A prokaryotic transcription factor ttg r-based unstable domain mutant and application thereof

Abstract

The present application belongs to the field of synthetic biology, and relates to a kind of unstable domain mutant based on prokaryotic transcription factor TtgR and its application.The present application constructs protein level reversible control switch by directional modification of prokaryotic transcription factor, and upgrades traditional transcription level regulation to protein level precise regulation.The regulation system (TtgR regulation system) has modular structure and wide adaptability: in specific embodiments, the coding sequence of reporter gene EGFP has been connected and modified into a fusion protein, and can precisely regulate the expression of EGFP under the induction of various small molecule ligands.The system can be applied to the operable connection of the coding sequences of various target proteins, including but not limited to CAR-T, CAR-NK related functional proteins, IL-2 and other cytokines, and other therapeutic proteins, functional proteins, etc.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and relates to an unstable domain mutant based on the prokaryotic transcription factor TtgR and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the biomedical field, particularly in cell therapy and synthetic biology, precise and reversible regulation of target protein expression levels is of great value. Among known technologies, destabilizing domains (DDs) have been developed as a general tool for regulating protein stability in mammalian cells. This system typically consists of a genetically engineered unstable domain and a small molecule ligand that can penetrate the cell membrane. In the absence of the ligand, the domain induces its fusion protein to degrade via the proteasome pathway; however, in the presence of the ligand, the fusion protein is stabilized, thereby achieving reversible regulation of target protein expression levels. For example, DHFR-based DDs can achieve stabilization regulation via trimethoprim (TMP). Although such systems have shown potential in regulating CAR-T cell activity, they still suffer from problems such as high background leakage expression and limited regulatory dynamic range.

[0004] Regulatory systems derived from the prokaryotic transcription factor TtgR have also been used for gene expression control. TtgR is a transcriptional repressor belonging to the TetR family in Pseudomonas bacteria, responding to a variety of hydrophobic small molecule ligands, including antibiotics (such as chloramphenicol and tetracycline) and plant-derived molecules (such as resveratrol and phlorizin). Researchers have developed a bifunctional transcriptional regulatory system, RES rep and RES ind, using the interaction between TtgR and resveratrol, which can regulate target genes such as CARs at the DNA transcriptional level. However, such transcriptional regulatory systems typically have slow response times and are significantly affected by intracellular metabolic pathways, limiting their application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a mutant of the unstable domain of the prokaryotic transcription factor TtgR and its applications. This invention constructs a protein-level reversible regulatory switch by directionally modifying a prokaryotic transcription factor, upgrading traditional transcriptional regulation to precise protein-level regulation. This regulatory system (TtgR regulatory system) has a modular structure and broad adaptability: in a specific embodiment, it has been modified into a fusion protein by linking it to the coding sequence of the reporter gene EGFP, and can precisely regulate EGFP expression after induction with various small molecule ligands. This system can be extended to the operable linking of coding sequences of various target proteins, including but not limited to CAR-T and CAR-NK related functional proteins, cytokines such as IL-2, and other therapeutic and functional proteins.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] Firstly, a mutant of the unstable domain of the prokaryotic transcription factor TtgR, wherein the unstable domain mutant has any one or more of the following mutations based on the prokaryotic transcription factor TtgR or its mutants:

[0008] a. The amino acid residue at position 200 is replaced by Y instead of D;

[0009] b. The amino acid residue at position 137 is replaced by C with R, and the amino acid residue at position 153 is replaced by Q with L;

[0010] c. The amino acid residue at position 17 is replaced with I and F, the amino acid residue at position 66 is replaced with L and V, and the amino acid residue at position 134 is replaced with V and F;

[0011] d. The amino acid residue at position 186 is replaced by Y, the amino acid residue at position 189 is replaced by V, and the amino acid residue at position 202 is replaced by P;

[0012] e. The amino acid residue at position 117 is replaced by C with W, and the amino acid residue at position 161 is replaced by E with D;

[0013] f. The amino acid residue at position 161 is replaced by E with D, and the amino acid residue at position 195 is replaced by V with L;

[0014] g. The amino acid residue at position 41 is replaced by A with S, the amino acid residue at position 117 is replaced by C with W, and the amino acid residue at position 161 is replaced by E with D;

[0015] h. The amino acid residues at positions 1 to 51 are missing.

[0016] Based on the prokaryotic transcription factor TtgR or its mutants, this invention further develops mutants with reversible regulation and high sensitivity and specificity, and proposes this invention based on the research results.

[0017] In some embodiments, the substitution is a conservative substitution. Those skilled in the art know that conservative substitutions can be performed on proteins or peptides without significantly affecting or altering their function or properties. Methods for replacing one amino acid residue in a protein / peptide chain with another amino acid residue are well known to those skilled in the art. For example, any amino acid residue in a protein / peptide chain can be modified (e.g., substituted) using standard techniques known in the art, such as error-prone PCR-mediated mutagenesis.

[0018] In some embodiments, the mutant of the prokaryotic transcription factor TtgR has any one of the following mutations:

[0019] (1) The amino acid residue at position 200 is replaced by Y instead of D;

[0020] (2) The amino acid residues at positions 1 to 51 are missing, and the amino acid residue at position 200 is replaced by Y instead of D;

[0021] (3) The amino acid residue at position 137 is replaced by C with R, and the amino acid residue at position 153 is replaced by Q with L;

[0022] (4) The amino acid residue at position 186 is replaced by Y with D, the amino acid residue at position 189 is replaced by V with G, and the amino acid residue at position 202 is replaced by P with L;

[0023] (5) The amino acid residue at position 41 is replaced by A with S, the amino acid residue at position 117 is replaced by C with W, and the amino acid residue at position 161 is replaced by E with D;

[0024] (6) The amino acid residue at position 41 is replaced by A with S, the amino acid residue at position 117 is replaced by C with W, the amino acid residue at position 161 is replaced by E with D, and the amino acid residue at position 200 is replaced by D with Y.

[0025] (7) The amino acid residues at positions 1 to 51 are deleted, the amino acid residue at position 117 is replaced by C with W, the amino acid residue at position 161 is replaced by E with D, and the amino acid residue at position 200 is replaced by D with Y.

[0026] (8) The amino acid residue at position 17 is replaced by I with F, the amino acid residue at position 66 is replaced by L with V, and the amino acid residue at position 134 is replaced by V with F;

[0027] (9) The amino acid residue at position 117 is replaced by C with W, and the amino acid residue at position 161 is replaced by E with D; or,

[0028] (10) The amino acid residue at position 161 is replaced by E with D, and the amino acid residue at position 195 is replaced by V with L.

[0029] In some implementations, mutants of the prokaryotic transcription factor TtgR have an amino acid sequence as shown in SEQ ID NO: 1, namely TtgRH67A.

[0030] In some embodiments, the unstable domain mutants are amino acid sequences as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8.

[0031] In a second aspect, a recombinant protein comprising the unstable domain mutant described in the first aspect of the invention, and one or more additional peptides linked to the unstable domain mutant.

[0032] In some embodiments, the additional peptide is directly linked to the mutant or linked to the mutant via a adapter. In some preferred embodiments, the additional peptide is linked to the mutant via a adapter. Suitable prior art adapters may consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, an adapter having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). In addition, other connectors may be used, such as those described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. Specifically, the connector is LEVD.

[0033] In some implementations, the additional peptide is linked to the N-terminus or C-terminus of the mutant.

[0034] In some embodiments, the recombinant protein comprises at least one, at least two, at least three, at least five or more additional peptides.

[0035] In some embodiments, the additional peptide is selected from signal peptides, tag peptides, folding motifs, detectable tags, and any combination thereof. In some preferred embodiments, the additional peptide is a detectable tag. Specifically, the additional peptide is a fluorescent protein. The fluorescent protein has the amino acid sequence shown in SEQ ID NO: 12.

[0036] Thirdly, a nucleic acid molecule comprising or encoding the unstable domain mutant described in the first aspect of the invention or the recombinant protein described in the second aspect of the invention.

[0037] Fourthly, a carrier comprising the nucleic acid molecule described in the third aspect of the present invention.

[0038] The vector of the present invention can be a cloning vector, a transfer vector, or an expression vector. In a preferred embodiment, the vector of the present invention is a plasmid.

[0039] Fifthly, a host cell comprising the nucleic acid molecule described in the third aspect of the invention or the vector described in the fourth aspect.

[0040] The host cells described in this invention include, but are not limited to, prokaryotic cells such as *E. coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells of this invention can also be cell lines, such as 293T cells. Without being bound by any theory, it is generally believed that the use of eukaryotic cells helps maintain the correct conformation of proteins and promotes protein folding. In some embodiments, the host cell is a CAR-T cell.

[0041] Sixthly, the use of an unstable domain mutant as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, a vector as described in the fourth aspect of the present invention, or a host cell as described in the fifth aspect of the present invention in the preparation of a biosensor or a drug.

[0042] A seventh aspect is a composition comprising CAR-T cells and a regulatory factor; said CAR-T cells comprising the nucleic acid molecule described in the third aspect of the invention or the vector described in the fourth aspect of the invention, thereby enabling expression of the unstable domain mutant described in the first aspect of the invention or the recombinant protein described in the second aspect of the invention; said regulatory factor is a ligand of said unstable domain mutant or recombinant protein.

