CN116675780A - Preparation of canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis - Google Patents

Preparation of canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis Download PDF

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Publication number
CN116675780A
CN116675780A CN202310648161.0A CN202310648161A CN116675780A CN 116675780 A CN116675780 A CN 116675780A CN 202310648161 A CN202310648161 A CN 202310648161A CN 116675780 A CN116675780 A CN 116675780A
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fgf
fusion protein
factor21
fibroblast growth
atopic dermatitis
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任桂萍
尹杰超
郭笑辰
孙文影
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Northeast Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention discloses a preparation method of a canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis. The invention provides a canine fibroblast growth factor21 (FGF 21) fusion protein, which consists of canine fibroblast growth factor21 (FGF 21) and a fusion tag, wherein the fusion tag is TAT or R11 or Fc; the three FGF-21 fusion proteins obtained by the invention can effectively improve the symptom of the atopic dermatitis of mice and reduce the expression level of related inflammatory factors; the three FGF-21 recombinant fusion proteins provided by the invention can be used as potential drugs for treating canine atopic dermatitis.

Description

Preparation of canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis
Technical Field
The invention relates to preparation of a canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis, belonging to the field of biotechnology.
Background
With the rising of the feeding amount of the pet dogs, the canine diseases are also more and more widely focused by people, and the increasingly developed industries and the aggravated pollution of living environment lead to the high incidence of allergic diseases of the dogs. Allergic diseases in dogs include atopic dermatitis, gastrointestinal inflammation and allergic reactions. Among them, canine Atopic Dermatitis (CAD) is widely prevalent worldwide, with up to 10% of dogs affected. Canine Atopic Dermatitis (AD) is a common hereditary inflammatory and pruritic allergic skin disease. Atopic dermatitis is seen in a very similar clinical presentation in different species, and unlike humans where respiratory symptoms may occur, dogs often remain in the cutaneous stage of the disease. However, many clinical and etiologic features are highly similar and CAD is often described as a model of human Atopic Dermatitis (AD). In clinical treatment, glucocorticoids, cyclosporine, oxatinib and the like are commonly used, and the medicines have great side effects after long-term use, and allergen immunotherapy with good treatment effect needs to detect the allergen of dogs and obtain allergen extracts, so that the method is complex in operation, poor in universality and difficult to use in a large amount. Therefore, there is a great clinical need for the development of anti-inflammatory drugs suitable for dogs, which have long-acting effects and less side effects, and which can treat atopic dermatitis.
Fibroblast growth factor21 (Fibroblast growth factor, FGF-21) is a member of the fibroblast growth factor family, which is of great interest because of its close association with glycolipid metabolism. In recent years, there have been studies showing that FGF-21 has anti-inflammatory effects in addition to the effects of combating obesity, improving insulin resistance and various metabolic diseases. FGF-21 can reduce inflammatory factors such as IL-1 beta, IL-6, TNF-alpha and the like in mouse serum, and inhibit the transcription and translation of NF-kappa B p 65.
FGF-21 has not been reported to have an ameliorating effect on canine atopic dermatitis. Therefore, the invention adopts a DNCB-induced atopic dermatitis mouse model to explore the pharmacodynamic effect of FGF-21 on canine atopic dermatitis. The laboratory finds that FGF-21-TAT, FGF-21-R11 and FGF-21-Fc can obviously reduce dermatitis score, improve dermatitis symptoms and effectively inhibit the expression of related inflammatory factors in animal models, and shows that the laboratory has the potential of treating atopic dermatitis diseases.
Disclosure of Invention
The invention aims to provide the preparation of a canine fibroblast growth factor21 fusion protein and the application thereof in treating atopic dermatitis. The fusion protein consists of canine fibroblast growth factor21 (FGF 21) and a fusion tag, wherein the fusion tag is TAT or R11 or Fc, and the fusion protein has important application in treating canine atopic dermatitis diseases.
Drawings
FIG. 1 identification of three FGF-21 fusion proteins after purification
FIG. 2 in vitro assay of three fusion protein Activity
FIG. 3 in vivo detection of three fusion protein Activity
FIG. 4 results of back H & E staining of AD mice
FIG. 5 results of H & E staining of ear of AD mice
FIG. 6 results of immunohistochemical analysis of TSLP protein in AD mouse tissue
FIG. 7 serum-related inflammatory factor ELISA results
Detailed Description
Example 1 acquisition of FGF21-TAT, FGF21-R11, FGF21-Fc Gene
TAT, R11 and Fc fragment of IgG are designed at 3' end of FGF21 gene, FGF-21 gene with cleavage sites BsaI and BamHI are designed at two ends, and three recombinant plasmids are synthesized by Shanghai Ind.
The newly synthesized FGF-21-TAT, FGF-21-R11 and FGF-21-Fc plasmids of Shanghai engineering and SUMO vectors stored in a laboratory were subjected to double digestion (100. Mu.L system) by restriction enzymes BsaI and BamHI, and the gel recovery products were ligated. After the reagents in the connection system are mixed uniformly, the connection is carried out in a metal water bath connector overnight. The following day, the ligation products were transferred into DH 5. Alpha. Competent cells by heat shock transformation, 200. Mu.L was plated on solid medium containing ampicillin resistance, incubated for 12-16h in a 37℃incubator, single colonies were picked up in 20mL of ampicillin resistant liquid medium in a sterile ultra-clean bench, and incubated overnight at 37℃in a shaker at 120 rpm. The vials were removed from the shaker and plasmids were extracted according to the plasmid extraction kit.
