Pharmaceutical composition and application thereof in treating central obesity

Abstract

The invention relates to the technical field of central obesity related research and treatment, and discovers that up-regulation of brain-derived neurotrophic factor (BDNF) expression in adipose tissue is closely related to central obesity resistance by constructing a central obesity mouse model. A BDNF fusion polypeptide composed of a TAT cell-penetrating peptide, a flexible connecting peptide and a BDNF mature peptide is designed and prepared, and the fusion polypeptide has a strong cell activity promoting effect at a cellular level, can effectively activate a BDNF signal channel and has stability superior to that of natural BDNF protein; the invention also discloses an anti-TrkB monoclonal antibody with high affinity and high titer. In-vivo experiments show that the BDNF fusion polypeptide and the composition of the BDNF fusion polypeptide and the anti-TrkB monoclonal antibody can effectively inhibit weight gain of central obesity mice, improve glucose metabolism disorder, increase serum BDNF level and activate a TrkB signal channel, the synergistic treatment effect of the composition is better, and a new direction and an experimental basis are provided for treatment of central obesity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition and its application in treating central obesity. Background Art

[0002] In recent years, the number of obese people in the world has exploded. Among them, central obesity has become a difficult problem that the medical community urgently needs to overcome due to its complex pathological mechanism and high difficulty in treatment. Central obesity is a metabolic disease characterized by abnormal accumulation of fat in the internal organs and abdomen. Its core mechanism is the dysfunction of the hypothalamic energy regulatory nuclei (ventromedial nucleus VMH, arcuate nucleus ARC, etc.), which leads to hyperphagia, reduced energy expenditure and lipid metabolism imbalance. Studies have shown that the imbalance of activity of the hypothalamic melanocortin system (POMC neurons) and neuropeptide Y (NPY) neurons is the key to the occurrence of obesity, and brain-derived neurotrophic factor (BDNF) can activate POMC neurons and inhibit NPY expression by binding to its receptor TrkB, thereby regulating energy homeostasis. Clinical data show that the levels of BDNF in the serum and hypothalamus of obese patients are significantly lower than those in healthy people. The defects in the BDNF / TrkB signaling pathway caused by gene mutations are highly correlated with obesity.

[0003] Current treatments for central obesity have obvious limitations. For example, although GLP-1 receptor agonists (such as liraglutide) can suppress appetite through peripheral pathways, they cannot directly target the central BDNF signaling pathway, and long-term use can easily cause gastrointestinal side effects such as nausea and vomiting; and natural BDNF protein has a short half-life (about 1-2 hours) and is difficult to penetrate the blood-brain barrier (BBB), resulting in limited efficacy when administered centrally. In recent years, researchers have attempted to deliver BDNF through gene therapy or nanocarriers, but still face problems such as high immunogenicity and complex preparation processes. In addition, the development of monoclonal antibodies against TrkB is mostly concentrated in the field of cancer, and no agonist antibodies have yet been used to treat obesity.

[0004] Therefore, it is urgent to develop a treatment plan that is highly effective, low-toxic, and can target the key pathological links of central obesity. Summary of the Invention

[0005] The present invention reveals the association between BDNF and central obesity through a series of experiments, and develops related therapeutic substances and compositions.

[0006] Therefore, the present invention discloses a BDNF fusion polypeptide in one aspect. The amino acid sequence of the fusion polypeptide consists of a TAT transmembrane peptide, a flexible linker peptide and a BDNF mature peptide in sequence. The specific amino acid sequence is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the TAT transmembrane peptide of the present invention is YGRKKRRQRRR, the amino acid sequence of the flexible connecting peptide is GPGGGGS, and the BDNF mature peptide is a human BDNF mature peptide, whose amino acid sequence corresponds to the 119 amino acids His129-Arg247 in UniProt number P23560.

[0008] In one aspect, the present invention further discloses a composition for treating central obesity, comprising an effective amount of the BDNF fusion polypeptide and an effective amount of an anti-TrkB monoclonal antibody.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the anti-TrkB monoclonal antibody of the present invention is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3.

[0010] Preferably, the dosage of the BDNF fusion polypeptide and anti-TrkB monoclonal antibody of the present invention is 5 mg / kg.

