Use of fgf-2 nanobodies in the preparation of a medicament for treating psoriasis

Abstract

The application discloses application of FGF-2 nanobody in preparation of a drug for treating psoriasis. The application takes IMQ-induced psoriasis mice as a model, finds that targeting FGF-2 can effectively inhibit abnormal proliferation expressed by the IMQ-induced psoriasis mice, and relieves the skin inflammation of the psoriasis mice. The application first expounds that taking FGF-2 as a target point can effectively relieve psoriasis, provides a brand-new treatment scheme for treatment of psoriasis, and also provides an important basis for subsequent treatment of psoriasis by taking growth factors as target points.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of FGF-2 nanobody in preparation of a medicine for treating psoriasis. BACKGROUND

[0002] Psoriasis is a common chronic inflammatory skin disease that affects 2-3% of the global population. Its characteristics are abnormal differentiation of keratinocytes and infiltration of inflammatory cells into the dermis and epidermis. The exact pathogenesis of psoriasis is not clear, but the main feature of psoriasis lesions is that keratinocytes proliferate excessively, long-term release of proinflammatory factors and antimicrobial peptides stimulate immune cells, activated immune cells release inflammatory factors to stimulate keratinocytes again, and so on, to cyclically induce psoriasis formation.

[0003] The biological agents for clinically treating psoriasis at the present stage mainly include TNF inhibitors (etanercept, adalimumab, infliximab), IL-12 / 23 inhibitors (ustekinumab), IL-17 inhibitors and IL-23 inhibitors (guselkumab). These four types of biological agents play a therapeutic role in the anti-inflammatory aspect by targeting inflammatory factors.

[0004] FGF-2 is a typical fibroblast proliferation and pro-angiogenic factor, and recombinant FGF-2 has been developed as an external medicine for treating refractory wounds, based on the pro-proliferative activity of FGF-2 on various skin cells.

[0005] At present, no research reports the effect of targeting FGF-2 on psoriasis. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide application of FGF-2 nanobody in preparation of a medicine for treating psoriasis.

[0007] The purpose of the present application is achieved by the following technical solution: application of FGF-2 nanobody in preparation of a medicine for treating psoriasis, which is the research result of the inventors who first found that targeting FGF-2 can effectively relieve psoriasis.

[0008] The amino acid sequence of the FGF-2 nanobody is as follows:

[0009] MLQESGGDLVQPGGSLRLSCEVSGSQFSINDMGWYRQAPGKRREVVAGISSVGRTMYGDPVKGRFTISRDNAKNMVYLQMNRLKPEDTAVYYCHLYGDYRGTGFWGKGTQVTVSSEPKTPKPHHHHHH.

[0010] The sequence of the nucleic acid encoding the FGF-2 nanobody is preferably as follows:

[0011] ATGCTGCAGGAGTCTGGGGGAGACTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGAAGTTTCTGGAAGCCAATTCAGTATCAATGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGCGACGCGAGGTGGTCGCAGGTATTAGTAGTGTTGGACGCACAATGTATGGAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGGTGTATCTGCAAATGAACAGACTGAAACCTGAGGACACGGCCGTCTATTACTGTCACCTTTATGGTGACTATAGGGGGACTGGTTTCTGGGGCAAGGGGACCCAGGTCACCGTCTCGTCGGAACCCAAGACACCAAAACCACATCATCACCATCACCATTAA.

[0012] The present application has the following advantages and effects relative to the prior art:

[0013] The present application takes imiquimod (IMQ)-induced psoriasis mice as a model to explore the therapeutic effect of FGF-2 nanobody on psoriasis. Experiments have proved that targeting FGF-2 can effectively inhibit the abnormal proliferation expressed by imiquimod (IMQ)-induced psoriasis mice, and relieve the skin inflammation of psoriasis mice. This study first describes that targeting FGF-2 can effectively relieve psoriasis, providing a new treatment scheme for the treatment of psoriasis, and also providing an important basis for the subsequent treatment of psoriasis by targeting growth factors. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the SDS-PAGE photo map of the small amount of expressed Nb FGF-2 (M-F3).

