A drug for treating inflammatory bowel disease

Abstract

The present invention discloses a drug for treating inflammatory bowel disease, wherein the drug is an extracellular vesicle loaded with a targeted drug and a Trim21 protein domain, wherein the targeted drug is capable of targeting the extracellular vesicle to CD4 + The targeted drug for T cells, wherein the Trim21 protein domain is a Trim21 protein domain lacking ubiquitination function. + The T cell targeted drug and the Trim21 protein domain lacking ubiquitination function are loaded into extracellular vesicles, and the targeted drug transports the entire drug-loaded extracellular vesicle to CD4 + T cells then compete with the intracellular host Trim21 for binding to FBXW7 through the Trim21 protein domain that lacks ubiquitination function, thereby antagonizing the effect of intestinal flora in promoting Th17 cell differentiation, thereby achieving the purpose of treating inflammatory bowel disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a medicine for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a common autoimmune disease of the digestive tract, including ulcerative colitis (UC) and Crohn's disease (CD). Th17 cells are CD4 + Under the combined action of IL-6 and TGF-β1, T cells are induced to differentiate into a group of effector T cells characterized by the secretion of high levels of cytokines such as IL-17A, IL-17F, IL-21, and IL-22. They are widely involved in the occurrence and development of diseases such as infection, tumors, and autoimmune diseases. Analysis of the composition of infiltrating cells in the intestinal tissue of IBD patients and research data from IBD mice have confirmed that effector Th17 cells play an important role in intestinal mucosal inflammation (Jun-Bin Yan, Min-Min Luo, Zhi-Yun Chen, Bei-Hui He. The Function and Role of the Th 1 7 / Treg Cell Balance in Inflammatory Bowel Disease. J Immunol Res 2020, 2020: 8813558.). IL-17A recruits neutrophils to the inflamed area and induces macrophages to release inflammatory factors, further exacerbating local inflammation (Veldhoen M. Interleukin 17 is a chief orchestrator of immunity. Nature immunology 2017, 18(6): 612-621.). The mammalian intestine is inhabited by microbiota, and the interaction between these microbiota and immune cells is crucial for maintaining intestinal immune homeostasis. Intestinal microbiota can promote Th17 cell differentiation and Th17 cytokine expression.

[0003] Extracellular vesicles (EVs) are small vesicles with a lipid bilayer structure secreted by living cells. They can be divided into four subgroups: exosomes, extranuclear particles, apoptotic bodies, and oncosomes. As natural endogenous drug carriers, they have unique advantages: their nanometer-scale diameter allows them to penetrate multiple barriers, including the blood-brain barrier; their complex protein and phospholipid bilayer structure effectively protects drugs from clearance by the body; and their low immunogenicity means they can be used as carriers to deliver drugs or therapeutic proteins without eliciting an immune response in the host. Summary of the Invention

[0004] This study found that intestinal flora promotes the in vitro differentiation of Th17 cells through the Trim21 / FBXW7 signaling axis. This study constructed a Trim21 domain (TΔR) lacking ubiquitination function. TΔR can effectively antagonize the binding of Trim21 and FBXW7, thereby inhibiting Th17 cell differentiation.

[0005] The present invention first provides the use of extracellular vesicles loaded with targeted drugs and Trim21 protein domains in the preparation of drugs for treating inflammatory bowel disease.

[0006] The targeted drug is capable of targeting the extracellular vesicles to CD4 + T cell targeted drugs,

[0007] The Trim21 protein domain is a Trim21 protein domain lacking ubiquitination function.

[0008] Preferably, the targeted drug is CD4-scFV, and the amino acid sequence is shown in SEQ ID No. 1;

[0009] The amino acid sequence of the Trim21 protein domain lacking ubiquitination function is shown in SEQ ID No. 2.

[0010] Preferably, the targeted drug and the Trim21 protein domain are both connected to a GPI anchor sequence, and the amino acid sequence of the GPI anchor sequence is shown in SEQ ID No.3.

[0011] The extracellular vesicles are exosomes, apoptotic bodies, oncosomes or extranuclear particles;

[0012] The extracellular vesicles used to load drugs are derived from milk, blood, saliva, urine or cerebrospinal fluid of humans or other mammals.

