Immunomodulator target spot and imaging method for in-situ detection of immunomodulator target spot

Abstract

The invention relates to the technical field of biological medicine, in particular to a class of immunomodulator targets and an imaging method for in-situ detection of the immunomodulator targets, and the class of immunomodulator targets comprise: a TP53RK target which is a target CRBN independent target in a single cell and comprises a gene sequence in SEQ ID No.1; the TP53RK target spot is subjected to substitution and / or addition and / or deletion of one or more residues, and the homology of the derivative gene sequence is 90% or above; and a downstream pathway of a TP53RK target spot, wherein the downstream pathway comprises one or more of a protein molecule RNASEK, a protein molecule TSPO2, a protein molecule PADI4 and a protein molecule PSMB9. The novel target TP53RK protein and downstream pathway key molecules provided by the invention provide a theoretical basis for diagnosis and treatment of immune and inflammation mediated diseases and research and development of related drugs; the single-molecule in-situ detection target of the effects of the ibodomide on targets of important immune cells and molecular signal channels and multiple downstream key molecules in a multi-tissue area immune microenvironment is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an imaging method for a class of immunomodulator targets and in-situ detection thereof. Background Art

[0002] Small molecule compound Iberdomide (Iberdomide, molecular formula C 25 H 27 N3O5, molecular weight 449.50) is a commonly used immunomodulatory drug with complex and difficult to grasp underlying molecular mechanisms. Ibodamide acts on target molecules and forms multimolecular complexes with other molecules, regulating multiple biological molecules downstream of the pathway through the complex. The known molecular signal is that ibodamide targets and binds to the E3 ubiquitin ligase CRBN protein to change its conformation, inducing the degradation of proteins such as transcription factors IKZF3 downstream of the reaction, thereby exerting immune function. The main challenges encountered at this stage are: in disease models with low expression or loss of CRBN, ibodamide treatment is still effective and the effect is within a measurable range, indicating that other targets and signaling pathways play a role; currently, high-resolution cryo-electron imaging methods can only be completed in vitro experiments, and the spatial resolution of small animal optics and CT imaging with a large field of view encounters the nanoscale limit, which makes it difficult to accurately depict the in situ spatial omics spectra of the targeted effects of small molecule drugs on the immune microenvironment in multiple tissue regions of disease models. Summary of the invention

[0003] Based on this, the present invention proposes a type of immunomodulator target and an imaging method for in situ detection thereof, which can be used as a new target for the immunomodulator small molecule compound ibodomide and has good practical application value.

[0004] According to a first aspect of the present invention, there is provided a class of immunomodulator targets, including TP53RK targets;

[0005] The TP53RK target is a single intracellular target CRBN-independent target, and the TP53RK target includes the gene sequence in SEQ ID No. 1;

[0006] The TP53RK target site is subjected to one or more residue substitutions and / or additions and / or deletions, and the derived gene sequence has a homology of more than 90%; and

[0007] The downstream pathway of the TP53RK target includes: protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9, and one or more of mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9.

[0008] According to an embodiment of the present invention, the downstream pathway of the TP53RK target includes:

[0009] The protein molecule RNASEK includes the protein sequence in SEQ ID No.2;

[0010] The protein molecule TSPO2 includes the protein sequence in SEQ ID No.3 and / or SEQ ID No.4;

[0011] The protein molecule PADI4 includes the protein sequence in SEQ ID No.5 and / or SEQ ID No.6;

[0012] The protein molecule PSMB9 includes the protein sequence in SEQ ID No.7 and / or SEQ ID No.8;

[0013] The protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 are derived protein sequences after one or more amino acid residues are replaced and / or added and / or deleted, and the homology is more than 90%;

[0014] The nucleic acid sequences corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 and the derived nucleic acid sequences which have undergone one or several residue substitutions and / or additions and / or deletions and have a homology of more than 90%; and

[0015] The mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9.

[0016] According to a second aspect of the present invention, there is provided an imaging method for in-situ detection, comprising:

[0017] Molecular detection of target CRBN-dependent pathway and substrate IKZF3 protein in single tissue cells; and / or

[0018] Based on the above-mentioned TP53RK target, and the detection of its downstream pathway protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9; and / or

[0019] Based on the above-mentioned TP53RK target, the mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 are detected.