[0011] In one aspect, the present invention also discloses a use of the BDNF fusion polypeptide in preparing a drug for treating central obesity.

[0012] In one aspect, the present invention also discloses a use of the anti-TrkB monoclonal antibody in the preparation of a drug for treating central obesity.

[0013] The beneficial effects of the present invention are summarized as follows:

[0014] 1. This study clarifies the close relationship between upregulated BDNF expression in adipose tissue and central obesity resistance, providing an important theoretical basis for a deeper understanding of the pathogenesis of central obesity and the exploration of therapeutic targets.

[0015] 2. Based on this, the present invention has developed a BDNF fusion polypeptide. By introducing TAT transmembrane peptide and flexible connecting peptide, the cellular uptake efficiency and stability are improved compared with the natural BDNF protein. It also exhibits stronger ability to promote cell viability and activate the BDNF signaling pathway at the cellular level, providing a new active substance for the treatment of central obesity.

[0016] 3. The anti-TrkB monoclonal antibody prepared by the present invention has high affinity and high titer, and has better performance than commercial antibodies, providing a powerful tool for targeting TrkB for central obesity treatment.

[0017] 4. In vivo experiments, the composition of the present invention, consisting of a BDNF fusion polypeptide and an anti-TrkB monoclonal antibody, demonstrated significant synergistic effects on weight control, improved glucose metabolism, and activation of the TrkB signaling pathway in mice with central obesity. This provides a new and effective strategy and drug development direction for the clinical treatment of central obesity and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE detection results of BDNF fusion peptide.

[0019] Figure 2 Western blot detection results of BDNF fusion peptide.

[0020] Figure 3 SDS-PAGE detection results of anti-TrkB monoclonal antibody.

[0021] Figure 4 Western blot test results of anti-TrkB monoclonal antibody (the molecular weight of TrkB recombinant protein is approximately 78kDa). DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Construction, expression and functional verification of BDNF fusion polypeptide

[0025] 1. Fusion polypeptide construction and design: Design a fusion polypeptide sequence as shown in SEQ ID NO.1, which consists of three parts:

[0026] (1) Human BDNF mature peptide (UniProt number: P23560, His129-Arg247, total 119aa);

[0027] (2) TAT transmembrane peptide (transmembrane transduction, improve cell uptake): YGRKKRRQRRR

[0028] (3) Flexible linker peptide (stability-enhancing peptide SA): GPGGGGS

[0029] The final fusion sequence is: TAT transmembrane peptide-flexible connecting peptide-BDNF mature peptide, and the specific amino acid sequence is shown in SEQ ID NO.1.

[0030] 2. Preparation and Identification of BDNF Fusion Peptides

[0031] 1. Experimental Materials

[0032] (1) Host cell: Escherichia coli BL21 (DE3).

[0033] (2) Expression vector: pET-28a(+).

[0034] (3) Reagents: restriction endonucleases (NdeI, XhoI), T4 DNA ligase, DNA polymerase, antibiotics (kanamycin), IPTG (isopropyl-β-D-thiogalactopyranoside), protein purification-related reagents (nickel column packing, elution buffer, etc.), SDS-PAGE-related reagents, Western blot-related reagents (primary antibody is anti-BDNF antibody, secondary antibody is HRP-labeled goat anti-rabbit IgG).

[0035] 2. Experimental Procedure

[0036] 2.1 Gene Synthesis and Vector Construction: Based on the amino acid sequence of the BDNF fusion polypeptide, a corresponding DNA sequence was designed through codon optimization. A gene synthesis company was commissioned to synthesize the DNA sequence, and NdeI and XhoI restriction sites were introduced at both ends. The synthesized DNA fragment and the pET-28a(+) vector were double-digested with NdeI and XhoI, respectively. The digestion system was as follows: 5 μg of DNA or vector, 2 μL of NdeI, 2 μL of XhoI, 5 μL of 10× digestion buffer, added with ddH2O to 50 μL, and digested at 37°C for 3 h. The digestion products were separated by agarose gel electrophoresis, and the target fragment and vector fragment were recovered using a gel recovery kit. The recovered target fragment and vector fragment were ligated using T4 DNA ligase. The ligation system was as follows: 3 μL of target fragment, 1 μL of vector fragment, 1 μL of T4 DNA ligase, 1 μL of 10× ligation buffer, added with ddH2O to 10 μL, and ligated at 16°C overnight. The ligation product was transformed into E. coli DH5α competent cells, plated on LB plates containing kanamycin (50 μg / mL), and cultured overnight at 37°C. Single colonies were selected for PCR identification, and positive clones were sent for sequencing verification.