[0013] Preferably, the targeted drug and Trim21 protein domain are saturated loaded into the extracellular vesicles, that is, during loading, the targeted drug and Trim21 protein domain are added in excess relative to the extracellular vesicles, and after loading, the excess unloaded targeted drug and Trim21 protein domain are separated out.

[0014] The present invention also provides a drug for treating inflammatory bowel disease, which is an extracellular vesicle loaded with a targeted drug and a Trim21 protein domain.

[0015] The targeted drug is capable of targeting the extracellular vesicles to CD4 + T cell targeted drugs,

[0016] The Trim21 protein domain is a Trim21 protein domain lacking ubiquitination function.

[0017] Preferably, the targeted drug is CD4-scFV, and the amino acid sequence is shown in SEQ ID No. 1;

[0018] The amino acid sequence of the Trim21 protein domain lacking ubiquitination function is shown in SEQ ID No. 2.

[0019] Preferably, the targeted drug and the Trim21 protein domain are both connected to a GPI anchor sequence, and the amino acid sequence of the GPI anchor sequence is shown in SEQ ID No.3.

[0020] The extracellular vesicles are exosomes, apoptotic bodies, oncosomes or extranuclear particles;

[0021] The extracellular vesicles used to load drugs are derived from milk, blood, saliva, urine or cerebrospinal fluid of humans or other mammals.

[0022] Preferably, the targeted drug and Trim21 protein domain are saturated loaded into the extracellular vesicles, that is, during loading, the targeted drug and Trim21 protein domain are added in excess relative to the extracellular vesicles, and after loading, the excess unloaded targeted drug and Trim21 protein domain are separated out.

[0023] Exosomes are small vesicles with a lipid bilayer structure, ranging in diameter from 30 to 150 nanometers, secreted by various living cells. Exosomes have become a promising drug delivery vehicle, garnering widespread attention from researchers due to their reduced immunogenicity, enhanced biocompatibility, increased engineering potential, and their ability to easily penetrate the blood-brain barrier and escape lysosomal degradation.

[0024] This application targets extracellular vesicles to CD4 +The T cell targeted drug and the Trim21 protein domain lacking ubiquitination function are loaded into extracellular vesicles, and the targeted drug transports the entire drug-loaded extracellular vesicle to CD4 + T cells then compete with the intracellular host Trim21 for binding to FBXW7 through the Trim21 protein domain that lacks ubiquitination function, thereby antagonizing the effect of intestinal flora in promoting Th17 cell differentiation, thereby achieving the purpose of treating inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 FBXW7 littermate controls (FBXW7 + / + ) mice CD4 + Results of the detection of T cell polarization ability toward Th17 cells, "***" indicates P < 0.001 (the same below).

[0026] Figure 2 Conditional knockout of FBXW7 (peripheral mature CD4 + FBXW7 deletion in T cells, FBXW7 - / - ) mice CD4 + The results of the T cell polarization ability test toward Th17 cells are shown in the figure, and "ns" indicates no statistical significance (the same below).

[0027] Figure 3 FBXW7 littermate controls (FBXW7 + / + ) mice CD4 + Western blot results of T cells after various treatments under anti-CD3 and anti-CD28 activation conditions, where a is the expression level of FBXW7 in T cells after the action of intestinal flora stimulants (GM-Lyts), b is the expression level of FBXW7 in T cells (normal and knockdown of Trim21) after the action of intestinal flora stimulants (GM-Lyts), and c is the expression level of FBXW7 in T cells (normal and deleted of Trim21) after the action of intestinal flora stimulants (GM-Lyts).

[0028] Figure 4 The body weights of mice with acute enteritis after administration of PBS, mEVs / αCD4, or mEVs / αCD4 / TΔR. “**” P < 0.01 (the same below).

[0029] Figure 5 The large intestine of mice with acute enteritis after administration of PBS, mEVs / αCD4, or mEVs / αCD4 / TΔR. (a) is a photo of the large intestine, and (b) is the statistics of the large intestine length.

