Preparation of canine fibroblast growth factor 21 fusion protein and its application in the treatment of atopic dermatitis

Abstract

This invention discloses the preparation of canine fibroblast growth factor 21 fusion proteins and their use in the treatment of atopic dermatitis. This invention provides canine fibroblast growth factor 21 (FGF21) fusion proteins, which are composed of canine fibroblast growth factor 21 (FGF21) and a fusion tag, which may be TAT, R11, or Fc. The three FGF-21 fusion proteins obtained by this invention can effectively improve the symptoms of atopic dermatitis in mice and reduce the expression levels of related inflammatory factors. The three recombinant FGF-21 fusion proteins provided by this invention can serve as potential drugs for the treatment of canine atopic dermatitis.

Description

Technical Field

[0001] This invention relates to the preparation of canine fibroblast growth factor 21 fusion protein and its use in the treatment of atopic dermatitis, belonging to the field of biotechnology. Background Technology

[0002] With the increasing number of pet dogs, canine diseases have received growing attention. The increasing industrial development and worsening environmental pollution have led to a high incidence of canine allergic diseases. Canine allergic diseases include atopic dermatitis, gastroenteritis, and allergic reactions. Among these, canine atopic dermatitis (CAD) is widespread globally, affecting up to 10% of dogs. Canine atopic dermatitis (AD) is a common, hereditary inflammatory and pruritic allergic skin disease. The clinical manifestations of atopic dermatitis are remarkably similar across different species. Unlike humans, who may exhibit respiratory symptoms, dogs typically remain at the dermal stage of the disease. However, the high degree of similarity in many clinical and etiological features often leads to CAD being described as a model for human atopic dermatitis (AD). Clinically, CAD is often treated with corticosteroids, cyclosporine, oxcalinib, etc. Long-term use of these drugs can cause significant side effects. Furthermore, more effective allergen immunotherapy requires identifying the canine allergen and obtaining allergen extracts, which is cumbersome, complex, and lacks versatility, making large-scale application difficult. Therefore, there is an urgent clinical need to develop anti-inflammatory drugs that are suitable for dogs, have long-lasting effects, few side effects, and can treat atopic dermatitis.

[0003] Fibroblast growth factor 21 (FGF-21) is a member of the fibroblast growth factor family and has attracted widespread attention due to its close relationship with glucose and lipid metabolism. Recent studies have shown that, in addition to combating obesity, improving insulin resistance, and alleviating various metabolic diseases, FGF-21 also possesses anti-inflammatory effects. FGF-21 can reduce inflammatory factors such as IL-1β, IL-6, and TNF-α in mouse serum and inhibit the transcription and translation of NF-κB p65.

[0004] Currently, no literature reports that FGF-21 has an ameliorative effect on canine atopic dermatitis. Therefore, this invention uses a DNCB-induced atopic dermatitis mouse model to investigate the pharmacodynamic effects of FGF-21 on canine atopic dermatitis. In this animal model, our laboratory found that FGF-21-TAT, FGF-21-R11, and FGF-21-Fc can significantly reduce dermatitis scores, improve dermatitis symptoms, and effectively inhibit the expression of related inflammatory factors, demonstrating their potential for treating atopic dermatitis. Summary of the Invention

[0005] The purpose of this invention is to provide the preparation of a canine fibroblast growth factor 21 fusion protein and its use in the treatment of atopic dermatitis. The fusion protein consists of canine fibroblast growth factor 21 (FGF21) and a fusion tag, which may be TAT, R11, or Fc, and these tags have important applications in the treatment of canine atopic dermatitis. Attached Figure Description

[0006] Figure 1 Identification of three FGF-21 fusion proteins after purification

[0007] Figure 2 In vitro detection of the activity of three fusion proteins

[0008] Figure 3 In vivo detection of the activity of three fusion proteins

[0009] Figure 4 H&E staining results on the back of AD mice

[0010] Figure 5 H&E staining results of ears in AD mice

[0011] Figure 6 Immunohistochemical results of TSLP protein in AD mouse tissues

[0012] Figure 7 Serum-associated inflammatory factor ELISA results Detailed Implementation

[0013] Example 1: Obtaining FGF21-TAT, FGF21-R11, and FGF21-Fc genes

[0014] Fc fragments of TAT, R11, and IgG were designed at the 3' end of the FGF21 gene, and restriction enzyme sites BsaⅠ and BamHI were designed at both ends of the FGF-21 gene. Three recombinant plasmids were synthesized at Shanghai Sangon Biotech Co., Ltd.