[0020] According to an embodiment of the present invention, the present invention includes:

[0021] Disease model mice were used to construct an inflammation mouse model;

[0022] The disease model mice are B6 mice, B6.Fas obtained by cross-breeding of mice. lpr Mouse and B6.Fas lpr .Crbn I391V Mice;

[0023] The inflammatory mouse model is treated with ibodamide: a mixed solution containing ibodamide is intraperitoneally injected into the inflammatory mouse model at a dose of 0.7 micrograms per day to obtain a treated mouse model;

[0024] The treated mouse model was sampled and the inflammation level was evaluated;

[0025] Spationomic drug testing was performed on tissues of the treated mouse model.

[0026] According to an embodiment of the present invention, sampling the therapeutic mouse model and evaluating the inflammation level include:

[0027] Extracting serum from the treated mouse model to complete enzyme-linked immunosorbent assay;

[0028] Extracting peripheral blood cells from the treated mouse model to complete nucleic acid molecule sequencing analysis; and

[0029] The treated mouse model was subjected to cardiac perfusion, and the tissues were extracted for hematoxylin-eosin staining and immunofluorescence imaging analysis.

[0030] According to an embodiment of the present invention, spatial omics drug detection is performed on the tissue of the therapeutic mouse model, including:

[0031] After incubating the tissue with the swelling monomer solution at 4°C for 30 minutes, the tissue was transferred into the gel chamber;

[0032] The incubated gel chamber is sealed and transferred to 37° C. for overnight incubation to gel the cells of the tissue to obtain a gelled sample;

[0033] The gelled sample is taken out, digested and washed, and then stored in a phosphate buffer containing 0.02% sodium azide at 4° C. to allow the sample to swell and obtain a sample to be tested;

[0034] The sample to be tested is subjected to spatial omics detection, and multiple rounds of detection and imaging are performed using a sequential imaging method.

[0035] According to an embodiment of the present invention, the mixed solution containing ibodomide includes the following components in terms of volume fraction:

[0036] Ibolidomide 0-2.7%, phosphate buffer 52.3%, polyethylene glycol 37.3%-40% and Tween-80 5%.

[0037] According to an embodiment of the present invention, the swelling monomer solution

[0038] The ingredients are sodium acrylate 0-30% w / v, acrylamide 8-15% w / v, dimethylacrylamide 0-4% v / v, N,N-methylenebisacrylamide 0.01%-0.05% w / v, sodium chloride 1-12% w / v, dissolved in PBS for storage, and before use, add ammonium persulfate 0.1-0.25% w / v, tetramethylethylenediamine 0.04% v / v, 4-hydroxytamoxifen 0.001% w / v and methacrolein 0.1%-0.25% v / v to initiate polymerization.

[0039] According to an embodiment of the present invention, the spatial omics detection of the sample to be tested includes: staining the sample to be tested:

[0040] When testing the target CRBN-dependent pathway and substrate IKZF3 protein molecules in single cells of tissues:

[0041] The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azide DNA sequences to obtain modified CRBN antibodies and IKZF3 antibodies; the drug was localized by ibodamide autofluorescence;

[0042] Using the CRBN antibody and the IKZF3 antibody to stain and mark the signaling pathway targets and downstream protein antibodies;

[0043] An ATP-targeted fluorescent probe is used to perform spatial quantitative monitoring of ATP in samples; a fluorescent probe targeting ubiquitin is used to perform spatial localization and quantitative detection of CRBN-mediated protein ubiquitination;

[0044] Wherein, the nanobody is modified with a coding substrate DNA sequence by click chemistry method.

[0045] According to an embodiment of the present invention, the spatial omics detection of the sample to be tested includes: staining the sample to be tested:

[0046] When testing the TP53RK target and its downstream pathway protein molecules RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9:

[0047] Phosphate-buffered saline was used to stain TP53RK, RNASEK, and TSPO2 antibodies, and drug localization was performed by ibodamide autofluorescence;

[0048] and / or

[0049] When the TP53RK target, the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 corresponding to the mRNA, sugars, mitochondria and lipid molecules are detected:

[0050] The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azidated DNA sequences to obtain modified TP53RK antibody, RNASEK antibody and TSPO2 antibody staining;

[0051] The signaling pathway targets and downstream protein antibodies were stained and marked using the TP53RK antibody, RNASEK antibody and TSPO2 antibody;

[0052] Wherein, the nanobody is modified with a coding substrate DNA sequence by click chemistry method.