[0030] Figure 6 Figure 3 Histological morphology of the large intestine of mice with acute enteritis after administration of PBS, mEVs / αCD4, or mEVs / αCD4 / TΔR.

[0031] Figure 7 IL-17A in intestinal lamina propria lymphocytes after administration of PBS, mEVs / αCD4 or mEVs / αCD4 / TΔR to mice with acute enteritis + CD4 + T cell ratio.

[0032] Figure 8 CD4 in the intestinal lamina propria of mice with acute enteritis after administration of PBS, mEVs / αCD4 or mEVs / αCD4 / TΔR + Binding of endogenous Trim21 or exogenous TΔR-flag to FBXW7 in T cells. DETAILED DESCRIPTION

[0033] Fbxw7 fl / fl The mice were purchased from The Jackson Laboratory (strain#: 017563, The Jackson Laboratory). LoxP sites were added to both ends of exons 5 and 6 of the Fbxw7 gene in the mice.

[0034] Distal Lck Cre mice were purchased from The Jackson Laboratory (strain#: 012837, The Jackson Laboratory). Cre recombinase in these mice is expressed in peripheral mature T cells.

[0035] Fbxw7 fl / fl Fbxw7 was obtained by crossing distal Lck Cre mice with Fbxw7 mice. fl / fl*distal Lck Cre Mice, designated as FBXW7- / - mice, have a phenotype of Fbxw7 protein knockdown in mature T cells.

[0036] Fbxw7 fl / f Mice were designated as FBXW7 + / + Mice with normal Fbxw7 protein in mature T cells.

[0037] Trim21 - / - Mice (S-KO-04542, Cyagen Biosciences) were purchased from Cyagen Biosciences. The phenotype of these mice is that they lack Trim21 protein.

[0038] GPI-modified CD4-scFV: Prepared by Pujian Biotechnology (Wuhan) Co., Ltd. (Pujian Biotechnology), the amino acid sequence upon expression is as follows: signal peptide-CD4 scFV-first linker-His tag-second linker-GPI anchor sequence, where the signal peptide sequence is: MVLQTQVFISLLLWISGAYG; the CD4 scFV sequence (CD4 targeting protein) is shown in SEQ ID No. 1; the first linker sequence is: GGGSGGGSGGGS; the His tag sequence is: HHHHHH; the second linker sequence is: GS; and the GPI anchor sequence is shown in SEQ ID No. 3. The amino acid sequence of the GPI-modified CD4-scFV upon expression is shown in SEQ ID No. 7. The signal peptide is removed from the expressed protein.

[0039] GPI-modified TΔR: Prepared by Pujian Biotechnology (Wuhan) Co., Ltd., the corresponding amino acid sequence upon expression is: signal peptide-Trim21 domain protein lacking ubiquitination function-first linker peptide-His tag-second linker peptide-GPI anchor sequence. The signal peptide sequence is: MVLQTQVFISLLLWISGAYG; the sequence of the Trim21 domain protein lacking ubiquitination function is shown in SEQ ID No. 2; the first linker peptide sequence is: GGGSGGGSGGGS; the His tag sequence is: HHHHHH; the second linker peptide sequence is: GS; and the GPI anchor sequence is shown in SEQ ID No. 3. The corresponding amino acid sequence of the GPI-modified TΔR upon expression is shown in SEQ ID No. 8. The signal peptide is removed from the expressed protein.

[0040] Example 1

[0041] Scrape FBXW7 + / + The intestinal contents of mice were centrifuged, large precipitates were removed, ultrasonically lysed, and filtered. The supernatant obtained was the intestinal flora stimulator (GM-Lyts). The concentration was determined using a BCA protein quantification kit (23225, ThermoScientific) for subsequent use.

