System for targeted degradation of RNF31 protein through selective autophagy and application thereof

Abstract

The invention belongs to the technical field of targeted protein degradation, and particularly relates to a system for targeted degradation of RNF31 protein through selective autophagy and application of the system. The protein targeted degradation system comprises a recombinant sequence comprising an LIR motif and a protein targeted binding sequence, wherein the protein targeted binding sequence is at the C terminal of the recombinant sequence. According to the invention, after a single-domain antibody specifically bound with an LIR motif DSFVEI derived from OPTN and a target protein RNF31 is recombined, RNF31 in a T cell can be specifically guided to lysosome for degradation through selective autophagy targeting; in-vitro experiments show that RNF31 in T cells is efficiently and accurately degraded in a targeted manner, and after RNF31 and CAR are introduced into the T cells, depletion of the CAR-T cells can be remarkably inhibited, durability of the CAR-T cells is enhanced, and then the in-vitro tumor cell killing effect of the CAR-T cells is enhanced, so that a novel targeted degradation tool is provided for T cell endogenous depletion-related proteins.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted protein degradation, and specifically relates to a system for targeted degradation of RNF31 protein via selective autophagy and application thereof. Background Art

[0002] CAR-T cell therapy (chimeric antigen receptor T cell immunotherapy) is a revolutionary immunotherapy technology that reprograms the patient's own T cells to recognize and attack their own tumor cells, showing great potential for personalized treatment. This technology has achieved remarkable results in the treatment of relapsed or refractory diffuse large B-cell lymphoma, acute lymphoblastic leukemia and multiple myeloma. CAR-T cell therapy can not only provide lasting anti-tumor effects, but also has a high degree of individualization, bringing new treatment options to patients. However, this technology also faces challenges such as safety, high treatment costs and tolerance of some patients to treatment. With the continuous advancement of science and technology and the accumulation of clinical practice, CAR-T cell therapy is expected to become an important breakthrough in the field of tumor treatment in the future, bringing more hope and possibilities to patients.

[0003] Lysosomes promote the degradation of intracellular proteins and organelles in three different ways: endocytosis, phagocytosis, and autophagy. Cells introduce extracellular material or membrane proteins into the cell through endocytosis. In phagocytosis, cells bind and engulf viruses, bacteria, or other large particles. Autophagy is a highly conserved cellular process in which misfolded or aggregated proteins, damaged organelles, and intracellular pathogens are cleared. The autophagic pathway includes three forms: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). During macroautophagy, dysfunctional proteins or organelles are recognized by autophagy receptors and selectively encapsulated in autophagosomes, which then fuse with lysosomes and their contents are degraded. In microautophagy, lysosomes directly engulf autophagic cargo and cause its degradation. In CMA, proteins are directed to lysosomes after binding to chaperone proteins and directly cross the lysosomal membrane for degradation. In recent years, with the in-depth study of the endosomal-lysosomal and autophagy-lysosomal degradation pathways, targeted protein degradation (TPD) strategies through the lysosomal pathway, such as LYTAC, AbTAC, ATTEC, AUTAC, bispecific aptamer chimeras, and AUTOTAC, have begun to attract attention. Compared with proteasome-based TPD, lysosome-based TPD can not only degrade certain intracellular proteins, but also has the potential to remove protein aggregates, damaged excess organelles, membranes, and extracellular proteins.

[0004] The LIR (LC3-interacting region) motif of selective autophagy is a specific sequence present in the protein structure that can interact with the shared autophagy component LC3 on the autophagy adaptor protein. This interaction enables the protein to be recognized and directed for degradation through the autophagy pathway. The LIR motif usually contains a specific conserved amino acid sequence, such as "WxxL" or "F / L / W / YxxL / I / V", which is the key structure for binding to LC3. The LIR motif plays a key role in the selective autophagy process. It can interact with LC3 family proteins (including LC3A, LC3B, LC3C and GABARAP) to mediate the selective degradation of specific proteins or organelles by cells. Many autophagy adaptor proteins, such as p62, NBR1, OPTN, etc., contain LIR motifs, and selectively recognize and capture specific waste proteins or organelles through the interaction between LIR motifs and LC3. Proteins containing LIR motifs can interact with LC3 family proteins to achieve recognition and targeted degradation of specific proteins. This mechanism provides researchers with a strategy to selectively regulate the degradation of target proteins, thereby modulating cellular metabolism and function.