[0015] Figure 2 is the expression and purification result map of Nb FGF-2 (M-F3); wherein, A is the affinity chromatography purification map, and B is the SDS-PAG photo map.

[0016] Figure 3 is the expression and purification result map of Nb FGF-2Figure (M-F3) shows the results of relieving IMQ-induced psoriasis in mice; wherein, A is a representative photograph of each group at day 7; B is the results of total PASI score of erythema, infiltration and scaling at day 1, 3, 5 and 7.

[0017] Figure 4 Nb is FGF-2 Figure (M-F3) shows the results of the influence of M-F3 on the histopathology of psoriatic skin tissue in mice; wherein, A is a skin tissue H&E section; B is a skin tissue Ki67 immunohistochemistry; C is a skin tissue CD31 immunohistochemistry; D is a column chart obtained by analyzing A, B and C; data is expressed as mean ± standard deviation (SD), *p<0.05, **p<0.01; ***p<0.001.

[0018] Figure 5 Nb is FGF-2 Figure (M-F3) shows the results of inhibiting IMQ-induced psoriasis-related inflammatory factors in mice; wherein, A-D are the skin of the mouse skin lesion site, RNA extraction for Q-PCR detection of inflammatory factors IL-1β, IL-6, IL-17 and IL-23 mRNA levels; data is expressed as mean ± standard deviation (SD), *p<0.05; **p<0.01; ***p<0.001.

[0019] Figure 6 Nb is FGF-2 Figure (M-F3) shows the results of inhibiting the expression of psoriasis-related proteins in the skin of mice.

[0020] Figure 7 Nb is FGF-2 Figure (M-F3) shows the results of inhibiting IMQ-induced psoriasis-related signaling pathways in mice. DETAILED DESCRIPTION

[0021] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto.

[0022] Example 1 pPICzαA-Nb FGF-2 (M-F3) Construction of recombinant plasmid

[0023] In our previous study, we immunized alpaca with FGF-2 (purchased from Gibco, 13256029) as antigen, and constructed FGF-2 nanobody phage library by phage display technology. We screened a strain of FGF-2 nanobody and constructed it in prokaryotic expression vector. In eukaryotic system, the expression of the F3 sequence may have glycosylation sites, and reducing the number of N-glycosylation sites in the exogenous protein may improve the level of its secretory expression. Therefore, we predicted the glycosylation sites of F3 sequence through the glycosylation prediction website (http: / / www.cbs.dtu.dk / services / NetNGlyc / ), changed the DNA sequence of the glycosylation site, and analyzed the Nb FGF-2 gene through NetNGlyc 1.0 Server (http: / / www.cbs.dtu.dk / services / NetNGlyc / ), and found that there was an N-glycosylation site in the sequence of Nb FGF-2 gene. After mutating the glycosylation site in the sequence, we obtained the following sequence (5'-3'), and sent it to Sangon Biotech for synthesis to obtain the recombinant plasmid pPICzαA-Nb FGF-2 (M-F3) cloned in pPICzαA vector by EcoR I / Not I.

[0024] The sequence of Nb FGF-2 (M-F3) is as follows:

[0025] ATGCTGCAGGAGTCTGGGGGAGACTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGAAGTTTCTGGAAGCCAATTCAGTATCAATGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGCGACGCGAGGTGGTCGCAGGTATTAGTAGTGTTGGACGCACAATGTATGGAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGGTGTATCTGCAAATGAACAGACTGAAACCTGAGGACACGGCCGTCTATTACTGTCACCTTTATGGTGACTATAGGGGGACTGGTTTCTGGGGCAAGGGGACCCAGGTCACCGTCTCGTCGGAACCCAAGACACCAAAACCACATCATCACCATCACCATTAA.