[0043] Methods that regulate CAR-T function, such as shortening CAR-T survival time and altering CAR expression levels, address the safety concerns of CAR-T therapy, including toxic side effects and targeted toxicity. However, current CAR-T therapies face the challenge of irreversible suicide switches leading to treatment interruption and tumor recurrence. This invention constructs a gene control switch based on a TtgR-based unstable domain mutant and utilizes food-grade small molecule ligands to achieve reversible regulation of CAR protein expression. This improves safety while avoiding treatment interruption, providing a new strategy for precise control of CAR-T therapy.

[0044] Eighthly, the use of the above-mentioned composition in the preparation of drugs for treating tumors, immunomodulatory drugs, metabolic regulation drugs, gene therapy drugs, and enzyme therapy drugs.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. The unstable domain mutants provided by this invention possess excellent sensitivity and specificity. Based on the TtgR unstable domain where the amino acid residue at position 67 is replaced by an A, the core performance of these mutants is significantly enhanced through a directed evolution strategy. These mutants exhibit high binding affinity and fast conformational response rates to various small molecule ligands (such as resveratrol and doxycycline), achieving reversible and highly sensitive "on-off" regulation at the protein level. Their protein expression dynamic range can reach up to 10⁶-fold, while background leakage is extremely low.

[0047] 2. The unstable domain mutant provided by this invention achieves safe and controllable precise regulation in CAR-T therapy. In advanced cell therapies such as CAR-T, this mutant successfully solves the problem of irreversible cell elimination and treatment interruption caused by traditional "suicide switches." By introducing this invention, the expression level of CAR proteins can be reversibly and precisely regulated using orally safe, food-grade small molecules (such as resveratrol) or drugs (such as doxycycline). When overactivation or severe toxic side effects occur, ligand withdrawal can rapidly reduce CAR-T cell activity to achieve safe control; and after the risk is eliminated, reintroduction of the ligand can restore its tumor-killing function. This dynamic and reversible "safety switch" mechanism can effectively manage toxicity without permanently losing therapeutic cells, greatly improving the safety window and clinical applicability of CAR-T therapy.

[0048] 3. The widespread application of the TtgR regulatory system includes, but is not limited to, two major areas. First, in the field of endocrine and metabolic diseases, it can precisely regulate metabolism-related proteins such as leptin, adiponectin, insulin, and GLP-1 to improve insulin resistance and provide stratified intervention for type I / II diabetes, adapting to physiological and dynamic precise regulatory needs. Second, in the field of tumor-related diseases, it can regulate CAR-T / CAR-NK functional elements, IL-2, IL1RN, CXCR4, etc., for tumor immunotherapy, tumor metastasis blocking, and tumor microenvironment inflammation management, covering the entire tumor treatment cycle and avoiding related side effects. Attached Figure Description

[0049] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0050] Figure 1 The bar chart shows the fluorescence intensity response of each mutant individual to resveratrol in the embodiments of the present invention.

[0051] Figure 2 The graphs show the time dependence of different mutant individuals on resveratrol in the embodiments of the present invention. A represents the "on-off-on" mode, and B represents the "off-on-off" mode.

[0052] Figure 3 The following is a graph showing the dose relationship between resveratrol and different mutant individuals in this invention (Western blot (WB) detection results, A is the Western blot graph, and B is the relative protein level bar graph).

[0053] Figure 4 These are images showing the transfection results of different cell lines in embodiments of the present invention;

[0054] Figure 5 The above are bar charts showing the fluorescence intensity detected 24 hours after adding different small molecule ligands to mutants 1-5 in this embodiment of the invention.

[0055] Figure 6 This is a bar chart showing the results of detecting the in vitro killing activity of resveratrol in dose-dependent regulation of BBZ19-CAR-TtgRH67A-1-5-T and BBZ19-CAR-TtgRH67A-3-14-T cells in this embodiment of the invention.

[0056] Figure 7 According to flow cytometry, after transient transfection of the TtgRH67A-1-5-IL-2 fusion recombinant plasmid in this embodiment of the invention, resveratrol can significantly improve IL-2 expression in the transiently transfected recombinant plasmid group.

[0057] Figure 8The bar chart shows the results of dose-dependent upregulation of IL-2 expression with increasing resveratrol concentration after lentivirus was stably transfected with the TtgRH67A-1-5-IL-2 fusion plasmid in this embodiment of the invention. Detailed Implementation

[0058] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0059] Example 1

[0060] 1. Constructing the TtgRH67A mutant library

[0061] (1) Synthesizing the sequence and designing primers: The wild-type TtgR used in this embodiment is a transcriptional repressor from Pseudomonas, whose small molecule ligands include antibiotics (such as chloramphenicol and tetracycline) and plant antimicrobial agents (such as quercetin, naringenin, and phloretin). In the screening stage, this embodiment selected a low concentration of resveratrol. In the experimental response stage, this embodiment selected plant-derived small molecule ligands resveratrol, quercetin, and phloretin, as well as the antibiotics doxycycline and chloramphenicol.

[0062] The study found that mutating His-67 of the TtgR protein to alanine (i.e., TtgRH67A) does not affect its binding effector, but reduces its DNA binding ability to one-third of that of the wild type. This indicates that this site specifically regulates DNA binding function, providing a key model for studying the interdomain regulatory mechanism of TtgR protein effector binding and DNA binding.

[0063] According to TtgRH67A (C. Daniels, A. Daddaoua, D. Lu, etc. Domain cross-talkduring effector binding to the multidrug binding TTGR regulator. J Biol Chem, 2010, 285(28): 21372-81) and PGK-EGFP (J. Sun, W. Zhang, Y. Zhao, etc. Conditional control of chimeric antigen receptor T-cell activity through adestabilizing domain switch and its chemical ligand. CytotherapyGenes were synthesized from the sequences of (2021, 23(12): 1085-1096) to obtain PuC57-TtgRH67A and PGK-EGFP-DHFR plasmids, and plasmid extraction was performed. Upstream and downstream primers were designed according to the sequences.

[0064] (2) Error-prone PCR: The above-mentioned PuC57-TtgRH67A plasmid was mutated using the error-prone PCR kit. The primers were designed and a 50 μL PCR amplification system was prepared as shown in Table 1.

[0065] Table 1 Error-prone PCR amplification systems

[0066]

[0067] Note: Target DNA refers to the DNA sequence to be amplified, not the total amount of DNA from the Pu57-TtgRH67A plasmid in the reaction. The upstream primer is: ATGGTGCGGAGAACAAAGGAAG, as shown in SEQ ID NO: 13; the downstream primer is: TTATCACTTCCGCAGGGCTGG, as shown in SEQ ID NO: 14.

[0068] Reaction conditions: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 10 min, 4℃ holding.

[0069] (3) Recovery: DNA fragments were identified by agarose gel electrophoresis under UV light. First, a 1% agarose gel was prepared. 0.25 g of agarose was weighed and placed in an Erlenmeyer flask. 25 mL of 1×TAE was added. The flask was heated in a microwave oven and boiled three times until the agarose was completely dissolved. 2.5 μL of EB substitute was added, and the mixture was shaken well and poured into the gel plate. After the gel solidified completely, the comb was removed vertically, and the gel and inner tank were placed in the electrophoresis tank. 1×TAE was added until the gel plate was submerged. Error-prone PCR products were added to the sample wells, and the gel was run at 120 V for 20 min. After electrophoresis, the gel was removed, and the presence or absence of bands was observed under UV light. The size of the DNA fragments was observed in nucleic acid electrophoresis, and the DNA in the gel was recovered using a kit. The mutated TtgRH67A fragment was recovered.

[0070] (4) Normal PCR: The TtgRH67A fragment, the product of the error-prone PCR mutation, was used as a template for normal PCR based on the error-prone PCR. Primers were redesigned and a 100 μL PCR amplification system was prepared as shown in Table 2.

[0071] Table 2. Conventional PCR amplification system

[0072]

[0073] Reaction conditions: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 1 min, 60℃ annealing for 1 min, 72℃ extension for 45 s, 30 cycles; 72℃ final extension for 10 min, and holding at 4℃; wherein, the upstream primer is: GATTATGTCGACATGGTGCGGAGAACAAAGGAAG, as shown in SEQ ID NO: 15; the downstream primer is: GATTATGATTATCACTTCCGCAGGGCTGG, as shown in SEQ ID NO: 16.

[0074] (5) Recovery: DNA fragments were identified by agarose gel electrophoresis under UV light. The ordinary PCR product was added to the sample well and the gel was run at 120V for 20 min. After electrophoresis, the gel was removed and the presence or absence of bands was observed under UV light. The size of the DNA fragments was observed and the DNA in the gel was recovered using the kit. The TtgRH67A fragment after ordinary PCR was recovered.

[0075] (6) Enzyme digestion reaction: Digest the vector plasmid and the target DNA using the required restriction endonuclease;

[0076] The enzyme digestion system for the vector plasmid is shown in Table 3.

[0077] Table 3. Enzyme digestion system for vector plasmids

[0078]

[0079] The enzyme digestion system for the target DNA is shown in Table 4.

[0080] Table 4. Enzyme digestion system for target DNA

[0081]

[0082] Enzyme digestion in a 37℃ water bath for 40 minutes.

