2 modular regulatory protein targeted degradation system had light time and intensity dependence for the degradation of exogenous d2EGFP. The experimental data is shown in the attached Figure 5 .

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

[0116] In this example, d2EGFP was used as a reporter gene to verify the light time and intensity dependence of GNCR1 modular regulatory protein targeted degradation system for degrading exogenous d2EGFP. The schematic diagram of the mechanism of action of the blue light modular regulatory protein targeted degradation system is shown in the attached Figure 2 . The specific steps are as follows:

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

[0118] Second step, cell inoculation. One day before transfection, HEK-293T cells were inoculated in a 24-well plate at a cell amount of 5×10 4 cells per well, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0119] Third step, plasmid transfection. The transfection system of this embodiment is divided into drug-added group, drug-free group and d2EGFP group. The drug-added group and the drug-free group are the same transfection group divided into two parts, transfected with pMD106, pMD108 and pMD44, and the plasmid amount is 150 ng, 150 ng and 15 ng; the d2EGFP group is transfected with pcDNA3.1, pWL115 and pMD44, and the plasmid amount is 150 ng, 150 ng and 15 ng; the above-mentioned plasmids in each group are pre-mixed with the transfection reagent PEI (the mass ratio of plasmid to PEI is 1:3) according to the total amount of 315 ng, and dissolved in 50 ul of serum-free and antibiotic-free DMEM. After 15 minutes of static culture, the DNA-PEI pre-mixed solution is added dropwise into each well of the cell. After transfection for 6 hours, 500 ul of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture is added for culture.

[0120] Fourth step, drug incubation. After 14-18 hours of medium replacement, the incubation time-dependent exploration group: the drug-added group is added with 5 uM of drug MK-5172, and the incubation time is 0-30 min. After the incubation is completed, the culture medium is immediately removed, washed with PBS for three times, and then fresh culture medium is added for culture. The incubation concentration-dependent exploration group: the drug-added group is added with 0-5 uM of drug MK-5172, and the incubation time is 30 min. After the incubation is completed, the culture medium is immediately removed, washed with PBS for three times, and then fresh culture medium is added for culture.

[0121] Fifth step, fluorescence analysis. After the last group of incubation is completed, the cells are immediately extracted for protein, and the degradation of the reporter protein is detected by Western-blot. Then the average fluorescence value is calculated and analyzed.

[0122] The Western-blot data statistical analysis shows that the GNCR1 modular regulatory protein targeted degradation system has illumination time and intensity dependence for the degradation of exogenous d2EGFP. The experimental data are shown in the description attached 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] In this embodiment, four endogenous proteins HER2, PD-L1, EGFR and cMYC are used as target proteins to verify the simultaneous degradation of different endogenous proteins in HeLa cells based on four modular regulatory protein targeted degradation systems. The specific steps are as follows:

[0125] First step, plasmid construction. The plasmid construction in this embodiment is shown in Table 1.

[0126] Second step, inoculate cells. HeLa cells were inoculated in 24-well plates at a cell amount of 5 x 10 4

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

[0128] Fourth step, drug or light treatment. After 14-18 hours of medium change, the dark group in the blue light group is cultured in the dark, and the light group is irradiated with blue light for 30 minutes; the drug treatment group is added with the corresponding drug for 30 minutes.

[0129] Fifth step, Western-blot analysis. Immediately after treatment, the cells are extracted for protein, and the degradation of the reporter protein is detected by Western-blot, and then the average fluorescence value is calculated and analyzed.

[0130] The Western-blot data statistical analysis shows that the four kinds of modular regulated protein degradation systems can simultaneously degrade different endogenous proteins in HeLa cells. The experimental data are shown in the attached Figure 7 .

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

[0132] ​The present embodiment takes four endogenous proteins HER2, PD-L1, EGFR, and cMYC as target proteins to verify the light intensity dependence of the blue light-based modular protein degradation system in degrading endogenous proteins HER2, PD-L1, EGFR, and c-Myc in HeLa cells. The specific steps are as follows:

[0133] Step 1, plasmid construction. The plasmid construction in the present embodiment is shown in Table 1.