[0026] Example 2: Transformation of pPICzαA-Nb FGF-2 (M-F3) plasmid

[0027] The synthesized pPICzαA-Nb FGF-2 The recombinant plasmid (M-F3) was linearized by QuickCut TM The linearized pPICzαA-Nb

[0028]

[0029] The above system was reacted in a water bath at 37°C for 1 h.

[0030] The linearized pPICzαA-Nb FGF-2 The recombinant plasmid (M-F3), Pichia pastoris GS115 competent cells (purchased from Beijing Huayueyang Biological Technology Co., Ltd., 88732), and a 0.2-cm electroporation cup (purchased from Bio-rad, Bio-rad 620) were placed on ice for 5 min. 5-10 μg of the linearized plasmid was added to each tube of competent cells, mixed, and then transferred to a pre-cooled electroporation cup, which was then placed in an ice bath for 10 min. An electroporator (Eppendorf) was used for electroporation at 1900 V and 5 ms. Immediately after electroporation, 1 mL of a 1 M sorbitol solution was added, and the mixture was incubated at 30°C for 2 h. 100 μL of the bacterial solution was spread on MD plates and YPD plates (50 μg / mL of bleomycin (purchased from Yuan Ye Biological Technology Co., Ltd., S24063-5 mg)) and incubated in a 30°C incubator for 2 d.

[0031] Example 3: Expression and identification of Nb FGF-2 (M-F3)

[0032] (1) Since the pPICzαA plasmid contains a Zeocin resistance gene, high-copy strains can be screened by increasing the concentration of Zeocin (bleomycin).

[0033] Single colonies were picked from the transformed YPD plates obtained in Example 2 using a sterile gun tip, diluted with 200 μL of sterile water, and then spotted on YPD plates containing 100 μg / mL of bleomycin and incubated in a 30°C incubator for 2-3 d. Single colonies were picked from the YPD plates containing 100 μg / mL of bleomycin using a sterile gun tip, diluted with 200 μL of sterile water, and then spotted on YPD plates containing 500 μg / mL of bleomycin and incubated in a 30°C incubator for 2-3 d. Single colonies were picked from the YPD plates containing 500 μg / mL of bleomycin using a sterile gun tip, diluted with 200 μL of sterile water, and then spotted on YPD plates containing 2 mg / mL of bleomycin and incubated in a 30°C incubator for 2-3 d.

[0034] (2) Small amount expression: positive yeast transformants on YPD plates containing 2 mg / mL of bleomycin were inoculated into YPD medium containing 1‰ v / v Amp (100 mg / mL, purchased from Solarbio, A8180) at 30°C, 220 rpm for 20 h. The bacterial solution was inoculated into 10 mL of BMGY medium at a ratio of 1:100 by volume, and cultured at 30°C, 220 rpm until the OD 600 = 1, 30°C, 220 rpm, and cultured for 96 h. Meanwhile, the same operation was performed on the Pichia pastoris transformed with the blank plasmid pPICZαA as a negative control. After centrifugation at 4000 rpm for 10 min, the bacterial body and the centrifuged medium were taken respectively, and the bacterial body was resuspended with PBS (1 g / 100 mL). 600 = 1, 30°C, 220 rpm, and cultured for 96 h. Meanwhile, the same operation was performed on the Pichia pastoris transformed with the blank plasmid pPICZαA as a negative control. After centrifugation at 4000 rpm for 10 min, the bacterial body and the centrifuged medium were taken respectively, and the bacterial body was resuspended with PBS (1 g / 100 mL).

[0035] (3) SDS-PAGE analysis of bacterial expression: the collected components whole bacteria (pPICzαA), fermentation medium (pPICzαA), whole bacteria (pPICzαA-Nb FGF-2 (M-F3)) and fermentation medium (pPICzαA-Nb FGF-2 (M-F3)) were subjected to SDS-PAGE using 10% SDS-PAGE precast gels (purchased from Invitrogen TM , NP0306BOX). 80 μL of each component was mixed with 20 μL of 5x loading buffer, and then placed in a 100°C metal bath for boiling for 10 min. 25 μL of the prepared sample was added to the precast gel well, and electrophoresis was performed at 70 V for 30 min to separate the sample to the separation gel. The voltage was adjusted to 120 V and the electrophoresis was continued until the indicator reached the bottom.