[0015] The newly synthesized plasmids FGF-21-TAT, FGF-21-R11, and FGF-21-Fc from Shanghai Sangon Biotech were double-digested with the laboratory-preserved SUMO vector using restriction endonucleases BsaⅠ and BamHI (100 μL system). The gel-recovered products were then ligated. After mixing the reagents in the ligation system, ligation was performed overnight in a metal water bath ligator. The next day, the ligation product was transformed into DH5α competent cells using a heat shock transformation method. 200 μL of the product was plated onto a solid medium containing ampicillin and incubated at 37°C for 12–16 h. Single colonies were picked in a sterile laminar flow hood and cultured overnight in a shaker at 37°C and 120 rpm. The vials were then removed from the shaker, and plasmids were extracted according to the plasmid extraction kit.

[0016] Example 2: Expression and preparation of fibroblast growth factor 21-TAT / R11 / Fc

[0017] (1) Induced expression

[0018] Three recombinant plasmids containing the correct sequences were transformed into the expression strain Rossetta(DE3) (Shanghai Baoman Biotechnology Co., Ltd., catalog number: 130558-10). Single colonies after transformation were inoculated into 5 mL of LB medium containing ampicillin (100 μg / mL) and cultured at 37°C for 12 h. Then, they were inoculated 1:100 into 500 mL of LB medium containing ampicillin (100 μg / mL) and cultured at 37°C for 2 h. When A600 = 0.5, IPTG was added to a final concentration of 0.5 mmol / L for induction. After 4 h of induction, the bacterial cells were harvested, sonicated, and centrifuged. Whole cells, supernatant, and precipitate were analyzed by 15% SDS-PAGE electrophoresis. The results showed that SUMO-FGF-21-R11 and SUMO-FGF-21-Fc expressed the target protein in soluble form, while SUMO-FGF-21-TAT expressed the target protein in precipitate form.

[0019] (2) Protein preparation

[0020] Following the optimal conditions for fusion protein expression described above, the culture volume was expanded, and bacterial cells were collected after induction of expression. SUMO-FGF-21-R11 and SUMO-FGF-21-Fc were lysed, centrifuged, and the supernatant was collected. The supernatant was first purified using DEAE Sepharose FF, eluted with 25 mmol / L Tris-HCl, 0.25 mol / L NaCl, pH 8.0. The eluted peak fraction was collected and subjected to Ni·NTA resin affinity chromatography. The Ni·NTA resin was equilibrated with buffer 50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, and 10 mmol / L imidazole. The lysate was then transferred to the equilibrated resin column. After loading, the column was washed with buffer 50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, and 50 mmol / L imidazole. Finally, the target protein was eluted with 50 mmol / L Tris-HCl buffer (pH 8.0), 0.5 mol / L NaCl, and 500 mmol / L imidazole. Each elution peak was collected and analyzed by SDS-PAGE. The results showed that the purified FGF-21-R11 and FGF-21-Fc protein bands were consistent with the expected sizes.

[0021] SUMO-FGF-21-TAT was lysed and denatured using a denaturing buffer (8 mol / L Urea, 0.1 mol / L Na2HPO4, 0.01 mol / L Tris, pH 7.5) to open the protein's spatial structure, allowing it to dissolve in the denaturing buffer. The dissolved protein was then slowly added dropwise to 10 times its volume of refolding buffer (10 mmol / L Tris-buffer, pH 8.0) to achieve a final concentration of 1.0 mg / mL. The protein was then dialyzed three times with PBS (pH 7.4) at 4°C for 8 hours each time. The dialyzed protein was concentrated using PEG8000, followed by affinity chromatography (using the same procedure as above). The purified protein was then analyzed by SDS-PAGE. The results showed that the size of the purified FGF-21-TAT protein band was consistent with the expected size.

[0022] 3. Activity detection of FGF21-TAT, FGF21-R11, and FGF21-Fc proteins

[0023] (1) Protein cell activity detection

[0024] S9 cells were starved for 12 hours. The purified FGF-21-TAT, FGF-21-R11, and FGF-21-Fc proteins were diluted with cell culture medium to a final concentration of 10, 100, and 1000 nmol / L. 1 ml of the diluted solution was added to each well of the differentiated adipocytes. At least three replicates were performed for each concentration.