[0037] 2.2 Protein expression and induction: The correctly sequenced recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB plates containing kanamycin (50 μg / mL), and cultured at 37°C overnight. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin (50 μg / mL), and culture it at 37°C and 220 rpm overnight. Take 1 mL of the overnight culture and transfer it to 100 mL of LB liquid medium containing kanamycin (50 μg / mL), and culture it at 37°C and 220 rpm until the OD 600 The expression of the protein was about 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and the expression was induced at 16°C and 180 rpm for 16 h.

[0038] 2.3 Protein purification: After induction of expression, the bacterial solution was centrifuged at 4°C and 8000rpm for 10 minutes to collect the bacteria. Resuspend the bacteria with an appropriate amount of binding buffer (20mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH 7.4) and ultrasonically disrupt (power 300W, working 3s, interval 5s, total 30min). The disrupted bacterial solution was centrifuged at 4°C and 12000rpm for 30 minutes to collect the supernatant. The supernatant was passed through a nickel column and the column was washed with binding buffer until the effluent OD 280 The target protein was eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.4) and the eluted peak was collected. The eluted protein was analyzed by SDS-PAGE to determine its purity and molecular weight.

[0039] 2.4 Protein Identification: Transfer proteins separated by SDS-PAGE to a PVDF membrane and block with 5% skim milk for 1 hour. Add anti-BDNF antibody (1:1000 dilution) and incubate overnight at 4°C. Wash three times with TBST (10 minutes each). Add HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) and incubate at room temperature for 1 hour. Wash three times with TBST (10 minutes each). Develop with ECL luminescent solution and expose to light to confirm that the protein is a BDNF fusion peptide.

[0040] 3. Experimental Results

[0041] 3.1 Protein expression and purification results: SDS-PAGE analysis showed that the bacterial solution after induced expression showed a clear band at about 15kDa, which is consistent with the theoretical molecular weight of BDNF fusion polypeptide. After nickel column purification, the purity of the target protein reached more than 90% ( Figure 1 shown).

[0042] 3.2 Protein identification results: Western blot results showed that a specific band appeared at about 15 kDa, further confirming that the purified protein was a BDNF fusion polypeptide ( Figure 2 shown).

[0043] 3. Verification of BDNF fusion peptide activity at the cellular level

[0044] 1. Experimental Materials

[0045] (1) Cell line: SH-SY5Y human neuroblastoma cells.

[0046] (2) Culture medium: DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0047] (3) Reagents: BDNF fusion peptide, natural BDNF protein (purchased from Huamei Biotechnology, product number CSB-EP002655HU(A4)), MTT reagent, CCK-8 reagent, BDNF receptor antagonist K252a, and POMC detection kit.

[0048] 2. Experimental Procedure

[0049] 2.1 Cell Culture: SH-SY5Y cells were seeded into culture flasks containing DMEM / F12 medium and cultured in a 37°C, 5% CO2 incubator. Cell passage or experiment was performed when the cell confluence reached 80%-90%.

[0050] 2.2 Cell viability assay: SH-SY5Y cells were plated at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 hours. Different concentrations (0, 10, 20, 50, and 100 ng / mL) of BDNF fusion peptide and native BDNF protein were added, with six replicates per well. Culture was continued for another 24 hours. 10 μL of 5 mg / mL MTT reagent was added to each well and cultured for another 4 hours. The supernatant was aspirated, and 150 μL of DMSO was added to each well. The cells were shaken for 10 minutes to fully dissolve the crystals. Absorbance was measured at 570 nm using a microplate reader to calculate cell viability.

