Use of cdc37 protein in preparation of treg cell disorder treatment product

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of CDC37 protein in preparation of Treg cell dysregulation disease treatment product. The application obtains a CDC37 protein with immunoregulatory function and high expression through mass spectrum analysis and bioinformatics analysis on proteins of lung stage child worms of Japanese blood flukes. The amino acid sequence of the CDC37 protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the gene coding the CDC37 protein is shown as SEQ ID NO. 2. Experimental results show that the CDC37 protein can realize the effect of non-hormone-dependent long-acting relief of Treg cell dysregulation diseases by inducing Treg cell expansion, inhibiting Th2 response and reducing airway hyperresponsiveness, and can be used for preparation of Treg cell dysregulation disease treatment product.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CDC37 protein in the preparation of therapeutic products for Treg cell disorders. Background Technology

[0002] Treg cell dysregulation refers to abnormalities in the number, function, or differentiation of regulatory T cells (Tregs), leading to a loss of normal regulatory function in the immune system and triggering a series of pathological states. Treg cells play a crucial role in maintaining immune tolerance, controlling inflammatory responses, and regulating immune homeostasis. When Treg cells are dysregulated, it can lead to allergic diseases (such as allergic asthma), autoimmune diseases (such as systemic lupus erythematosus), or certain infectious diseases. For example, in allergic asthma, insufficient Treg cell function fails to suppress the overactivation of Th2 cells, resulting in chronic inflammation and airway hyperresponsiveness; while in systemic lupus erythematosus, a decrease in the number or impairment of Treg cells fails to suppress the activation of autoreactive T cells and B cells, leading to the production of autoantibodies and multi-organ damage.

[0003] Currently, clinically available products for treating Treg cell dysregulation mainly include glucocorticoids and monoclonal antibody biologics. While glucocorticoids, as first-line anti-inflammatory drugs, have broad-spectrum immunosuppressive effects, long-term use can lead to local and systemic side effects, such as growth inhibition in children, osteoporosis, metabolic disorders, and increased susceptibility to infections. More importantly, some patients respond poorly to hormones, exhibiting "hormone resistance," thus limiting treatment options. Although monoclonal antibody biologics achieve "precision targeted" therapy, they suffer from drawbacks such as blocking only a single inflammatory factor, short-lived efficacy, and frequent relapses, which also limit their widespread application.

[0004] Recent studies have revealed that certain parasite-derived crude extracts can significantly alleviate Treg cell dysregulation through mechanisms such as inducing regulatory T cells and inhibiting Th2-type immune responses, demonstrating unique immunomodulatory potential. However, while these parasite-derived crude extracts can modulate immunity, they suffer from complex compositions, lack of standardization, difficulty in drug development, and potential biosafety risks. Therefore, there is an urgent need to develop a highly effective, safe, and non-hormonal treatment product for Treg cell dysregulation diseases that provides sustained immune homeostasis. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an application of CDC37 protein in the preparation of therapeutic products for Treg cell disorder. CDC37, a protein secreted by the lung larval stage of Schistosoma japonicum, can persistently regulate immune balance through non-hormonal means. When used to prepare therapeutic products for Treg cell disorder, the resulting parasite-derived protein biological agents have the advantages of clearly defined components, precise targeting, safety, stability, and mass production capability.

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

[0007] This invention provides the application of CDC37 protein in the preparation of therapeutic products for Treg cell disorders, wherein the amino acid sequence of CDC37 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding CDC37 protein is shown in SEQ ID NO.2.

[0008] Furthermore, the product uses CDC37 protein as the active ingredient and is supplemented with a pharmaceutically acceptable carrier or excipient.

[0009] Furthermore, the content of CDC37 protein in the product is 0.01g / L to 0.05g / L.

[0010] Furthermore, the carrier is a microsphere delivery system; the excipient is any one or more of fillers, stabilizers, diluents, and adjuvants.

[0011] Furthermore, the microsphere delivery system uses one or more of polylactic acid-glycolic acid copolymer, chitosan, gelatin, hyaluronic acid, and sodium alginate as carrier materials.

[0012] Furthermore, the diluent is a PBS solution.

[0013] Furthermore, the Treg cell disorder is allergic asthma or systemic lupus erythematosus.

