WO2023134121A1

WO2023134121A1 - Use of deubiquitinating enzyme in preparation of drug for preventing or treating acute graft-versus-host disease and graft-versus-leukemia

Info

Publication number: WO2023134121A1
Application number: PCT/CN2022/100866
Authority: WO
Inventors: 徐杨; 程巧; 吴德沛; 刘吟; 郑慧; 朱婷婷
Original assignee: 苏州大学
Priority date: 2022-01-13
Filing date: 2022-06-23
Publication date: 2023-07-20
Also published as: CN114191544A

Abstract

Provided is a use of a deubiquitinating enzyme BRCC3 in the preparation of a drug for preventing or treating acute graft-versus-host disease (aGVHD) and graft-versus-leukemia (GVL). A use of a deubiquitinating enzyme BRCC3 in the preparation of a drug for preventing and treating graft-versus-host disease is researched and explored by means of animal experiments. The results show that BRCC3 has the effect of inhibiting aGVHD after allogeneic hematopoietic stem cell transplantation, can inhibit donor T cell activation and the functions of T cells and inhibit proliferation of macrophages, down-regulates the ubiquitylation of TRIM25 by a proteasome pathway, stabilizes the protein level of TRIM25, then promotes secretion of IFNβ, inhibits occurrence and development of aGVHD, retains a GVL effect, and thus has great application prospects.

Description

Application of deubiquitinase in preparation of drugs for preventing or treating acute graft-versus-host disease and graft-versus-leukemia

technical field

The invention belongs to the field of biomedicine, and specifically relates to the application of deubiquitinating enzyme BRCC3 in the preparation of drugs for preventing or treating acute graft-versus-host disease and graft-versus-leukemia.

Background technique

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently an important method for the treatment of malignant hematological diseases, some genetic diseases and bone marrow failure diseases (Figure 1), mainly through donor-derived transplants Mediating graft-versus leukemia (GVL) plays a key role in treating hematological malignancies and preventing relapse after transplantation. However, donor-derived T cells in these grafts can also induce graft-versus-host disease, especially acute graft-versus host disease (aGVHD), which is currently the most serious complication restricting allo-HSCT One of the main causes of morbidity and mortality after transplantation, mainly involving the skin, liver and gastrointestinal tract, target organs can be alone or in combination with other organs. The incidence of aGVHD with obvious clinical manifestations is between 10% and 80%. Due to different transplantation methods, the average incidence is about 40%, especially aGVHD with hormone resistance, which seriously affects the quality of life and prognosis of patients. Therefore, it is extremely important to find effective means to prevent and treat aGVHD.

Ubiquitination is a kind of protein post-translational modification, which regulates the properties or functions of proteins, participates in cell signal transduction, transcription regulation, DNA damage repair, cell cycle and other processes, and is reversibly and precisely regulated by deubiquitinating enzymes. Stabilize the stability of protein in the body and maintain cell function. The human deubiquitinating enzyme BRCC36, and its murine homologue BRCC3, belong to the JAMM protease family and contain a Zn ²⁺ domain. The core catalytically active subunit of the enzyme complex is involved in processes such as inflammasome activity, interferon signaling, and DNA damage repair. There have been previous reports about the mechanism by which BRCC36 regulates the interferon signaling pathway and then affects the immune response. The envelope receptor IFNAR1 of the type I interferon IFNβ is regulated by the ubiquitination modification of the E3 ubiquitin ligase SCFβ ^-Trcp . The BRISC deubiquitinase complex can specifically cleave the ubiquitin chain binding of IFNAR1 and inhibit its degradation process, thereby promoting the cell's response to type I interferon, which provides new opportunities for BRCC3 to prepare drugs to prevent cancer and autoimmune diseases treatment method.

The pathogenesis of aGVHD is complicated, and the treatment effect is even worse. Steroid-resistant aGVHD patients have a poor prognosis, and long-term use of steroids will cause adverse reactions such as diabetes, infection, osteoporosis, and abnormal mental status, which will affect the quality of life of patients, and in severe cases will endanger There is an urgent need to find out effective methods to prevent and treat aGVHD. And there is no previous report that BRCC3 prevents and treats aGVHD.

Ubiquitination modification is a kind of protein post-translational modification, similar to methylation, phosphorylation, acetylation, etc., involved in cell signal transduction, transcription regulation, DNA damage repair, cell cycle, cell apoptosis, vesicle transport and other physiological process. Deubiquitinases can reversibly regulate the ubiquitination process, and together with the ubiquitination system constitute a complex network covering almost all cellular functions. In recent years, the research on targeting the ubiquitination modification process has achieved certain results. The human deubiquitinating enzyme BRCC36 and its mouse homologue BRCC3 are metalloprotein deubiquitinating enzymes containing Zn ²⁺ domains, which have been reported to affect immune responses by regulating interferon signaling pathways. Cancer and autoimmune diseases offer new treatments. However, its role in aGVHD has not been reported so far.

Contents of the invention

The invention explores the application of a deubiquitinating enzyme BRCC3 in the preparation of drugs for preventing and treating graft-versus-host disease and graft-versus-leukemia through animal experiments.

The invention provides the application of the deubiquitinating enzyme BRCC3 in the preparation of drugs for preventing or treating acute graft-versus-host disease and graft-versus-leukemia.

Preferably, the drug is used for inhibiting aGVHD in mice.

Preferably, the drug is used to inhibit the activation of T cells in the target organ.

Preferably, the drug is used to inhibit the secretion of IFNγ by T cells and inhibit the function of T cells.

Preferably, the drug is used to inhibit the production of macrophages in the target organ.

Preferably, the drug is used to up-regulate the protein level of E3 ubiquitin ligase Trim25.

Preferably, the drug is used to down-regulate the ubiquitination of the E3 ubiquitin ligase Trim25 through the proteasome pathway, and remove the ubiquitin levels at positions K48 and K63.

Preferably, the drug is used to promote the secretion of IFNβ.

Preferably, the drug is used to inhibit the growth of leukemia cells in vivo while inhibiting aGVHD, and retain the GVL effect.

The present invention also provides a drug for preventing or treating acute graft-versus-host disease and graft-versus-leukemia, the drug comprising deubiquitinating enzyme BRCC3.

Further, the dosage of the deubiquitinating enzyme BRCC3 is 40-60 μg/ml.

Further, the administration route of the drug includes one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection and intramuscular injection.

Compared with the prior art, the technical solution of the present invention has the following advantages:

In the present invention, BRCC3 has the effect of inhibiting acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation; it can inhibit the activation of donor T cells and the function of T cells, and inhibit the proliferation of macrophage cells; through the proteasome pathway, it can down-regulate the function of Trim25 Ubiquitination modification stabilizes the protein level of Trim25, thereby promoting the secretion of IFNβ and inhibiting the occurrence and development of aGVHD; while retaining the GVL effect.

Description of drawings

Fig. 1 is the graph of survival curve (A), body weight change (B) and aGVHD score (C) of GVHD model mice after transplantation.