[0083] (7) Recovery: Perform agarose gel electrophoresis, add the enzyme digestion product to the sample well, run the gel at 120V for 20min; after the electrophoresis is completed, take out the gel, observe whether there are bands under UV light and observe the size of the target DNA fragment in nucleic acid electrophoresis, use the kit to recover the DNA in the gel, and recover the TtgRH67A fragment after ordinary PCR.

[0084] (8) Ligation: Prepare 6 ligation systems according to the ratio of vector to target gene 1:5, and use T4 DNA ligase to ligate the mutated TtgRH67A gene and PGK vector fragment, and incubate at 22℃ for 2h.

[0085] (9) Transformation: Gently add the ligation product to 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 37℃ for 5 min, and then incubate on ice for 2 min. After returning to room temperature, add 500 μL of LB medium (ammonia-free) and activate on a shaker at 30℃ and 180 rpm for 1 h. Centrifuge all 6 systems and discard the supernatant. Resuspend the resuspended liquid in a total of 1 mL of LB medium (ammonia-free). Transfer 250 μL of the resuspended liquid to a 15 mL centrifuge tube, add 5 mL of LB medium (ammonia-free) to the centrifuge tube, and incubate on a shaker at 30℃ and 180 rpm for 5-6 h. Then transfer the liquid from the 15 mL centrifuge tube to 4 conical flasks, add 200 mL of LB medium (ammonia-free), and incubate on a shaker at 30℃ and 180 rpm for 12-14 h.

[0086] (10) Serial dilution counting: After 12-14 hours, a portion of the bacterial culture was taken from the mixture of the four conical flasks, and 100 μL was taken for serial dilution counting: 100 μL of bacterial culture was added to 900 μL of LB medium, mixed well, and then another 100 μL of bacterial culture was taken and added to 900 μL of LB medium for serial dilution, to obtain a 10-fold dilution. 4 -10 9 After the bacterial culture was prepared, 100 μL of each culture was spread onto LB agar plates. The plates were then inverted and incubated overnight at 37°C. After 18 hours, the bacterial count was performed and diluted 10⁻⁶ times. 4 -10 9 The number of colonies after each run was used to estimate the order of magnitude of the mutant library. The number of mutants in one run of the mutant library is approximately 1.5 × 10⁻⁶. 11 .

[0087] (11) Plasmid preparation and sequencing verification: 18h, use a pipette tip to pick up and dilute 10 5 Twenty-four single colonies from the plate were added to LB medium (ampicillin resistant) and incubated overnight by shaking. Twenty-four plasmids were extracted, and their accuracy was verified by agarose gel electrophoresis with XbaI and SalI restriction enzymes, followed by sequencing verification. The mutation rate of the first-round mutant library was estimated based on the mutation frequency of the 24 plasmids. The mutation rate of the mutants in the first-round mutant library was approximately 3.54 / kb.

[0088] (12) Large-scale plasmid extraction and strain preservation: Centrifuge 10 mL of bacterial culture from the mixture in 4 conical flasks, resuspend in 3 mL LB, and add 3 mL of 30% glycerol for preservation. Finally, extract plasmids from the mixture in the remaining 4 conical flasks according to the instructions of the endotoxin-free plasmid large-scale extraction kit. Detect the plasmid concentration using Nanodrop and store at -20℃ for later use. Name the plasmid PGKC-EGFP-TtgRH67A-library1.

[0089] (13) Construction of the second-round error-prone TtgRH67A mutant library: The above-mentioned PGKC-EGFP-TtgRH67A-library1 plasmid was subjected to a second mutation using an error-prone PCR kit. The second mutation can increase the mutation rate of the mutant library. The product recovered from the ordinary PCR gel obtained from the first-round mutant library was used as the DNA template for error-prone PCR again. After gel recovery, ordinary PCR was performed to obtain the PGKC-EGFP-TtgRH67A-library2 plasmid in the same way. Its order of magnitude is 2.4 × 10⁻⁶. 11 The mutation rate is 6.94 / kb.

[0090] (14) Construction of a three-round error-prone TtgRH67A mutant library: The above-mentioned PGKC-TtgRH67A-library2 plasmid was further mutated using an error-prone PCR kit. The product recovered from the ordinary PCR gel obtained in the second round of mutant library was used as the DNA template for error-prone PCR again. After gel recovery, ordinary PCR was performed to obtain the PGKC-EGFP-TtgRH67A-library3 plasmid in the same way. Its order of magnitude is 8.8 × 10⁻⁶. 11 The mutation rate is 11.02 / kb.

[0091] 2. Lentiviral infection of Jurkat cells and detection of their response to the small molecule ligand resveratrol.

[0092] 2.1 Lentiviral Packaging:

[0093] (1) Cell preparation: Take out the frozen 293ft cells and quickly place them in a 37℃ water bath until completely thawed. Prepare complete culture medium: 90% high glucose DMEM medium, 10% fetal bovine serum and 1% penicillin-streptomycin (100×); one day before packaging, ensure that the density of 293 cells per dish reaches 1×10⁻⁶. 7 indivual.

[0094] (2) Change the medium: Change the medium 2 hours before packaging the virus to ensure that each dish contains 8-10 mL of culture medium.

[0095] (3) System preparation: Single-plate system: 2 μg PACK2 plasmid, 6 μg PACK8 plasmid, 8 μg PGKC-EGFP-TtgRH67A-library1 target plasmid, 2 mL of DN (dihydrogenase), and 35 μL PEI. First add the DN solution, then add the plasmid, and finally mix with PEI. Use a 1 mL pipette tip to mix thoroughly and let stand for 10 min. If you need to package 3 plates of virus, prepare the system to 3.5 times its original volume. Add the excess solution evenly to each culture plate, adding 2 mL of the system to each plate.

[0096] (4) Culture and virus collection: After adding the system, the cells were placed in an incubator and cultured for 72 hours. Then, the cell supernatant was collected. Using a 1 mL pipette, the mixture was slowly and evenly added to the surface of the prepared 293ft cells. The cells were carefully transferred to a cell culture incubator. 293ft cells are adherent cells. When the virus was packaged, the confluence of 293ft cells in the culture dish was about 70%-80%. The target plasmid contained EGFP. The observation of green fluorescence under an inverted fluorescence microscope indicated that the target plasmid had been successfully transferred into the 293ft cells and expressed. The green fluorescence coverage rate was 60% or higher, indicating that the packaging effect was good. The packaged virus supernatant could be collected.

[0097] (5) Preliminary centrifugation and filtration: The collected supernatant was centrifuged at 3000 rpm and 4℃ for 15 min, and the virus supernatant was retained and filtered using a 0.45 μm filter membrane to remove bacteria and cell debris;

[0098] (6) Precipitation of PEG8000: Add 0.3 times the volume of PEG8000 to the supernatant, shake well to mix, and let stand in a refrigerator at 4°C for more than 3 hours (overnight is not recommended).

[0099] (7) High-speed centrifugation: Centrifuge at 3000g (maximum speed of centrifuge) for 45min-1h (approximately 30mL per tube);

[0100] (8) Discarding supernatant and resuspending: After centrifugation, discard the supernatant and centrifuge at maximum speed for 2 minutes to completely remove the residual supernatant. Resuspend the virus with PBS or use differential centrifugation. Aliquot the resuspended virus into 50 μL / tube and store at -80℃ to avoid repeated freeze-thaw cycles.

[0101] 2.2. Lentiviral infection of Jurkat cells

[0102] (1) Thawing frozen Jurkat cells: Take out the frozen Jurkat cells and quickly place them in a 37°C water bath until completely thawed. Add 3 mL of complete culture medium to a 15 mL centrifuge tube, transfer the completely thawed Jurkat cell suspension into the tube, centrifuge at 1000 rpm for 7 min, discard the supernatant, and prepare the complete culture medium: 90% 1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin (100×), add 1 mL of complete culture medium to resuspend the liquid, and plate the thawed Jurkat cells into plates, 1×10⁶ cells per well in a 6-well plate. 7 cell.

[0103] (2) Take the packaged Library-1 and 2 groups of lentivirus suspensions, place them at 4℃ to thaw, add 6μL, 12μL and 28μL of one group of virus to each well, mix in a figure-eight pattern, set up two replicates, and add culture medium to 1.5mL.

[0104] (3) Transfer the well plate to an incubator at 37°C and 5% CO2 and continue culturing. Replenish the culture medium to 3 mL every 2 days and observe the cell growth status under a microscope.

[0105] (4) On the 3rd day after Jurkat cells were infected with lentivirus, the transfection rate of Jurkat cells was detected. Cells were blown evenly in each well, and 300 μL of cell suspension was taken. After centrifugation at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 300 μL of PBS. EGFP fluorescence was detected by flow cytometry.

[0106] 2.3 Response of Jurkat cells to the small molecule ligand resveratrol after transfection with the mutant library plasmid

[0107] (1) The Jurkat cells packaged with lentivirus were plated in 24-well plates, with 4 × 10⁶ cells per well. 5 cell.

[0108] (2) Prepare a 50 mM solution of resveratrol powder with DMSO. Divide the 24-well plate into a blank control group, a 0 μM resveratrol group, a 20 μM resveratrol group and a 50 μM resveratrol group. Dilute the prepared 50 mM solution 10 times. Add 4 μL of resveratrol solution and 6 μL of DMSO to the 20 μM group, add 10 μL of resveratrol solution to the 50 μM group, add 10 μL of DMSO to the 0 μM resveratrol group, and do not add DMSO to the blank control group. Add culture medium to each well to 1 mL.