[0014] Furthermore, the product can induce Treg cell proliferation, inhibit Th2 responses, or reduce airway hyperresponsiveness.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention addresses the problems of existing immunomodulatory drugs, such as significant side effects, short-lived efficacy, single target, and frequent relapse. For the first time, it isolates and identifies the juvenile worm secreted protein CDC37 from the protoprotein of the lung stage of Schistosoma japonicum, which possesses immunomodulatory properties and can be used to prepare therapeutic products for Treg cell disorders. The amino acid sequence of CDC37 protein is shown in SEQ ID NO.1. CDC37 can target the inflammatory pathways of asthma and systemic lupus erythematosus, precisely intervene in the immune response, and fundamentally improve the symptoms of immune dysregulation. Specifically: (1) As a juvenile worm secreted protein, CDC37 protein can persistently regulate immune balance through non-hormonal means, avoiding the significant side effects of hormonal drugs; (2) CDC37 protein can induce Treg cell proliferation, inhibit Th2 response, and reduce airway hyperresponsiveness. Through multi-target synergy, it can treat Treg cell disorders, improve the effectiveness and persistence of treatment, reduce the relapse rate of the disease, and overcome the problems of short-lived efficacy and frequent relapse caused by the single target of monoclonal antibody biological agents. (3) Compared with the complex and difficult-to-standardize insect-derived crude extracts, CDC37 has a clear composition, is safe and stable, and is suitable for large-scale preparation. It can accelerate the clinical translation and industrialization process, provide patients with safer, more effective and economical treatment options, and has significant advantages in clinical translation and industrialization.

[0017] To fully realize the potential of the CDC37 protein, a directed evolution and intelligent protein design strategy was employed to optimize its key functional sites, thereby enhancing its immunomodulatory activity. Combined with liquid-liquid phase separation (LLPS) technology, drug stability and delivery efficiency were improved, ensuring long-lasting in vivo efficacy. A microsphere delivery system was constructed using CDC37 to further enhance protein stability and targeting, achieving slow drug release. By inducing Treg cell-mediated immune memory, it can persistently regulate immune homeostasis in a non-hormonal manner, achieving long-term symptom relief and significantly reducing the need for repeated medication. This approach can accelerate clinical translation and industrialization, providing patients with safer, more effective, and more economical treatment options. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Based on the principal component analysis results, the variance contribution rate of the first principal component (PC1) is 52.4%, the variance contribution rate of the second principal component (PC2) is 16.9%, and the cumulative contribution rate is 69.3%.

[0020] Figure 2 This is a volcano plot showing the differential protein levels between Schistosoma juveniles and cercariae on day 4 post-infection. The differential threshold is |10g FC|≥1 and FDR<0.05; red dots represent upregulated proteins, and blue dots represent downregulated proteins.

[0021] Figure 3 This is a volcano plot showing the differential protein levels between Schistosoma juveniles and cercariae on day 7 post-infection. The differential threshold is |10g FC|≥1 and FDR<0.05; red dots indicate upregulation and blue dots indicate downregulation.

[0022] Figure 4 This is a volcano plot showing the differentially expressed proteins between Schistosoma juveniles on day 4 and day 7 post-infection. The differential threshold is |10g FC| ≥ 1 and FDR < 0.05; red dots indicate upregulation and blue dots indicate downregulation.

[0023] Figure 5 The results of GO-Biological Process enrichment analysis (Top1000) of Schistosoma juveniles on day 4 post-infection are shown. The node size represents the protein quantity, and the color corresponds to -log10 (P-value).

[0024] Figure 6 The results of GO-Biological Process enrichment analysis (Top1000) of Schistosoma juveniles on day 7 post-infection are shown. The node size represents the protein quantity, and the color corresponds to -log10 (P-value).

[0025] Figure 7 Prediction of CDC37 signal peptide.

[0026] Figure 8 The effect of CDC37 intervention on OVA-induced Treg cells in mouse lung tissue is shown in the error bar, which represents the standard error. *** express P <0.001. Figure 8 In the figure, A represents the CD4 content in the lung tissue of mice in the control and experimental groups. + CD25 + FoxP3 + The average percentage of Treg cells. Figure 8 B in the text is CD4 + CD25 + FoxP3 + Statistical chart of Treg cell counts (n=5).