[0042] Example 2

[0043] Using in vitro induced differentiation system, FBXW7 was detected - / - and FBXW7 + / + mice CD4 + T cell polarization ability to Th17 cells. - / - and FBXW7 + / +One mouse each, spleens and lymph nodes from the two groups of mice were obtained. The specific steps are as follows:

[0044] T cell sorting: (1) Take the spleen and lymph nodes of the mouse and squeeze them with a syringe piston to make a tissue suspension; (2) Transfer them to a 15 ml conical test tube and allow large pieces to settle to the bottom of the test tube or filter through a nylon filter to obtain a single cell suspension; (3) Centrifuge the cell suspension at 1500 rpm and 4°C for 5 minutes and discard the supernatant; (4) Resuspend the sample in 2 ml PBS buffer, take 10 μl of 3% glacial acetic acid to dilute, and count the cells; (5) Centrifuge the cells again, discard the supernatant, and resuspend the cells in sorting buffer according to the count results to adjust the density to 1×10 8 (6) CD4 negative selection kit (EasySepTM Mouse CD4 + CD4 T Cell Isolation Kit, #19852A) was used to separate + T cells were then sorted using a Biotin sorting kit (EasySepTM Mouse Biotin Positive Selection Kit, #18556) to obtain CD4 + CD62L + T cells, i.e. CD4 + T cells.

[0045] In vitro differentiation of T cells: In a 96-well plate, dilute anti-CD3 and anti-CD28 with autoclaved PBS to a final concentration of 2 μg / ml, coat the plate at 200 μl / well, and incubate at 37°C for more than 2 hours. + T cells, 4 × 10 5 Th17 cells were induced to differentiate at 10 μg / well. The Th17 cell differentiation conditions were as follows: 10 μg / ml anti-IFNγ (BE0054, Bio Xcell), 10 μg / ml anti-IL4 (BE0045, Bio X cell), 20 ng / ml IL-6 (130-094-065, Miltenyi Biotec) and 10 ng / ml TGF-β (130-095-067, Miltenyi Biotec). Plating was performed on day 0 of differentiation. At the same time, 10 μg / ml intestinal flora stimulator was added or not on day 0 (the method for obtaining intestinal flora stimulator was the same as in Example 1). On day 4, cells were added with Cell Stimulation Cocktail (00-4975-03, Invitrogen) and the cells were subjected to the treatment for another 4 hours before samples were collected and IL-17A was detected by flow cytometry. + CD4+ T cells.

[0046] Flow cytometry: Wash cells once with PBS, centrifuge, and resuspend in PBS. Add CD4-PE flow cytometry antibody (12-0041-82, Invitrogen) and incubate on ice or at 4°C in the dark for 30 min. Neutralize with PBS and centrifuge twice. Discard the supernatant. Add 200 μL of IC fixation buffer (00-8222-49, Invitrogen) to each tube and incubate at room temperature in the dark for 20 min. Neutralize with 1× permeabilization buffer (00-8333-56, Invitrogen) and centrifuge. Discard the supernatant and resuspend the cells in 100 μL of 1× permeabilization buffer. Add IL-17A-APC flow cytometry antibody (17-7177-81, Invitrogen) and incubate at 4°C in the dark for 30 min. Neutralize with 1× permeabilization buffer and wash twice. The supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS. Flow cytometric analysis was performed using a NovoCyte flow cytometer (ACEA), and the data were analyzed using FlowJo software.

[0047] The results showed that intestinal flora promoted FBXW7 + / + mice CD4 + T cells differentiate into Th17 cells ( Figure 1 ), without affecting FBXW7 - / - mice CD4 + The ability of T cells to differentiate into Th17 cells Figure 2 ), indicating that the process of intestinal flora promoting Th17 cell differentiation is dependent on FBXW7 protein.

[0048] Example 3

[0049] FBXW7 littermate controls (FBXW7 + / + ) mice CD4 + Western blot analysis of T cells after various treatments in the activated state with anti-CD3 and anti-CD28.

[0050] get CD4 +T cells (the method is the same as that in Example 2) were stimulated with 10 μg / ml of an intestinal flora stimulator (the method for obtaining the intestinal flora stimulator is the same as that in Example 1) under Th17 cell induction conditions. After 48 hours, the cells were collected, centrifuged, washed with PBS, and then added with protein lysis buffer 1× cell lysis buffer (#9803, Cell Signaling Technology) and phenylmethylsulfonyl fluoride (PMSF, #8553, Cell Signaling Technology). The cells were placed on ice for 30 minutes and then centrifuged at 12000 g at 4°C for 15 minutes. The supernatant was added with 5× SDS loading buffer and boiled in a boiling water bath for 5 minutes to prepare a protein sample.