[0053] It can be seen from the above technical solutions that the TP53RK target and spatial omics detection method based on ibodomide provided by the present invention has the following beneficial effects:

[0054] The present invention proposes a new target TP53RK protein and key molecules of the downstream pathway of the small molecule compound ibodamide, providing a theoretical basis for the diagnosis and treatment of immune and inflammatory mediated diseases and the development of related drugs, and proposes a spatial omics detection method based on expansion microscopy technology and sequential imaging methods to achieve the single-molecule in situ detection goal of ibodamide's effects on the targets of important immune cells and molecular signaling pathways and multiple downstream key molecules in the immune microenvironment of multiple tissue regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure is a flow chart of a target detection method according to an embodiment of the present invention.

[0056] Figure 2 This is a flow chart of spatial omics imaging according to an embodiment of the present invention.

[0057] Figure 3 Schematic diagram of sequential imaging in spatial omics according to an embodiment of the present invention.

[0058] Figure 4 This is a schematic diagram of increasing the size of tissue protein signals by expansion microscopy according to an embodiment of the present invention.

[0059] Figure 5 This is a schematic diagram of increasing the size of nucleic acid molecule signals by expansion microscopy according to an embodiment of the present invention. DETAILED DESCRIPTION

[0060] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0061] According to a first aspect of the present invention, there is provided a class of immunomodulator targets, including TP53RK targets;

[0062] The TP53RK target is a single intracellular target and a CRBN-independent target. The TP53RK target includes the gene sequence in SEQ ID No. 1;

[0063] A derived gene sequence with more than 90% homology after one or several residues are substituted and / or added and / or deleted in the TP53RK target site; and

[0064] The downstream pathway of the TP53RK target includes: protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9, and one or more of mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9.

[0065] According to an embodiment of the present invention, the downstream pathway of the TP53RK target includes:

[0066] The protein molecule RNASEK includes the protein sequence in SEQ ID No. 2;

[0067] The protein molecule TSPO2 includes the protein sequence in SEQ ID No.3 and / or SEQ ID No.4;

[0068] The protein molecule PADI4 includes the protein sequence in SEQ ID No.5 and / or SEQ ID No.6;

[0069] The protein molecule PSMB9 includes the protein sequence in SEQ ID No.7 and / or SEQ ID No.8;

[0070] Protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9 are derived protein sequences with one or more amino acid residues substituted and / or added and / or deleted, and the homology is more than 90%;

[0071] Nucleic acid sequences corresponding to protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9 and derived nucleic acid sequences having one or several residues substituted and / or added and / or deleted and having a homology of more than 90%; and

[0072] The mRNA, carbohydrates, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9.

[0073] Among them, the protein molecule TSPO2 SEQ ID No. 3 is the human protein molecule TSPO2;

[0074] Protein molecule TSPO2 SEQ ID No. 4 is mouse protein molecule TSPO2;

[0075] Protein molecule PADI4 SEQ ID No. 5 is human protein molecule PADI4;

[0076] Protein molecule PADI4 SEQ ID No. 6 is mouse protein molecule PADI4;

[0077] Protein molecule PSMB9 SEQ ID No.7 is human protein molecule PSMB9;

[0078] Protein molecule PSMB9 SEQ ID No. 8 is the mouse protein molecule PSMB9.

[0079] According to a second aspect of the present invention, there is provided an imaging method for in-situ detection, comprising:

[0080] Molecular detection of target CRBN-dependent pathway and substrate IKZF3 protein in single tissue cells; and / or

[0081] Based on the above-mentioned TP53RK target, and the detection of its downstream pathway protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9; and / or

[0082] Based on the above-mentioned TP53RK target, the mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecules RNASEK, TSPO2, PADI4 and PSMB9 were detected.