EXAMPLE 2 expression and preparation of fibroblast growth factor 21-TAT/R11/Fc
(1) Induction of expression
Three recombinant plasmids containing the correct sequences were transformed into the expression strain Rossetta (DE 3) (Shanghai Bowman Biotechnology Co., ltd., catalog No. 130558-10). The transformed single colonies were inoculated into 5mL of LB medium containing ampicillin (100. Mu.g/mL), cultured at 37℃for 12 hours, inoculated into 500mL of LB medium containing ampicillin (100. Mu.g/mL) at 1:100, cultured at 37℃for 2 hours, induced by adding IPTG to a final concentration of 0.5mmol/L when A600=0.5, harvested after 4 hours, sonicated, centrifuged, and 15% SDS-PAGE analysis was performed on whole bacteria, supernatant and pellet, respectively. As a result, it was found that SUMO-FGF-21-R11 and SUMO-FGF-21-Fc were expressed as a target protein in a soluble form, and SUMO-FGF-21-TAT was expressed as a target protein in a precipitated form.
(2) Protein preparation
According to the optimal conditions for the expression of the fusion protein, the culture volume is enlarged, bacterial cells are collected after induced expression, SUMO-FGF-21-R11 and SUMO-FGF-21-Fc are crushed, supernatant is centrifugally collected, DEAE Sepharose FF is purified, and 25mmol/L Tris-HCl,0.25mol/L NaCl and pH8.0 are used for elution. Collecting the adsorption peak component, performing Ni.NTA resin affinity chromatography, balancing Ni.NTA resin with 50m mol/L Tris-HCl buffer solution, pH8.0,0.5 mol/LNaCl and 10m mol/L imidazole, draining the lysate to a well-balanced resin column, and washing with 50m mol/L Tris-HCl buffer solution, pH8.0,0.5mol/L NaCl and 50m mol/L imidazole after loading. Finally, 50m mol/L Tris-HCl buffer, pH8.0,0.5mol/L NaCl,500m mol/L imidazole was used to elute the target protein. The elution peaks were collected and examined by SDS-PAGE. The results showed that the band sizes of the purified FGF-21-R11, FGF-21-Fc proteins were consistent with the expected sizes.
SUMO-FGF-21-TAT is subjected to bacterial disruption and denaturation, the protein space structure is opened through denaturation of denaturation liquid (8 mol/L Urea,0.1mol/LNa2HPO4,0.01mol/L Tris, pH 7.5), so that the protein is dissolved in the denaturation liquid, the dissolved protein is slowly added dropwise into 10-time volume renaturation liquid (10 mmol/L Tris-buffer, pH 8.0) so that the final concentration reaches 1.0mg/mL, and the solution is dialyzed 3 times at 4 ℃ through PBS (pH 7.4) for 8 hours each time. The dialyzed proteins were concentrated using PEG8000, and the concentrated proteins were subjected to affinity chromatography, as described above, and the purified proteins were 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.
3. Activity detection of FGF21-TAT, FGF21-R11, FGF21-Fc protein
(1) Protein cell level activity assay
S9 cells were starved for 12h, purified FGF-21-TAT, FGF-21-R11, FGF-21-Fc protein was diluted with cell culture medium to a final concentration of 10, 100, 1000nmol/L, and dilutions of different concentrations were added to differentiated mature adipocytes at 1ml per well. At least 3 replicate wells were provided for each concentration.
Detection of glucose concentration: after incubating the cells for 24 hours in the above treatments, 2. Mu.L of the supernatant medium was placed in 200. Mu.L of a glucose test solution to determine the glucose content. Each concentration was repeated at least 3 times, and after 5 to 10 minutes of reaction at 37℃the OD value was measured at a wavelength of 500 nm. The glucose consumption rate of the cells was calculated as follows, and the experimental results were analyzed using statistics.
And calculating the concentration of the residual glucose in the culture solution, wherein the formula is as follows:
glucose concentration (mmol/L) =od Sample of /OD Standard of ×5.55mmol/L
The consumption rate of the cells on glucose is calculated, and the formula is as follows:
cell glucose consumption rate (%) = [ (C) Blank glucose -C Administration of glucose )/C Blank glucose ]×100％。
After S9 cells are treated for 24 hours by FGF-21 proteins with different concentrations, the glucose content in the culture medium is detected by a glucose detection kit of a micro-GOD-POD method, and the statistical analysis result shows that three FGF-21 proteins can obviously promote the glucose absorption level of the cells, thereby indicating that the three FGF-21 proteins have better biological activity.
(2) In vivo Activity assay of FGF-21 protein
C57BL/6J mice were purchased from Gibbs laboratory animal technology Co., ltd, and the laboratory mice were randomly divided into 4 groups of 5 mice each. 20C 57BL/6 mice are intraperitoneally injected with 80mg/kg Streptozotocin (STZ), a diabetes model is established, the successfully modeled diabetes model mice are selected and randomly divided into a model group, an FGF-21-TAT group, an FGF-21-R11 group and an FGF-21-Fc group, 5 mice in each group are used, and a blank control group (n=5) is established. Mice in the FGF-21-TAT group, the FGF-21-R11 group and the FGF-21-Fc group are subjected to intraperitoneal injection of three FGF-21 proteins at a dose of 1mg/kg every day under the conditions of no fasting and no water, and the continuous injection is carried out for 7 days; normal and model mice were injected daily with physiological saline for 7 days. Early 8 a day: 00 injections and blood glucose measurements prior to administration