Figure 2 is the H&E staining images of skin, lung and intestinal tissues of mice in Vector group and BRCC3 group.

Figure 3 is a graph showing the inhibitory effect of BRCC3 on T cell activation.

Figure 4 is a graph showing the inhibitory effect of BRCC3 on T cell function.

Figure 5 is a graph showing the inhibition of BRCC3 on macrophages.

Figure 6 is a graph showing the inhibitory effect of BRCC3 on the protein level of E3 ubiquitin ligase Trim25.

Fig. 7 is a graph showing the inhibitory effect of BRCC3 on the ubiquitination modification level of Trim25 protein.

Fig. 8 is a graph showing that BRCC3 regulates the ubiquitination level of Trim25 through the proteasome pathway.

Fig. 9 is a graph showing the effect of BRCC3 on promoting the transcriptional activity of IFNβ.

Fig. 10 is a graph showing the inhibitory effect of knocking down BRCC3 on the expression of IFNβ in macrophages.

Fig. 11 is a graph showing the effects of BRCC3 on the expression of IFNβ and the secretion of inflammatory factors in macrophages.

Fig. 12 is a graph showing the promoting effect of BRCC3 on the expression of IFNβ in mice.

Figure 13 is a graph showing the survival curve (A), body weight change (B) and aGVHD score (C) of GVHD/GVL model mice after transplantation.

Fig. 14 is an imaging diagram of a mouse in vivo and a statistical diagram of quantified leukemia cells.

Fig. 15 is a quantitative diagram of the fluorescence value in the small living body imaging.

In the figure, * represents P<0.05, ** represents P<0.01, *** represents P<0.001.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

Example 1 CaCl ₂ method to prepare DH5α competent

Prepare 2 bottles of 200ml 0.1M CaCl ₂ in a sterile ultra-clean bench, and pre-cool on ice before use after autoclaving; take the DH5α monoclonal strains stored at -80°C, dip and smear them on the sterilized place without antibiotics Solid LB medium, culture in a water-proof constant temperature incubator at 37°C for 12-16h until colonies are formed; select a single colony on the solid LB medium, inoculate into 5ml of fresh LB medium after sterilization without antibiotics, and place it on a horizontal shaker at 37°C , shake at 250rpm overnight (12-16h); take 50μl of the above bacterial solution into 100ml of fresh LB medium sterilized without antibiotics, place on a horizontal shaker at 37°C, shake at 250rpm for 3h; put the above bacterial solution on ice for 30min, pour into In a pre-cooled 50ml sterile centrifuge tube, put it in an ice bath for 10min, 3500rpm, and centrifuge at 4°C for 15min; discard the supernatant, and resuspend the bacteria with 25ml (or 1/3 volume of the original bacterial solution) ice-cooled 0.1M CaCl ₂ , Ice bath for 10min, 3500rpm, centrifuge at 4°C for 10min; discard the supernatant, resuspend with 50ml ice-cold 0.1M CaCl ₂ , ice bath for 20min, 3500rpm, centrifuge at 4°C for 10min; discard the supernatant, resuspend with 5ml (or original bacterial solution 1/15 volume) pre-cooled 0.1M CaCl ₂ (containing 20% glycerol) to resuspend the pellet; each 100 μl was dispensed into a sterile 1.5ml EP tube, and stored in a -80°C refrigerator for later use. Competence detection: Take competent bacteria and smear them on the LB medium plate containing A ⁺ /K ⁺ , incubate in a water-proof constant temperature incubator at 37°C for 12-16 hours, and observe whether the competent bacteria are contaminated.

The extraction of embodiment 2 plasmid

Plasmid transformation: Take 50 μl of DH5α competent bacteria and immediately put it on ice to melt for 6-8 minutes; absorb 1 μl of the target plasmid and add it to the competent bacteria, blow it gently, and put it in an ice bath for 15-30 minutes; put it in a water bath/metal bath at 42°C , heat shock for 90s, put it on ice immediately, let it stand for 2-3min; add 200μl sterilized fresh LB medium, place it on a horizontal shaker at 37°C, shake at 250rpm for 30-45min; centrifuge at 3000rpm for 10min, suck off 100μl Clear, gently blow evenly, and apply on the sterilized A ⁺ solid LB medium plate; after the bacterial solution is absorbed, place the LB culture plate in a constant temperature and humidity incubator at 37°C for overnight cultivation until colonies are formed (12-16h).

Single-clonal amplification: Pick a single transformed colony and add it to 3ml of sterilized fresh LB medium containing ampicillin, shake at 37°C and 250rpm for 2-3h; take 1ml of the amplified bacterial liquid and use Axygen small extraction reagent Box Description Extract the plasmid (can be omitted); take 1ml of the amplified bacterial liquid into 400ml of sterilized fresh LB medium containing 100μg/ml A+, place on a horizontal shaker at 37°C, shake at 250rpm overnight (12-16h ); Bacteria collection: 3400rpm, centrifuge at 4°C for 30min, discard the supernatant, the precipitate obtained is the desired cell, and freeze at -20°C.

Kangwei high-purity plasmid extraction reagent: Add all RNase to Buffer P1, store at 4°C for later use, add ethanol to Buffer PW, and store at room temperature; place the frozen bacteria at room temperature until it melts, add 15ml of Buffer P1 Resuspend the bacteria and blow them evenly; add 15ml of Buffer P2, gently invert 8-10 times up and down to fully lyse the bacteria, let stand at room temperature for 3-5min (no more than 5min), the solution becomes clear and viscous; add Buffer P3 15ml, immediately upside down and gently upside down 8-10 times, white flocculent precipitate appears, let it stand for 5min, 12000g, 4 ℃ high-speed centrifugation for 10min; filter with 200 mesh nylon membrane to get the supernatant, discard the lysed bacteria; Pour it into the endotoxin removal filter (FQ), slowly push the push handle to filter, and collect it in a self-prepared 50ml centrifuge tube; column balance: add 2ml Buffer PS to the adsorption column (DQ) that has been loaded into the collection tube, Centrifuge at 12000g at 4°C for 2 minutes at high speed, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube; pass through the column: transfer the filtered liquid in the 50ml centrifuge tube to the adsorption column, centrifuge at 12000g at 4°C 2min, discard the waste liquid, pass through the column several times, each time the volume of the adsorption column does not exceed 10ml; add 10ml of Buffer PW, 12000g, to the adsorption column, centrifuge at 4°C for 2min, discard the waste liquid; repeat this step once; Put the adsorption column back into the collection tube, centrifuge at 12000g at 4°C for 5 minutes at high speed, discard the waste liquid; place it at room temperature for 5 minutes, dry the remaining rinse solution thoroughly, add 1ml of sterilized double-distilled water preheated in an oven at 65°C, Let stand at room temperature for 5 minutes, centrifuge at 12,000g at 4°C for 5 minutes at high speed; add the liquid in the collection tube to the middle of the adsorption column membrane again, centrifuge at 12,000g at 4°C for 5 minutes; detect the plasmid concentration with a spectrophotometer, and freeze at -20°C; enzyme digestion After the identification is correct, the plasmid is sent for sequencing verification.