[0083] The present invention proposes a spatial omics detection method based on expansion microscopy and sequential imaging for the above-mentioned new targets and multiple types of downstream biological molecules, detects the above molecules at the in vivo single-molecule scale, reveals the actual interaction between the drug and the target and downstream biological molecules, and analyzes the mechanism of action of ibodomide. Specifically, the method first sequences and analyzes the nucleic acid molecules of whole blood cells, expands the physical size of biological molecules in single cells of tissues through expansion microscopy, and increases the fluorescence imaging resolution of biological molecules such as nucleic acids and lipids to 20 nanometers, so as to achieve single-molecule detection of the above molecules; applies the sequential imaging method to the expanded biological samples to achieve single-molecule detection of protein molecules with a resolution of 0.1 nanometers; combines the autofluorescence of ibodomide to achieve spatial omics of in situ single-molecule detection of drug molecules, targets and their downstream biological molecules, analyzes the mechanism of action of drugs, and assists in drug application, mechanism research and new drug development.

[0084] The detection method can act on known targets and downstream molecules of ibodomide, such as CRBN and its pathway molecule IKZF3, and can also act on potential new targets and their downstream molecules, such as the new target TP53RK proposed in the present invention and its downstream protein molecules RNASEK, TSPO2, PADI4, PSMB9 and related nucleic acids, lipid molecules, etc.

[0085] According to an embodiment of the present invention, Figure 1 As shown, including:

[0086] Disease model mice were used to construct an inflammation mouse model;

[0087] Among them, the disease model mice used were B6 mice and B6.Fas obtained by cross-breeding of mice. lpr Mouse and B6.Fas lpr .Crbn I391V Mice;

[0088] Treating the inflammatory mouse model with ibodamide: intraperitoneally injecting a mixed solution containing ibodamide into the inflammatory mouse model at a dose of 0.7 micrograms per day to obtain a treated mouse model;

[0089] The treated mouse models were sampled and their inflammation levels assessed;

[0090] Spatiomics drug testing of tissues in treated mouse models.

[0091] like Figure 1 As shown, for the constructed inflammatory model mice, CRBN humanized and non-humanized mice were treated with ibodamide, and the drug treatment effect and in vivo mechanism of action were analyzed through inflammation evaluation and spatial omics detection.

[0092] According to an embodiment of the present invention, samples are collected from the treatment mouse model and the inflammation level is evaluated, including:

[0093] The serum of the treated mouse model was extracted to complete the ELISA test;

[0094] Extract peripheral blood cells from the treated mouse model to complete nucleic acid molecular sequencing analysis; and

[0095] The treated mouse models were perfused cardiacly, and tissues were extracted for hematoxylin-eosin staining and immunofluorescence imaging analysis.

[0096] According to an embodiment of the present invention, Figure 2 As shown, spatial omics drug testing of tissues in treated mouse models includes:

[0097] After incubation of the tissue with the swelling monomer solution at 4°C for 30 min, it was transferred into the gel chamber;

[0098] The incubated gel chamber was sealed and transferred to 37°C for overnight incubation to gel the cells of the tissue and obtain a gelled sample;

[0099] The gelled sample was taken out, digested, washed, and stored in a phosphate buffer containing 0.02% sodium azide at 4°C to allow swelling, thereby obtaining a sample to be tested;

[0100] The samples to be tested are subjected to spatial omics detection and multiple rounds of detection and imaging are performed using sequential imaging methods.

[0101] like Figure 2 As shown, for tissue section samples, the physical size of biomolecules interacting with multiple drugs is preserved and isotropically enlarged based on expansion microscopy, and is displayed simultaneously at the same location by multiple staining methods.

[0102] According to an embodiment of the present invention, a mixed solution containing ibolidomide includes a mixed solvent and ibolidomide dissolved in the mixed solvent;

[0103] According to an embodiment of the present invention, the mixed solution containing ibodomide includes the following components in terms of volume fraction:

[0104] Ibolidomide 0-2.7%, phosphate buffer 52.3%, polyethylene glycol 37.3%-40% and Tween-80 5%.