[0051] 2.3 BDNF signaling pathway activation detection: SH-SY5Y cells were plated at 1×10 5Cells were seeded at a density of 100 ng / mL in 6-well plates and cultured for 24 hours. 100 ng / mL of BDNF fusion peptide, native BDNF protein, and an equal volume of culture medium were added (control group). A BDNF receptor antagonist K252a treatment group was also established (pretreatment with K252a for 30 minutes followed by addition of BDNF fusion peptide). Each group was cultured for an additional 24 hours in triplicate. Cells were harvested, total protein was extracted, and protein concentration was determined using the BCA assay. POMC expression levels were determined using a POMC detection kit according to the kit's instructions.

[0052] 3. Experimental Results

[0053] 3.1 Cell viability test results As shown in Table 1, both BDNF fusion polypeptide and natural BDNF protein can promote the viability of SH-SY5Y cells, and the BDNF fusion polypeptide has a stronger effect in promoting cell viability.

[0054] Table 1 Cell viability test results

[0055]

[0056] 3.2 BDNF signaling pathway activation test results As shown in Table 2, BDNF fusion polypeptide can significantly activate the BDNF signaling pathway and promote the expression of POMC, and this effect can be inhibited by the BDNF receptor antagonist K252a.

[0057] Table 2 BDNF signaling pathway activation detection results

[0058]

[0059] 4. Stability Assessment

[0060] The fusion protein was cultured in PBS at 37°C for 24, 48, and 72 hours, and the residual protein was detected. The results are as follows:

[0061] (1) The residual proportions of the fusion polypeptide were: 92.1% at 24 h, 79.6% at 48 h, and 65.3% at 72 h;

[0062] (2) The residual proportions of native BDNF protein were: 64.6% at 24 h, 45.2% at 48 h, and 38.4% at 72 h;

[0063] The above results show that the fusion peptide is significantly superior to the natural BDNF protein.

[0064] Example 2: Preparation and testing of anti-TrkB monoclonal antibodies

[0065] 1. Experimental Materials

[0066] 1. Experimental animals: 6-8 week old female Balb / c mice.

[0067] 2. Cell line: SP2 / 0 myeloma cells.

[0068] 3. Reagents: Complete Freund's adjuvant, incomplete Freund's adjuvant; TrkB antigen protein (ab132927) (purity >95%); polyethylene glycol (PEG, MW 1500); HAT medium (containing hypoxanthine, aminopterin, and thymidine), HT medium (containing hypoxanthine and thymidine), RPMI-1640 medium; fetal bovine serum (FBS); goat anti-mouse IgG-HRP secondary antibody; TMB colorimetric solution, stop solution (2M H2SO4); Protein A affinity chromatography column packing; SDS-PAGE and Western blot reagents; commercial anti-TrkB monoclonal antibody (ab134155).

[0069] 2. Experimental steps

[0070] 1. Antibody Preparation

[0071] (1) Animal immunization: TrkB antigen protein was mixed with complete Freund's adjuvant in equal volumes, and after sufficient emulsification, multiple subcutaneous injections were performed on Balb / c mice, with each mouse receiving an injection dose of 100 μg of antigen protein. Three weeks later, TrkB antigen protein was mixed with incomplete Freund's adjuvant in equal volumes and emulsified, and a second subcutaneous multiple injection was performed, with each mouse receiving an injection dose of 50 μg of antigen protein. Thereafter, booster immunizations were performed every two weeks, for a total of three booster immunizations, with each dose of 50 μg of antigen protein, all emulsified with incomplete Freund's adjuvant. Seven days after the last booster immunization, blood was collected from the mouse tail vein, serum was separated, and the serum antibody titer was detected by indirect ELISA. When the antibody titer reached the requirement (>1:10 5 ), cell fusion was performed.

[0072] (2) Cell fusion: The spleen of the immunized mouse was taken, rinsed with sterile PBS, ground into a single cell suspension, and filtered through a 200-mesh cell sieve to collect the spleen cells. At the same time, SP2 / 0 myeloma cells in the logarithmic growth phase were taken and washed twice with PBS. The spleen cells and SP2 / 0 myeloma cells were mixed in a ratio of 10:1, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. In a 37°C water bath, 1 mL of preheated 50% PEG 1500 solution was slowly added while gently stirring for 1 minute. Then, 1 mL of RPMI-1640 medium was slowly added within 1 minute, followed by 9 mL of RPMI-1640 medium within 2 minutes to terminate the effect of PEG. Centrifuged at 1200 rpm for 5 minutes, discarded the supernatant, and the cells were resuspended in HAT medium and inoculated into a 96-well cell culture plate with 100 μL per well. The plates were cultured in a 37°C, 5% CO2 incubator.