[0109] (3) Transfer the well plate to an incubator at 37°C and 5% CO2, continue culturing and observe the cell growth status under a microscope.

[0110] (4) Three days later, the fluorescence intensity of Jurkat cells in each group was observed under a fluorescence microscope and flow cytometry was performed. Cells were blown evenly in each well, 200 μL of cell suspension was taken, centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. EGFP fluorescence was detected by flow cytometry.

[0111] All data obtained from flow cytometry assays were processed using FlowJo V10 software.

[0112] 3. Flow sorting:

[0113] (1) Sample preparation: Add resveratrol to the Jurkat cell culture flask infected with lentivirus to make the resveratrol concentration 50 μM. Transfer the culture flask to an incubator at 37℃ and 5% CO2 and culture the cells for 36 h. Then transfer all the cells and culture medium to centrifuge tubes and centrifuge at 1000 rpm for 5 min at room temperature. Remove the supernatant and prepare the flow cytometry collection solution: 90% 1640 medium, 10% fetal bovine serum and 2% penicillin, streptomycin and gentamicin triple antibodies (100×).

[0114] (2) Cell collection: Combine the suspended cells in the supernatant, wash twice with pre-cooled PBS, and adjust the cell concentration to 2×10⁻⁶ cells with cell sorting medium containing 2% penicillin, streptomycin, gentamicin, and triple antibodies (100×). 6 Cells / mL, add 5μL of 7-AAD staining to every 100μL of cell suspension, incubate at room temperature in the dark for 15min. After incubation, add an appropriate amount of buffer to the cell suspension to dilute the unbound staining solution, and then transfer the cell suspension to the sample tube of the flow cytometer.

[0115] (3) Initial screening: Cells were cultured with 50 μM resveratrol for 36 h and sorted using CytoFLEX SRT flow cytometer. Dead cells (7-AAD positive, emitting red fluorescence) and live cells (7-AAD negative, no red fluorescence) were distinguished based on fluorescence signal intensity. Live cells were first selected by flow cytometer. Plasmids PGKC-EGFP-TtgRH67A-library1-3 all carry EGFP fluorescent label. Cells that strongly express EGFP were selected first, and the screened cells were collected into centrifuge tubes containing 5 mL of collection solution.

[0116] (4) Cell culture: Centrifuge 5 mL of the collected solution containing the selected cells in the centrifuge tube at 1000 rpm for 7 min, discard the supernatant, and prepare the triple antibody complete culture medium: 90% 1640 medium, 10% fetal bovine serum and 2% penicillin, streptomycin and gentamicin triple antibodies (100×), add 1 mL of triple antibody complete culture medium to resuspend the liquid, transfer it to a 12-well plate for culture, transfer the 12-well plate to a 37℃, 5% CO2 incubator, continue to culture, replenish the culture medium every 2 days and observe the cell growth status under a microscope, and transfer it to a small bottle for culture after the cell status improves.

[0117] (5) Secondary screening: After culturing cells for 5-7 days and the cell viability is higher than 90%, a second screening is carried out. After culturing in a complete medium containing three antibodies without resveratrol for 36 hours, flow cytometry is performed to select 7-AAD negative live cells and cells that weakly express EGFP. The screened cells are collected and cultured for a longer period.

[0118] (6) Three rounds of screening: In the third round of screening, cells that strongly express EGFP were selected by culturing with 50 μM resveratrol for 36 h.

[0119] 4. Detect the response of flow-cytosorized cells to the small molecule ligand resveratrol.

[0120] (1) After each round of cell sorting, the cells were plated into 24-well plates, with 4 × 10⁶ cells per well. 5 cell.

[0121] (2) The 24-well plate was divided into a blank control group, a resveratrol 20μM group and a resveratrol 50μM group. The corresponding volume of resveratrol was added to each well and the culture medium was replenished to 1 mL.

[0122] (3) Transfer the well plate to an incubator at 37°C and 5% CO2, continue culturing and observe the cell growth status under a microscope.

[0123] (4) Three days later, the fluorescence intensity of Jurkat cells in each group was observed under a fluorescence microscope and flow cytometry was performed. Cells were blown evenly in each well, 200 μL of cell suspension was taken, centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. EGFP fluorescence was detected by flow cytometry.

[0124] 5. Screening for TtgRH67A mutant individuals from the third-round screening mutation library.

[0125] (1) Extraction of genomic DNA: After sorting, allow the cells to recover in the culture medium. Once the cell viability is higher than 90%, extract genomic DNA from the cells in the fourth round of screening according to the instructions of the DNA extraction kit. The integrity of the DNA is detected by agarose gel electrophoresis. The absorbance of the DNA at wavelengths of 260 nm and 280 nm is measured using a UV spectrophotometer. The OD260 / OD280 ratio is calculated to evaluate the purity of the DNA. The pure DNA ratio should be between 1.8 and 2.0.

[0126] (2) PCR amplification: In order to isolate individual mutants from the genomic DNA library, primers are used to amplify the gene by PCR. The specific steps are the same as step (4) in “1. Constructing the TtgRH67A mutant library”.

[0127] (3) Detect and recover the product. Identify the DNA fragment by agarose gel electrophoresis under UV light. Add the PCR product to the sample well and run the gel at 120V for 20min. After electrophoresis, remove the gel and observe for bands under UV light. Observe the size of the DNA fragment and use the kit to recover the DNA in the gel. Recover the PCR product.

[0128] (4) Enzyme digestion reaction: The vector plasmid and the target DNA are digested using the required restriction endonucleases XbaI and SalI.

[0129] (5) Recovery: Perform agarose gel electrophoresis, add the enzyme digestion product to the sample well, run the gel at 120V for 20min; after the electrophoresis is completed, take out the gel, observe whether there are bands under UV light and observe the size of the target DNA fragment in nucleic acid electrophoresis, and use the kit to recover the DNA in the gel.

[0130] (6) Ligation: Prepare a ligation system according to the ratio of vector to target gene 1:5, and use T4 DNA ligase to ligate the mutant TtgRH67A gene and PGK-EGFP vector fragment after the fourth round of screening, and incubate at 22℃ for 2h.

[0131] (7) Transformation: Gently add the ligation product into 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 37℃ for 5 min, and then incubate on ice for 2 min. After returning to room temperature, add 500 μL of LB medium (ampicillin-free) and activate on a shaker at 30℃ and 180 rpm for 1 h.

[0132] (8) Spreading: Spread 100 μL of LB medium onto a plate, then invert the plate and grow it overnight in a 37°C incubator.

[0133] (9) Plasmid preparation and sequencing verification: 18h, use a pipette tip to pick up and dilute 10 5 Twenty-four single colonies from a plate were added to LB medium (ampicillin resistant) and incubated overnight by shaking. The accuracy was then verified by agarose gel electrophoresis with XbaI and SalI restriction enzymes, followed by sequencing verification. A total of 102 plasmids were extracted in batches, of which 24 plasmids were correctly ligated and contained mutations.

[0134] 6. Screening for individuals with higher responsiveness to the small molecule ligand resveratrol from TtgRH67A mutant individuals.

[0135] (1) Cell plating: P293 cells were resuspended in complete culture medium, and the cells were counted. Cell suspension was added to each well of a 24-well plate to make the number of cells per well approximately 1 × 10⁻⁶. 5 Add 500 μL of preheated complete culture medium to each well, then gently shake the culture plate to distribute the cells evenly. Place the 24-well plate in a 37°C incubator for incubation.

[0136] (2) Liposome transient transfection: Before transfection, preheat the serum-free medium (Opti-MEM) at 37°C. In sterile centrifuge tubes, perform the following operations: Tube A: Take 2 μg of plasmid, add 50 μL of preheated serum-free medium, and mix gently. Tube B: Take an appropriate amount of liposome transfection reagent (according to the instructions, Lipofectamine 3000 is generally used for 1 μg of plasmid transfection with 3 μL of reagent), add 50 μL of preheated serum-free medium, mix gently, and incubate at room temperature for 5 min. Mix the above solutions in tube A containing plasmid and tube B containing liposomes, mix gently, and incubate at room temperature for 20 min to form a transfection complex. Remove the 24-well plate from the incubator, add the prepared transfection complex dropwise to the corresponding wells, gently shake the plate to mix, and return the 24-well plate to the 37°C, 5% CO2 incubator for overnight incubation. Two replicates are set up for each of the 24 mutant plasmids.

[0137] (3) Flow cytometry detection of transfection rate: 24 h later, the cells in each well were centrifuged in a centrifuge tube (1000 rpm) for 10 min, the culture medium containing the transfection complex was aspirated and discarded, and the cells were resuspended in 100 μL of complete culture medium. 50 μL of cell suspension in each well was transferred to an EP tube, and after resuspending in PBS, the fluorescence intensity of the cells was detected by flow cytometer.