[0105] According to an embodiment of the present invention, the swelling monomer solution

[0106] The ingredients are sodium acrylate 0-30% w / v, acrylamide 8-15% w / v, dimethyl acrylamide 0-4% v / v, N,N-methylenebisacrylamide 0.01%-0.05% w / v, sodium chloride 1-12% w / v, dissolved in PBS for storage, and ammonium persulfate 0.1-0.25% w / v, tetramethylethylenediamine 0.04% v / v, 4-hydroxytamoxifen 0.001% w / v, methacrolein 0.1%-0.25% v / v are added before use to initiate polymerization. According to an embodiment of the present invention, spatial omics detection of the sample to be tested includes: staining the sample to be tested:

[0107] When testing the target CRBN-dependent pathway and substrate IKZF3 protein molecules in single cells of tissues:

[0108] The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azide DNA sequences to obtain modified CRBN antibodies and IKZF3 antibodies; the drug was localized by ibodamide autofluorescence;

[0109] CRBN antibody and IKZF3 antibody were used to stain and mark signaling pathway targets and downstream proteins;

[0110] An ATP-targeted fluorescent probe is used to perform spatial quantitative monitoring of ATP in samples; a fluorescent probe targeting ubiquitin is used to perform spatial localization and quantitative detection of CRBN-mediated protein ubiquitination;

[0111] Among them, the nanoantibody is modified with a coding substrate DNA sequence through click chemistry.

[0112] According to an embodiment of the present invention, performing spatial omics detection on a sample to be tested includes: staining the sample to be tested:

[0113] When testing the TP53RK target and its downstream pathway protein molecules RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9:

[0114] Phosphate-buffered saline was used to stain TP53RK, RNASEK, and TSPO2 antibodies, and drug localization was performed by ibodamide autofluorescence;

[0115] and / or

[0116] When detecting the mRNA, sugars, mitochondria and lipid molecules corresponding to the TP53RK target, protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9:

[0117] The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azidated DNA sequences to obtain modified TP53RK antibody, RNASEK antibody and TSPO2 antibody staining;

[0118] TP53RK antibody, RNASEK antibody and TSPO2 antibody staining were used to mark signal pathway targets and downstream protein antibodies;

[0119] Among them, the nanoantibody is modified with a coded base DNA sequence by click chemistry. The molecular glue drug ibodomide produces spontaneous fluorescence under laser and can be located without special treatment.

[0120] The expansion microscopy technique is characterized by its ability to simultaneously fix multiple biological molecules such as intracellular drugs, proteins, lipids, sugars and nucleic acids, isotropically expand their size, and allow them to be stained and observed; it increases the spatial size of tissues and molecules tenfold, and can image lipids, nucleic acids, sugars and other molecules with a resolution of 20 nanometers, and achieve single-molecule detection of the above molecules.

[0121] Sequential imaging technology achieves 0.1 nanometer resolution imaging of protein molecules and detects protein target and drug interactions in situ in cells at a single molecule. In the present invention, sequential imaging targets the target protein through nanoantibodies, and the nanoantibodies are modified with encoded substrate DNA sequences by click chemistry methods. The characteristic is that the sequences on adjacent nanoantibodies are different, so adjacent nanoantibodies emit light in different imaging rounds. After the nanoantibody binds to the target protein, a fluorescent probe DNA sequence targeting the substrate DNA sequence is added to the imaging buffer. The characteristic of the probe is that it can dynamically bind and separate from the substrate DNA sequence, and emit fluorescence when bound, thereby achieving dynamic fluorescent point flashing to form a light spot. The center of mass of the light spot is determined by computational reconstruction to overcome the optical diffraction limit. Several substrate DNA sequences are modified on the nanoantibody so that the nanoantibodies adjacent to the target protein do not emit light at the same time, and the spatially overlapping fluorescent groups are separated in time.

[0122] The spatial omics imaging technology combines expansion microscopy and sequential imaging technology, with a sensitivity of 6.82 pixels per micron; the protein imaging resolution is 0.1 nanometers, and the imaging resolution of lipids, nucleic acids, sugars and other molecules is 20 nanometers, which meets the requirements of single-molecule detection; the imaging time of a single fluorescent channel is 1.3 seconds per square micron. The spatial omics technology described in the present invention combines protein immunofluorescence imaging, nucleic acid encoding imaging, lipid imaging and other technologies for simultaneous imaging, and simultaneously realizes single-molecule detection of drugs, targets and important molecules in multiple pathways, and jointly analyzes the mechanism of drug action.