[0027] Figure 9 The effect of CDC37 intervention on the expression of Treg-related factors in OVA-induced mouse lung tissue. Figure 9A, B, C, D, and E in the table represent the mRNA levels of IL-4, IL-5, IL-13, IL-10, and TGF-β in mouse lung tissue detected by qPCR, respectively; data are expressed as relative expression levels (2^(...). –ΔΔCt ) indicates mean ± standard error; ns indicates P ≥0.05, * express P <0.05, ** express P <0.01, *** express P <0.001. Figure 9 In the table, F, G, and H represent the concentrations of IL-4, IgG, and IL-10 proteins in lung tissue detected by ELISA, respectively; data are expressed in pg / mL, mean ± standard error; ns represent... P ≥0.05, *** express P <0.001.

[0028] Figure 10 Transcriptome analysis of the CDC37-regulated Treg mechanism. Figure 10 The result of principal component analysis is A, n=3. Figure 10 B in the table represents the immune regulation-related GO biological processes that were significantly enriched by CDC37 intervention (Top 20). Detailed Implementation

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1: Application of CDC37 protein in the preparation of therapeutic products for Treg cell disorder diseases

[0031] 1. Collection of lung-stage juvenile worms

[0032] The lung-stage juvenile worms were collected using the portal vein perfusion method. Each mouse was infected with 2000-3000 cercariae, and euthanized 4 days after infection. After euthanizing the infected mice, the thoracic and abdominal cavities were opened to expose and sever the portal vein. A syringe was inserted into the portal vein at the thoracic vertebrae, and pre-cooled 0.9% physiological saline containing heparin sodium (0.1 mg / mL) was perfused. This perfusion was repeated 3-4 times until no worms remained in the mouse's blood vessels. After perfusion, intact lung tissue was collected and washed three times with RPMI 1640 medium containing 5% fetal bovine serum and antibiotics (1:100) to remove peripheral blood. The lung tissue was minced and incubated at 37°C in a 5% CO2 incubator for 3 hours. The sample was filtered through a 400-mesh sterile metal sieve, and the residue and filtrate were collected. The residue was resuspended in RPMI 1640 medium preheated to 37°C and incubated at 37°C for 1 hour to further separate juvenile worms. The filtrate was centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the precipitate was resuspended in RPMI 1640 medium preheated to 37°C and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the filtration and centrifugation steps were repeated, the supernatant was discarded, and the precipitate was mixed with the residue. The sample was centrifuged using a density gradient centrifugation medium (400g, 20 minutes), and the lower layer was retained. RPMI medium containing 5% fetal bovine serum was added to the lower layer. The 1640 culture medium was incubated in a 37℃, 5% CO2 incubator for 2 hours. Finally, sterile double-distilled water was added to dissolve the red blood cells, centrifuged at 1500 rpm for 3 minutes, the juvenile worms were collected, and they were quickly frozen in liquid nitrogen and stored at -80℃ for subsequent experiments.

[0033] 2. Preparation of total protein samples from lung-stage juvenile worms

[0034] The protein samples of juvenile worms were prepared using the FASP (Filter-Assisted Sample Preparation) method. Juvenile worm samples were resuspended at a 1:10 ratio in SDT lysis buffer (0.1% SDS, 50 mM Tris-HCl, pH 7.5, 0.1 M DTT, with added PMSF), and incubated at 95°C for 5 min to fully lyse the protein. The samples were then sonicated on ice for 10 seconds each time, repeated 3 times, followed by centrifugation at 16000g for 5 min to clarify the lysate. 30 µL of the protein extract was taken and mixed with 200 µL of urea buffer UA (8 M urea, 0.1 M Tris-HCl, pH 8.5), then added to an ultrafiltration tube and centrifuged at 14000g for 40 min, repeated once, and the filtrate was discarded. 100 µL of IAA buffer (0.05 M iodoacetamide, urea buffer) was added, mixed well, and incubated for 5 min, followed by centrifugation at 14000g for 30 min. 100 µL of urea buffer UA (8 M urea, 0.1 M Tris-HCl, pH 8.5) was added. Centrifuge at 14000g for 40 min (8.0), repeat this step twice; add 40 µL of UB containing Lys-C (enzyme-protein ratio 1:50), mix well, and incubate overnight; transfer the ultrafiltration device to a new collection tube, add 120 µL of 0.05M NH4HCO3 aqueous solution and trypsin (enzyme-protein ratio 1:100), mix well, and incubate at room temperature for 4 h; centrifuge at 14000g for 40 min, add 50 µL of 0.5M NaCl, centrifuge at 14000g for 20 min using the ultrafiltration device, and collect the filtrate (i.e., protein); acidify the flow-through to pH 2 with CF3COOH and desalt using an SPE extraction column, and collect the desalted peptide eluent.