[0025] Glucose concentration detection: After incubating cells for 24 hours, 2 μL of the supernatant culture medium was added to 200 μL of glucose detection solution to determine the glucose content. Each concentration was repeated at least 3 times. After reacting at 37℃ for 5–10 minutes, the OD value was measured at a wavelength of 500 nm. The glucose consumption rate of cells was calculated as follows, and the experimental results were analyzed statistically.

[0026] The formula for calculating the residual glucose concentration in the culture medium is:

[0027] Glucose concentration (mmol / L) = OD 样品 / OD 标准 ×5.55mmol / L

[0028] The formula for calculating the rate of glucose consumption by cells is:

[0029] Cellular glucose consumption rate (%) = [(C 空白葡萄糖 -C 给药葡萄糖 ) / C 空白葡萄糖 ×100%.

[0030] After treating S9 cells with different concentrations of FGF-21 protein for 24 hours, the glucose content in the culture medium was detected using a miniaturized GOD-POD glucose assay kit. Statistical analysis showed that the three FGF-21 proteins significantly promoted glucose uptake in the cells, indicating that the three FGF-21 proteins have good biological activity.

[0031] (2) In vivo activity assay of FGF-21 protein

[0032] C57BL / 6J mice were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. The mice were randomly divided into four groups of five mice each. Twenty C57BL / 6 mice were injected intraperitoneally with 80 mg / kg streptozotocin (STZ) to establish a diabetic model. Successfully modeled diabetic mice were randomly divided into four groups: model group, FGF-21-TAT group, FGF-21-R11 group, and FGF-21-Fc group, with five mice in each group. A blank control group (n=5) was also established. Under conditions of no fasting and no water restriction, mice in the FGF-21-TAT, FGF-21-R11, and FGF-21-Fc groups were injected intraperitoneally with one of the three FGF-21 proteins at a dose of 1 mg / kg daily for 7 consecutive days. Mice in the normal and model groups were injected intraperitoneally with saline daily for 7 consecutive days. Injections were administered at 8:00 AM daily, and blood glucose levels were measured before administration.

[0033] The results showed that after 7 days of treatment with the three FGF-21 proteins, the blood glucose levels in mice were significantly lower than those in the model group, while the blood glucose levels in the model group continued to rise, reaching 27.3±0.25 mmol / L at the end of the experiment. After treatment with FGF-21 proteins, the blood glucose levels in mice continued to decrease. The blood glucose levels in the FGF-21-TAT group decreased from 20.2±0.37 mmol / L to 13.6±0.16 mmol / L, the blood glucose levels in the FGF-21-R11 group decreased from 19.8±0.31 mmol / L to 13.8±0.15 mmol / L, and the blood glucose levels in the FGF-21-Fc group decreased from 20.9±0.26 mmol / L to 13.6±0.24 mmol / L. Compared with the model group, the blood glucose levels were significantly lower (p<0.01), indicating that the FGF-21-TAT, FGF-21-R11, and FGF-21-Fc proteins have good biological activity and can be used for subsequent experiments.

[0034] Example 3: Study on the effect of FGF21-TAT / R11 / Fc on improving atopic dermatitis

[0035] 1. Establishment and treatment of atopic dermatitis mouse model

[0036] Forty-eight 6-8 week old SPF-grade female Balb / c mice were randomly divided into six groups of eight each: normal control group, model group, FGF-21-TAT, FGF-21-R11, FGF-21-Fc, and positive control group (tacrolimus). A matrix solution was prepared using acetone and olive oil (3:1), and the chemical hapten 1-chloro-2,4-dinitratenzene (DNCB) was diluted to 1% DNCB and 0.5% DNCB. Twenty-four hours before modeling, hair was removed from the backs of the mice using a depilatory cream. During the sensitization phase, in week 1, every two days, 150 μL of a 1% DNCB solution prepared with a base solution (acetone / olive oil = 3:1) was applied to multiple spots on the shaved backs of mice using a micropipette, and then spread evenly with a sterile cotton swab. Additionally, 50 μL of the 1% DNCB solution was applied to the inner and outer surfaces of the right ear of the experimental mice, and 50 μL of the DNCB-free base solution was applied to the left ear. Mice in the normal control group had the base solution applied to their backs and right ears. In weeks 2-4, every two days, 150 μL of a 0.5% DNCB solution prepared with a base solution (acetone / olive oil = 3:1) was applied to multiple spots on the shaved backs of mice using a micropipette, and then spread evenly with a sterile cotton swab. Additionally, 50 μL of the 0.5% DNCB solution was applied to the inner and outer surfaces of the right ear of the experimental mice, and 50 μL of the DNCB-free base solution was applied to the left ear. Mice in the normal control group had the base solution applied to their backs and right ears. During weeks 2-4, mice were treated by intraperitoneal injection of FGF-21-TAT, FGF-21-R11, and FGF-21-Fc proteins (1 mg / kg) every two days. Mice in the model group were injected with an equal volume of PBS. Mice in the positive control group (Tacrolimus) were treated with 0.02 mg of tacrolimus ointment. The normal control group was not treated.