[0073] (3) Hybridoma cell screening: Starting from the third day after fusion, observe the cell growth and replace the HAT medium in time. On the 7th to 10th day after fusion, when the hybridoma cells grow to 1 / 3-1 / 2 of the well bottom area, take the culture supernatant and use the indirect ELISA method to detect the secretion of anti-TrkB antibodies. The specific operation is as follows:

[0074] Dilute TrkB antigen protein to 1 μg / mL in coating buffer (0.05 M carbonate buffer, pH 9.6) and add 100 μL per well to a 96-well microtiter plate for overnight coating at 4°C. The next day, discard the coating buffer and wash three times with PBST (PBS containing 0.05% Tween-20) for 5 minutes each. Add 200 μL of 5% skim milk to each well and block at 37°C for 1 hour. Discard the blocking buffer and wash three times with PBST for 5 minutes each. Add 100 μL of hybridoma cell culture supernatant and incubate at 37°C for 1 hour. Wash three times with PBST for 5 minutes each. Add 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody and incubate at 37°C for 1 hour. Wash five times with PBST for 5 minutes each. Add 100 μL of TMB color development solution to each well and develop for 15 minutes at room temperature in the dark. Add 50 μL of stop solution to terminate the reaction and measure the absorbance at 450 nm using a microplate reader. Select hybridoma cell wells with absorbance values ​​significantly higher than the negative control wells for the next subcloning step.

[0075] (4) Subcloning: Subclone positive hybridoma cells using the limiting dilution method. Dilute the positive hybridoma cells with HT medium to a cell suspension of 1 cell / well, 3 cells / well, and 5 cells / well, respectively, and inoculate 100 μL per well in a 96-well cell culture plate. Culture the plates in a 37°C, 5% CO2 incubator. After 7-10 days of culture, collect the culture supernatant and test the secretion of anti-TrkB antibodies using an indirect ELISA method. Select the wells with monoclonal growth and antibody-positive cells for expansion culture.

[0076] (5) Antibody production and purification: The hybridoma cells stably secreting anti-TrkB monoclonal antibodies obtained by screening were inoculated into the abdominal cavity of Balb / c mice, with 1×10 6 -5×10 6 cells. After 7-10 days, collect the mouse ascites. Dilute the ascites 1-2 times with PBS, centrifuge at 12000rpm for 30 minutes, and take the supernatant. Pass the supernatant through a Protein A affinity chromatography column, equilibrate the column with binding buffer (20mM sodium phosphate buffer, pH 7.0), and then elute the antibody with elution buffer (0.1M glycine-HCl buffer, pH 3.0). Collect the elution peak and neutralize it to pH 7.0 with neutralization buffer (1M Tris-HCl, pH 9.0). Detect the purity of the antibody by SDS-PAGE, and determine the concentration of the antibody by BCA assay.

[0077] 2. Antibody testing

[0078] (1) Antibody specificity identification (Western blot): TrkB antigen protein and other unrelated proteins were separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 1 hour, and then diluted anti-TrkB monoclonal antibodies of the present invention and commercial anti-TrkB monoclonal antibodies were added, respectively, and incubated at 4°C overnight. Washed with PBST three times, each time for 10 minutes. Diluted goat anti-mouse IgG-HRP secondary antibody was added and incubated at room temperature for 1 hour. Washed with PBST five times, each time for 10 minutes. Developed with ECL luminescent solution, exposed and developed, and observed whether bands appeared only at the position corresponding to the TrkB antigen protein, and compared the clarity and intensity of the bands.