[0134] Step 2, cell inoculation. One day before transfection, HEK-293T cells were inoculated in a 24-well plate at a cell amount of 5x10 4 cells per well, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0135] Step 3, plasmid transfection. The transfection system of the present embodiment is divided into HER2 group, PD-L1 group, EGFR group, and cMyc group. The HER2 group was transfected with pMD99 and pWH62, and the amount of each plasmid was 150 ng; the PD-L1 group was transfected with pMD99 and pWH63, and the amount of each plasmid was 150 ng; the EGFR group was transfected with pMD99 and pWH64, and the amount of each plasmid was 150 ng; the cMyc group was transfected with pMD99 and pMD145, and the amount of each plasmid was 150 ng; the above plasmids of each group were pre-mixed with transfection reagent PEI (plasmid to PEI mass ratio 1:3) at a total amount of 300 ng, and dissolved in 50 ul of serum-free and antibiotic-free DMEM. After 15 minutes of standing, the DNA-PEI pre-mix was added dropwise to each well of cells. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin, and streptomycin mixture was added for culture.

[0136] Step 4, light irradiation. After 14-18 hours of medium change, the time-dependent group: the dark group was placed in the dark, and the light group was placed under LED irradiation at a wavelength of 460 nm and an intensity of 1000 μW / cm 2 for 0-30 min, and immediately placed in the dark after light treatment. The intensity-dependent group: the dark group was placed in the dark, and the light group was placed under LED irradiation at a wavelength of 460 nm and an intensity of 0-1000 μW / cm 2 for 30 min, and immediately placed in the dark after light treatment.

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

[0138] Western blot analysis showed that the blue light modular regulation protein-targeting degradation system exhibited light intensity- and light time-dependent degradation effects on endogenous proteins HER2, PD-L1, EGFR, and c-Myc. Detailed experimental data can be found in the instruction manual. Figure 8 , 9 .

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

[0140] This embodiment uses three endogenous proteins, PD-L1 and c-Myc, as target proteins to verify the effectiveness of the blue light-based modular regulation protein targeted degradation system in B16F10. Luc Degradation of tumor-associated proteins inhibits tumor growth in C57BL / 6JGpt mice after melanoma cell transplantation. See the instruction manual for details. Figure 10 The specific steps are as follows:

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

[0142] The second step is cell culture. Before tumor formation, B16F10 cells are cultured. Luc Cells were cultured and expanded in DMEM complete medium until each mouse carried 5 × 10⁶ cells. 5 Total number of cells.

[0143] The third step involves subcutaneous tumor implantation in mice. After collecting the cells by low-speed centrifugation, B16F10 cells are injected using a syringe. Luc Cells were distributed at a rate of 5 × 10⁶ per mouse. 5 The amount of cells was injected subcutaneously into the back of mice.

[0144] The fourth step is grouping and delivery of the degradation system. In this example, mice were divided into a blue light group, a dark group, a PBS group, and an empty virus group. The blue light group was injected with 5 × 10⁻⁶ ppm of the degradation system. 6 TU-mediated viral degradation and 30 minutes of blue light therapy daily; the dark group received 5×10 6 TU virus with degradation system, protected from light; PBS group injected with equal volume of PBS; empty virus group injected with 5×10 6 TU is a virus that does not have 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. The tumor size was monitored when it grew to 1500 mm². 3 The mice were then sacrificed for analysis.

[0146] Step 6, analysis of therapeutic effect. After the experiment, the tumor size was analyzed by Prism, the fluorescence intensity was analyzed by live imaging analysis, and the tumor tissue was immediately extracted for protein, the endogenous protein degradation was detected by Western-blot, then the mean fluorescence value was analyzed.