[0035] 3. iTRAQ tag

[0036] 100 µg of the desalted peptide mixture was dissolved in 150 µL of 0.5 M TEAB buffer, and iTRAQ reagent (each reagent was dissolved in 70 µL of acetonitrile) was added. The mixture was incubated at room temperature in the dark for 1 h to complete the labeling. The labeled peptide mixtures were pooled, dried to near dryness using a vacuum centrifuge, resuspended in 0.5 M TEAB buffer, and then subjected to subsequent mass spectrometry analysis.

[0037] 4. HPLC-MS / MS mass spectrometry detection

[0038] The LC system is calibrated using a DIA (Data Independent Acquisition) analysis workflow based on a spectral library, combined with the iRT (Internal Retention Time) standard. The Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Germany) was equipped with an EASY-nLC 1200 liquid chromatography system and a manually packed analytical column (75 µm inner diameter × 25 cm, 2.4 µm ReproSil-Pur 120 C18-AQ, Dr. Maisch GmbH, Germany). Mobile phase A was MilliQ water containing 0.1% formic acid, and mobile phase B was 80% acetonitrile containing 0.1% formic acid. The elution gradient was set to 120 min: 0 min–3 min, 2%–5% B; 4 min–103 min, 5%–35% B; 104 min–108 min, 35%–44% B; 109 min–110 min, 44%–100% B; 111 min–120 min, maintaining 100% B, with a flow rate of 150 nL / min.

[0039] Library construction samples were analyzed using DDA (Data-Dependent Acquisition) mode, with a first-stage mass spectrometry resolution of 60,000 m / s and a second-stage mass spectrometry resolution of 15,000 m / s, a cycle time of 3 seconds, and HCD fragmentation. Experimental samples were analyzed using DIA mode, with a first-stage mass spectrometry resolution of 120,000 m / s and a second-stage mass spectrometry resolution of 30,000 m / s. A full first-stage mass spectrometry scan was performed within a mass-to-charge ratio (m / z) range of 350–1200, followed by 12 second-stage mass spectrometry scans with windows set as follows: m / z 350–650 (one window every 25 m / z) and m / z 650–850 (one window every 25 m / z).

[0040] 5. Bioinformatics Analysis

[0041] The raw DIA data was matched with the spectral library built using the DDA mode using Spectronaut X software (Biognosys). The retention time window was optimized by iRT (internal retention time) calibration, and strict thresholds of peptide Q value < 0.01 and protein FDR < 1% were set to ensure the reliability of the identification.

[0042] Differentially expressed proteins were screened based on fold change (FC≥1.5) and ANOVA p<0.05 criteria, and significantly differentially expressed molecules were visualized using volcano plots.

[0043] Figure 1Principal component analysis (PCA) of the proteome of Schistosoma japonicum juvenile worms revealed that cercarial proteins and juvenile worm proteins could be well distinguished. The total protein clusters of juvenile worms at 4 days (pulmonary stage) and 7 days (late pulmonary stage) post-infection were relatively close, possibly due to the similarity of proteins secreted at these two stages. However, volcano plots, using statistical thresholds (|log2FC|≥1 and FDR<0.05), further screened for differentially expressed proteins between the two stages, clearly showing that immune and immune-regulatory effector molecules were more enriched in the pulmonary juvenile worm group. Figures 2-4 ).