[0005] Based on the results obtained in multiple clinical trials, the first CAR-T cell therapy product (CTL-019) was used for patients with relapsed / refractory B-cell acute lymphoblastic leukemia (r / r B-ALL), and since then, several similar products have been approved. Overall, the complete remission (CR) rate of patients treated with CD19 CAR-T is 30-70%, and in some trials, this rate exceeds 90%. The fact that these products were produced using different technical solutions shows that CAR-T cells have a significant efficiency in the treatment of lymphoblastic leukemia. CAR-T cells have also been tried for the treatment of solid tumors, but the efficacy is less than that of lymphoblastic leukemia. Therefore, there is an urgent need to further optimize CAR-T therapy to solve the existing problems, especially the practical problems such as limited persistence of solid tumors, high relapse rate and insufficient cytotoxicity.

[0006] In view of this, it is extremely urgent to develop a protein degradation tool based on the LIR motif that can target and degrade exhaustion-related proteins in T cells. Summary of the invention

[0007] The purpose of the present invention is to provide a system for selective autophagy-targeted degradation of RNF31 protein and its application. The present invention provides a new strategy for endogenous protein degradation by recombining LIR motifs with endogenous protein targeting binding sequences, and after the targeting sequences bind to substrate proteins in T cells, the LIR motifs can guide endogenous proteins to be degraded in lysosomes via the selective autophagy pathway.

[0008] Therefore, to achieve the above objectives, the first objective of the present invention is to provide a recombinant plasmid, wherein the recombinant plasmid comprises a recombinant sequence having a LIR motif and a protein targeting RNF31 sequence.

[0009] Furthermore, in the above technical solution, the protein targeting RNF31 sequence is at the C-terminus of the recombinant sequence, its amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown as SEQ ID NO.2.

[0010] EVQLLESGGGLVQPGGSLRLSCAASGFTFNDYAMGWVRQAPGKGLEWVSAISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSEMAVGDFDYWGQGTLVTVSS (SEQ ID NO.1); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACGACTATGCCATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTAGTAGCACATACTACGC AGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGGGAGCGAGATGGCAGTGGGTGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ IDNO.2).

[0011] Furthermore, in the above technical solution, the LIR motif is the LIR motif of the OPTN protein, whose amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.4.

[0012] DSFVEI (SEQ ID NO. 3); GATTCCTTTGTTGAAATT (SEQ ID NO. 4).

[0013] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant plasmid, using FV185 as a plasmid vector, recombining the protein targeting RNF31 sequence with the LIR sequence, and obtaining the OPTNR recombinant sequence specifically targeting RNF31 after transcription and translation, thereby obtaining a recombinant plasmid.

[0014] Specifically, in the recombinant plasmid (OPTNR recombinant sequence), the LIR sequence is a single group or multiple groups of repeated LIR motifs, preferably two groups of repeated LIR motifs, such as 2×LIR-RNF31.

[0015] The third object of the present invention is to provide a system for targeted degradation of RNF31 protein via selective autophagy, comprising any of the recombinant plasmids described above.

[0016] The fourth object of the present invention is to provide an application of the above-mentioned system for targeted degradation of RNF31 protein through selective autophagy in the preparation of CAR-T cell anti-tumor drugs.

[0017] The present invention has the following beneficial effects: The present invention utilizes the ability of LIR to interact with the shared autophagy component LC3 on the autophagy adaptor protein to achieve selective recognition and capture of specific waste proteins or organelles. By recombining the LIR motif (DSFVEI) derived from OPTN and the protein-targeted RNF31 sequence, RNF31 in T cells can be specifically targeted to lysosomes for degradation through selective autophagy, which can effectively downregulate the expression of exhaustion-related proteins in CAR-T cells, providing a new strategy for inhibiting the exhaustion of CAR-T cells to enhance the efficacy of anti-tumor treatment.

