Preparation of canine fibroblast growth factor 21 fusion protein and its use in treating atopic dermatitis

Abstract

The application discloses preparation of a canine fibroblast growth factor 21 fusion protein and application thereof in treating atopic dermatitis. The application provides a caninized fibroblast growth factor 21 (FGF21) fusion protein, which is composed of a caninized fibroblast growth factor 21 (FGF21) and a fusion tag, and the fusion tag is TAT or R11 or Fc. The three kinds of FGF-21 fusion proteins obtained by the application can effectively improve the symptoms of atopic dermatitis of mice and reduce the expression level of related inflammatory factors. The three kinds of FGF-21 recombinant fusion proteins provided by the application can be used as potential drugs for treating atopic dermatitis of dogs.

Description

TECHNICAL FIELD

[0001] The present application 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

[0002] With the increase of pet dogs, canine diseases have also been widely concerned by people, and the development of industry and the intensification of environmental pollution have led to the high incidence of canine allergic diseases. Canine allergic diseases include atopic dermatitis, gastrointestinal inflammation and allergic reactions. Among them, canine atopic dermatitis (CAD) is widespread throughout the world, affecting up to 10% of dogs. Canine atopic dermatitis (AD) is a common genetic inflammatory and pruritic allergic skin disease. Atopic dermatitis in different species can be observed to have very similar clinical manifestations, unlike humans who can have respiratory symptoms, dogs usually stay in the skin stage of the disease. However, the high similarity of many clinical and pathogenic characteristics also makes CAD often described as a model of human atopic dermatitis (AD). Clinically, glucocorticoids, cyclosporine, oclacitinib and other drugs are often used to treat CAD, and long-term use of these drugs can have significant side effects. Allergen immunotherapy, which has better therapeutic effect, needs to detect the allergen of the dog and obtain the allergen extract, which is complicated and difficult to use in large quantities. Therefore, there is an urgent need in the clinic to develop anti-inflammatory drugs suitable for dogs, with long-term effect and fewer side effects, which can also treat atopic dermatitis.

[0003] Fibroblast growth factor 21 (FGF-21) is a member of the fibroblast growth factor family, which is closely related to glycolipid metabolism and has been widely concerned by people. In recent years, studies have shown that FGF-21 has anti-inflammatory effects in addition to resisting obesity, improving insulin resistance and various metabolic diseases. 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, there is no literature report that FGF-21 has an improvement effect on canine atopic dermatitis. Therefore, the present application uses a DNCB-induced atopic dermatitis mouse model to explore the pharmacodynamic effect of FGF-21 on canine atopic dermatitis. In animal models, our laboratory found that FGF-21-TAT, FGF-21-R11 and FGF-21-Fc can significantly reduce the dermatitis score, improve the symptoms of dermatitis and effectively inhibit the expression of related inflammatory factors, showing their potential to treat atopic dermatitis. SUMMARY

[0005] The application aims to provide a canine fibroblast growth factor 21 fusion protein preparation and its use in treating atopic dermatitis. The fusion protein is composed of a canine-derived fibroblast growth factor 21 (FGF21), a fusion tag, and a TAT or R11 or Fc fusion tag, which has important application in treating canine atopic dermatitis diseases. BRIEF DESCRIPTION OF DRAWINGS

[0006] Figure 1 Post-purification identification of three FGF-21 fusion proteins

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

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

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

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

[0011] Figure 6 TSLP protein immunohistochemical results in the tissues of AD mice

[0012] Figure 7 ELISA results of serum-related inflammatory factors DETAILED DESCRIPTION

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

[0014] The TAT, R11, and IgG Fc fragment were designed at the 3' end of the FGF21 gene, and the enzyme digestion sites Bsa I and BamH I were designed at both ends of the FGF-21 gene. The three recombinant plasmids were synthesized by Shanghai Biosciences.

[0015] The newly synthesized FGF-21-TAT, FGF-21-R11, and FGF-21-Fc plasmids of Shanghai Biosciences were double-digested with restriction enzymes Bsa I and BamH I (100 μL system), and the gel recovery product was ligated. After mixing the reagents in the ligation system, overnight ligation was performed in a metal water bath ligation instrument. The next day, the ligation product was transformed into DH5α competent cells using the heat shock transformation method, 200 μL was taken and plated on solid medium containing ampicillin resistance, and incubated in a 37°C incubator for 12-16 h. Single colonies were picked on a sterile clean bench in 20 mL ampicillin-resistant liquid medium, and incubated at 37°C on a shaker at 120 rpm overnight. The vial was removed from the shaker, and the plasmid was 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] The three recombinant plasmids containing the correct sequence were transformed into the expression strain Rossetta (DE3) (Shanghai Boman Biotechnology Co., Ltd., Catalog No: 130558-10). The transformed single colonies were inoculated into 5 mL of LB medium containing ampicillin (100 μg / mL) and incubated at 37°C for 12 h. The culture was inoculated into 500 mL of LB medium containing ampicillin (100 μg / mL) at a ratio of 1:100 and incubated at 37°C for 2 h. When the A600 was 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 and subjected to ultrasonic disruption followed by centrifugation. The whole bacteria, supernatant and precipitate were subjected to 15% SDS-PAGE electrophoresis analysis, respectively. The results showed that the target proteins of SUMO-FGF-21-R11 and SUMO-FGF-21-Fc were expressed in a soluble form, and the target protein of SUMO-FGF-21-TAT was expressed in a precipitated form.