[0044] Given that lung-stage juvenile worms can suppress host immunity, this study systematically screened immune-regulating effector molecules at the GO-BP Top 10,000 level. Figures 5-6 The results showed that the "regulation of immune response" functional node was specifically enriched at 4 days post-infection (pulmonary phase), and CDC37 was one of the juvenile worm-derived proteins detected at this node. However, at 7 days post-infection (late pulmonary phase), CDC37 was not detected in the BP-top1000 enrichment results, suggesting that its phase-specific expression is highly associated with immune regulation. Combined with extracellular prediction and evidence of non-classical exosome secretion, CDC37 was identified as a key effector molecule in pulmonary immune regulation. Figure 7 ).

[0045] 6. Protein Expression and Purification

[0046] The amino acid sequence of the CDC37 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the CDC37 protein is shown in SEQ ID NO.2.

[0047] SEQ ID NO.1:

[0048] MSCLNYSKWDHIEVSDDEDDTHPNIDTPSLFRWRHQARLERDAAWKKEREEFELNYKSFLTKYNESQQKLNKARENNADNIQELQKDFDKLEVEAKEWLVKETEMKKKERLRPLNIDTICKEGKSKTIINSPKLKYEETVDSSANSEEAAVNRLKDFVNKHNKAIRKFGLFQKPLDSQNYLVENPYLVCEETANQLVIWCIDLAMEEKFELMQHVSHQCIVMQFMLELAKSLKVDPRACIRPFFAKFMNPEPEYQKAFNDELSAFRERIRARAKVRLDEAMMEIEEEEKQKRLGPGGLDPVEVFDSLPANLQECFEKKDVELLKTVLCSMDPQQAEYHMKRCVDSGLWVDNAQEQQGGEVDESLPNSDNTGNDDATTIQQTDEKPADNSA。

[0049] SEQ ID NO.2:

[0050]

[0051] cDNA was obtained by reverse transcription of the genome of Schistosoma japonicum juvenile worms, according to CDC37 Gene sequence, design amplification CDC37 The primer set for the CDS region of the gene is shown in SEQ ID NO.3 for the upstream primer and SEQ ID NO.4 for the downstream primer. Using cDNA as a template, PCR amplification was performed according to the following reaction system and amplification program to obtain the PCR product.

[0052] SEQ ID NO.3: 5'-ATGTCTTGTCTTAATTATAGCAAGT-3';

[0053] SEQ ID NO. 4: 5'-TTAAGCACTGTTATCAGCTGGTTTT-3'.

[0054] Each 25µL reaction system contains: 2µL cDNA, 0.5µL upstream primer, 0.5µL downstream primer, 12.5µL enzyme, and DEPC water to bring the total to 25µL.

[0055] PCR amplification program: 98℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 55℃ annealing for 30 s, 72℃ extension for 1 min 20 s, 28 cycles; 72℃ final extension for 10 min, store at 4℃.

[0056] PCR products were purified using a purification kit (Zhuangmeng Biotechnology); restriction enzyme (TAKARA) was used. Xho I and Hind III. The purified PCR product and cloning vector pET28a were double-digested with enzymes, and the digested DNA fragments were purified using a gel extraction kit (Zhuangmeng Biotechnology). The purified insert fragment and linearized vector pET28a were ligated overnight at 16°C using T4 DNA ligase (TAKARA). The ligation mixture was transformed into competent *E. coli* DH5α cells (TAKARA), and transformants were selected on LB agar plates containing ampicillin. Positive clones were obtained by colony PCR and sequencing verification, and the recombinant plasmid pET28a- was extracted. CDC37 .

[0057] The recombinant plasmid pET28a- CDC37 Transformed into *E. coli* BL21 cells (TAKARA) using the standard bacterial heat shock transformation method. Single colonies were selected and inoculated into LB medium containing ampicillin, and cultured overnight at 37°C with shaking at 200 rpm. The overnight culture was then transferred 1:100 to fresh LB medium containing ampicillin and cultured at 37°C until the optical density (OD) at 600 nm was reached. 600The concentration reached 0.6; protein expression was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM. The culture was then incubated at 37°C for 4 h; the cell pellet was harvested by centrifugation at 4500 rpm and 4°C for 10 min; the cells were lysed on ice using lysis buffer (500 mM NaCl, 20 mM Tris, pH 7.5, with added PMSF) (120 W, sonication for 5 s, pause for 8 s, 15 min), centrifuged at 14500 rpm and 4°C for 30 min, filtered through a 0.22 μm or 0.45 μm filter membrane, and the supernatant and pellet were collected.