[0051] The protein samples were electrophoresed on a 10% SDS-PAGE gel at a constant voltage of 80 V until the prestained protein marker bands were separated, then the voltage was increased to 160 V. The electrophoresis was stopped when the blue band of the sample loading reached the bottom. The proteins in the gel were transferred to a PVDF membrane at a constant current of 300 mA for 90 minutes and blocked with 5% skim milk powder at room temperature for 1 hour. After washing the membrane three times with PBST, Fbxw7 (40-1500, ThermoFisher) and β-Actin (3700, Cell Signaling technology) primary antibodies were added and incubated overnight at 4°C. The next day, the membrane was washed three times with PBST, anti-rabbit IgG (Cat. No. 7074, Cell Signaling technology) was added and incubated at room temperature for 1 hour. The membrane was washed three times with PBST, and ECL substrate was added and exposed to the Tanon imaging analysis system for imaging.

[0052] Western blot results showed that intestinal flora reduced the expression of FBXW7 in T cells ( Figure 3 a).

[0053] Example 4

[0054] FBXW7 littermate controls (FBXW7 + / + ) mice CD4 + Western blot analysis of T cells after various treatments in the activated state with anti-CD3 and anti-CD28.

[0055] get CD4 +T cells (using the same method as in Example 2) were transfected with Trim21 siRNA (sc-140349, Santa Cruz Biotechnology) using Mirus transfection reagent. Under Th17 cell induction conditions, 10 μg / ml of intestinal flora stimulator (method for obtaining intestinal flora stimulator in Example 1) was added for stimulation. After 48 hours, the cells were collected and prepared into protein samples using the same method as in Example 3, run on a gel, and exposed.

[0056] After knocking down the expression of Trim21 protein in T cells, intestinal flora stimulator (GM-Lyts) no longer reduced the expression of FBXW7 in T cells ( Figure 3 b).

[0057] Example 5

[0058] FBXW7 littermate controls (FBXW7 + / + ) mice and Trim21- / - mice CD4 + Western blot analysis of T cells after various treatments in the activated state with anti-CD3 and anti-CD28.

[0059] get CD4 + T cells (using the same method as in Example 2) were stimulated by adding 10 μg / ml of intestinal flora stimulator (the method for obtaining intestinal flora stimulator was the same as in Example 1) under the induction conditions of Th17 cells. After 48 hours, the cells were collected and prepared into protein samples using the same method as in Example 3, and then run on a gel and exposed.

[0060] After knocking out Trim21 protein in T cells, the intestinal flora no longer reduces the expression of FBXW7 in T cells ( Figure 3 c).

[0061] Example 6

[0062] Construction of an IBD acute enteritis model (Jiang L. EpCAM-dependent extracellular vesicles from intestinal epithelial cells maintain intestinal tract immune balance. Nature Communications 2016, 7: 13045.): Dextran sulfate sodium salt (DSS, 9011-18-1, MP Biomedicals) was prepared into a 2% (mass-volume ratio) concentration in drinking water and given to FBXW7 littermate controls (FBXW7 + / +) Mice were fed with the above-mentioned 2% DSS for 11 consecutive days.

[0063] Example 7

[0064] Extraction of milk-derived exosomes: Freshly harvested raw milk was centrifuged at 10,000 × g for 20 minutes to remove lipids. The supernatant was collected, diluted with an equal volume of water, and the pH was adjusted to 4.5 with 6N HCl. The pH-adjusted supernatant was centrifuged at 6,000 × g for 20 minutes (two centrifugations). The collected supernatant was filtered through 0.45 μm and 0.22 μm filters and finally centrifuged at 100,000 × g for 90 minutes. The resulting pellet was milk-derived exosomes (mEVs).

[0065] Example 8

[0066] Preparation of mEVs / aCD4: Equal amounts of GPI-modified CD4-scFV (Pujian Bio) were incubated with mEVs (mEVs were obtained as in Example 7) at 37°C for 3 hours to transfer the GPI-modified CD4-scFV to the mEVs (mEVs / αCD4). The mEVs / αCD4 were then washed by centrifugation at 10,000 × g for 30 minutes (twice).