Example 3 Extraction of minicircle plasmids

The eukaryotic expression plasmid can be transformed into a microcircle plasmid after transformation and induction, which can be stably expressed in vivo without being degraded. It is injected into mice by the HGT method, resulting in stable and high expression of the target gene in the mouse, and because it does not contain bacterial DNA The backbone will not cause immunogenic reactions, and is suitable for in vivo experiments to construct mouse models for overexpression systems.

3.1 Transformation of microcircle plasmids: take 50 μl of ZYCY10P3S2T E.coli competent bacteria, and immediately put them on ice to melt for 6-8 minutes; absorb 1 μl of microcircle plasmids and add them to the competent bacteria, blow gently, and put them in ice bath for 15-30 minutes; In a water bath/metal bath at 42°C, heat shock for 90s, put it on ice immediately, and let it stand for 2-3min; add 200μl sterilized fresh LB medium, place on a horizontal shaker at 37°C, shake at 250rpm for 30-45min; 3000rpm Centrifuge for 10 minutes, suck off 100 μl of supernatant, blow gently and spread it on the sterilized K+ solid LB medium plate; after the bacterial solution is absorbed, place the LB culture plate in a constant temperature and humidity incubator at 37°C to incubate overnight until colonies are formed (12-16h).

3.2 Induction of microcircle plasmids: prepare 30% L-Arabinose solution and 5% NaOH solution, filter and sterilize with a 0.22 μm sterile filter; pick a single transformed colony and add 2 ml of sterilized fresh solution containing 100 μg/ml K ⁺ In TB medium, shake at 37°C and 250rpm for 2-3h; pipette 100μl of activated bacterial solution into 400ml of sterilized fresh TB medium containing 100μg/ml K ⁺ , and add 40ml of sterilized phosphate buffer, Place on a horizontal shaker at 37°C, shake at 250rpm overnight (12-14h); add 400ml of sterilized fresh LB medium containing 100μg/ml K ⁺ and 4ml of 30% L-Arabinose solution; measure the pH of the bacterial solution, if the pH< 7, continue to add 4ml 5% NaOH solution, adjust to weak alkaline, place on a horizontal shaker at 37°C, shake at 250rpm for 2h; continue to add 4ml 30% L-Arabinose, test the pH of the bacterial solution, if the pH<7, continue Add 4ml of 5% NaOH solution, adjust to weak alkaline, place on a horizontal shaker at 37°C, shake at 250rpm for 2h; repeat this step once; collect bacteria: 3400rpm, centrifuge at 4°C for 30min, discard the supernatant, and freeze the bacteria at -20°C The body can improve the extraction efficiency.

3.3 Invitrogen large-scale pumping kit large-scale plasmid extraction: put the frozen bacteria at room temperature until they melt, add 120ml of Solution I solution to resuspend the bacteria, blow well to avoid residual clumps, and dissolve the bacteria; add Solution II Solution 120ml, mix gently, so that the bacteria are fully lysed to a clear and slightly viscous state; add Solution III solution 120ml, mix gently, a white flocculent precipitate appears; Keep the supernatant for the required nuclear content after lysis; place the balance column system from bottom to top in the order of Erlenmeyer flask-mouth card-balance column, add 30ml EQ1 solution to the balance column, and drop by drop to balance Column; slowly pour the supernatant nuclear content obtained after centrifugal filtration into the balance column, drop by drop to absorb the nuclear content in the supernatant; add 60ml of W8 solution into the balance column, drop by drop Rinse the equilibrated column; transfer the equilibrated column to a clean round-bottomed tube, add 15ml of E4 solution, and collect the eluate in the round-bottomed tube; add 10.5ml of isopropanol to the round-bottomed tube, and gently invert to mix. Centrifuge at 12000g at 4°C for 30min at high speed, discard the supernatant; add 5ml of absolute ethanol to the round bottom tube, centrifuge at 12000g at 4°C for 6min at high speed, discard the supernatant carefully; place in a ventilated place to dry thoroughly at room temperature, add 1ml of oven pre- Heat sterilized double distilled water at 65°C to resuspend the pellet and mix thoroughly; use a spectrophotometer to detect the concentration of the plasmid, and freeze it at -20°C; digest and run agarose electrophoresis to identify the successful construction of the microcircle plasmid.

Example 4 Transient transfection expression of target gene

(1) Transient system:

(2) Inoculate cells according to the specifications of the orifice plate, add complete medium, and place in a 37°C, CO ₂ incubator to cultivate to 80%;

(3) Prepare the following solutions under sterile conditions:

Solution A: dilute the plasmid to be transfected with serum-free medium;

Solution B: dilute the transfection reagent with serum-free medium (plasmid: transfection reagent ratio is 1:2);

(4) Gently mix A and B solutions, and let stand at room temperature for 10 minutes;

(5) Abandon the original cell medium and replace it with a serum-free medium;

(6) Add the mixed AB solution dropwise to each well, shake gently and mix well, and culture in a CO ₂ incubator at 37°C for 6-8 hours; discard the transfection solution, replace it with a complete medium to continue the culture, Collect the results after 48-72 hours for further testing.

Example 5 Dual luciferase reporter experiment

Prepare 1×PLB, LAR II and 1×Stop Solution reagents according to the instructions; use the transient transfection method to prepare samples, discard the medium, and transfer to EP tubes; wash twice with 1×PBS, centrifuge at 450g, 4°C for 5min, Discard the supernatant; add 100 μl of 1×PLB lysate, place on a horizontal shaker and lyse for 15 minutes; take 20 μl of the lysate to a 96-well flat-bottomed plate, and blow well; add 50 μl of fluorescent substrate solution LAR II at room temperature in the dark , use a microplate reader to detect the fluorescence value of luciferase; quickly add 50 μl 1×Stop Solution, and immediately use a microplate reader to detect the fluorescence value of renila; use the ratio of the fluorescence values of the two groups to compare the expression of the reporter gene between each group.

Example 6 Determination of the relative expression level of the target gene at the transcription level

6.1 RNA extraction (Trizol)

Take the spleen, liver, lung and small intestine tissues of the transplanted mice and place them in a nuclease-free EP tube, add 300 μl of Trizol reagent, and use a tissue grinding gun to grind the cells thoroughly; continue to add 700 μl of Trizol reagent, blow until no adhesion, and place on ice Let stand for 5 minutes; add 200 μl chloroform, vortex vigorously for 15 seconds, and let stand at room temperature for 5 minutes; centrifuge at 12,000 g at 4°C for 15 minutes, transfer the supernatant to another prepared nuclease-free EP tube, and add an equal volume of isopropyl Alcohol, mix upside down, stand at room temperature for 10min; centrifuge at 12000g, 4°C for 10min, white precipitate at the bottom can be seen, discard the supernatant; add 1ml of pre-cooled 75% ethanol (prepared with DEPC water), point to mix, 8000rpm, 4 Centrifuge at ℃ for 5 minutes, and discard the supernatant; repeat this step; hang upside down to dry, and add 20-50 μl of DEPC water preheated in an oven to dissolve the precipitate after the ethanol has evaporated; use a spectrophotometer to detect the plasmid concentration, and the 260/280 value is 1.8 -2.0, 260/230 greater than 2.0 indicates good purity; freeze at -80°C.