[0058] Protein purification was performed using a nickel column method. One mL of purification nickel beads was added to a pre-equilibrated nickel column, followed by 20 mL of the lysis buffer described above. 25 mL of bacterial supernatant was mixed with the equilibrated nickel beads and incubated at 4°C for 1 hour in a shaker. After incubation, 25 mL of wash buffer (500 mM NaCl, 20 mM Tris, 2 mM imidazole) was added, 5 mL at a time, and the wash buffer was collected. Finally, 1 mL of elution buffer (500 mM NaCl, 20 mM Tris, 600 mM imidazole) was added for elution, repeating 8 times. The eluent was collected as the target protein. The entire purification process was performed on ice to ensure protein stability.

[0059] 7. Cell sorting

[0060] Uses EasySep™ Mouse Naïve CD4 + T Cell Isolation Kit (Catalog:#19765) for sorting mouse Naive CD4 + T cells. Adjust the sample size to 1×10⁻⁶. 8 At a concentration of 0.1 mL to 2 mL, add 10 µL / mL FCR Blocker and 50 µL / mL Isolation Cocktail, incubate at room temperature for 7.5 min, add 50 µL / mL Depletion Cocktail, incubate at room temperature for 5 min, vortex for 30 s, add 75 µL / mL Rapid Spheres, and incubate at room temperature for 2.5 min. Bring the total volume to 2.5 mL and gently mix three times. Place the tube in an EasySep™ magnetic incubator (Catalog: #18000) and incubate for 2.5 min. Finally, slowly pour the purified T cells from the tube into a new centrifuge tube.

[0061] 8. Establish an OVA-induced asthma mouse model

[0062] The mouse model of allergic asthma was induced using ovalbumin (OVA), and the experimental grouping and treatment methods are as follows.

[0063] ① Normal control group (Naive): Mice were not sensitized or challenged in any way. On day 0 and day 14, 100 μL of PBS solution was injected intraperitoneally. On day 20 and day 22, each mouse was nebulized with 5 mL of PBS solution as a control for nebulization intervention.

[0064] ② CDC37 monotherapy group (Naive+CDC37): Mice were not sensitized or challenged in any way. On day 0 and day 14, 100 μL of PBS solution was injected intraperitoneally. On day 20 and day 22, each mouse was nebulized with 100 μg of CDC37 protein solution (100 μg of CDC37 protein dissolved in 5 mL of PBS solution).

[0065] ③OVA model control group (OVA):

[0066] Sensitization: On days 0 and 14, 50 μL of OVA solution (10 μg OVA dissolved in 50 μL PBS) and 50 μL of Alum (aluminum hydroxide gel) were injected intraperitoneally.

[0067] Stimulation: From day 21 to 24, nebulize 1% OVA solution (1 g OVA + 100 mL PBS) for 30 min daily;

[0068] Intervention: On days 20 and 22, each mouse was nebulized with 5 mL of PBS solution (without CDC37 protein) as a control for the treatment intervention.

[0069] ④OVA model group + CDC37 protein treatment group (OVA + CDC37):

[0070] Sensitization and challenge: Same as the OVA model control group (intraperitoneal injection of OVA + Alum on days 0 and 14; nebulized 1% OVA for 30 min on days 21-24).

[0071] Intervention: On days 20 and 22, each mouse was nebulized with 5 mL of CDC37 protein solution (100 μg of CDC37 protein dissolved in 5 mL of PBS solution).

[0072] ⑤ Positive drug control group of OVA model group + dexamethasone treatment (OVA+Dxms):

[0073] Sensitization and challenge: Same as the OVA model group (intraperitoneal injection of OVA+Alum on days 0 and 14; nebulization of 1% OVA for 30 min on days 21-24).

[0074] Intervention: On days 20 and 22, each mouse was injected intraperitoneally with dexamethasone (5mg:5mL from Ruicheng County Fanghong Animal Pharmaceutical Co., Ltd.) (100μL / mouse).

[0075] Mice in each group were sacrificed on day 25, and samples (serum, lung tissue, bronchoalveolar lavage fluid) were collected, flash-frozen in liquid nitrogen, and stored at -80°C.