[0138] (4) Treatment with resveratrol: The remaining 50 μL of cell suspension was cultured for a longer period. The 24-well plate was divided into a blank control group, a 0 μM resveratrol group, a 20 μM resveratrol group and a 50 μM resveratrol group. The prepared 50 mM solution was diluted 10 times. 4 μL of resveratrol solution and 6 μL of DMSO were added to the 20 μM group. 10 μL of resveratrol solution was added to the 50 μM group. 10 μL of DMSO was added to the 0 μM resveratrol group. No DMSO was added to the blank control group. The culture medium was added to each well to 1 mL and 1000 μL of 1640 complete culture medium was added to each well. The cells were then cultured at 37 °C in a 5% CO2 incubator for 24 h.

[0139] (5) Flow cytometry detection: After 24 hours, the 24-well plate was removed from the incubator, and the cell suspension in each well was transferred to a flow cytometer. The fluorescence intensity of the cells was detected by the flow cytometer. A suitable detection channel was set up, data was collected, and mutant individuals with high resveratrol response were screened out.

[0140] 7. Lentiviral packaging and transfection:

[0141] (1) Select mutant individuals with high resveratrol response, package them with lentivirus and transfect them into Jurkat cells. Other steps are the same as in “2.2. Lentiviral infection of Jurkat cells”.

[0142] (2) Treatment with resveratol: Further confirm the response of Jurkat cells transfected with mutant individuals to the small molecule ligand resveratol, and prepare Western Blot cell samples. Other steps are the same as those in “2.3. Response of Jurkat cells transfected with mutant library plasmid to the small molecule ligand resveratol”.

[0143] (3) Detection of fluorescence intensity in PGK-EGFP-TtgRH67A cells:

[0144] In the short-term resveratrol concentration increase assay, PGK-EGFP-TtgRH67A cells were divided into 5 groups on day 5 after transfection. They were treated with 0, 5, 10, 15, 20, 30, 40, 50, and 100 μM resveratrol for 24 h, respectively. After centrifugation, the cells were resuspended in PBS for analysis. In the resveratrol elimination assay, 20 μM resveratrol was added to the culture medium on day 3 after PGK-EGFP-TtgRH67A cell transfection. After 24 h, cells were collected, washed twice with PBS, and cultured in resveratrol-free medium. Cells were collected at 0, 1, 2, 4, 6, 16, and 24 h for analysis.

[0145] 8. Western Blot qualitative comparison of mutant individuals with proteins:

[0146] (1) Sample preparation: Prepare cell samples for Western blotting, seed Jurkat cells, count cells, and add cell suspension to each well of a 6-well plate to make the cell count approximately 5 × 10⁶ cells per well. 5 In one group, resveratrol was added to a concentration of 50 μM and withdrawn after 24 hours. In another group, no resveratrol was added. In the last group, resveratrol was added when the first group withdrew the drug and the cells were cultured for 24 hours. Three groups of cells were obtained: one group without drug, one group with drug for 24 hours, and one group with drug for 24 hours and then withdrawn for 24 hours.

[0147] Centrifuge the cells and discard the cell culture medium. Wash the cells twice with pre-chilled PBS to remove residual culture medium. Add 200 μL of pre-chilled RIPA lysis buffer (containing protease inhibitors and 10 μL PMSF), mix well, and lyse on ice for 30 min. Transfer the lysis buffer to an EP tube, centrifuge at 15000g for 15 min at 4°C, and transfer the supernatant to a new EP tube. This is the total cell protein extract, which can be stored at -80°C for later use.

[0148] (2) Protein quantification: Protein quantification was performed using the BCA method. Following the instructions of the BCA protein quantification kit, the standard protein (BSA) of known concentration was serially diluted to prepare a standard curve. The protein sample to be tested was appropriately diluted and added to a 96-well plate along with the standard. BCA working solution was added, and the plate was incubated at 37°C for 30 min. The absorbance at 562 nm was measured using a microplate reader. The protein concentration of the sample to be tested was calculated based on the standard curve.

[0149] (3) SDS-PAGE gel electrophoresis: Select a separating gel concentration of 10% according to the molecular weight of the target protein. Add acrylamide solution, Tris-HCl buffer (pH 8.8), SDS, ammonium persulfate, TEMED, etc. in sequence according to the ratio, mix quickly, pour the gel, leave space for the stacking gel, seal the gel with water, and wait for the separating gel to polymerize (about 30-60 min). Remove the sealing liquid, prepare the stacking gel (usually 5%), add the stacking gel solution, insert the comb, and wait for the gel to polymerize (about 30 min).

[0150] (4) Sample loading and electrophoresis: Mix the protein sample with 5×SDS loading buffer at a ratio of 4:1 and boil at 95℃ for 10 min to denature the protein. Adjust the loading amount according to the protein quantification results, loading 10-30 μg of protein per well, and add protein marker as a molecular weight reference. Mount the gel into the vertical electrophoresis tank, add electrophoresis buffer, and electrophores at a constant voltage of 60V until the protein enters the separating gel (about 30 min). Then adjust the voltage to 120V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the gel (about 1-2 h).

[0151] (5) Wet transfer: Place the gel and PVDF membrane sequentially into the transfer clamp, sandwiched between the sponge and filter paper, taking care to avoid air bubbles. Place the transfer clamp into the transfer tank containing transfer buffer and transfer at a constant current of 100V for 2 hours under ice bath conditions.

[0152] (6) Blocking: After the transfer is completed, the PVDF membrane is placed in a solution containing 5% milk powder and incubated overnight on a decolorizing shaker at room temperature to block the non-specific binding sites on the membrane.

[0153] (7) Primary antibody incubation: After blocking, wash the membrane three times with TBST (Tris-buffered saline containing 0.1% Tween-20), 5-10 min each time. Dilute the primary antibody with blocking buffer according to the dilution ratio recommended in the primary antibody instructions. Add the diluted primary antibody to the incubation chamber, place the membrane in the chamber, and incubate overnight at 4°C or at room temperature.

[0154] (8) Secondary antibody incubation: Recover the primary antibody and wash the membrane three times with TBST, 5-10 min each time. Dilute the secondary antibody with blocking buffer according to the dilution ratio recommended in the secondary antibody instructions. Add the diluted secondary antibody to the incubation box and incubate slowly with shaking on a decolorizing shaker at room temperature for 2 h.

[0155] (9) Colorimetric Development and Result Analysis: Chemiluminescence (ECL): Wash the membrane three times with TBST, 5-10 min each time. According to the ECL luminescence solution instructions, mix equal volumes of solution A and solution B, and add them evenly to the membrane, reacting for 1-5 min. Expose the membrane to a chemiluminescence imaging system for imaging, and analyze the expression level and molecular weight of the target protein based on the band brightness and position. Use software such as ImageJ for grayscale analysis to perform semi-quantitative analysis of protein expression.

[0156] 9. Regulation of different TtgR-EGFP mutant individuals in Jurkat T and other cell lines

[0157] (1) Viral infection and cell model construction: PGK-EGFP-TtgRH67A-1-5, PGK-EGFP-TtgRH67A-3-14 and other viruses were packaged with lentiviruses and infected Jurkat cells to construct cell models infected with several mutants.

[0158] (2) Time-dependent experiment: “On-Off-On” and “Off-On-Off” modes were set up. Jurkat cells infected with mutants were treated with 50 μM resveratrol and 2 μg / mL doxycycline in different time sequences and the drug response (change in fluorescent labeling) and the withdrawal effect (recovery of cell state after drug withdrawal) were compared.

[0159] (3) Dose-dependent experiment: Set up resveratrol concentration groups of 0, 10, 20, 30, 40, 50, 60 and 100 μM, and apply them to cells infected with mutants. Observe cell responsiveness (fluorescent labeling, stability) and withdrawal efficiency (state changes after drug withdrawal). Record fluorescence changes under a fluorescence microscope and analyze the dose-effect relationship.

[0160] (4) Transfection experiments of different cell lines: 293FT, HeLa, HT1080 and Jurkat cell lines were selected and transfected with the same amount of virus. 0μM and 50μM resveratrol treatment groups were set up, and the fold change of EGFP induction was detected to compare the differences in cell line response.

[0161] 10. Regulation of TtgR-1-5-EGFP / TtgR-1-5-C-EGFP mutant individuals by different small molecule ligands

[0162] (1) Viral infection and cell model construction: Jurkat cells were infected with lentiviruses PGK-EGFP-TtgRH67A-1-5 to construct cell models infected with several mutants.

[0163] (2) Time-dependent experiment: Jurkat cells infected with mutants were given and withdrawn with doxycycline at 4 μg / mL, chloramphenicol at 20 μg / mL, phloretin at 50 μM and quercetin at 50 μM in different time sequences. The drug response (change in fluorescent labeling) and the withdrawal effect (recovery of cell state after withdrawal) were compared.

[0164] 11. Regulation of primary T cells by different TtgR-EGFP mutant individuals

[0165] (1) Viral infection and cell model construction: Lentiviral viruses such as PGK-EGFP-TtgRH67A-1-5 and PGK-EGFP-TtgRH67A-3-14 were packaged and infected primary T cells to construct cell models infected with several mutants.

[0166] (2) Time-dependent experiment: “On-Off-On” and “Off-On-Off” modes were set up, and 50 μM resveratrol was used in both modes. Jurkat cells infected with mutants were added and withdrawn in different time sequences to compare drug response (change in fluorescent labeling) and withdrawal effect (recovery of cell state after withdrawal).