[0018] In vitro experiments showed that RNF31 in T cells was efficiently and accurately targeted for degradation, and after being introduced into T cells in combination with CAR, it could significantly inhibit the exhaustion of CAR-T cells, enhance the persistence of CAR-T cells, and thus enhance its in vitro tumor cell killing effect. At the same time, nude mouse orthotopic glioma constructed based on U251-luciferase-CD19-EGFP (hereinafter referred to as U251-CD19) glioma cells achieved better efficacy after CAR-T cell treatment in the RNF31 knockdown group, significantly inhibited the occurrence and development of glioma, and further improved the survival prognosis of glioma mice. These will provide a new targeted degradation tool for proteins related to endogenous exhaustion of T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the sequence combination of OPTNR, OPTNA and OPTNV of the present invention.

[0020] Figure 2 Schematic diagram of the OPTNR of the present invention guiding the degradation of target protein.

[0021] Figure 3 The gel electrophoresis diagrams are of the protein level of RNF31 detected after the T cells of the present invention were transferred with empty vector, OPTNR, OPTNA and OPTNV.

[0022] Figure 4 This is a comparison chart of the mRNA levels of RNF31 detected after the T cells of the present invention were transferred with an empty vector, OPTNR, OPTNA and OPTNV.

[0023] Figure 5 This is a comparison chart of the differences in cell release factors between the CAR-T cells of the present invention and CAR-knock-down RNF31 (KB)-T (hereinafter referred to as CAR-KR-T) cells.

[0024] Figure 6 This is a comparison chart of the RNF31 protein expression in the CAR-T cells and CAR-KR-T cells of the present invention.

[0025] Figure 7 This is a comparison chart of the cytotoxic activity of CAR-T or CAR-KR-T of the present invention against U251 luciferase at different E:T ratios.

[0026] Figure 8 This is a comparison chart of the cytotoxic activity of CAR-T or CAR-KR-T of the present invention on U251 luciferase at different time points.

[0027] Fig. 9 This is a comparison chart of the remaining proportion of U251 cells after co-culture of CAR-T or CAR-KR-T and U251-EGFP cells at different E:T ratios in the present invention.

[0028] Fig.10 This is a comparison chart of the remaining proportions of U251 cells at different time points after the CAR-T or CAR-KR-T of the present invention and U251-CD19 cells were co-cultured at a ratio of 0.2:1; Fig.11 The figure is a flow chart of the experiment of the nude mouse intracranial orthotopic glioma model of the present invention.

[0029] Fig.12 This is the intracranial bioluminescence image of the nude mouse on the 28th day after intracranial tumor formation.

[0030] Fig.13 This is a comparison chart of the intracranial bioluminescence intensity of four groups of mice on the 28th day of the present invention.

[0031] Fig.14 This is a comparison chart of the survival status of mice monitored by the present invention after T cell injection. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified. The methods not specifically described are all carried out with reference to conventional methods in the art.

[0033] Among them, the lentiviral plasmid empty vector FV185 was purchased from Tianjin Sheweisi; male nude mice were purchased from Nanjing Jicui Yaokang.

[0034] The LIR motif (derived from OPTN protein, a single group of amino acid sequence as shown in SEQ ID NO.3)-RNF31 Single domain antibody (amino acid as shown in SEQ ID NO.1) polypeptide designed by the present invention can target RNF31 protein in T cells. The present invention takes two groups of LIR motifs as an example, and the obtained 2×OPTN (LIR)-RNF31 is hereinafter referred to as OPTNR, the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.6. The polypeptide can target RNF31 protein in T cells. The inventor has successfully verified that it can significantly reduce the level of RNF31 protein in T cells and CAR-T cells without significantly changing the transcription level of RNF31.