6.2 Reverse transcription of RNA into cDNA (10μl system)

Prepare working solution ① (6 μl) according to the obtained RNA concentration:

Place in a PCR instrument/water bath at 65°C for 5 minutes;

Prepare working solution ② (4μl):

Place in a PCR instrument/water bath at 42°C for 1h, 70°C for 10min, and freeze at -20°C.

6.3 Real-time fluorescent quantitative PCR (Realtime PCR)

Amplify the reverse-transcribed cDNA by PCR to prepare a quantitative PCR system (20 μl):

Quantitative PCR reaction conditions: 95°C for 30s, (95°C for 5s, 60°C for 30s) × 40 cycles;

Using GAPDH/Actin as an internal reference, the relative expression of the target gene was analyzed using the ΔΔCt method.

Example 7 Determination of the relative expression level of the target gene at the protein level

7.1 Preparation of protein samples

Pre-cool the desktop refrigerated centrifuge to 4°C, heat the metal bath to 100°C for use; discard the original cell culture medium, wash the cells twice with 1×PBS solution, centrifuge at 450g for 5 minutes at 4°C, and discard the supernatant; prepare protein lysate : Prepare PMSF protease inhibitor: RIPA strong lysate at a ratio of 1:100, mix gently, and place on ice for later use; add protein lysate according to the amount of cells, place on ice, and use an ultrasonic cell disruptor to fully lyse the protein ; 12000g, centrifuge at 4°C for 15min, transfer the supernatant to a new EP tube; detect the protein concentration with Bradford protein quantification kit; add 6×Loading Buffer to the protein sample, blow or vortex to mix, cook the protein in a metal bath at 100°C 10min; 12000g was spun off, cooled on ice for 2-3min, and stored at -20°C.

7.2 Western Blot

According to the molecular weight of the protein, prepare the corresponding concentration of SDS-PAGE separating gel and stacking gel, put the gel plate into the electrophoresis tank, add the diluted 1×Running Buffer electrophoresis liquid, add protein marker and sample respectively; electrophoresis: run in the stacking gel When gelling, set the electrophoresis condition as a voltage of 80V, 20min, until the sample enters the separation gel, adjust the voltage to 120V, 90min, and stop the electrophoresis when the bromophenol blue is almost at the bottom of the gel; Add the Transfer Buffer into the transfer membrane tank, prepare PVDF of the size corresponding to the size of the separation gel (the size of the whole plate is 8×6.5cm), soak in anhydrous methanol for 15s to activate; put the PVDF membrane, filter paper, and sponge pad together into the Transfer Equilibrate in Buffer for 15-20min, trim excess stacking gel at the same time, separate gel plate and colloid in Transfer Buffer; put sponge pad, filter paper, glue, PVDF on the transfer film plate in sequence from cathode to anode (black to white) Membrane, filter paper, sponge pad, drive away air bubbles in turn, clamp the transfer plate, put it into the transfer tank, put ice packs around it, set the current at 200mA constant current transfer for 90min; seal: after the transfer is completed, trim the PVDF membrane, Add blocking solution (5% skimmed milk/BSA) prepared with 1×TBST, place on a shaker to block at room temperature for 1 hour or overnight at 4°C; for primary antibody incubation: discard the blocking solution, rinse with 1×TBST and discard Go, add the primary antibody solution diluted with 3% BSA, incubate overnight (12-16h) at 4°C in a shaker to avoid air bubbles; wash the membrane: recover the primary antibody solution, wash the membrane 3 times with 1×TBST, 10min each time; secondary antibody Incubation: Discard TBST, add secondary antibody solution diluted with 3% BSA (1:10000), place on a shaker and incubate at room temperature for 1 hour; wash membrane: recover primary antibody solution, wash membrane 3 times with 1×TBST, 10 minutes each time; Development: Under the condition of room temperature and dark, the supersensitive ECL solution A: B solution is prepared according to the ratio of 1:1, and evenly dropped on the film, and the CLiNX chemiluminescence imager is used to develop and expose to take pictures.

Example 8 Immunoprecipitation/co-immunoprecipitation (IP/CO-IP)

Prepare samples by transient transfection method, discard the medium, transfer to EP tube; wash twice with 1×PBS, centrifuge at 450g, 4°C for 5min, discard supernatant; prepare protein lysate: PMSF protease inhibitor: PI Cocktail: IP lysate is prepared according to the ratio of 1:1:100, mixed gently, and placed on ice for later use; add 300 μl of lysate to ice bath, place on a horizontal shaker and lyse for 30 min; Transfer the colorless lysate to a new EP tube (nuclease-free EP tube); add IP antibody at a ratio of 1:100, take 30 μl as Input, seal it with parafilm, and incubate overnight at 4°C on an inverting shaker; Cool 1% NP40 Washing Buffer and centrifuge, take 40μl/sample of ProteinA/G beads, add 1ml Washing Buffer to wash the beads, centrifuge at 12000g, 4°C for 2min, discard the supernatant, repeat washing 2 times; add the same volume as the original sample Resuspend the beads in Washing Buffer, mix well, take 40 μl and quickly add to each sample, seal with parafilm, incubate at 4°C on an overturning shaker for 2 hours; centrifuge at 12,000g at 4°C for 5 minutes, discard the supernatant, and wash 3 times with Washing Buffer , discard the supernatant as much as possible; add Washing Buffer 40μl and 1×Loading Buffer 8μl, 100 ℃ metal bath for 10 min;

Example 9 Preparation of mouse tissue cells

9.1 Preparation of mouse bone marrow cell suspension

Mice were sacrificed by cervical dislocation, soaked in 75% alcohol solution for disinfection; the femur, tibia and spine of both lower limbs of the mouse were taken, excess muscle tissue was removed, placed in a mortar filled with 1640 medium, and gently squeezed and ground with a grinding pestle until most of the cells are removed; filter the cell suspension obtained through 200-mesh nylon mesh into a clean 50ml centrifuge tube, centrifuge at 1300rpm, 4°C for 8min, and discard the supernatant; use 5ml 1×RBC lysate per mouse After adding the proportion of mice, fully resuspend, lyse at room temperature for 7 minutes, add 30-40ml 1640 medium to stop lysis, centrifuge at 1300rpm, 4°C for 8 minutes, discard the supernatant; resuspend cells with an appropriate amount of 1×PBS/1640 medium , filtered through a 200-mesh nylon mesh into a clean 50ml centrifuge tube, centrifuged at 1300rpm at 4°C for 8min, discarded the supernatant, and washed to filter dead cell clumps; according to the ratio of 1ml 1×PBS solution/mouse, fully Resuspend the cells, adjust the concentration of the cell suspension after counting on a counting plate/counter, and place on ice for use.