[0167] (3) Dose-dependent experiment: Set up resveratrol concentration groups of 0, 10, 20, 30, 40, 50, 60 and 100 μM, and apply them to cells infected with mutants. Observe cell responsiveness (fluorescent labeling, stability) and withdrawal efficiency (state changes after drug withdrawal). Record fluorescence changes under a fluorescence microscope and analyze the dose-effect relationship.

[0168] Experimental results:

[0169] 1. Construction of the PGK-EGFP-TtgRH67A mutant library

[0170] In this embodiment, TtgR was selected as a transcriptional repressor from Pseudomonas, whose small molecule ligands include antibiotics (such as chloramphenicol and tetracycline) and small molecules derived from plants (such as quercetin, naringenin, and phloretin). The PuC57-TtgRH67A sequence was synthesized according to the DNA sequence in a published article. The above PuC57-TtgRH67A plasmid was mutated using an error-prone PCR kit. After gel recovery, the product was subjected to conventional PCR. The mutated TtgRH67A was digested with XbaI and SalI and ligated into the PGKC vector plasmid to construct the mutant library plasmid PGK-EGFP-TtgRH67A-library1-3. The resulting PGKC-EGFP-TtgRH67A-library1 plasmid was approximately 1.5 × 10⁻⁶. 11The mutation rate is approximately 3.54 / kb; the size of the PGKC-EGFP-TtgRH67A-library2 plasmid is approximately 2.4 × 10⁻⁶. 11 The mutation rate is approximately 6.94 / kb; the order of magnitude of the PGKC-EGFP-TtgRH67A-library3 plasmid is approximately 8.8 × 10⁻⁶. 11 The mutation rate is approximately 11.02 / kb.

[0171] 2. Screening for mutant individuals in PGK-EGFP-TtgRH67A-library1-3 that are highly sensitive to resveratrol.

[0172] Lentiviral packaging of PGK-EGFP-TtgRH67A-library1-3 was used to infect Jurkat cells, and their response to the small molecule ligand resveratol was detected. Red represents uninfected Jurkat cells, blue represents Jurkat cells infected with PGKC-EGFP-TtgRH67A-library1-3 virus, and green represents Jurkat cells infected with PGKC-EGFP-TtgRH67A-library1-3 virus and then treated with resveratol to a final concentration of 50 μM.

[0173] Jurkat cells infected with PGK-EGFP-TtgRH67A-library1-3 lentivirus were screened by FACS flow cytometry. Through three rounds of alternating "drug stimulation-withdrawal recovery" screening, a mutant cell population with significantly enhanced resveratrol response was successfully enriched. After the first round of resveratrol treatment (final concentration 50 μM) for 24 h, strongly EGFP-positive cells were sorted by flow cytometry. After 24 h of culture following drug withdrawal, weakly EGFP-positive cells were screened, and then treated again with resveratrol (final concentration 50 μM) for 24 h, followed by strongly EGFP-positive cell sorting by flow cytometry. The final mutant cell population showed significantly increased EGFP expression under resveratrol stimulation compared to the initial cells, indicating a higher sensitivity and responsiveness to resveratrol.

[0174] By extracting plasmid DNA, 100 colonies were selected after plate transformation, plasmids were extracted, and the plasmids were transiently transformed into Jurkat cells using lipo3000. The responsiveness of each plasmid in the PGK-EGFP-TtgRH67A-library1-3 mutant library to a final concentration of 50 μM resveratrol after viral transfection was detected, and individuals that were more sensitive to resveratrol were screened out.

[0175] After multiple rounds of comparison, mutant individuals 1-5, 2-2, 2-21, and 3-14 were all unstable domain mutants with good responsiveness. When resveratrol was added, the mutants became stable and expressed high levels of fluorescent labeling. When resveratrol was removed, the mutants became unstable and expressed low levels of fluorescent labeling. Among them, mutant individuals 1-5 and 3-14 showed more outstanding performance and better responsiveness to resveratrol.

[0176] like Figure 1 As shown, Lipo3000 transfection verification: Compared with wild type (WT), the fluorescence intensity of mutant individuals 1-5, 2-2, 2-21, 2-22 and 3-14 was significantly increased after the addition of a final concentration of 50 μM resveratrol, indicating that these clones are more sensitive and more stable in response to the ligand.

[0177] 3. Obtain individuals with unstable domain mutations

[0178] Based on the above experiments, unstable domain mutants PGK-EGFP-TtgRH67A-1-5, 2-2, 2-21, and 3-14 were obtained, and unstable domain mutants PGK-EGFP-TtgRH67A-1-5+3-14, 1-5+3-14-C-terminus, and 1-5-C-terminus were synthesized. The C-terminus (carboxyl terminus) of the prokaryotic transcription factor TtgR is a ligand-responsive domain, possessing ligand-binding ability and specifically sensing and binding to various small molecule ligands in the environment. To reduce immunogenicity, PGK-EGFP-TtgRH67A-1-5+3-14-C-terminus and 1-5-C-terminus were mutants consisting only of the C-terminus. Lentiviral packaging of PGK-EGFP-TtgRH67A-1-5, 2-2, 2-21, 3-14, 1-5+3-14, 1-5+3-14-C-terminus, 1-5-C-terminus and wild-type WT TtgRH67A was used to infect Jurkat cells to observe the responsiveness of different mutant individuals to different small molecule ligands.

[0179] 3.1 Time dependence of resveratrol and doxycycline on different mutant individuals:

[0180] Using "on-off-on" and "off-on-off" modes, with resveratrol at a final concentration of 50 μM and doxycycline at a final concentration of 2 μg / ml, the responses and withdrawal effects of several mutants (PGK-EGFP-TtgRH67A-1-5, 2-2, 2-21, 3-14, 1-5+3-14, 1-5+3-14-C-terminus, 1-5-C-terminus) and wild-type WT TtgRH67A to Jurkat cells were compared under different treatment sequences. All mutants showed some responsiveness; fluorescence intensity increased with the addition of resveratrol and decreased with the removal of resveratrol. Figure 2 As shown in A and B, PGK-EGFP-TtgRH67A-1-5 and its ligand-binding domain 1-5-C-terminus showed good responses, while the unstable domains 1-5+3-14 and 1-5+3-14-C-terminus synthesized based on 1-5 and 3-14 showed poor responses. The C-terminus of the ligand-binding domain may play a key role in maintaining transcription factor function. Mutants containing only the C-terminus (such as 1-5-C-terminus) and mutants with the complete TtgRH67A structure showed comparable efficacy in drug response and other functional aspects. To reduce immunogenicity, the transcription factor sequence can be truncated and optimized, retaining only its C-terminus (ligand-response domain).

[0181] 3.2 Dose-dependent effects of resveratrol on individuals with different mutations:

[0182] Eight resveratrol concentration groups (0, 10, 20, 30, 40, 50, 60, and 100 μM) were established. At different doses, the cellular responsiveness and retreat efficiency differed after mutant infection. Fluorescence intensity increased with increasing resveratrol concentration and decreased with decreasing resveratrol concentration. Fluorescence microscopy revealed fluorescence changes in different cell groups at different resveratrol concentrations; fluorescence intensity increased with increasing resveratrol concentration and decreased with decreasing resveratrol concentration.

[0183] 3.3 Dose-dependent Western blot results of resveratrol in individuals with different mutations:

[0184] Western blot results showed that, for all the selected unstable domains, the band of the fusion protein TtgRH67A-EGFP became significantly brighter at a final resveratrol concentration of 50 μM. The band darkened significantly after the addition and subsequent removal of resveratrol. Figure 3 As shown in A and B in the diagram.

[0185] The eGFP band signal in groups 1-5 was stronger than that in groups 3-14, and the GADPH (internal control) band was relatively uniform. This indicates that after 24 hours of resveratrol addition and 24 hours of drug withdrawal, mutant individuals 1-5 showed higher eGFP expression and better response to drug withdrawal.

[0186] 3.4 Transfection results of different cell lines:

[0187] In addition to infecting Jurkat cells, three cell lines—293FT, HeLa, and HT1080—were transfected with the same viral load and treated with 0 μM and 50 μM resveratrol (RES) to detect the EGFP fold induction. The unstable domain of PGK-EGFP-TtgRH67A-1-5 in this experiment showed certain regulatory effects in multiple cell lines, such as... Figure 4 As shown.

[0188] 3.5 Regulation of TtgR-1-5-EGFP in mutant individuals by different small molecule ligands:

[0189] Testing revealed that doxycycline, chloramphenicol, phlorizin, and quercetin all significantly regulated the TtgR-1-5-EGFP mutant: fluorescence intensity was significantly enhanced after drug administration and significantly weakened after drug withdrawal. Figure 5 As shown in the figure, resveratrol and doxycycline showed the best fluorescence response, with their fluorescence intensity variation and response stability being superior to other ligands. This provides crucial data support for further analysis of the interaction mechanism and functional applications of small molecule ligands and mutants.

[0190] The amino acid sequence of TtgRH67A is as follows:

[0191] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLDMLRLSPALRK, as SEQ IDNO: 1 is shown.

[0192] The amino acid sequences of TtgRH67A mutants 1-5 are as follows:

[0193] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLYMLRLSPALRK, as in SEQ IDNO: 2 shown.

[0194] The amino acid sequence of the TtgRH67A mutant 1-5-C-terminus is as follows:

[0195] FNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTRRINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLYMLRLSPALRK, as shown in SEQ ID NO: 3.