[0037] 2. Detection of relevant dermatitis indicators

[0038] During the modeling process, the skin lesions on the backs of mice were assessed before each sensitization. The severity of the skin lesions was assessed using four symptoms: edema; erythema / hemoptysis; epidermal peeling; scaling / dryness; and lichenification. Each clinical symptom was graded as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe), with half-point increments (0.5 points) between each symptom score. The scoring principle was: 0 points: no sign; 1 point: the sign exists but requires careful observation to see; 2 points: the sign is immediately visible; 3 points: the sign is very obvious. Three observers outside this experiment independently observed and combined the symptom scores for each mouse using the above scoring criteria. Before each sensitization, the weight of the mice was measured using a balance, and the thickness of the ears was measured using a micrometer, including the thickness of the left ear (non-sensitized) and the right ear (sensitized). The difference between each measurement (difference = right ear thickness - left ear thickness) was taken to assess the degree of ear swelling, and the results were compared through statistical analysis. Dermatitis scoring results showed that 7 days after sensitization, erythema, edema, scarring, and lichenification lesions appeared on the back skin of mice in all groups, consistent with the symptoms of atopic dermatitis. Compared with the model group, the dermatitis score of the FGF-21-R11 group was significantly lower on day 14 (p<0.05), the dermatitis score of the FGF-21-Fc group was extremely significantly lower on day 14 (p<0.01), and the dermatitis score of the FGF-21-Fc group was significantly lower on day 14 compared with the FGF-21-TAT group (p<0.05). Compared with the Tacrolimus group, there was no significant difference in dermatitis scores on day 28 during treatment among the FGF-21-TAT group, FGF-21-R11 group, and FGF-21-Fc group (p>0.05). Ear thickness measurements showed that on days 24 and 28, the degree of ear swelling in mice in the FGF-21-TAT, FGF-21-R11, and FGF-21-Fc groups was significantly reduced (p<0.01). Compared with the Tacrolimus group, the degree of ear swelling in mice in the FGF-21-R11 group was significantly reduced on day 20 (p<0.05). This indicates that the three proteins were more effective than Tacrolimus in reducing ear swelling.

[0039] 3. Histopathological examination

[0040] After the experiment, blood was collected from mice in EP tubes, incubated at 4°C for 30 minutes, and centrifuged at 3500 rpm for 15 minutes at 4°C. Serum was then pipetted into EP tubes and stored at -80°C. A portion of skin from the mouse's back and ear tissue was wrapped in aluminum foil and stored at -80°C; another portion was placed in 4% paraformaldehyde (prepared with PBS), sealed, and fixed at room temperature for at least 48 hours. The fixed tissue was then trimmed to a thickness of 2-3 mm and an area of ​​1 × 1 cm. 2Tissue blocks were collected and placed in an embedding cassette. After rinsing with tap water for at least 12 hours to remove paraformaldehyde, the tissues were dehydrated, cleared, impregnated with paraffin, and embedded to prepare tissue sections. The tissue sections were then subjected to H&E staining and immunohistochemistry.

[0041] 4. Detection of relevant cytokines

[0042] The protein expression levels of IL-1β, IL-4, IL-13, IL-17A, IFN-γ, TNF-α and total IgE in mouse serum were detected using an ELISA kit.

Claims

1. The use of the canine fibroblast growth factor 21 fusion protein shown in Sequence 1 or its encoding gene or an expression cassette containing the encoding gene, recombinant vector, transgenic cell line or recombinant bacteria in the preparation of a product, wherein the function of the product is as follows (I): (I) Treatment of canine atopic dermatitis.

Images