[0123] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0124] Example 1

[0125] Expansion microscopy spatial omics detection of the molecular glue drug ibodomide acting on two types of signaling pathways in a mouse nephritis model, including: nucleic acid molecular sequencing in whole blood cells, target CRBN-dependent pathways in single tissue cells and substrate IKZF3 protein molecular detection;

[0126] Detection of CRBN-independent target TP53RKa, and its downstream signaling protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9; detection of mitochondria and lipids related to pathway function; localization of DNA staining in the nucleus, etc. Figure 1 As shown, the following steps are included:

[0127] S1, Construction of mouse model of inflammation.

[0128] Nephritis was induced by injecting sheep anti-rat glomerular basement membrane serum into mice. The disease model mice were B6 mice and 4-week-old B6.Fas mice obtained by cross-breeding. lpr Mouse and B6.Fas lpr .Crbn I391V Mice. Three groups of mice were set up, namely, CRBN humanized mice, CRBN non-humanized mice, and control group B6 mice. Each group of mice was divided into two groups: a drug-treated group and a blank group. All six groups of mice were injected with 0.4 ml of serum per 100 g of body weight through the ophthalmic vein.

[0129] S2, model mice were treated with ibodomide.

[0130] Starting from the second day after serum injection, ibodamide was dissolved in a mixed solution of 2.7% dimethyl sulfoxide, 52.3% phosphate buffer, 40% polyethylene glycol and 5% Tween-80. The mice in the treatment group were intraperitoneally injected with 0.7 micrograms of the drug every day for two weeks, and the mice in the blank group were intraperitoneally injected with an equal amount of solvent solution without the drug every day.

[0131] S3, sampling and assessment of inflammation levels.

[0132] After two weeks of treatment, serum was extracted from all mice for enzyme-linked immunosorbent assay, nucleic acid molecular sequencing analysis was performed using peripheral blood cells, cardiac perfusion was performed and spleen, lymph nodes, kidneys, blood vessels and other tissues were extracted for hematoxylin-eosin staining and immunofluorescence imaging analysis to evaluate and analyze the level of inflammation.

[0133] S4, Spationomic drug testing.

[0134] like Figure 2 As shown, the expansion microscopy procedure was performed as follows.

[0135] 1. Incubate the tissue with the swelling monomer solution (components are dimethyl acrylamide, sodium acrylate, acrylamide, N,N-methylenebisacrylamide, sodium chloride, dissolved in PBS for storage, and ammonium persulfate, tetramethylethylenediamine, 4-hydroxytamoxifen and methacrolein are added before use to initiate polymerization) at 4°C for 30 minutes, and then transfer the cells and the incubation solution to the 0.1 mm thick gel chamber. After the incubation, seal the gel chamber and transfer it to 37°C for overnight incubation to allow the cells to gel.

[0136] 2. The gelled samples were removed from the chamber and incubated in homogenization buffer for digestion. The digested samples were washed three times with phosphate buffer on a shaker at room temperature for 15 minutes each time and then stored in phosphate buffer containing 0.02% sodium azide at 4°C.

[0137] 3. After digestion, the samples were expanded and subjected to spatial omics detection.

[0138] The gel was trimmed to a suitable shape and washed three times with phosphate buffered saline for 15 min.

[0139] Prepare nanobodies for protein molecular sequence imaging. Nanobodies are prepared and purchased by commercial companies. The nanobodies are thawed on ice, and then a twenty-fold excess molar amount of maleimide-PEG4-DBCO linker is added and reacted on ice for 2 hours. The nanobodies are centrifuged and the unreacted linker is removed by buffer exchange into phosphate buffer. The modified nanobodies are reacted with a five-fold excess molar amount of azidated DNA sequences at 4°C overnight. Unbound proteins and free DNA are removed by anion exchange chromatography. In this example, there are three azidated DNA sequences, namely R1 (SEQ ID No. 9), R2 (SEQ ID No. 10) and R3 (SEQ ID No. 11). The corresponding fluorescent complementary chains are r1 (TCCTCCT), r2 (GAAGAAG) and r3 (TCTCTCT), which are labeled with the same channel fluorescence. For different antibodies below, their fluorescent complementary chains are labeled with different channel fluorescence.