[0196] The amino acid sequence of TtgRH67A mutant 2-2 is as follows:

[0197] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDRHKGITLALANAVRRGLLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLDMLRLSPALRK, as SEQ IDNO: 4 is shown.

[0198] The amino acid sequence of TtgRH67A mutant 2-21 is as follows:

[0199] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVYLLVDVEKWVDTGLDMPRLSPALRK, as SEQ IDNO: 5 shown.

[0200] The amino acid sequence of TtgRH67A mutant 3-14 is as follows:

[0201] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELSGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKWEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDADRAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLDMLRLSPALRK, as SEQ IDNO: 6.

[0202] The amino acid sequence of the TtgRH67A mutant 1-5+3-14 is as follows:

[0203] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELSGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKWEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDADRAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLYMLRLSPALRK, as in SEQ IDNO: 7 is shown.

[0204] The amino acid sequence of the TtgRH67A mutant 1-5+3-14-C-terminus is as follows:

[0205] FNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTRRINEILHHKWEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDADRAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLYMLRLSPALRK, as shown in SEQ ID NO: 8.

[0206] The amino acid sequence of TtgRH67A mutant 2-10 is as follows:

[0207] MVRRTKEEAQETRAQIFEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSVAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAFLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLDMLRLSPALRK, as in SEQ IDNO: 9 is shown.

[0208] The amino acid sequence of TtgRH67A mutant 3-1 is as follows:

[0209] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKWEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDADRAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLDMLRLSPALRK, as SEQ IDNO: 10 shown

[0210] The amino acid sequence of TtgRH67A mutant 3-2 is as follows:

[0211] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDADRAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWLDTGLDMLRLSPALK, as in SEQ IDNO: 11 shown

[0212] The amino acid sequence of EGFP is:

[0213] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK, as SEQ ID NO: 12 is shown.

[0214] EGFP is linked to TtgRH67A or its mutants via the LEVD adapter, with EGFP located at the N-terminus of TtgRH67A or its mutants. TtgRH67A mutants 1-7 are formed by replacing the W amino acid residue at position 178 of the TtgRH67A sequence with C.

[0215] Example 2

[0216] 1. Construction of CAR-T cells with TtgR mutant:

[0217] BBZ19-CAR-TtgRH67A-1-5-T cells and BBZ19-CAR-TtgRH67A-3-14-T cells were constructed from mutant individuals 1-5 and 3-14 screened in Example 1.

[0218] 2. Lentiviral packaging:

[0219] This step in this embodiment is the same as the "2.1 Lentiviral Packaging" step in Embodiment 1.

[0220] 3. Preparation of T lymphocytes:

[0221] (1) Take a 50mL centrifuge tube, add Ficoll and fresh blood in a ratio of 4:3, centrifuge at 400g speed and 1 for 40min.

[0222] (2) Take the white membrane layer, wash it 3 times with phosphate-buffered saline (PBS) (pH 7.2), resuspend the cells in 1 mL PBS and mix well. Take 20 μL of the cell suspension into a 1.5 mL EP tube, add 380 μL of X-VIVO15 medium to dilute and mix well. Take 20 μL of the diluted cell suspension into another new 1.5 mL EP tube, add 20 μL of AOPI and mix well. Add the mixture to a counting chamber for cell counting.

[0223] (3) Based on the number of cells, per 1×10 7 Add 20μL LCD3 to each cell + / CD4 + T-cell positive sorting magnetic beads, per 1×10 7 Add 80 μL of PBS buffer to each cell, mix well, and incubate at 4°C for 15 min.

[0224] (4) Add 20 mL of PBS buffer and centrifuge at 400 g for 10 min.

[0225] (5) Discard the supernatant, add 6 mL of PBS to resuspend the cells and mix well. Place the sorting column on the sorting rack and pass 5 mL of phosphate buffer through the column to equilibrate once.

[0226] (6) Add the PBMC suspension from step (4) to the sorting column. After sorting, wash twice with 1 mL of phosphate buffer. Remove the sorting column from the sorting rack and add 5 mL of PBS buffer to wash out CD3. + / CD4 + T cells.

[0227] (7) Centrifuge at 1500 rpm for 10 min and discard the supernatant.

[0228] (8) The prepared T lymphocytes were resuspended and counted in 1 mL of X-VIVO15T cell culture medium containing 100 U / mL IL-2. 20 μL of cell suspension was taken, 20 μL of AOPI was added and mixed, and 20 μL was taken onto a counting plate for cell counting.

[0229] (9) Every 10 6 Add 25 μL of CD3 / CD28 activation magnetic beads to each cell, mix well to activate T lymphocytes, and adjust the cell concentration to 2 × 10⁶ cells / mL. 6 cells / mL

[0230] (10) Place T lymphocytes in a 24-well plate, add X-VIVO15T cell culture medium containing 100 U / mL IL-2 to 500 μL, and incubate overnight in an incubator.

[0231] 4. Lentiviral infection of T lymphocytes:

[0232] (1) CD3-positive T lymphocytes were obtained by magnetic bead sorting and resuspended in 1 mL of T cell culture medium containing X-VIVO 15 and 100 U / mL for counting. The cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 5 / mL, per 10 6 Add 25 μL of CD3 / CD28 activation beads to each cell and mix well to activate T lymphocytes. Seed 500 μL / well in a 24-well plate and incubate overnight.

[0233] (2) RetroNectin coating: 2 μg / cm 2 As required, the RetroNectin solution was diluted with PBS, and 500 μL was added to each well of a 24-well plate and incubated overnight at 4°C. The next day, the RetroNectin was discarded, and the plate was blocked with PBS containing 2% BSA for 0.5 h, followed by washing with PBS.

[0234] (3) Take the packaged lentivirus supernatant and thaw it at 4°C. Add the viral supernatant to a 24-well plate coated with RetroNectin at an MOI of 30. Centrifuge at 3000 rpm and 4°C for 2 h. Discard the viral supernatant, add activated T lymphocytes, and centrifuge at 3000 rpm and 12°C for 2 h. Continue culturing at 37°C and 5% CO2 in an incubator, replenishing the culture medium every 2 days.

[0235] 5. Detection of CAR expression on the surface of BBZ19-TtgRH67A-1-5-CAR-T and BBZ19-CAR-TtgRH67A-3-14-T

[0236] (1) Take 0.5g BSA + 10mL PBS to prepare a 5% BSA solution; dilute it 10 times when needed to obtain a 0.5% BSA solution for subsequent cell washing and antibody dilution.

[0237] (2) Take T cells, at a dose of 5 × 10 5 -1×10 6 Collect cells; wash cells twice with 1 mL of 0.5% BSA solution and discard the supernatant; resuspend cells in 100 μL of 0.5% BSA solution.

[0238] (3) Add WHITLowLinker (2 μL per tube) to the cell suspension and incubate on ice in the dark for 1 h.

[0239] (4) Wash twice with 1 mL of 0.5% BSA solution; add 200 μL PBS to resuspend the cells and prepare for detection; set the APC channel (detection wavelength 585), use untransfected samples as BLANK (blank control), and group (transfected + drug, etc.) for detection.

[0240] 6. Cell proliferation experiment:

[0241] (1) Effect of TtgRH67A mutant domain insertion on CAR-T cell proliferation: Homologous T lymphocytes were isolated and divided into four groups: BBZ19-TtgRH67A-1-5-CAR-T, BBZ19-CAR-TtgRH67A-3-14-T, BBZ19-CAR-T, and UDT cells. 10 μL of T cells were added to each well of a 24-well plate. 5 Cell culture (DAY 0). CAR-TtgRH67A-T and BBZ19-CAR-T cells were obtained by lentiviral transfection, and then all four groups continued to be cultured under identical conditions. Cell counts were performed on days 4, 6, 8, 10, and 14 to obtain cell assay data.

[0242] (2) Effect of resveratrol on T cell proliferation: After T lymphocytes isolated from the same batch were plated, they were treated with either the carrier drug or 50 μM resveratrol and cultured for a period of time. Changes in cell proliferation level were detected periodically.

[0243] 7. Cytotoxicity assay:

[0244] Raji-Luc and K562-Luc cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. Raji-Luc cells were cultured in a complete medium containing RPMI-1640, 10% fetal bovine serum, and 1% penicillin-drug antibiotics. K562-Luc cells were cultured in a complete medium containing IMDM, 10% fetal bovine serum, and 1% penicillin-drug antibiotics. Both were cultured and passaged at 37°C in a 5% CO2 incubator. 1×10⁶ cells were then cultured and passaged. 5Raji-Luc or K562-Luc cells were seeded as target cells in 96-well plates, and 150 μL of X-VIVO15 medium was added to each well. Simultaneously, effector T cells were added to the target cells according to the designed effector-to-target ratio (E:T), and resveratrol was added to final concentrations of 0, 20 μM, and 50 μM, respectively, with three replicates for each concentration. After co-culturing the two cell types for 24 h, the cell suspension was collected by centrifugation at 500g for 8 min, and the supernatant was discarded. 5× cell lysis buffer was diluted to 1× with PBS, and 50 μL was added to each tube of cells. The cells were then pipetted and lysed for 30 min at room temperature. 40 μL of the lysis product was transferred to a bioluminescent detection plate, and 50 μL of luciferase substrate was added to each well using an automated microplate reader. The luciferase activity in the remaining cells was then detected. The relative T cell killing rate was calculated as 100 × [1 - (fluorescence intensity of BBZ19-CAR-TtgRH67A-T group / fluorescence intensity of UDT group)].