[0147] The Western-blot detection, Prism and live imaging fluorescence data statistical analysis showed that the blue light modular regulation protein targeted degradation system could degrade the endogenous proteins PD-L1 and c-Myc in B16F10 melanoma cells, and inhibit the growth of tumor cells. Luc The melanoma cells transplanted into C57BL / 6J Gpt mice can simultaneously achieve degradation of endogenous proteins PD-L1 and c-Myc, and achieve the effect of inhibiting tumor growth. The experimental data are shown in the attached drawings of the specification. Figure 11 .

[0148] Table 1 shows the plasmid construction table

[0149]

[0150] The protection scope of the present application is not limited to the above embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended 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) MHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETESDSAGSAGSAGSGSMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.2: Amino acid sequence of pMag-Linker-Ab (anti-HER2) MHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETEAQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG SEQ ID NO. 3: Amino acid sequence of ABI-Linker-Ab (anti-EGFP) TRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKSDSAGSAGSAGSGSMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFF.

[0153] GIPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG

[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] SQEAREGEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQ

[0226] KSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQE

[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 regulatable protein targeted degradation system, characterized in that, The system is based on the modified protein ΔTrim21 degradation system, and is induced by blue light, abscisic acid, Danoprevir and / or MK-5172. The system is based on the modified protein ΔTrim21 degradation system, and the binding of the target protein and the modified protein ΔTrim21 is controlled by introducing and modifying a controllable protein dimerization system to perform modularly controllable protein targeted 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. The system includes a blue light-regulated pMag / nMag system, 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; and / or, it includes ChemΔ2Trim-TPD, OptoΔ2Trim-TPD. The system includes a target protein recognition module and an E3 ubiquitinase-based degradation module; the modules are formed by splitting and recombining the controllable protein dimerization system and the ΔTrim21 domain; The target protein recognition module is assembled by the C-terminal of a target protein-specific antibody (Ab) through a flexible amino acid linker (Linker) and one of the proteins of the protein dimerization system, including: pMag-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 1; pMag-Linker-Ab (anti-HER2), the amino acid sequence of which is shown as SEQ ID NO. 2; ABI-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 3; NS3a-Linker-Ab (anti-EGFP), the amino acid sequence of which is shown as SEQ ID NO. 4; and / or, NS3a-Linker-Ab (anti-HER2), the amino acid sequence of which is shown as SEQ ID NO. 5; The E3 ubiquitinase-based degradation module is assembled by the C-terminal of the other protein of the protein dimerization system through a flexible amino acid linker (Linker) and Δ2Trim21, including: nMag-Linker-Δ2Trim21, the amino acid sequence of which is shown as SEQ ID NO. 7; PYL1-Linker-Δ2Trim21, the amino acid sequence of which is shown as SEQ ID NO. 8; DNCR2-Linker-Δ2Trim21, the amino acid sequence of which is shown as SEQ ID NO. 9; and / or, GNCR1-Linker-Δ2Trim21, the amino acid sequence of which is shown as SEQ ID NO. 10; Wherein, the amino acid sequence of the Δ2Trim21 is as shown in SEQ ID NO. 6; the Δ2Trim21 is formed by deleting the PRYSPRY domain on the basis of ΔTrim21; wherein, the PRYSPRY domain is the binding domain of IgG-Fc fragment; compared with the wild type Trim21, the Δ2Trim21 protein is reduced by 50%; Wherein, the flexible amino acid connecting peptide (Linker) includes (GS) n, and the amino acid sequence is as shown in SEQ ID NO. 11-15; The target protein degradation based on the ΔTrim21 can precisely regulate the degradation of exogenous proteins including fluorescent proteins and enzyme proteins, and endogenous proteins including HER2, PDL1 and EGFR, and the amino acid sequence is as shown in SEQ ID NO. 16-20.

2. The use of the modularly regulated protein-targeting degradation system according to claim 1 for degrading tumor-associated proteins, inhibiting tumor growth; characterized in that, It is 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.