The results show that after 7 days of treatment by three FGF-21 proteins, the blood sugar of the mice is obviously reduced compared with that of a model group, the blood sugar level of the model group is continuously increased, and the blood sugar reaches 27.3+/-0.25 mmol/L after the experiment is finished; the blood sugar of the mice continuously decreases after being treated by the FGF-21 protein, the blood sugar of the mice in the FGF-21-TAT group decreases from 20.2+/-0.37 mmol/L to 13.6+/-0.16 mmol/L, the blood sugar of the mice in the FGF-21-R11 group decreases from 19.8+/-0.31 mmol/L to 13.8+/-0.15 mmol/L, and the blood sugar of the mice in the FGF-21-Fc group decreases from 20.9+/-0.26 mmol/L to 13.6+/-0.24 mmol/L, compared with the model group, the blood sugar is obviously decreased (p < 0.01), which indicates that the biological activity of the FGF-21-TAT, FGF-21-R11 and FGF-21-Fc proteins is good, and the mice can be used for subsequent experiments.
EXAMPLE 3 investigation of the Effect of FGF21-TAT/R11/Fc on improving atopic dermatitis
1. Atopic dermatitis mouse model establishment and treatment
48 female Balb/c mice of SPF class 6-8 weeks old were randomly divided into 6 groups of 8: normal control, model, FGF-21-TAT, FGF-21-R11, FGF-21-Fc, and positive drug (Tacrolimus Tacrolimus). Mixing with acetone and olive oil (3:1) to obtain matrix solution, and diluting chemical hapten 1-chloro-2, 4-dinitrobenzene (1-chloro-2, 4-dinitratenzene, DNCB) into 1% DNCB and 0.5% DNCB. The back hair of the mice was removed 24h before molding using depilatory cream. In the mould sensitization stage, at week 1, sucking 150 mu L of 1% DNCB solution prepared by matrix liquid (acetone/olive oil=3:1) by using a micropipette every 2 days, multi-point coating on the back dehairing place of a mouse, uniformly coating by using a sterile cotton swab, point-coating 50 mu L of 1% DNCB solution on the inner and outer surfaces of the right ear of the experimental mouse, and coating 50 mu L of DNCB-free matrix liquid on the left ear; the matrix liquid is smeared on the back and the right ear of the normal mice. Sucking 150 μl of 0.5% DNCB solution prepared from matrix solution (acetone/olive oil=3:1) by using a micropipette every 2 days in week 2-4, applying to the back dehairing place of the mice at multiple points, uniformly applying with a sterile cotton swab, and applying 50 μl of 0.5% DNCB solution to the inner and outer surfaces of the right ear of the experimental mice at points, and applying 50 μl of DNCB-free matrix solution to the left ear; the matrix liquid is smeared on the back and the right ear of the normal mice. On weeks 2-4, mice were treated, groups of FGF-21-TAT, FGF-21-R11, FGF-21-Fc were given intraperitoneal injections of FGF-21-TAT, FGF-21-R11, FGF-21-Fc protein (1 mg/kg) each every two days, model groups were given equal amounts of PBS, positive group (Tacrolimus) mice were coated with 0.02mg Tacrolimus ointment, and the normal groups were not treated.
2. Related dermatitis index detection
During the molding process, mice were evaluated for back skin lesions prior to each sensitization. Skin lesion severity was estimated by 4 symptoms, edema; erythema/hemorrhage; peeling off the epidermis; crumb/dry; lichenification, each clinical symptom was scored in half, i.e., 0.5 points, on a scale of 0 (none), 1 (mild), 2 (moderate) and 3 (severe) between the scores of each symptom. The scoring principle is 0 score, namely that no sign appears; 1 minute, the sign exists, but the sign can be seen through careful observation; 2 minutes, the sign can be seen immediately; 3 minutes, the sign is very obvious. Three observers outside the experiment independently observe and aggregate the symptom scores for each mouse with the above scores as criteria. Before each sensitization, the body weight of the mice was measured by a balance and the ear thickness of the mice was measured by a micrometer thickness gauge, including the thickness of the left ear (non-sensitization) and the thickness of the right ear (sensitization), the difference value (difference value=thickness of the right ear-thickness of the left ear) was taken after each measurement, and the ear swelling degree of the mice was evaluated and compared by statistical analysis. Dermatitis scoring results show that after sensitization for 7 days, the back skin of each group of mice has erythema, edema, scars and lichen lesions, which accords with the symptoms of atopic dermatitis. The dermatitis score was significantly reduced on day 14 for the FGF-21-R11 group (p < 0.05), compared to the model group, the dermatitis score was significantly reduced on day 14 for the FGF-21-Fc group (p < 0.01), compared to the FGF-21-TAT group, the dermatitis score was significantly reduced on day 14 (p < 0.05); there was no significant difference in dermatitis scores (p > 0.05) on day 28 during treatment of FGF-21-TAT, FGF-21-R11, and FGF-21-Fc groups compared to Tacrolimus. Ear thickness measurements showed that the ear swelling levels of mice in groups FGF-21-TAT, FGF-21-R11, FGF-21-Fc were significantly reduced (p < 0.01) on days 24 and 28, and that the ear swelling levels of mice in group FGF-21-R11 were significantly reduced (p < 0.05) on day 20 compared to Tacrolimus. The three proteins are shown to have better therapeutic effect on relieving ear swelling than Tacrolimus.
3. Histopathological detection
After the experiment, the mice were bled in EP tubes and allowed to stand at 4℃for 30 minutes and centrifuged at 3500r/min at 4℃for 15 minutes. The pipette aspirates serum into the EP tube and stores at-80 ℃. Wrapping a part of back skin and ear tissue of a mouse with tinfoil, and storing at-80deg.C; the other part is put into 4% paraformaldehyde (PBS for preparation), and the mixture is sealed and fixed for more than 48 hours at room temperature. Trimming the fixed tissue to a thickness of 2-3mm and an area of 1X 1cm 2 The tissue blocks are placed in an embedding box, are washed for more than 12 hours by tap water, and after paraformaldehyde is removed, the tissue is dehydrated, transparent, waxed and embedded to prepare tissue slices. Tissue sections were subjected to H&E staining and immunohistochemistry.
4. Related cytokine detection
The protein expression levels of IL-1 beta, IL-4, IL-13, IL-17A, IFN-gamma, TNF-alpha and total IgE in the serum of mice were detected by ELISA kit.