[0067] Example 9

[0068] Preparation of mEVs / αCD4 / TΔR: Equal amounts of GPI-modified TΔR (Pujian Bio) and mEVs / αCD4 (mEVs / αCD4 obtained as in Example 8) were incubated at 37°C for 3 hours to transfer the GPI-modified TΔR to the mEVs / αCD4 (mEVs / αCD4 / TΔR). The mEVs / αCD4 / TΔR was then washed by centrifugation at 10,000 × g for 30 minutes (twice).

[0069] Example 10

[0070] An IBD mouse model was constructed according to Example 6. PBS, 200 μg mEVs / αCD4 or mEVs / αCD4 / TΔR were gavaged every two days. The body weight of mice in each group was observed and recorded from day 0 to day 11.

[0071] The results showed that compared with the IBD mice in the mEVs / αCD4 group, the weight loss of IBD mice in the mEVs / αCD4 / TΔR group was significantly attenuated ( Figure 4 ).

[0072] Example 11

[0073] An IBD mouse model was constructed according to Example 6. PBS and 200 μg mEVs / αCD4 or mEVs / αCD4 / TΔR were gavage-administered every two days. On day 11, the large intestine tissues of mice in each group were collected, photographed, and the intestinal length was measured.

[0074] The results showed that compared with the IBD mice in the mEVs / αCD4 group, the IBD mice in the mEVs / αCD4 / TΔR group had less shortening of the intestinal length ( Figure 5 a, Figure 5 b).

[0075] Example 12

[0076] An IBD mouse model was constructed according to Example 6. PBS and 200 μg mEVs / αCD4 or mEVs / αCD4 / TΔR were gavage-administered every two days. On day 11, the large intestine tissues of mice in each group were stained with hematoxylin-eosin (HE).

[0077] The results showed that compared with the IBD mice in the mEVs / αCD4 group, the IBD mice in the mEVs / αCD4 / TΔR group had fewer inflammatory cells infiltrating in the intestinal tissue and less tissue damage ( Figure 6 ).

[0078] Example 13

[0079] An IBD mouse model was established as described in Example 6. PBS and 200 μg of mEVs / αCD4 or mEVs / αCD4 / TΔR were administered orally every two days. On day 11, intestinal lamina propria lymphocytes were isolated from each group of mice using the following procedures:

[0080] The small intestine of each group of mice was isolated, and the intestinal Pan's lymph nodes (Payers' Patches) and connective tissue were removed. The intestine was cut longitudinally, and the intestinal contents were cleaned. The small intestine was cut into 0.5-1.0 cm segments and placed in Buffer A (DMEM + 5 mM EDTA + 0.145 mg / ml DTT + 4% FCS + 0.2% HEPES) (repeatedly pipetted for 10 minutes, and the intestinal segments were collected), Buffer B (DMEM + 2 mM EDTA + 4% FCS + 0.2% HEPES) (repeatedly pipetted for 5 minutes, and the intestinal segments were collected), and Buffer C (DMEM + 50 mg / ml DNase I + 75 mg / ml collagenase II + 8% FCS) (repeatedly pipetted for 5 minutes, and the filtrate was collected). The final filtrate was neutralized with Buffer D (DMEM + 4% FCS + 0.2% HEPES). The filtrate was centrifuged and the resulting cell pellet was separated using 40% and 80% Percoll. The intermediate layer obtained after centrifugation was the intestinal lamina propria lymphocytes. The proportion of Th17 cells in the intestinal lamina propria lymphocytes extracted from each group of mice was analyzed by flow cytometry as described in Example 2.