9.2 Preparation of mouse splenocyte suspension

Mice were sacrificed by cervical dislocation, soaked in 75% alcohol solution for disinfection; the whole spleen of mice was taken, placed in a cell culture dish filled with 1640 medium, and gently squeezed and ground with a glass slide until all the cells were ground out; Filter the ground cell suspension through a 200-mesh nylon mesh into a clean 50ml centrifuge tube, centrifuge at 1300rpm at 4°C for 8min, discard the supernatant; add 3ml 1×RBC Lysis Solution/mouse to fully resuspend, Lyse at room temperature for 5 minutes, add 30-40ml 1640 medium to stop the lysis, centrifuge at 1300rpm, 4°C for 8 minutes, discard the supernatant; resuspend the cells with an appropriate amount of 1×PBS/1640 medium, filter through a 200-mesh nylon mesh until clean In a 50ml centrifuge tube, centrifuge at 1300rpm, 4°C for 8min, discard the supernatant, rinse and filter the dead cell mass; add 0.5-1ml 1×PBS or Staining Buffer solution/mouse, fully resuspend the cells, and count Adjust the concentration of the cell suspension after counting on the plate/counter, and place it on ice for use.

9.3 Preparation of mouse liver lymphocyte suspension

Mice were sacrificed by cervical dislocation, soaked in 75% alcohol solution for disinfection; cut open the thoracic and abdominal cavity, thoracic septum, and right atrial appendage of the mouse layer by layer, sucked 1×PBS with a 10ml syringe, and then inserted the needle tip into the left ventricle with a thin needle. Inject PBS to wash out the blood in the liver until the liver turns white; take out the mouse liver, place it in a cell culture dish filled with 1640 medium, squeeze and grind it gently with a glass slide until all the cells are grinded; grind the obtained Filter the cell suspension through a 200-mesh nylon mesh into a clean 50ml centrifuge tube, centrifuge at 1300rpm at 4°C for 6min, discard the supernatant; resuspend the cells in 5ml of 40% Percoll lymphocyte separation medium, centrifuge at 2000rpm for 20min at room temperature, and increase speed 6 Slow down to 0 density gradient centrifugation, pour off the upper two layers gently, and only keep the bottom layer of liver lymphocytes; add 5ml 1×RBC lysate/mouse, mix well, lyse at room temperature for 5min, add 30- Stop lysis with 40ml 1640 medium, centrifuge at 1300rpm, 4°C for 8min, discard the supernatant; resuspend the cells with 4ml 1640 medium, wash once, centrifuge at 1300rpm, 4°C for 6min, discard the supernatant; use 1ml Staining Buffer/mouse After adding the ratio, resuspend the cells and count them with a counter for use.

9.4 Preparation of mouse lung lymphocyte suspension

The mice were sacrificed by cervical dislocation, soaked in 75% alcohol solution for disinfection; the thorax and thoracic septum of the mice were cut layer by layer, the lungs of the mice were taken out, and placed in a cell culture dish equipped with 1640 medium (containing 10% FBS). Squeeze and grind gently with a glass slide until all the cells are grinded out; filter the cell suspension obtained by grinding through a 200-mesh nylon mesh into a clean 50ml centrifuge tube, centrifuge at 1300rpm at 4°C for 6min, discard the supernatant; use 40% Resuspend the cells in 5ml of Percoll Lymphocyte Separation Medium, centrifuge at room temperature for 20min at 2000rpm, increase speed 6 and decrease speed 0 density gradient centrifugation, pour off the upper two layers gently, and only keep the bottom layer of liver lymphocytes; according to 5ml 1×RBC lysate Mix well after adding the ratio, lyse at room temperature for 5 minutes, add 30-40ml 1640 medium to stop lysis, centrifuge at 1300rpm at 4°C for 8min, discard the supernatant; resuspend cells with 40ml 1640 medium, wash once, and centrifuge at 1300rpm at 4°C After 6 minutes, discard the supernatant; add 1ml of Staining Buffer/mouse, resuspend the cells, and count them with a counter before use.

9.5 Preparation of mouse small intestinal epithelial lymphocyte suspension

The mice were sacrificed by cervical dislocation, soaked in 75% alcohol solution for disinfection; the abdominal cavity of the mice was cut open layer by layer, the intestines of the mice were taken out, the small intestine between the lower duodenum and the ileocecal was cut off, and placed in a culture chamber equipped with 1640 In a cell culture dish containing 10% FBS; use a 10ml syringe to draw 1×PBS repeatedly to rinse the intestinal tract until the intestinal tract is rinsed clean and there is no excrement left in the tube, then cut the small intestine longitudinally with scissors and cut it into 3-5mm After crushing, transfer to a 50ml centrifuge tube; add 50ml of intestinal digestion solution, 37°C, 250rpm horizontal shaker for 45min; filter the digestion solution through a 200-mesh nylon mesh into a 50ml centrifuge tube, centrifuge at 1300rpm, 4°C for 6min, discard Supernatant; take the precipitated part, resuspend the cells with 5ml of 40% Percoll lymphocyte separation medium, centrifuge at room temperature for 20min at 2000rpm, increase speed 6 and decrease speed 0 density gradient centrifugation, pour off the upper two layers gently, and only keep the bottom layer Liver lymphocytes: Add 5ml 1×RBC lysate and mix thoroughly, lyse at room temperature for 5 minutes, add 30-40ml 1640 medium to stop lysis, centrifuge at 1300rpm, 4°C for 8min, discard supernatant; reconstitute with 40ml 1640 medium Suspend the cells, wash once, centrifuge at 1300rpm, 4°C for 6min, discard the supernatant; add 1ml Staining Buffer/mouse and resuspend, count with a counter and set aside for use.

Example 10 Magnetic bead sorting cells (positive selection)

Obtain and count mouse spleen cell suspension as above, centrifuge at 1300rpm, 4°C for 5min; add sorting buffer, adjust the final concentration to 1× ¹⁰⁸ cells/ml, transfer to sterile flow sorting tube; Add Rat serum thawed in advance at a ratio of 50 μl/ml; add Isolation Cocktail at a ratio of 50 μl/ml, blow it repeatedly, and let it stand for 7.5 minutes; add Depletion Cocktail at a ratio of 50 μl/ml, blow it repeatedly, and let it stand for 2.5 minutes; Fully oscillate the Rapid Spheres on the vortex for 1 min; quickly add the vortexed Rapid Spheres to each sample at a ratio of 75 μl/ml, blow repeatedly, and let stand for 2.5 min; fill up the volume to 2.5 ml with sorting buffer, Repeatedly blow gently 2-3 times; place the flow sorting tube in the magnet and let it stand for 2.5 minutes; prepare another sterile 50ml centrifuge tube, and pour the positive cell suspension after flow sorting into the centrifuge tube ; Add 2.5ml of sorting buffer to the flow sorting tube, gently blow it 2-3 times repeatedly, put it in the magnet and let it stand for 2.5min, pour the cell suspension of the positive sorting monkey into the centrifuge tube at an angle, this step Repeat 3-5 times; centrifuge the sorted cells in the centrifuge tube at 1300 rpm at 4°C for 5 min, discard the supernatant, resuspend and count, adjust the concentration, and place on ice for later use.