[0245] 8. Cytokine detection:

[0246] On day 8 after T lymphocyte infection with lentivirus, cells were collected, washed twice with IL-2-free X-VIVO medium, and cultured overnight in X-VIVO-only medium. On day 2, cultured Raji cells and various T cell types (including blank T cell UDT, BBZ19-CAR-TtgRH67A-T, and conventional BBZ19-CAR-T cells) were collected by centrifugation. X-VIVO medium was added to bring the final cell concentration to 10⁻⁶. 7 Raji cells and T cells were seeded in 96-well round-bottom plates at an effector-to-target ratio of 2 × 10⁶ / mL and cultured. 5 Different concentrations of resveratrol were added to each well, ensuring a total solution volume of 200 μL for each well. After 24 hours, the supernatant was collected, and the secretion levels of IFN-γ and IL-2 in the supernatant were measured according to the instructions of the Human IFN-γ and IL-2 ELISA Kit.

[0247] Experimental results:

[0248] 1. Construction and killing of BBZ19-CAR-TtgRH67A-1-5-T cells

[0249] 1.1 The TtgRH67A1-5 unstable domain regulatory system can regulate the membrane expression level of BBZ19-CAR molecules via RES.

[0250] Mutant individuals 1-5 and 3-14 were selected to construct BBZ19-CAR-TtgRH67A-1-5-T and BBZ19-CAR-TtgRH67A-3-14-T cells based on the DD-CAR sequence structure. Using a specific method (Whitelowlinker antibody), the effect of different concentrations of resveratrol (RES) on the membrane expression of BBZ19-CAR molecules was detected. RES concentration gradients of 0, 10, 20, 30, 40, 50, 60, and 100 μM were set, and CAR molecule expression was analyzed by relative fluorescence intensity. The results showed that with increasing RES concentration, the membrane expression level of BBZ19-CAR molecules gradually stabilized, while the relative fluorescence intensity showed an increasing trend. This indicates that the TtgRH67A1-5 unstable domain regulatory system can regulate the membrane expression of BBZ19-CAR molecules in dependence on RES concentration, and the higher the RES concentration, the more significant the promoting effect on CAR molecule expression.

[0251] 1.2 Resveratrol can dose-dependently regulate the in vitro killing activity and cytokine secretion of BBZ19-CAR-TtgRH67A-1-5-T cells and BBZ19-CAR-TtgRH67A-3-14-T cells.

[0252] Using Raji-LUC cells as target cells, BBZ19-CAR-TtgRH67A-1-5-T cells and BBZ19-CAR-TtgRH67A-3-14-T cells were co-incubated with target cells. The cytotoxic effect was detected by fluorescein assay, and the secretion levels of IFN-γ and IL-2 were also measured. Results showed that with increasing resveratrol concentration, the cytotoxic activity of CAR-T cells against Raji-LUC cells gradually increased, exhibiting a clear dose-dependent effect. Regarding cytokine secretion, the concentrations of IFN-γ and IL-2 increased with increasing RES concentration, reaching a peak at a resveratrol concentration of 60 μM. This suggests that RES can effectively activate the cytokine secretion pathway of BBZ19-CAR-TtgRH67A-T cells, enhancing their anti-tumor immune effect. Figure 6 As shown.

[0253] 1.3 Insertion of the TtgRH67A unstable domain does not affect the state of CAR-T cells.

[0254] BBZ19-CAR-TtgRH67A-1-5-T cells, BBZ19-CAR-TtgRH67A-3-14-T cells, conventional BBZ19-CAR-T cells, and untreated T cells (UDT) were cultured under the same conditions, and cell proliferation and apoptosis were dynamically observed. The results showed no significant difference in proliferation capacity among the three groups (P>0.05). Apoptosis was detected on day 15 post-transfection. The apoptosis rates of BBZ19-CAR-TtgRH67A-1-5-T cells, BBZ19-CAR-TtgRH67A-3-14-T cells, conventional BBZ19-CAR-T cells, and UDT cells were similar, indicating that the insertion of the TtgRH67A unstable domain did not significantly affect the proliferation and apoptosis of CAR-T cells, and the cell state was stable.

[0255] 1.4 The small molecule ligand resveratrol does not affect the state of T cells.

[0256] T cells from the same batch were treated with 50 μM resveratrol and adjuvant, respectively, and cultured under the same conditions to detect cell proliferation and apoptosis. The results showed that the proliferation rate of T cells in the resveratrol-treated group was slightly slower than that in the non-resveratrol group in the early stage, but there was no significant difference in the later stage. Apoptosis staining on day 13 of culture showed no significant difference in the apoptotic state of the two groups of cells, indicating that resveratrol has no significant toxicity to T cells and does not affect the normal state of T cells.

[0257] Example 3

[0258] 1. Recombinant plasmid construction and lentivirus packaging

[0259] A recombinant expression plasmid, TtgRH67A-1-5-IL-2, containing the TtgR regulatory system (mutants 1-5) and interleukin-2 (IL-2) fusion protein, was constructed and packaged lentivirally to obtain recombinant lentiviral particles carrying the TtgR unstable domain-IL-2 fusion gene. Other steps were the same as in "2.1. Lentiviral Packaging".

[0260] 2. Flow cytometry to verify the effectiveness of recombinant cell expression

[0261] The plasmid was transiently transfected into HEK293-t cells. Recombinant 293 cells were collected on day 2 post-transfection. After induction with 50 μM resveratrol for 24 hours, on day 3, the cells were washed and resuspended in PBS, fixed in fixative at room temperature in the dark for 20 minutes, treated with 1× permeabilizing agent, and centrifuged. Then, IL-2 antibody was added and incubated at room temperature in the dark for 20 minutes. After washing and resuspending with 1× permeabilizing agent, flow cytometry analysis was performed. The results showed that the expression level of IL-2 in 293 cells transfected with 50 μM resveratrol was significantly higher than that in cells transfected without resveratrol, confirming that the TtgR regulatory system can effectively mediate IL-2 expression. Figure 7As shown.

[0262] 3. ELISA assay to verify the regulatory role of resveratrol

[0263] HEK293-t cells were transfected with lentivirus and then cultured. Different concentration gradients of resveratrol were added to the culture medium, and the cells were cultured in DMEM for 24 hours. The supernatant was then collected. The IL-2 secretion level in the supernatant was detected using an IL-2 ELISA kit. The results showed that resveratrol significantly regulated IL-2 secretion in recombinant HEK293-t cells, and the secretion level showed a controllable change with resveratrol concentration. This confirms that the TtgR regulatory system with an unstable domain fused to a single IL-2 coding sequence exhibits efficient and precise regulatory effects. Figure 8 As shown. The other steps are the same as those in "8. Cytokine Detection" of Example 2.

[0264] The amino acid sequences of TtgRH67A mutants 1-5 are as follows:

[0265] MVRRTKEEAQETRAQIIEAAERAFYKRGVARTTLADIAELAGVTRGAIYWHFNNKAELVQALLDSLAETHDHLARASESEDEVDPLGCMRKLLLQVFNELVLDARTR RINEILHHKCEFTDDMCEIRQQRQSAVLDCHKGITLALANAVRRGQLPGELDAERAAVAMFAYVDGLIRRWLLLPDSVDLLGDVEKWVDTGLYMLRLSPALRK, as in SEQ IDNO: 2 shown.

[0266] The amino acid sequence of IL-2 is as follows:

[0267] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTVD as shown in SEQ ID NO: 17

[0268] IL-2 is linked to TtgRH67A or its mutants via the adapter VD, and IL-2 is located at the N-terminus of TtgRH67A or its mutants.

[0269] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant of the unstable domain based on the prokaryotic transcription factor TtgR, characterized in that, The unstable domain mutant, based on the prokaryotic transcription factor TtgR mutant, undergoes any of the following mutations: (1) The amino acid residue at position 200 is replaced by Y instead of D; (2) The amino acid residue at position 41 is replaced by S with A, the amino acid residue at position 117 is replaced by W with C, and the amino acid residue at position 161 is replaced by D with E; The amino acid sequence of the mutant prokaryotic transcription factor TtgR is shown in SEQ ID NO:

1.

2. A recombinant protein, characterized in that, It comprises the unstable domain mutant of claim 1, and one or more additional peptides linked to the unstable domain mutant; the additional peptides are selected from signal peptides, tag peptides, detectable tags, and any combination thereof.

3. A nucleic acid molecule characterized by, It contains or encodes the unstable domain mutant of claim 1 or the recombinant protein of claim 2.

4. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. The use of the unstable domain mutant of claim 1, the recombinant protein of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, or the host cell of claim 5 in the preparation of a biosensor responsive to resveratrol and doxycycline.

7. A composition characterized in that, The invention includes CAR-T cells and regulatory factors; the CAR-T cells contain the nucleic acid molecule of claim 3 or the vector of claim 4, thereby enabling them to express the unstable domain mutant of claim 1 or the recombinant protein of claim 2; the regulatory factor is resveratrol.

8. Use of the composition of claim 7 in the preparation of a medicament for treating lymphoma.

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