[0035] MDSFVEIDSFVEISGGSSGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFNDYAMGWVRQAPGKGLEWVSAISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSEMAVGDFDYWGQGTLVTVSS* (SEQ ID NO.5); ATGGATTCCTTTGTTGAAATTGATTCCTTTGTTGAAATTTCTGGTGGTTCTTCTGGTGGTTCTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACGACTATGCCATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTAT TAGTGGTAGTGGTAGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGGGAGCGATGGGTGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCATAA (SEQ ID NO.6).

[0036] At the same time, the inventors also used OPTN-AKTin (OPTNA) targeting AKT, the amino acid sequence of which is shown in SEQ ID NO.7, and OPTN-VhhGFP4 (OPTNV) targeting GFP, the amino acid sequence of which is shown in SEQ ID NO.8 as controls for OPTNR. The schematic diagram of the combination of the sequence and control sequence of the degradation system applied to degrade RNF31 is shown in Figure 1 shown.

[0037] Schematic diagram of the mechanism of RNF31 degradation in cells Figure 2 As shown, first, the protein of interest (POI) binds to the RNF31 antibody domain in the OPTNR molecule, and then the DSFVEI sequence in OPTNR specifically binds to the phagophore. After completing the cargo recruitment, the phagophore develops into an autophagosome and fuses with the lysosome to form an autolysosome, which then degrades RNF31. The application of this degradation tool is expected to downregulate the expression of the exhaustion-related protein RNF31 in CAR-T cells, inhibit the exhaustion of CAR-T cells, and enhance the efficacy of anti-tumor therapy.

[0038] The present invention is described in further detail below in conjunction with embodiments: Example 1: Acquisition of protein targeting RNF31 sequence and construction of luciferase-CD19-EGFP recombinant plasmid The empty lentiviral plasmid vector FV185 was purchased from Tianjin Sheweisi. The LIR motif DSFVEI of the proven effective OPTN protein was used for recombination with the single-domain antibody sequence targeting RNF31. Two groups of LIR motifs were used as an example (2×OPTN(LIR)-RNF31), and the obtained recombinant sequence was named OPTNR. At the same time, the designed OPTN-AKTin (OPTNA) targeting AKT and the OPTN-VhhGFP4 (OPTNV) targeting GFP were combined with short peptides as comparisons. The above fragments and luciferase-CD19-EGFP sequences were directly synthesized by Jinweizhi. Then, the synthesized fragments were recombined with the empty vector using the recombination kit of Novizan. The OPTNR / OPTNA / OPTNV fragments were synthesized by Jinweizhi and recombined into the vector in a similar manner to obtain a recombinant plasmid with a protein targeting binding sequence.

[0039] During in vitro verification, the above-mentioned recombinant plasmid, psPAX2 and pMD2.G (psPAX2 and pMD2.G are lentiviral packaging plasmids from addgene) were transferred into 293T according to the 2:1.5:1 transient co-transfection method to obtain recombinant lentivirus. The lentiviral supernatant secreted by 293T was collected and filtered through a 0.22μm filter membrane, then packaged and stored in a -80℃ refrigerator for later use.

[0040] Example 2: Isolation, purification, activation and expansion of T cells Take 3-5 mL of peripheral blood from healthy volunteers, separate lymphocytes and monocytes from the whole blood using human peripheral blood lymphocyte separation solution within 2 hours of in vitro excretion, and then purify CD3+T cells using a T cell purification kit. Adjust the separated CD3+T cells to 10 ^6 The cells were mixed with CD3 / CD28+ magnetic beads at a density of about 100 / mL in 1640 complete medium containing IL-2 for activation and amplification. During the amplification process, the density was carefully observed and the cells were passaged.

[0041] Example 3: Verification of RNF31 degradation efficiency Primary T cells were isolated from the whole blood of healthy adult volunteers. When preparing to transfect T cells, the well plate to be used was treated with poly-lysine for 2 hours, washed with PBS, and then treated with Retronectin and Lamimin overnight to promote T cell adhesion and enhance the transfection efficiency of T cells. The CD3+T cells obtained in Example 2 were transferred to a 24-well plate and adjusted to an appropriate density. The lentivirus and PolyBrene transfection reagent prepared in Example 1 were added to the culture medium. After 24-48 hours, it was replaced with normal 1640 complete culture medium for culture and screened with blasticidin. When the screened T cells grow to an appropriate density, protein and total RNA can be extracted. After the protein concentration is determined by the BCA method, the difference in RNF31 protein levels between the empty load, OPTNR, OPTNA and OPTNV groups can be detected by protein imprinting experiments. The results are shown in the following table. Figure 3 As shown, from Figure 3 The results showed that only OPTNR could specifically reduce the level of RNF31 protein in T cells.