[0076] 9. Flow cytometry analysis

[0077] Tissue samples from the OVA-induced asthma mouse model were minced and placed in a 40μm filter, then thoroughly ground into a 15mL centrifuge tube. The tube was washed with 2mL of 1×PBS, centrifuged at 500g, 4℃ for 8min, and the supernatant was discarded. 2mL of 1× erythrocyte lysis buffer was added, and the mixture was repeatedly pipetted and mixed. After standing for 2min, 2mL of 1×PBS was added, and the tube was centrifuged at 500g, 4℃ for 8min, and the supernatant was discarded. 5mL of PBS containing 2% FBS was added, and the tube was blocked on ice in the dark for 30min, centrifuged at 500g, 4℃ for 8min, and the supernatant was discarded. 100μL of PBS and surface antibody were added, and the tube was blocked on ice in the dark for 30min. 1mL of PBS was then used to clean the tube. Wash the antibody with PBS, centrifuge at 500g, 4℃ for 8 min, and discard the supernatant. Add 100μL of fixative and incubate on ice in the dark for 30 min. Add 1mL of cell permeabilization buffer, centrifuge at 500g, 4℃ for 8 min, and discard the supernatant. Resuspend the cells in 100μL of cell permeabilization buffer, mix well by pipetting and aspiration, add 2μL of intracellular antibody, and incubate on ice in the dark for 30 min. Wash the antibody with 1mL of cell permeabilization buffer, centrifuge at 500g, 4℃ for 8 min, and discard the supernatant. Resuspend the cells in 500μL of PBS, filter through a cell filter gauze into a flow cytometer, and analyze and record 100,000 cells.

[0078] like Figure 8 The flow cytometry results shown indicate that, compared to the OVA sensitization group (OVA group), CDC37 protein treatment significantly upregulated the proportion and number of Treg cells in mouse lung tissue.

[0079] 10. Cytokine Analysis

[0080] Two methods, real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA), were used to quantify cytokines in mouse tissues.

[0081] For qPCR analysis, total RNA was extracted using the Trizol reagent method, and the RNA concentration was determined using a NanoDrop spectrophotometer. Hifair® qPCR SYBR Green Master Mix kit was used to reverse transcribe RNA into cDNA. The total reaction volume was 20 μL, including 10 μL SYBR Mix, 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL cDNA, and RNase-free H2O to make up the difference. The amplification program was set as follows: 95℃ pre-denaturation for 30 s; 40 cycles: 95℃ for 5 s denaturation, 60℃ for 30 s annealing / extension; melting curve 95℃-65℃-95℃, fluorescence was collected every 0.1℃. Each sample was in triplicate, with GAPDH as an internal control. After amplification, 2^( –ΔΔCt The relative expression levels of the target genes IL-4, IL-5, IL-13, IL-10, and TGF-β were calculated using the method.

[0082] The primer sequence information for amplifying IL-4, IL-5, IL-13, IL-10, and TGF-β is as follows.

[0083] IL-4 upstream primer: 5'-GGTCTCAACCCCCAGCTAGT-3', SEQ ID NO.5;

[0084] IL-4 downstream primer: 5'-GCCGATGATCTCTCTCAAGTGAT-3', SEQ ID NO.6;

[0085] IL-5 upstream primer: 5'-TCAGGGGCTAGACATACTGAAG-3', SEQ ID NO.7;

[0086] IL-5 downstream primer: 5'-CCAAGGAACTCTTGCAGGTAAT-3', SEQ ID NO.8;

[0087] IL-13 upstream primer: 5'-GGAGCTGAGCAACATCACACA-3', SEQ ID NO.9;

[0088] IL-13 downstream primer: 5'-GGTCCTGTAGATGGCATTGCA-3', SEQ ID NO.10;

[0089] IL-10 upstream primer: 5'-CACTACCAAAGCCACAAAGCA-3', SEQ ID NO.11;

[0090] IL-10 downstream primer: 5'-AGGAGTCGGTTAGCAGTATGTT-3', SEQ ID NO.12;

[0091] TGF-β upstream primer: 5'-GCTGAACCAAGGAGACGGAA-3', SEQ ID NO.13;

[0092] TGF-β downstream primer: 5'-GGGCTGATCCCGTTGATTTC-3', SEQ ID NO.14.