[0079] (2) Antibody affinity determination: The affinity of the antibody was determined using the ELISA competitive method. The details are as follows:

[0080] TrkB antigen protein was diluted to 1 μg / mL with coating buffer, and 100 μL was added to each well of a 96-well ELISA plate and coated at 4°C overnight. The next day, the coating solution was discarded and the plate was washed 3 times with PBST, each time for 5 minutes. 200 μL of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The blocking solution was discarded and the plate was washed 3 times with PBST, each time for 5 minutes. The anti-TrkB monoclonal antibody of the present invention and the commercial anti-TrkB monoclonal antibody were diluted with PBS to different concentrations (e.g., 10 -5 -10 -12 M), and then with equal amounts of TrkB antigen protein at different concentrations (such as 10 -5 -10 -12 M) and incubate at 37°C for 1 hour. Add the mixed solution to the ELISA plate coated with TrkB antigen protein, 100 μL per well, and incubate at 37°C for 1 hour. Wash with PBST 3 times, 5 minutes each time. Add 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody and incubate at 37°C for 1 hour. Wash with PBST 5 times, 5 minutes each time. Add 100 μL of TMB colorimetric solution to each well and develop at room temperature in the dark for 15 minutes. Add 50 μL of stop solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm using an enzyme-labeled instrument. Based on the absorbance values, draw the competitive inhibition curves of the antibody of the present invention and the commercial antibody, and calculate the affinity constant (Kd) of the antibody.

[0081] (3) Antibody titer determination: The antibody titer was determined by indirect ELISA. TrkB antigen protein was diluted to 1 μg / mL with coating buffer, and 100 μL was added to each well of a 96-well enzyme-labeled plate and coated at 4°C overnight. The next day, the coating solution was discarded and the plate was washed three times with PBST, each time for 5 minutes. 200 μL of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The blocking solution was discarded and the plate was washed three times with PBST, each time for 5 minutes. The anti-TrkB monoclonal antibody of the present invention and the commercial anti-TrkB monoclonal antibody were diluted in PBS in multiple ratios (such as 1:100, 1:200, 1:400...), 100 μL was added to each well, and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, each time for 5 minutes. 100 μL of the diluted goat anti-mouse IgG-HRP secondary antibody was added and the plate was incubated at 37°C for 1 hour. The plate was washed five times with PBST, each time for 5 minutes. Add 100 μL of TMB colorimetric solution to each well and develop at room temperature in the dark for 15 minutes. Terminate the reaction by adding 50 μL of stop solution and measure the absorbance at 450 nm using a microplate reader. The highest dilution with an absorbance greater than 2.1 times that of the negative control well is used as the antibody titer.

[0082] 3. Experimental Results

[0083] 1. Antibody Production and Purification Results

[0084] (1) Anti-TrkB monoclonal antibody of the present invention: SDS-PAGE detection showed that the purified antibody presented a single band with a purity of >90% ( Figure 3 The antibody concentration was determined by BCA assay and was 3.23 mg / mL.

[0085] (2) Antibody specificity identification results (Western blot): The anti-TrkB monoclonal antibody of the present invention showed a clear and strong band at the position corresponding to the TrkB antigen protein, with little background interference ( Figure 4 shown).

[0086] (3) Antibody affinity determination results: The affinity constant (Kd) of the anti-TrkB monoclonal antibody of the present invention is 5×10-11 M, while the affinity constant (Kd) of the commercial anti-TrkB monoclonal antibody is 2×10-10 M. The results show that the affinity of the anti-TrkB monoclonal antibody of the present invention is significantly higher than that of the commercial antibody.

[0087] (4) Antibody titer determination results: The antibody titer of the anti-TrkB monoclonal antibody of the present invention is 1:10 6 The titer of commercial anti-TrkB monoclonal antibody is 1:10. 5 Therefore, the titer of the anti-TrkB monoclonal antibody of the present invention is 10 times that of the commercial antibody.

[0088] IV. Sequence Determination of the Anti-TrkB Monoclonal Antibody of the Present Invention

[0089] Through detection, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TrkB monoclonal antibody of the present invention are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0090] These results demonstrate that the anti-TrkB monoclonal antibodies of the present invention have lower affinity constants and higher potencies, meaning they can bind to the TrkB antigen protein at lower concentrations and with greater binding capacity. This can reduce the amount of antibody used and lower costs in practical applications. Therefore, the anti-TrkB monoclonal antibodies of the present invention outperform commercially available antibodies in terms of performance and have broader application prospects.

[0091] Example 3: In vivo experiments of the composition

[0092] 1. Experimental Materials

[0093] 1. Experimental Animals: 60 6-week-old male C57BL / 6 mice, weighing 18-20 g. Mice were housed in a specific pathogen-free (SPF) animal room at a temperature of (22±2)°C, a humidity of 50%-60%, and a 12-h light / 12-h dark cycle, with free access to food and water.