The establishment of embodiment 11 mouse model

Hydrodynamic gene transfer (HGT) refers to injecting 2 ml of a solution containing 100 μg of eukaryotic expression plasmid into the mouse body through the tail vein of the mouse quickly (5-8 s), so as to transfer the plasmid to the liver-based It is highly expressed in a variety of organs, so that it can be stably expressed in vivo, and this technology is used to construct a mouse model of overexpression plasmid.

11.1 Syngeneic/allogeneic transplantation mouse model

A 6-8 week old BALB/c (H2K ^d background, female) mouse was used as a donor mouse, and a 6-8 week old BALB/c (H2K ^d background, female) mouse was used as a recipient mouse to construct an isogenic mouse model. The allogeneic mouse model was constructed by using 6-8 weeks old C57BL/6 (H2K ^b background, female) mice as donor mice and 6-8 weeks old BALB/c (H2K ^d background, female) mice as recipient mice. Pretreatment conditions: start to drink sterile acidified water containing gentamicin sulfate on the 7th day before transplantation, and undergo 650cGy X-ray irradiation for myeloablation within 4-12 hours before transplantation (325cGy/time, interval 2h, dose rate 500cGy/min) . Graft preparation: Extract donor mouse bone marrow cells (1×10 ⁷ /monkey) and whole spleen cells (4×10 ⁶ /monkey) on the day of transplantation, and prepare a cell suspension with a final volume of 200 μl/monkey with 1×PBS. Put it on ice, and inject it into recipient mice through tail vein within 24 hours. (This is a control experiment)

11.2 aGVHD mouse model

The allogeneic aGVHD mouse model was constructed by using 6-8 weeks old C57BL/6 (H2K ^b background, female) mice as donor mice and 6-8 weeks old BALB/c (H2K ^d background, female) mice as recipient mice. Pretreatment conditions: drinking sterile acidified water containing gentamicin sulfate on the 7th day before transplantation, injecting eukaryotic expression plasmids through HGT on the 3rd day before transplantation, and demyelinating by 650cGy X-ray irradiation within 4-12 hours before transplantation ( 325cGy/time, interval 2h, dose rate 500cGy/min). Graft preparation: Extract donor mouse bone marrow cells (1×10 ⁷ /monkey) and whole spleen cells (4×10 ⁶ /monkey) on the day of transplantation, and prepare a cell suspension with a final volume of 200 μl/monkey with 1×PBS. Put it on ice, and inject it into recipient mice through tail vein within 24 hours.

11.3 aGVHD/GVL mouse model

6-8 weeks old C57BL/6 (H2K ^b background, female) mice were used as donor mice, 6-8 weeks old BALB/c (H2Kd background, female) mice were used as recipient mice, and luciferase-labeled mice were infused at the same time The leukemia cell line A20-luciferase was used to construct the allogeneic aGVHD/GVL mouse model. Pretreatment conditions: drinking sterile acidified water containing gentamicin sulfate on the 7th day before transplantation, injecting eukaryotic expression plasmids through HGT on the 3rd day before transplantation, and demyelinating by 650cGy X-ray irradiation within 4-12 hours before transplantation ( 325cGy/time, interval 2h, dose rate 500cGy/min). Graft preparation: On the day of transplantation, extract donor mouse bone marrow cells (1×10 ⁷ /body) and whole spleen cells (4×10 ⁶ /body), and at the same time collect A20-luciferase cells (1×10 ⁷ /body), and use 1 ×PBS was prepared into a cell suspension with a final volume of 200 μl/mouse, placed on ice, and injected into recipient mice via tail vein within 24 hours.

Example 12 Mixed Lymphocyte Reaction Model (MLR)

Splenocytes from 6-8-week-old C57BL/6 (H2K ^b background, female) donor mice were used as stimulatory cells to stimulate CD3 ⁺ T cells obtained from 6-8-week-old BALB/c ( H2K ^d background, female) recipient mice reacted, and an in vivo MLR model was constructed. Pretreatment conditions: drink sterile acidified water containing gentamycin sulfate on the 7th day before reinfusion, inject the eukaryotic expression plasmid through HGT on the 3rd day, and irradiate the myeloablation with 650cGy X-rays within 4-12h (325cGy/time , interval 2h, dose rate 500cGy/min). Preparation of cells for reinfusion: Obtain CD3 ⁺ T cells (2×10 ⁶ per mouse) from donor mice through the above steps, wash them twice with 1×PBS, and prepare a cell suspension with a final volume of 200 μl per mouse with 1×PBS solution, placed on ice, and injected into recipient mice via the tail vein within 24 hours.

Embodiment 13 aGVHD scoring standard

From the day after the allogeneic hematopoietic stem cell transplantation, the mice were weighed and recorded every 2-3 days, and the aGVHD score of the mice was scored according to the indicators of mouse weight loss, posture, activity, body hair texture and skin integrity. Record.

Table 1-1 Standard table of aGVHD clinical score in mice after allogeneic hematopoietic stem cell transplantation

Example 14 Mouse Histopathological Detection and Damage Evaluation

On the 10th day after the allogeneic hematopoietic stem cell transplantation, the liver, lung, small intestine, and skin tissues of the mice were collected respectively, placed in 50ml centrifuge tubes containing 20ml of 4% paraformaldehyde solution and fixed overnight, and the tissues of the mice were prepared respectively. HE-stained sections were magnified at 200 or 400 times using an upright fluorescent microscope to observe the degree of pathological damage of these tissues and take pictures.

Damage assessment: skin GVHD pathology mainly observes hair follicle destruction, subcutaneous fat consumption, subcutaneous collagen deposition and other indicators; liver GVHD pathology observes lymphocyte infiltration in the portal area, bile duct necrosis, and focal necrosis of liver cells; Inflammatory cell infiltration around the lymphatic vessels, destruction of alveolar structure; small intestinal GVHD pathological observation of intestinal mucosal damage and ulceration, blunt villi, shedding of cell debris in the lumen, inflammatory cell infiltration in the lamina propria, loss of goblet cell degeneration and apoptosis, and crypt epithelium Apoptosis, crypt loss, etc.

Example 15 In vivo imaging detection of mice

One week after allogeneic hematopoietic stem cell transplantation, chloral hydrate reagent was injected intraperitoneally, and in vivo imaging was performed under anesthesia in mice to monitor the leukemia condition of recipient mice after transplantation.