[0140] The samples were stained with antibodies for two types of signaling pathway targets and downstream proteins. They were stained with the modified CRBN antibody and IKZF3 antibody as described above; TP53RKA antibody, RNASEK antibody, and TSPO2 antibody (stock solution 1:1500 dissolved in phosphate buffer). They were kept in the dark at 4°C overnight and washed with phosphate buffer for 15 minutes 3 times.

[0141] Mitochondrial and lipid staining related to the function of target molecules to detect mitochondrial and subcellular localization. Staining was performed using a commercially available mitochondrial lipid detection kit.

[0142] For DNA staining and cell localization, Hoechest 33258 (1:1000 in phosphate buffer) was used to stain the nucleus at room temperature for 30 minutes, and the sample was washed with phosphate buffer for 15 minutes three times. The sample was soaked in double distilled water at room temperature for 1 hour three times and the water was changed each time to allow the sample to fully swell, and it was kept away from light during the period.

[0143] Imaging was performed under a Leica DMi8 inverted confocal microscope at 100x magnification. The samples were placed in imaging buffer and divided into three rounds of detection according to the fluorescent complementary chains. Different types of mixed complementary chains were added to the buffer in each round: R1 and R2 were added in the first round; R2 and R3 were added in the second round; and R1 and R3 were added in the third round. Detection was performed, and 40,000 frames of images were acquired in each round of imaging. Sequential imaging calculation processing was performed to obtain spatial omics images. Ibodomide, two types of signaling pathway targets and downstream molecules, organelle interactions, complex molecular structure and subcellular location were analyzed.

[0144] like Figure 3 As shown, in the spatial omics technology described in the present invention, proteins are imaged sequentially through sequential imaging technology to achieve sequential flashing of multiple adjacent fluorescent spots on a single protein molecule, and adjacent fluorescent spots are separated by computational methods, with the resolution increased to 0.1 nanometers, revealing the protein molecular mechanism of drug action in the body.

[0145] Figure 4 and Figure 5 It shows that the vascular tissue maintains its original structure and increases in size isotropically. The purple signal shows that the dense and unclear mRNA signal before expansion can be clearly located after expansion.

[0146] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. 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 class of immunomodulatory agent targets, characterized in that: Including TP53RK targets; The TP53RK target is a single intracellular target CRBN-independent target, and the TP53RK target includes the gene sequence in SEQ ID No. 1; The TP53RK target site is subjected to one or several residue substitutions and / or additions and / or deletions, and the derived gene sequence has a homology of more than 90%; as well as The downstream pathway of the TP53RK target includes: protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and protein molecule PSMB9, and one or more of mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9.

2. The immunomodulatory agent target according to claim 1, characterized in that: The downstream pathways of the TP53RK target include: The protein molecule RNASEK includes the protein sequence in SEQ ID No.2; The protein molecule TSPO2 includes the protein sequence in SEQ ID No.3 and / or SEQ ID No.4; The protein molecule PADI4 includes the protein sequence in SEQ ID No.5 and / or SEQ ID No.6; The protein molecule PSMB9 includes the protein sequence in SEQ ID No.7 and / or SEQ ID No.8; The protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 are derived protein sequences after one or more amino acid residues are replaced and / or added and / or deleted, and the homology is more than 90%; The nucleic acid sequences corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 and the derived nucleic acid sequences which have undergone one or several residue substitutions and / or additions and / or deletions and have a homology of more than 90%; and The mRNA, sugars, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9.

3. An imaging method for in-situ detection, characterized in that: include: Molecular detection of target CRBN-dependent pathway and substrate IKZF3 protein in single tissue cells; and / or Based on the detection of the TP53RK target as described in claim 1 or 2, and its downstream pathway protein molecule RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9; and / or Based on the TP53RK target described in claim 1 or 2, the mRNA, carbohydrates, mitochondria and lipid molecules corresponding to the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 are detected.

4. The imaging method for in-situ detection according to claim 3, characterized in that: include: Disease model mice were used to construct an inflammation mouse model; The disease model mice are B6 mice, B6.Fas obtained by cross-breeding of mice. lpr Mouse and B6.Fas lpr .Crbn I391V Mice; The inflammatory mouse model is treated with ibodamide: a mixed solution containing ibodamide is intraperitoneally injected into the inflammatory mouse model at a dose of 0.7 micrograms per day to obtain a treated mouse model; The treated mouse model was sampled and the inflammation level was evaluated; Spationomic drug testing was performed on tissues of the treated mouse model.