[0019] (2) Protein preparation

[0020] According to the optimal conditions for expression of the fusion protein described above, the culture volume was expanded, and the bacterial cells were collected after induction. The supernatant of SUMO-FGF-21-R11 and SUMO-FGF-21-Fc was collected after disruption and centrifugation. DEAE Sepharose FF purification was performed, and elution was performed with 25 mmol / L Tris-HCl, 0.25 mol / L NaCl, pH 8.0. The fractions of the adsorption and desorption peaks were collected, and Ni-NTA resin affinity chromatography was performed. The resin was equilibrated with buffer 50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, 10 mmol / L imidazole, and the lysate was introduced into 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, 50 mmol / L imidazole. Finally, the target protein was eluted with buffer 50 mmol / L Tris-HCl, pH 8.0, 0.5 mol / L NaCl, 500 mmol / L imidazole. The elution peaks were collected and subjected to SDS-PAGE detection. The results showed that the sizes of the FGF-21-R11 and FGF-21-Fc protein bands after purification were consistent with the expected sizes.

[0021] SUMO-FGF-21-TAT was denatured after breaking the bacteria, and the protein spatial structure was opened by denaturation with a denaturation solution (8 mol / L urea, 0.1 mol / L Na2HPO4, 0.01 mol / L Tris, pH 7.5) to dissolve the protein in the denaturation solution. The dissolved protein was slowly added dropwise into 10 times the volume of a refolding solution (10 mmol / L Tris-buffer, pH 8.0) to achieve a final concentration of 1.0 mg / mL, and was dialyzed against PBS (pH 7.4) at 4°C for 3 times, each time for 8 hours. The dialyzed protein was concentrated using PEG8000, and the concentrated protein was subjected to affinity chromatography according to the above steps. The purified protein was subjected to SDS-PAGE analysis. 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 level activity detection

[0024] S9 cells were starved for 12 h, and 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. Different concentrations of the diluents were added to the differentiated mature adipocytes at a volume of 1 ml per well. At least 3 replicate wells were set for each concentration.

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

[0026] The residual glucose concentration in the culture solution was calculated according to the formula:

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

[0028] The glucose consumption rate of the cells was calculated according to the formula:

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

[0030] After S9 cells were treated with different concentrations of FGF-21 protein for 24 h, the glucose content in the culture medium was detected by micro-GOD-POD method glucose detection kit. Statistical analysis showed that the three kinds of FGF-21 protein could significantly promote the glucose absorption level of the cells, indicating that the three kinds of FGF-21 protein had good biological activity.

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

[0032] C57BL / 6J mice were purchased from Changchun Yis Experimental Animal Technology Co., Ltd. The experimental mice were randomly divided into 4 groups, 5 in each group. Eighty mg / kg streptozotocin (STZ) was injected intraperitoneally into 20 C57BL / 6 mice to establish a diabetic model. The successful diabetic model mice were randomly divided into a model group, an FGF-21-TAT group, an FGF-21-R11 group, and an FGF-21-Fc group, 5 in each group, and a blank control group (n=5) was set up. Under the conditions of not fasting and not water restriction, the FGF-21-TAT group, the FGF-21-R11 group, and the FGF-21-Fc group of mice were injected intraperitoneally with 1 mg / kg of the three kinds of FGF-21 protein every day, for 7 consecutive days; the normal group and the model group of mice were injected intraperitoneally with normal saline every day, for 7 consecutive days. Injection was performed at 8:00 every day, and blood glucose was measured before administration

[0033] The results showed that after treatment with the three kinds of FGF-21 protein for 7 days, the blood glucose of the mice was significantly decreased compared with the model group. The blood glucose level of the model group continued to rise, and reached 27.3±0.25 mmol / L at the end of the experiment. After treatment with the FGF-21 protein, the blood glucose of the mice continued to decrease. The blood glucose of the FGF-21-TAT group of mice decreased from 20.2±0.37 mmol / L to 13.6±0.16 mmol / L, the blood glucose of the FGF-21-R11 group of mice decreased from 19.8±0.31 mmol / L to 13.8±0.15 mmol / L, and the blood glucose of the FGF-21-Fc group of mice decreased from 20.9±0.26 mmol / L to 13.6±0.24 mmol / L. Compared with the model group, the blood glucose was significantly reduced (p<0.01), indicating that the FGF-21-TAT, FGF-21-R11, and FGF-21-Fc proteins had good biological activity and could be used for subsequent experiments.