(1) Inject 40 μl of chloral hydrate intraperitoneally into mice, and fix the mice on the imaging operation platform after anesthesia;

(2) intraperitoneally inject 100 μg/mouse of fireflying substrate into mice, and let stand for 5 minutes;

(3) Shoot with a small animal in vivo imager, and evaluate the tumor burden according to the fluorescence intensity in the mouse.

Example 16 Detection of Immune Cell Phenotype by Flow Cytometry

16.1 Membrane staining

Plate according to about 1×10 ⁶ cells/well, add 200 μl of Staining Buffer to resuspend the cells after centrifugation, centrifuge at 1300 rpm, 4°C for 5 minutes, discard the supernatant; prepare Fc Block anti-mouse CD16/32Ab according to 1:300, add to each well 100μl, 4°C to block non-specific antigen binding for 15min, replenish Staining Buffer 100μl, 1300rpm, centrifuge at 4°C for 5min, discard supernatant; prepare cell surface marker antibody according to 1:300, add 100μl to each well, incubate at 4°C in the dark for 30min, replenish Staining Buffer 100μl, centrifuge at 1300rpm for 5min at 4°C, discard the supernatant; add 200μl of Staining Buffer to resuspend and wash once, centrifuge at 1300rpm for 5min, discard the supernatant; add 120μl of Staining Buffer to resuspend on the machine for flow cytometry detection.

16.2 Treg cell staining

Plate according to about 1× ¹⁰⁷ cell number/well, after centrifugation, add 100 μl of Staining Buffer to resuspend the cells, centrifuge at 1300 rpm, 4°C for 5 minutes, discard the supernatant; envelope staining is the same as above; Fixation/Permeailization Concentrate: Diluent is prepared according to the ratio of 1:3 Fixation/permeabilization working solution, add 100 μl to each well, incubate at room temperature for 30 minutes in the dark; dilute Permeabilization Buffer (10×) with ddH ₂ O to prepare permeabilization buffer, add 100 μl to each well, centrifuge at 1300 rpm for 5 minutes at 4°C, discard the supernatant ; Prepare Foxp3 antibody with permeabilization buffer at a ratio of 1:150, add 100 μl to each well, incubate at 4°C in the dark for 30 minutes, replenish 100 μl of permeabilization buffer, centrifuge at 1300 rpm, 4°C for 5 minutes, discard the supernatant; add 200 μl of permeation buffer Resuspend and wash once, centrifuge at 1300rpm for 5min, discard the supernatant; add 120μl of Staining Buffer to resuspend on the machine, and perform flow cytometry detection.

16.3 Intracellular Staining

Plate according to about 1× ¹⁰⁷ cells/well, add 200 μl intracellular stimulation solution to resuspend the cells after centrifugation, stimulate for 4-6 hours in a CO ₂ incubator at 37°C, centrifuge at 1300 rpm for 5 minutes at 4°C, discard the supernatant; add Staining Buffer Resuspend 200μl and wash once, centrifuge at 1300rpm for 5min, discard the supernatant; envelope staining is the same as above; add Fixation and Permeailization Solution 100μl, 4°C in the dark and fix the ruptured membrane for 30min; dilute Permeailization Buffer (10×) with ddH ₂ O to prepare membrane rupture Add 100 μl of buffer solution to each well, centrifuge at 1300 rpm for 5 minutes at 4°C, and discard the supernatant; prepare intracellular antibody with permeabilization buffer at a ratio of 1:150, add 100 μl per well, incubate at 4°C in the dark for 30 minutes, and replenish permeabilization buffer 100 μl, centrifuge at 1300 rpm, 4°C for 5 min, discard the supernatant; add 200 μl of permeabilization buffer to resuspend and wash once, centrifuge at 1300 rpm for 5 min, discard the supernatant; add 120 μl of Staining Buffer to resuspend on the machine, and perform flow cytometry detection.

Example 17 Ubiquitinomics sequencing

On the 9th day after the allogeneic hematopoietic stem cell transplantation, the liver tissue of the mice was collected and frozen in liquid nitrogen, and the non-labeled protein quantification technology was used for Label-free quantitative proteomics analysis of the whole protein ubiquitination modification, and the change of the ubiquitination level between the groups was compared .

Embodiment 18 statistical analysis

Use unpaired t-test to compare the data between two groups of samples, and compare the means of multiple groups of samples with analysis of variance; when P<0.05, it means there is a statistical difference, represented by *, when P<0.01, it is represented by **, P<0.001 When indicated by ***. The log-rank test was used to compare the survival data, and the Kaplan-Meier curve was drawn. Analyze and draw using GraphPad Prism 8 software. The data of the flow cytometry results were analyzed and drawn using Eisen NovoExpress flow cytometer analysis software and Flowjo software.

Effect evaluation 1 Effect of drug treatment on aGVHD in mice

On the 3rd day before transplantation, each recipient mouse was injected with minicircle-BRCC3 100 μg/2ml or control plasmid mini-circle 100 μg/2ml through the rat tail vein by HGT, and the survival status of the two groups of mice was observed and recorded every day after transplantation. Body weight changes and aGVHD scores of mice were recorded every 2-3 days. After TBI pretreatment, all the mice showed radiation stiffness, decreased food and water intake, and lost weight about one week after transplantation. The mice in the control Vector group died 8 days after transplantation. Observe aGVHD manifestations such as arched back, hair loss, and activity, and record body weight changes. Compared with the Vector group, the weight loss and aGVHD score of the mice in the BRCC3 group were lower, as shown in Figure 1. On the 14th day after transplantation, the skin, lung and intestinal tissues of the recipient mice were collected for pathological H&E staining. The results indicated that the composition of each layer of skin and the distribution of subcutaneous fat in the BRCC3 group were relatively complete, the alveolar structure was less damaged, the bronchi at all levels were less congested, the intestinal mucosa was damaged, cell debris, and lumen shedding were less, and the integrity of villi was better. The infiltration of inflammatory cells in these parts was less, and the degree of pathological damage of each target organ was significantly alleviated, as shown in Figure 2.

Effect Evaluation 2 Inhibitory Effect of Drugs on T Cell Activation

On the 10th day after transplantation, the mice were sacrificed by cervical dislocation, and the spleen, lung, liver and small intestinal epithelial lymphocytes of the mice were extracted, and the immune cell phenotype was detected by flow cytometry. The results showed that in the mouse spleen and various target organ tissues, the ratio and absolute number of CD69 ⁺ CD4 ⁺ , CD69 ⁺ CD8 ⁺ T cells in the BRCC3 group were significantly decreased, suggesting that BRCC3 can inhibit the activation of T cells in aGVHD mice, see Fig. 3.