[0093] For ELISA analysis, serum or tissue homogenates were prepared according to the instructions of NOVUS Biologicals ELISA kits (IL-10: VAL605; IL-4: VAL603) and Elabscience (IgE: E-EL-M3107), and the supernatant was used for ELISA detection.

[0094] like Figure 9 As shown, qPCR analysis revealed that compared to the Naive group, the OVA group showed significantly increased IL-4, IL-5, and IL-13 mRNA levels. Furthermore, the OVA+CDC37 group significantly alleviated this upward trend in both factors compared to the OVA group, suggesting that CDC37 may inhibit the Th2 / Treg transcriptional response. Compared to the Naive group, the OVA group showed a significant decrease in TGF-β mRNA levels, and the OVA+CDC37 group significantly alleviated this downward trend in both factors. ELISA analysis showed that the OVA group had significantly higher IL-4 and IgG protein levels than the Naive group, while after CDC37 intervention, both IL-4 and IgG protein levels significantly decreased.

[0095] In summary, CDC37 may alleviate OVA-induced allergic airway inflammation by downregulating the IL-4 / TGF-β axis and inhibiting IgG production, thereby weakening Treg-mediated immune regulation.

[0096] 11. Transcriptome analysis of the CDC37-regulated Treg mechanism

[0097] The experimental steps are as follows.

[0098] Naive CD4 + T cell sorting: Single-cell suspensions of spleen cells from 6-8 week old C57BL / 6 female mice were collected and sorted according to EasySep™ Mouse Naive CD4. + T Cell Isolation Kit instruction manual sorting.

[0099] Treg polarization and CDC37 intervention: 24-well plates pre-coated with antibiotic-containing medium, 1×10⁻⁶ mmol / L added to each well. 5 Naive CD4 + T cells, Treg induction system: IL-2 (10 ng / mL), TGF-β (5 ng / mL). The experimental group was supplemented with CDC37 protein (10 μg) and labeled as the CDC37 group, while the control group (Naive group) was supplemented with an equal volume of PBS; the cells were co-cultured at 37℃ and 5% CO2 for 7 days, with half the medium being changed and the corresponding factors and proteins being added every 3 days.

[0100] Sample collection: On day 7 of culture, the cell pellet was collected by centrifugation, washed twice with cold PBS, flash-frozen in liquid nitrogen and stored at -80°C, and sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for sequencing.

[0101] Reference transcriptome sequencing: Figure 10 The PCA plot showed that the Naive group, the CDC37 group, and the group along PC1 (57% explanation) were clearly separated, suggesting that CDC37 intervention significantly remodeled the overall transcriptional profile of Treg polarized cells; the top 20 enriched entries in GO-BP were concentrated on "negative regulation of T cell activation", "regulation of cytokine production" and "regulation of RNA stability", indicating that CDC37 may weaken the Treg immunosuppressive program through post-transcriptional and signal inhibition modules.

[0102] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0103] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of CDC37 protein in the preparation of therapeutic products for Treg cell disorder diseases, characterized in that, The amino acid sequence of the CDC37 protein is shown in SEQ ID NO.1; the Treg cell disorder is allergic asthma; the allergic asthma is induced by ovalbumin.

2. The application according to claim 1, characterized in that, The product uses CDC37 protein as the active ingredient and is supplemented with pharmaceutically acceptable carriers or excipients.

3. The application according to claim 2, characterized in that, The CDC37 protein content in the product is 0.01 g / L to 0.05 g / L.

4. The application according to claim 2, characterized in that, The carrier is a microsphere delivery system; the excipients are any one or more of fillers, stabilizers, diluents and adjuvants.

5. The application according to claim 4, characterized in that, The microsphere delivery system uses one or more of the following as carrier materials: polylactic acid-glycolic acid copolymer, chitosan, gelatin, hyaluronic acid, and sodium alginate.

6. The application according to claim 4, characterized in that, The diluent is a PBS solution.

7. The application according to claim 1, characterized in that, The product can induce Treg cell proliferation, inhibit Th2 response, or reduce airway hyperresponsiveness.

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