[0081] The results showed that compared with the IBD mice in the mEVs / αCD4 group, the number of Th17 cells in the intestine of the IBD mice in the mEVs / αCD4 / TΔR group was reduced ( Figure 7 )

[0082] Example 14

[0083] The IBD mouse model was constructed according to Example 6. PBS and 200 μg mEVs / αCD4 or mEVs / αCD4 / TΔR were gavaged every two days. On the 11th day, the intestinal lamina propria lymphocytes of each group of mice were sorted (the method was the same as that of Example 13). Then, the CD4 + T cells (the method is the same as in Example 2), and finally the binding of endogenous Trim21 or exogenous TΔR-flag to FBXW7 is analyzed by immunoprecipitation. The immunoprecipitation method is briefly described as follows:

[0084] The two groups of intestinal lamina propria lymphocytes CD4 +T cells were lysed on ice for 30 minutes using Western and IP lysis buffer (P0013, Beyotime Biotechnology). The supernatant was collected after centrifugation at 12,000 rpm for 10 minutes at 4°C. One-tenth to one-fifth of the total lysate was used for protein input (i.e., lysate) detection. Antibody FBXW7 (40-1500, ThermoFisher) was added to the remaining lysate and incubated overnight at 4°C on a rotary shaker. The next day, Protein A / G plus Agarose (sc-2003, Santa Cruz Biotechnology) was added and incubated at 4°C on a rotary shaker for 4 hours. After centrifugation at 1,000 rpm at 4°C for 30 seconds, the supernatant was discarded and the Protein A / G plus Agarose was collected. The Protein A / G plus Agarose was then washed three times with IP lysis buffer, each time for 10 minutes at 4°C on a rotary shaker. The washed Protein A / G was then added with 2× SDS protein loading buffer and boiled in a boiling water bath for 5 minutes to prepare a protein sample. The prepared sample was run and exposed according to Example 3.

[0085] The results showed that compared with the IBD mice in the mEVs / αCD4 group, the IBD mice in the mEVs / αCD4 / TΔR group had a significantly higher intestinal CD4 + In T cells, exogenous TΔR-flag binds more to FBXW7, while endogenous Trim21 binds weakly to FBXW7, indicating that exogenous TΔR-flag competes with endogenous Trim21 for binding to FBXW7 ( Figure 8 ).

Claims

1. Application of extracellular vesicles loaded with targeted drugs and Trim21 protein domains in the preparation of drugs for the treatment of inflammatory bowel disease; The targeted drug is capable of targeting the extracellular vesicles to CD4 + A T cell targeted drug, wherein the targeted drug is CD4-scFV, the amino acid sequence of which is shown in SEQ ID No. 1; The Trim21 protein domain is a Trim21 protein domain lacking ubiquitination function, and the amino acid sequence of the Trim21 protein domain lacking ubiquitination function is shown in SEQ ID No. 2; The targeted drug and the Trim21 protein domain are both connected with a GPI anchor sequence, and the amino acid sequence of the GPI anchor sequence is shown in SEQ ID No.

3.

2. The use according to claim 1, characterized in that The extracellular vesicles are exosomes, apoptotic bodies, oncosomes or extranuclear particles; The extracellular vesicles used to load drugs are derived from milk, blood, saliva, urine or cerebrospinal fluid of humans or other mammals.

3. The use according to claim 1, characterized in that The targeted drug and the Trim21 protein domain are saturatedly loaded into the extracellular vesicles.

4. A drug for treating inflammatory bowel disease, characterized in that: The drug is an extracellular vesicle loaded with a targeted drug and a Trim21 protein domain. The targeted drug is capable of targeting the extracellular vesicles to CD4 + A T cell targeted drug, wherein the targeted drug is CD4-scFV, the amino acid sequence of which is shown in SEQ ID No. 1; The Trim21 protein domain is a Trim21 protein domain lacking ubiquitination function, and the amino acid sequence of the Trim21 protein domain lacking ubiquitination function is shown in SEQ ID No. 2; The targeted drug and the Trim21 protein domain are both connected with a GPI anchor sequence, and the amino acid sequence of the GPI anchor sequence is shown in SEQ ID No.

3.

5. The drug for treating inflammatory bowel disease according to claim 4, characterized in that The extracellular vesicles are exosomes, apoptotic bodies, oncosomes or extranuclear particles; The extracellular vesicles used to load drugs are derived from milk, blood, saliva, urine or cerebrospinal fluid of humans or other mammals.

6. The drug for treating inflammatory bowel disease according to claim 4, characterized in that The targeted drug and the Trim21 protein domain are saturatedly loaded into the extracellular vesicles.

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