[0036] (4) The PAGE gel was soaked in Coomassie Brilliant Blue solution (Solarbio, 419B025), and then dyed on a shaker for 1 h. The decolorizing liquid (purchased from Biyun Tian, P0017C) was used to decolorize until clear protein bands were visible. Figure 1 The fermentation medium (pPICzαA-Nb FGF-2 (M-F3) showed obvious bands near 15 kDa, which was consistent with the theoretical molecular weight, while the other lanes showed no obvious bands, indicating that the recombinant bacteria successfully expressed the target protein.

[0037] (5) Expression and purification. The same method as above was used to scale up to 1 L BMMY for induction expression. After induction expression, the bacterial solution was centrifuged at 10,000 rpm for 60 min, and the collected fermentation medium was purified by Ni-NTA affinity chromatography on an AKT purification instrument (GE). The nickel column (purchased from GE, 10230759) was washed with ultrapure water at a flow rate of 2 mL / min for 4 column volumes. The column was washed with 1xPBS at a flow rate of 2 mL / min for 4 column volumes. The column was equilibrated with 20 mM imidazole at a flow rate of 1 mL / min for 4 column volumes. The purified sample stored at 4°C was loaded at a flow rate of 1 mL / min. After loading, the column was equilibrated with 20 mM imidazole at a flow rate of 1 mL / min. The protein was eluted with 50 mM, 100 mM, 300 mM, and 500 mM imidazole, respectively, and each elution peak was collected and stored at 4°C at a flow rate of 2 mL / min. The column was washed with 0.5 M NaOH for 3 column volumes, and then washed with ultrapure water at a flow rate of 2 mL / min. The column was sealed with 20% ethanol for 6 column volumes, and then removed and stored at 4°C.

[0038] (6) SDS-PAGE analysis of the purification results. SDS-PAGE electrophoresis was performed using a precast gel. The prepared sample components (fermentation medium, purification breakthrough liquid, 50 mM imidazole eluate, 100 mM imidazole eluate, 300 mM imidazole eluate, and 500 mM imidazole eluate) were taken 25 μL and added to the precast gel well. Electrophoresis was performed at 70 V for 30 min to separate the sample into the separation gel, and then the voltage was adjusted to 120 V for continuous electrophoresis until the indicator reached the bottom. The electrophoresed PAGE gel was soaked in Coomassie Brilliant Blue solution and placed on a shaker for staining for 1 h, and then destained with destaining solution until clear protein bands were visible. Figure 2 As shown, obvious bands were found near 15 kDa in the fermentation medium and 300 mM imidazole eluate lanes, indicating that the target protein was eluted in the 300 mM imidazole eluate. The purified protein solution was concentrated by ultrafiltration using an ultrafiltration tube to a final volume of 5 mL. Each tube was divided into 1.5 mL centrifuge tubes at 1 mL, and the protein concentration was determined by the BCA method, with a yield of 30 mg / L. The expression amount was increased by about 12 times compared with Escherichia coli.

[0039] Example 4: Effect of FGF-2 nanobody on IMQ-induced psoriasis in mice

[0040] BALB / c mice (purchased from Zhuhai Boshitong Biotechnology Co., Ltd., license number SCXK (Guangdong) 2020-0051) were purchased and raised for 3 days to adapt to the environment before starting the experiment. They were raised in a SPF level animal room with 12 h light and dark alternation per day.

[0041] A 2cm x 3cm area of ​​hair was shaved off the back using a hair removal machine, and the remaining soft hair was removed with hair removal cream. The hair removal cream should not remain on the back for more than 2 minutes to avoid damaging the back and affecting the experiment. The hair-removed mice were randomly divided into 5 groups: a blank control group, an IMQ+PBS group, an IMQ+M-F3 (5mg / kg) group, an IMQ+M-F3 (20mg / kg) group, and an IMQ+Calcipotriol (calcipotriol ointment) group, with 5 mice in each group, and the groups were labeled. M-F3 was Nb prepared in Example 3. FGF-2 (M-F3) protein.