[0094] 2. Reagents: BDNF fusion polypeptide was prepared according to the method in Example 2; anti-TrkB monoclonal antibody was prepared according to the method in Example 3; TrkB and p-TrkB antibodies and corresponding secondary antibodies (abcam); RIPA lysis buffer, BCA protein quantification kit and other kits were all commercial products.

[0095] 2. Experimental steps

[0096] 1. Animal grouping: 60 male C57BL / 6 mice were randomly divided into 4 groups, with 15 mice in each group:

[0097] (1) Normal diet group (Group 1): The mice were given a normal diet for 8 weeks.

[0098] (2) Model control group (Group 2): The mice were given a high-fat diet for 8 weeks.

[0099] (3) Fusion peptide group (Group 3): The mice were fed a high-fat diet and orally administered BDNF fusion peptide at a dose of 10 mg / kg daily between 9 and 10 a.m. for 8 weeks. A dedicated mouse gavage needle was used to ensure accurate delivery of the drug to the stomach.

[0100] (4) Combination group (Group 4): Mice were fed a high-fat diet and received intraperitoneal injections of BDNF fusion peptide and anti-TrkB monoclonal antibody at a dose of 5 mg / kg every Monday and Thursday at 9-10 a.m. for 8 weeks. During the intraperitoneal injection, mice were placed in a Trendelenburg position, and the needle was inserted into the abdomen at a 30° angle to ensure that the drug was injected into the peritoneal cavity.

[0101] 2. Monitoring indicators and methods

[0102] (1) Weekly body weight and food intake monitoring: At a fixed time each week, weigh the mice using an electronic balance, record the feed consumption of each cage of mice in the previous week, and calculate the average daily food intake of each mouse.

[0103] (2) Fasting blood glucose test: In the eighth week of the experiment, mice were fasted for 12 h (water was not allowed), and their fasting blood glucose was tested by tail tip blood sampling using a blood glucose meter. 2-3 μL of blood was collected from each mouse for testing.

[0104] (3) Sample collection and processing: At the end of the 8th week of the experiment, mice were fasted for another 12 hours, and blood was collected from the eyeballs. The blood was collected in anticoagulant tubes, centrifuged at 3000 rpm for 15 minutes at 4°C, and the serum was separated and stored in a -80°C refrigerator for testing. After blood collection, the mice were quickly killed, and the hypothalamic tissue was isolated on ice, weighed, and placed in a cryovial. It was quickly frozen in liquid nitrogen and transferred to a -80°C refrigerator for subsequent Western blot analysis.

[0105] 3.Indicator detection method

[0106] (1) Serum BDNF detection: Follow the instructions of the BDNF detection kit and calculate the serum BDNF concentration based on the standard curve.

[0107] (2) Detection of p-TrkB / TrkB ratio in hypothalamic tissue:

[0108] Protein extraction: Remove the hypothalamic tissue, add an appropriate amount of RIPA lysis buffer, homogenize and lyse on ice, centrifuge at 4°C and 12,000 rpm for 15 min, collect the supernatant, and measure the protein concentration using a BCA protein quantification kit.

[0109] Western blot analysis: Protein samples were separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 1 hour, and TrkB and p-TrkB primary antibodies (diluted 1:1000) were added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 minutes each, and the corresponding secondary antibody (diluted 1:5000) was added and incubated at room temperature for 1 hour. The membrane was washed again with TBST three times for 10 minutes each, and finally developed using a chemiluminescence imaging system. The grayscale values ​​of the bands were analyzed using ImageJ software, and the p-TrkB / TrkB ratio was calculated.

[0110] 3. The experimental results are shown in Table 3.

[0111] 1. Body Weight and Food Intake Analysis: The weight gain of mice in Group 2 was significantly higher than that in Group 1 (P<0.01), indicating that the high-fat diet successfully induced obesity (i.e., central obesity) in mice. The weight gain of mice in Groups 3 and 4 was significantly lower than that in Group 2 (P<0.05 and P<0.01). The weight gain of the combined treatment group was 68% lower than that in Group 2, indicating that both BDNF fusion peptide and its combination with anti-TrkB monoclonal antibody can effectively inhibit weight gain caused by central obesity, and the combined treatment effect is more superior. In terms of food intake, the average daily food intake of Groups 3 and 4 was also significantly lower than that of Group 2 (P<0.05 and P<0.01), indicating that both interventions may reduce energy intake by regulating appetite, thereby controlling weight.