5. The imaging method for in-situ detection according to claim 4, characterized in that: The treated mouse model was sampled and the level of inflammation was assessed, including: Extracting serum from the treated mouse model to complete enzyme-linked immunosorbent assay; Extracting peripheral blood cells from the treated mouse model to complete nucleic acid molecule sequencing analysis; and The treated mouse model was subjected to cardiac perfusion, and the tissues were extracted for hematoxylin-eosin staining and immunofluorescence imaging analysis.

6. The imaging method for in-situ detection according to claim 4, characterized in that: Perform spatial omics drug testing on tissues of the treated mouse model, including: After incubating the tissue with the swelling monomer solution at 4°C for 30 minutes, the tissue was transferred into the gel chamber; The incubated gel chamber is sealed and transferred to 37° C. for overnight incubation to gel the cells of the tissue to obtain a gelled sample; The gelled sample is taken out, digested and washed, and then stored in a phosphate buffer containing 0.02% sodium azide at 4° C. to allow the sample to swell and obtain a sample to be tested; The sample to be tested is subjected to spatial omics detection, and multiple rounds of detection and imaging are performed using a sequential imaging method.

7. The imaging method for in-situ detection according to claim 4, characterized in that: The mixed solution containing ibolidomide comprises the following components in terms of volume fraction: Ibolidomide 0-2.7%, phosphate buffer 52.3%, polyethylene glycol 37.3%-40% and Tween-80 5%.

8. The imaging method for in-situ detection according to claim 4, characterized in that: The swelling monomer solution The composition is sodium acrylate 0-30% w / v, acrylamide 8-15% w / v, dimethylacrylamide 0-4% v / v, N,N-methylenebisacrylamide 0.01%-0.05% w / v, sodium chloride 1-12% w / v, dissolved in PBS for storage, and before use, ammonium persulfate 0.1-0.25% w / v, tetramethylethylenediamine 0.04% v / v, 4-hydroxytamoxifen 0.001% w / v and methacrolein 0.1%-0.25% v / v are added to initiate polymerization.

9. The imaging method for in-situ detection according to claim 6, characterized in that: The spatial omics detection of the sample to be tested includes: staining the sample to be tested: When testing the target CRBN-dependent pathway and substrate IKZF3 protein molecules in single cells of tissues: The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azide DNA sequences to obtain modified CRBN antibodies and IKZF3 antibodies; the drug was localized by ibodamide autofluorescence; Using the CRBN antibody and the IKZF3 antibody to stain and mark the signaling pathway targets and downstream protein antibodies; An ATP-targeted fluorescent probe is used to perform spatial quantitative monitoring of ATP in samples; a fluorescent probe targeting ubiquitin is used to perform spatial localization and quantitative detection of CRBN-mediated protein ubiquitination; Wherein, the nanobody is modified with a coding substrate DNA sequence by click chemistry method.

10. The imaging method for in-situ detection according to claim 6, characterized in that: The spatial omics detection of the sample to be tested includes: staining the sample to be tested: When testing the TP53RK target and its downstream pathway protein molecules RNASEK, protein molecule TSPO2, protein molecule PADI4 and / or protein molecule PSMB9: Phosphate-buffered saline was used to stain TP53RK, RNASEK, and TSPO2 antibodies, and drug localization was performed by ibodamide autofluorescence; and / or When the mRNA, sugar, mitochondria and lipid molecules corresponding to the TP53RK target, the protein molecule RNASEK, the protein molecule TSPO2, the protein molecule PADI4 and the protein molecule PSMB9 are detected: The nanobodies modified with maleimide-PEG4-DBCO linker were reacted with azidated DNA sequences to obtain modified TP53RK antibody, RNASEK antibody and TSPO2 antibody staining; The signaling pathway targets and downstream protein antibodies were stained and marked using the TP53RK antibody, RNASEK antibody and TSPO2 antibody; Wherein, the nanobody is modified with a coding substrate DNA sequence by click chemistry method.

Images