3. A method of modularly regulated protein targeted degradation, comprising, In the method, the system as claimed in claim 1 is used, blue light, abscisic acid, Danoprevir and / or MK-5172 are used as inducers, the degradation system of the modified protein ΔTrim21 is used as the basis, the combination of the target protein and the modified protein ΔTrim21 is controlled by introducing and modifying the controllable protein dimerization system, and the modularly controllable protein targeted degradation is carried out in the mammalian cells.

4. The use of the modularly regulated protein targeted degradation method of claim 3 in degrading tumor associated proteins, inhibiting tumor growth; characterized in that, It is 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.

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

6. The use of the eukaryotic expression vector of claim 5 in degrading tumor-related proteins and inhibiting tumor growth. It is 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.

7. A method for regulating a target protein degradation control system based on a protein dimerization system, characterized by, The method includes the following steps: (1) constructing the system as claimed in claim 1 in a host cell eukaryotic plasmid expression vector; (2) introducing the expression vector into the host cell and coding gene expression; (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.

8. The use of the method of claim 7 for degrading tumor-related proteins and inhibiting tumor growth. It is 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.

9. A method of degrading tumor-associated proteins to inhibit tumor growth in a melanoma cell-implanted mouse using the system according to claim 1, characterized by, The method includes: (1) Constructing the system as claimed in claim 1; wherein it is a blue light module regulated protein targeted degradation system; (2) Preparing a lentivirus vector expressing the blue light module regulated protein targeted degradation system; (3) Constructing a melanoma cell transplanted mouse; (4) Targeted specific degradation of endogenous proteins in tumors to inhibit tumor proliferation by delivering the blue light module regulated protein targeted degradation system through a lentivirus vector.

10. A modularly regulatable protein-targeting degradation device, comprising, It comprises the system as claimed in claim 1.

11. The use of the device according to claim 10 for degrading tumor-related proteins, inhibiting tumor growth; characterized in that, It is applied to regulating the degradation of target proteins in various mammalian cells; wherein the mammalian cells include HEK-293T, HEK-293A, HeLa, hMSC-TERT.

12. A method of modularly regulated protein targeted degradation, comprising, In the method, the device as claimed in claim 10 is used to control the binding of target proteins to the modified protein ΔTrim21 by introducing and modifying a controllable protein dimerization system based on the modified protein ΔTrim21 degradation system, and the modularly regulated protein targeted degradation is carried out in mammalian cells using blue light, abscisic acid, Danoprevir, and / or MK-5172 as inducers.

13. A eukaryotic expression vector of a programmable regulated protein targeting degradation system, characterized in that, The expression vector comprises the device as claimed in claim 10, which comprises: a eukaryotic expression vector of a blue light regulated target protein degradation control device; a eukaryotic expression vector of an 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.

14. The use of the eukaryotic expression vector of claim 13 in degrading tumor-related proteins and inhibiting tumor growth. It is applied to regulating the degradation of target proteins in various mammalian cells; wherein the mammalian cells include HEK-293T, HEK-293A, HeLa, hMSC-TERT.

15. 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 device as claimed in claim 10 in a host cell eukaryotic plasmid expression vector; (2) Introducing the host cell through the expression vector and encoding gene expression; (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.

16. The use of the method for regulating the degradation control system of a target protein based on a protein dimerization system according to claim 15 in degrading a tumor-related protein, inhibiting tumor growth. It is applied to regulating the degradation of target proteins in various mammalian cells; wherein the mammalian cells include HEK-293T, HEK-293A, HeLa, hMSC-TERT. ​ 17. A method of inhibiting tumor growth in a melanoma cell-implanted mouse by degrading tumor-associated proteins using the device according to claim 10, characterized in that, The method comprises: (1) Constructing the device as claimed in claim 10; wherein it is a blue light module regulated protein targeted degradation system; (2) Preparing a lentivirus vector expressing the blue light module regulated protein targeted degradation system; (3) Constructing a melanoma cell transplanted mouse; (4) Targeted specific degradation of endogenous proteins in tumors to inhibit tumor proliferation by delivering the blue light module regulated protein targeted degradation system through a lentivirus vector.

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