[0112] 2. Fasting blood glucose analysis: Fasting blood glucose levels in Group 2 were significantly higher than those in Group 1 (P < 0.01), reflecting abnormal glucose metabolism in mice with central obesity. Fasting blood glucose levels in Groups 3 and 4 were significantly lower than those in Group 2 (P < 0.05 and P < 0.01, respectively). Blood glucose levels in the combined treatment group approached those in Group 1, indicating that BDNF fusion peptide and its combination with anti-TrkB monoclonal antibody therapy can help improve central obesity-induced glucose metabolism disorders, with combined treatment being more effective in regulating blood glucose.

[0113] 3. Analysis of Serum BDNF Levels: Serum BDNF levels in Group 2 were significantly lower than those in Group 1 (P < 0.01), consistent with previous studies demonstrating decreased BDNF levels in obese humans or animal models. Serum BDNF levels in Groups 3 and 4 were significantly higher than those in Group 2 (P < 0.05 and P < 0.01, respectively). The combined treatment group had serum BDNF levels close to those in Group 1, demonstrating that BDNF fusion peptide and its combination with anti-TrkB monoclonal antibody therapy can effectively elevate serum BDNF levels, with the combined treatment having a more pronounced effect on BDNF levels.

[0114] 4. p-TrkB / TrkB Ratio Analysis: The p-TrkB / TrkB ratio in hypothalamic tissue of Group 2 was significantly lower than that of Group 1 (P<0.01), indicating that central obesity inhibits activation of the TrkB signaling pathway. The p-TrkB / TrkB ratios in Groups 3 and 4 were significantly higher than those in Group 2 (P<0.05 and P<0.01, respectively). The p-TrkB / TrkB ratio in the combined treatment group was significantly higher than that in Group 1 (P<0.05), suggesting that BDNF fusion peptide and its combination with anti-TrkB monoclonal antibody therapy can activate the TrkB signaling pathway, and that the combined treatment has a stronger activation effect on the TrkB pathway. This may be an important mechanism for the superior effect of combined treatment in improving body weight, blood glucose, and other indicators.

[0115] Table 3 Summary of experimental results

[0116]

[0117] (*P < 0.05 vs. Group 2; **P < 0.01 vs. Group 2)

[0118] In summary, the combined use of BDNF fusion peptide and anti-TrkB monoclonal antibody has a significant synergistic effect in improving the body weight, blood glucose metabolism and activating the TrkB signaling pathway in obese mice induced by central obesity, providing a strong experimental basis for the treatment of central obesity.

[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A BDNF fusion polypeptide, characterized in that: The amino acid sequence of the fusion polypeptide consists of a TAT transmembrane peptide, a flexible connecting peptide and a BDNF mature peptide in sequence, and the specific amino acid sequence is shown in SEQ ID NO.

1.

2. The BDNF fusion polypeptide according to claim 1, characterized in that The amino acid sequence of the TAT transmembrane peptide is YGRKKRRQRRR, the amino acid sequence of the flexible connecting peptide is GPGGGGS, and the BDNF mature peptide is a human BDNF mature peptide, the amino acid sequence of which corresponds to the 119 amino acids His129-Arg247 in UniProt number P23560.

3. A composition for treating central obesity, characterized in that: The composition comprises an effective amount of the BDNF fusion polypeptide according to claim 1 and an effective amount of an anti-TrkB monoclonal antibody.

4. The composition according to claim 3, characterized in that The amino acid sequence of the heavy chain variable region of the anti-TrkB monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

3.

5. The composition according to claim 3, characterized in that The dosage of the BDNF fusion polypeptide and the anti-TrkB monoclonal antibody was 5 mg / kg.

6. Use of the BDNF fusion polypeptide according to claim 1 in the preparation of a medicament for treating central obesity.

7. Use of the anti-TrkB monoclonal antibody according to claim 4 in the preparation of a medicament for treating central obesity.

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