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

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

[0036] Forty-eight 6-8 week old SPF female Balb / c mice were randomly divided into 6 groups, 8 in each group: normal control group, model group, FGF-21-TAT, FGF-21-R11, FGF-21-Fc, positive drug group (Tacrolimus). The chemical hapten 1-chloro-2,4-dinitrobenzene (DNCB) was diluted into 1% DNCB and 0.5% DNCB using acetone olive oil (3:1) mixed into a substrate solution. 24 hours before modeling, the mouse back hair was removed using depilatory cream. In the modeling and sensitization stage, in the first week, 150 μL of 1% DNCB solution prepared by the substrate solution (acetone / olive oil=3:1) was taken using a micropipette every 2 days, and was applied to the mouse back hair removal site in multiple points, and was evenly applied with a sterile cotton swab, and 50 μL of 1% DNCB solution was applied to the inner and outer surfaces of the right ear of the experimental mouse, and 50 μL of DNCB substrate solution was applied to the left ear; the normal group of mice was applied with the substrate solution on the back and right ear. In the second to fourth weeks, 150 μL of 0.5% DNCB solution prepared by the substrate solution (acetone / olive oil=3:1) was taken using a micropipette every 2 days, and was applied to the mouse back hair removal site in multiple points, and was evenly applied with a sterile cotton swab, and 50 μL of 0.5% DNCB solution was applied to the inner and outer surfaces of the right ear of the experimental mouse, and 50 μL of DNCB substrate solution was applied to the left ear; the normal group of mice was applied with the substrate solution on the back and right ear. In the second to fourth weeks, the mice were treated, and the FGF-21-TAT, FGF-21-R11, FGF-21-Fc groups of mice were injected intraperitoneally with FGF-21-TAT, FGF-21-R11, FGF-21-Fc protein (1 mg / kg) every other day, the model group of mice was injected with an equal amount of PBS, and the positive drug group (Tacrolimus) of mice was applied with 0.02 mg of Tacrolimus ointment, and the normal group was not treated.

[0037] 2. Detection of related dermatitis indicators

[0038] During the molding process, the back skin of mice was evaluated before each sensitization. The severity of skin lesions was evaluated by estimating 4 symptoms: edema; erythema / bleeding; epidermal exfoliation; scaling / dryness; lichenification, each clinical symptom was scored according to 0 (none), 1 (mild), 2 (moderate) and 3 (severe) grades, and each symptom score could be half a grade, i.e. 0.5 points. The scoring principle: 0 points: no sign; 1 point: the sign exists, but it can be seen only by careful observation; 2 points: the sign can be seen immediately; 3 points: the sign is very obvious. Three observers independent of this experiment observed and added up the symptom scores of each mouse according to the above scoring criteria. Before each sensitization, the body weight of mice was measured by using a balance and the ear thickness of mice was measured by using a micrometer gauge, including the left ear (non-sensitized) and the right ear (sensitized) thickness, the difference value (difference value = right ear thickness - left ear thickness) was taken after each measurement to evaluate the degree of ear swelling of mice, and the comparison was made by statistical analysis. The dermatitis score results showed that after 7 days of sensitization, the back skin of mice in each group appeared erythema, edema, scar and lichenoid lesions, which was consistent with the symptoms of atopic dermatitis. Compared with the model group, the dermatitis score of FGF-21-R11 group on day 14 was significantly reduced (p<0.05), and the dermatitis score of FGF-21-Fc group on day 14 was extremely significantly reduced (p<0.01), and compared with FGF-21-TAT group, the dermatitis score of FGF-21-Fc group on day 14 was significantly reduced (p<0.05); compared with Tacrolimus group, there was no significant difference in dermatitis score of FGF-21-TAT group, FGF-21-R11 group and FGF-21-Fc group on day 28 during the treatment process (p>0.05). The ear thickness measurement results showed that on days 24 and 28, the ear swelling degree of FGF-21-TAT, FGF-21-R11 and FGF-21-Fc groups was extremely significantly reduced (p<0.01), and compared with Tacrolimus group, the ear swelling degree of FGF-21-R11 group on day 20 was significantly reduced (p<0.05). It showed that the therapeutic effect of three kinds of proteins on reducing ear swelling was better than that of Tacrolimus.

[0039] 3. Histopathological examination

[0040] After the end of the experiment, the blood of mice was taken into EP tube, and 4℃ for 30 minutes, 4℃ centrifugation 3500r / min, 15min. Pipette the serum in EP tube, and store at -80℃. Part of the mouse back skin and ear tissue was wrapped with tin paper and stored at -80℃; another part was put into 4% paraformaldehyde (PBS preparation), sealed and fixed at room temperature for 48h or more. The fixed tissue was trimmed to a thickness of 2-3mm and an area of 1x1cm 2The tissue block was placed in an embedding box and washed with tap water for more than 12 h. After removing the paraformaldehyde, the tissue was dehydrated, transparentized, immersed in wax, and embedded to prepare tissue sections. The tissue sections were subjected to H&E staining and immunohistochemistry.

[0041] 4. Detection of related cytokines

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

Claims

1. The use of a caninized fibroblast growth factor 21 fusion protein shown in SEQ ID NO: 2 or a gene encoding the same or an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacteria comprising the gene in the preparation of a product, wherein the function of the product is as follows (I): (I) treating canine atopic dermatitis disease.

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