Effect evaluation 3 drugs used to inhibit T cell function

Simulating the MLR reaction in vivo, fixing intracellular staining after membrane rupture, and detecting the secretion of IL-4, IL-17A, IFNγ and TNFα in CD4 ⁺ T cells and CD8 ⁺ T cells, it was found that overexpression of BRCC3 can significantly inhibit CD4 ⁺ , CD8 ⁺ T cells secrete IFNγ, suggesting that BRCC3 may inhibit the function of T cells by significantly down-regulating the secretion of IFNγ by T cells, as shown in Figure 4.

Effect evaluation 4 The effect of drugs on other immune cells

On the 10th day after transplantation, the mice were killed by cervical dislocation, and the spleen, lung, liver and small intestinal epithelial lymphocytes of the mice were extracted, and flow cytometry was used to detect the expression of other immune cell subsets in the spleen and each target organ of the mice. It was found that in the mouse spleen and various target organ tissues, the proportion of F4/80 ⁺ CD11b ⁺ labeled macrophages in the BRCC3 overexpression group decreased significantly, as shown in Figure 5.

Effect evaluation 5 drugs used to up-regulate the protein level of E3 ubiquitin ligase Trim25

On the 9th day after transplantation, the liver tissues of the mice were collected, and the label-free quantitative proteomics analysis of the whole protein ubiquitination modification was performed using the non-labeled protein quantification technology. On the other hand, we transfected BRCC3 and control plasmids in 293T cells, lysed and extracted proteins, and performed mass spectrometry analysis by IP-MS. The results of combined ubiquitinomics sequencing and mass spectrometry analysis screened out 58 substrate proteins whose ubiquitin level decreased more than twice and could interact with BRCC3, three of which were involved in the immune regulation signaling pathway. Further, by transfecting shBRCC3 and control plasmids in 293T cells, Western Blot method was used to detect the changes of three potential substrate proteins of 14-3-3, TPP2 and Trim25, and the substrates of BRCC3 were screened out. The results showed that after knocking down BRCC3, the protein levels of 14-3-3 and TPP2 did not change significantly, while the protein level of E3 ubiquitin ligase Trim25 decreased significantly, as shown in Figure 6.

Effect evaluation 6

293T cells were transfected with HA-Ub, HA-K48, HA-K63, and shBRCC3 or control plasmids, and the cells were collected 72 hours after transfection, lysed and extracted, and Trim25 (EFP) was immunoprecipitated with Protein A/G beads. Western blot method was used to detect the changes of HA-Ub. It was found that after knocking down BRCC3, the levels of total ubiquitin, K48 ubiquitin and K63 ubiquitin of endogenous Trim25 all increased, as shown in Figure 7. However, with the addition of the proteasome inhibitor MG132, the deubiquitination of BRCC3 was blocked and the ubiquitination level of Trim25 was restored, see Figure 8.

Effect evaluation 7 Drugs used to reduce the ubiquitination modification level of Trim25 protein

Through dual luciferase reporter experiments, design luciferase reporter plasmid P125, transfect P125, Renila, FH-BRCC3 or control plasmids in 293T cells, collect cells 48 hours after transfection, use dual fluorescent reporter kit to process samples, and Fluorescence detection of luciferase was performed using a microplate reader. It was found that overexpression of BRCC3 can significantly promote the transcriptional activity of IFNβ, as shown in FIG. 9 . The macrophage cell line Raw264.7 was transfected with shBRCC3 or control plasmid, and the cells were collected 72 hours after transfection, RNA was extracted and reverse-transcribed, and the expression level of IFNβ was detected by Realtime PCR. It can be found that after knocking down BRCC3, the expression of IFNβ The level dropped significantly, see Figure 10. Correspondingly, we transfected FH-BRCC3 or control plasmids in the macrophage cell line Raw264.7 cells, collected cells 48 hours after transfection, extracted RNA by Trizol method and performed reverse transcription, and detected the expression level of IFNβ by Realtime PCR. After overexpression of BRCC3, the expression level of IFNβ was significantly enhanced, while the expression levels of inflammation-related cytokines IL-1β, IL-6 and IL-12P40 were significantly decreased, as shown in Figure 11. In the liver tissue of mice, the expression level of IFNβ in the BRCC3 overexpression group was significantly increased, as shown in FIG. 12 .

Efficacy evaluation 8 The effect of drugs on the GVL effect while reducing GVHD

The allogeneic aGVHD/GVL mouse model was constructed, and the living conditions of the two groups of mice were observed and recorded every day after transplantation. The mice in the control Vector group died 12 days after transplantation, and all died by about 31 days, and 31 days after the observation point Nearly half of the mice in the BRCC3 group survived. The weight change and aGVHD score of the mice were recorded every 2-3 days. Compared with the control group, the weight loss level and aGVHD score of the mice in the BRCC3 overexpression group were lower, as shown in FIG. 13 . Using mouse in vivo imaging technology, 3 weeks after transplantation, chloral hydrate reagent was injected intraperitoneally, the mice were anesthetized in advance, and fireflyin substrate was injected intraperitoneally, and the tumor burden was evaluated by detecting the fluorescence value in the mice with an in vivo imager. The results showed that after overexpression of BRCC3, the distribution of A20-luciferase in mice was significantly reduced, and the statistical results were significant, as shown in Figure 14 and Figure 15.

In summary, the present invention evaluates the effect of deubiquitinating enzyme BRCC3 on GVHD and GVL in mouse GVHD model and GVHD/GVL model. We found that BRCC3 has the function of inhibiting acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation; it can inhibit the activation and function of donor T cells, and inhibit the proliferation of macrophage cells; through the proteasome pathway, down-regulate the ubiquitous expression of Trim25 Trim25 can be modified to stabilize the protein level of Trim25, thereby promoting the secretion of IFNβ and inhibiting the occurrence and development of aGVHD; meanwhile, the GVL effect is retained.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims

Application of the deubiquitinating enzyme BRCC3 in the preparation of drugs for preventing or treating acute graft-versus-host disease and graft-versus-leukemia.
The use according to claim 1, characterized in that the drug is used to inhibit the activation of T cells in target organs.
The use according to claim 1, characterized in that the drug is used to inhibit the secretion of IFNγ by T cells.
The use according to claim 1, characterized in that the drug is used to inhibit the production of macrophages in target organs.
The use according to claim 1, characterized in that the drug is used to up-regulate the protein level of E3 ubiquitin ligase Trim25.
The application according to claim 1, characterized in that the drug is used to down-regulate the ubiquitination of the E3 ubiquitin ligase Trim25 through the proteasome pathway, and remove the ubiquitin levels at positions K48 and K63.
The use according to claim 1, characterized in that the drug is used to promote the secretion of IFNβ.
A medicine for preventing or treating acute graft-versus-host disease and graft-versus-leukemia, characterized in that the medicine includes deubiquitinating enzyme BRCC3.
The medicine according to claim 8, characterized in that the dosage of the deubiquitinating enzyme BRCC3 is 40-60 μg/ml.
The medicine according to claim 8, characterized in that, the route of administration of the medicine comprises one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection and intramuscular injection.