[0042] The extracted total RNA was reverse transcribed to obtain cDNA, and then RNF31 detection primers (forward primer-SEQ ID NO.9: 5'-GAGCCCCGAAACTACCTCAAC-3'; reverse primer-SEQ ID NO.10: 5'-CTTGACACCACGCCAGTACC-3') were added to each group of samples for real-time quantitative PCR to detect the changes in RNF31 mRNA levels. The results are shown in Figure 4 As shown. Figure 4 The results showed that there was no significant difference in the transcription level of RNF31 between each group. The above experiment was repeated three times.

[0043] In order to verify the difference in RNF31 levels between CAR-T and CAR-KR-T cells, the inventors selected a suitable empty vector and recombined the CAR fragment (nucleotide sequence as shown in SEQ ID NO.11) synthesized by GENEWISE into the empty vector. The CAR-KR fragment (nucleotide sequence as shown in SEQ ID NO.12) was further recombined with the OPTNR fragment on the synthesized CAR plasmid. After packaging these two plasmids into lentivirus, they were transfected into T cells according to the above method. After screening and amplification, the protein was extracted to detect the RNF31 protein level. The results are as follows Figure 5 As shown. Figure 5 The results show that the RNF31 protein level in CAR-KR-T cells is significantly lower than that in CAR-T cells.

[0044] Example 4: Verification of the in vitro killing effect of CAR-KR-T cells Taking the human glioma U251 cell line as an example of the target cell, the constructed luciferase-P2A-CD19-EGFP plasmid (nucleotide sequence as shown in SEQ ID NO.13) was first packaged into a lentivirus, transfected into U251 cells, and then screened and amplified to obtain the stable transfected strain U251-CD19.

[0045] Take 100 μL of the supernatant of the cultured CAR-T and CAR-KR-T cells, and use Elisa detection kits for IL-2 and IFNγ (IL-2 and IFNγ are two T cell activation indicators) to detect the difference in the levels of IL-2 and IFNγ secreted by the two T cells. The results are as follows: Figure 6 As shown, from Figure 6 It can be seen that CAR-KR-T cells secrete higher levels of IL-2 and IFNγ.

[0046] In order to test the in vitro killing effect of CAR-KR-T cells, CAR-T / CAR-KR-T cells were co-cultured with U251-CD19 cells at various ratios of 0.05:1, 0.1:1, 0.2:1, 0.5:1, 1:1, and 2:1 for 24 h. After the culture, the cells were lysed, and fluorescein potassium salt was added to the lysate to detect the cell-specific killing effect under different co-culture conditions. The results are shown in the figure. Figure 7 As shown, from Figure 7 The results show that the killing effect of CAR-KR-T cells on glioma cells is significantly better than that of CAR-T cells at all co-culture ratios.

[0047] In addition, the ratio of CAR-T / CAR-KR-T to U251-CD19 cells was 0.2:1, and the cells were lysed at 0, 0.5, 1, 2, 3, 4, 5, and 6 days of co-culture, and fluorescein potassium salt was added to the lysate to detect the changes in the tumor cell killing efficiency of CAR-T / CAR-KR-T cells over time. The results are shown in Figure 8 As shown, from Figure 8 The results show that when the co-culture ratio is fixed (0.2:1), CAR-KB-T cells show a more excellent tumor cell killing effect in the first 5 days of culture.

[0048] At the same time, the cell killing experiment based on real-time cell number does not require cell lysis. The suspended T cells and adherent U251-CD19 cells in the co-cultured cells are directly digested, resuspended and mixed, and then the proportion of glioma cells is detected by flow cytometry. The results are as follows Figure 9-10 As shown, the results showed that CAR-KR-T cells can kill tumor cells faster and more persistently than CAR-T cells.