Claims (5)

1. A canine fibroblast growth factor21 (FGF 21) fusion protein, wherein the fusion protein consists of canine fibroblast growth factor21 (FGF 21) and a fusion tag, and the fusion tag is TAT or R11 or Fc;
the fusion protein is (a) or (b) or (c): (a) a protein consisting of amino acid residues 1 to 193 from the N terminal of a sequence 1 in a sequence table, (b) a protein consisting of amino acid residues 1 to 193 from the N terminal of a sequence 2 in the sequence table, and (c) a protein consisting of amino acid residues 1 to 416 from the N terminal of a sequence 3 in the sequence table.
2. A gene encoding the fusion protein of claim 1.
3. The gene of claim 2, wherein:
in the gene, the DNA molecule encoding the canine fibroblast growth factor21 (FGF 21) fusion protein is (1) or (2) or (3) as follows: (1) a DNA molecule shown in nucleotide numbers 1-582 from the 5' end of the sequence 4 in the sequence table, (2) a DNA molecule shown in nucleotide numbers 1-582 from the 5' end of the sequence 5 in the sequence table, and (3) a DNA molecule shown in nucleotide numbers 1-1251 from the 5' end of the sequence 6 in the sequence table.
4. An expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising a gene according to any one of claims 2 to 3.
5. Use of the fusion protein of claim 1, the gene of claim 2 or 3, or the expression cassette, recombinant vector, transgenic cell line or recombinant bacterium of claim 4 in the preparation of a product; the functions of the product are as follows (I): and (I) treating canine atopic dermatitis diseases.
CN202310648161.0A 2023-06-02 2023-06-02 Preparation of canine fibroblast growth factor21 fusion protein and application thereof in treating atopic dermatitis Pending CN116675780A (en)

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