[0049] Example 5: Verification of the in vivo anti-tumor immune efficacy of CAR-KR-T cells 48 male nude mice were purchased from Nanjing Jicui Pharmaceutical and cultured in an SPF environment for the fourth week. With the help of a stereotaxic instrument, 10 μg of 1 ... ^5 U251-CD19 cells were added. On the third day of tumor formation, the nude mice were divided into four groups, with 12 mice in each group. PBS, T cells, CAR-T cells, and CAR-KR-T cells were injected into each group of nude mice through the tail vein and re-injected once seven days later. On the 28th day of tumor formation, small animal in vivo imaging was performed to detect the occurrence and development of intracranial tumors. Each nude mouse was intraperitoneally injected with 100g:1mL fluorescein potassium salt. After filming, the culture was continued for 50 days and the experiment was terminated. During the nude mouse experiment, the survival status of each group of nude mice was closely observed. The animal experimental process of the nude mouse intracranial in situ glioma model is as follows Fig.11 As shown, PBS / T / CAR-T / CAR-KR-T cells were injected into mice through the tail vein 3 days after modeling and divided into four groups accordingly. In vivo imaging of small animals was performed on the 28th day of modeling to detect the growth of intracranial gliomas. Fig.12 , Fig.13 As shown, the survival status of nude mice was recorded until the 50th day of modeling ( Fig.14 ). The above results show that both CAR-T and CAR-KR-T cell groups can achieve significant anti-tumor efficacy, and the anti-tumor effect of the CAR-KR-T group is significantly better than that of the CAR-T group.

[0050] In summary, the protein targeted degradation system of the present invention recombines the LIR motif derived from the OPTN protein with the endogenous RNF31 single domain antibody sequence, and uses it to stably express in CAR-T cells to specifically regulate the degradation of endogenous RNF31 protein, which can significantly reduce the endogenous RNF31 protein level in CAR-T cells. This study provides a targeted degradation system that can specifically target and degrade RNF31 protein in T cells, which has a significant degradation effect on endogenous RNF31 protein in CAR-T cells, enabling CAR-T cells to have stronger anti-tumor efficacy, and provides a new strategy for the development of CAR-T cell anti-tumor therapy.

[0051] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant plasmid, characterized in that: The recombinant plasmid comprises a recombinant sequence having an LIR motif and a protein targeting RNF31 sequence.

2. A recombinant plasmid according to claim 1, characterized in that: The protein targeting RNF31 sequence is at the C-terminus of the recombinant sequence, and its amino acid sequence is shown in SEQ ID NO.

1.

3. A recombinant plasmid according to claim 1, characterized in that: The LIR motif is the LIR motif of the OPTN protein, and its amino acid sequence is shown in SEQ ID NO.

2.

4. The method for constructing a recombinant plasmid according to any one of claims 1 to 3, characterized in that: Using FV185 as a plasmid vector, the protein targeting RNF31 sequence and the LIR sequence were recombined, and after transcription and translation, the OPTNR recombinant sequence specifically targeting RNF31 was obtained, thereby obtaining a recombinant plasmid.

5. The method for constructing a recombinant plasmid according to claim 4, characterized in that: In the recombinant plasmid, the LIR sequence is a single or multiple repeated LIR motifs, preferably two repeated LIR motifs.

6. The method for constructing a recombinant plasmid according to claim 5, characterized in that: In the recombinant plasmid, the LIR sequence is two sets of repeated LIR motifs.

7. A system for targeted degradation of RNF31 protein via selective autophagy, characterized in that: The protein targeted degradation system comprises the recombinant plasmid described in any one of claims 1 to 3 or the recombinant plasmid constructed by the construction method of any one of claims 4 to 6.

8. Use of the system for selectively autophagically targeting and degrading RNF31 protein as claimed in claim 7 in the preparation of CAR-T cell anti-tumor drugs.

Images