[0042] Except for the blank control group, the hair-removed areas on the backs of other mice were treated with 5% IMQ ointment, 62.5 mg / day, for 6 consecutive days. Six hours after each IMQ ointment application, the mice were administered medication via subcutaneous injection. The IMQ+PBS group received PBS, the IMQ+M-F3 (5 mg / kg) group received M-F3 (5 mg / kg), the IMQ+M-F3 (20 mg / kg) group received M-F3 (20 mg / kg), and the IMQ+Calcipotriol group received Calcipotriol ointment (purchased from Dalix) according to the manufacturer's instructions. The back areas of the mice were photographed for 7 consecutive days to observe the skin condition.

[0043] Figure 3 The results in A showed that on day 7, IMQ induced extensive psoriasis on the back of mice, and M-F3 effectively alleviated IMQ-induced psoriasis symptoms in mice, with the therapeutic effect being concentration-dependent. We also used the PASI score to assess erythema, scaling, and infiltration. Figure 3 As shown in Figure B, regarding erythema, both high and low concentrations of M-F3 had lower PASI scores than the positive control drug calcipotriol. Regarding scaling, M-F3 had higher PASI scores than calcipotriol. Regarding infiltration, the M-F3 (20 mg / kg) group had a lower PASI score than calcipotriol, while the M-F3 (5 mg / kg) group had a higher PASI score. From the total PASI score, it can be concluded that M-F3 (20 mg / kg) was more effective than calcipotriol in alleviating IMQ-induced psoriasis in mice, while the therapeutic effect of M-F3 (5 mg / kg) was lower than that of calcipotriol.

[0044] Example 5

[0045] I. HE staining of mouse skin tissue

[0046] (1) Washing: After fixing with 4% paraformaldehyde (purchased from Biosharp, BL539A) for 24 hours, the tissue was removed and rinsed slowly with water.

[0047] (2) Dehydration: The tissues were dehydrated in 50%, 70%, 80%, 90%, 95% and 100% ethanol (purchased from Aladdin, E111991) for 1 h at each concentration.

[0048] (3) Transparency: Since pure alcohol and paraffin (purchased from Xiyareagent, D14847) are incompatible, it is necessary to replace the alcohol in the tissue. First, the tissue was immersed in a solution of alcohol: xylene (purchased from Aladdin, X112051) at 1:1 for 1 h, and then transferred to xylene.

[0049] (4) Wax immersion: Replace xylene with paraffin, immerse the tissue in a paraffin cup containing equal amounts of xylene and paraffin, and place it in a 60°C incubator for 1.5 h. Then transfer it to a melted paraffin cup and place it in a 60°C incubator for 3 h.

[0050] (5) Embedding: Pour the tissue and paraffin into cold water to cool, and remove it after complete solidification.

[0051] (6) Sectioning: Use a paraffin microtome to section the embedded tissue, 3-6 μm per section, and place it in warm water at 45°C to flatten. Then pick it up and place it on a glass plate to flatten, and place it in a 45°C oven to dry.

[0052] (7) De-waxing: Place the sections in xylene I and xylene II for 5 min each.

[0053] (8) Hydration: Then place them in 100%, 95%, 80%, 70%, 60% and 50% ethanol for 5 min each. Rinse the sections with ultrapure water.

[0054] (9) Staining: Place the sections in hematoxylin dye (purchased from Biyun Tian, C0105S) in the HE staining kit for 5 min

[0055] (10) Differentiation and rinsing: Immerse the sections in 1% hydrochloric acid ethanol for 30 s, and then rinse with ultrapure water for 15 min.

[0056] (11) Counterstaining: Place the sections in eosin dye (purchased from Biyun Tian, C0105S) in the HE staining kit for 1 min.

[0057] (12) Dehydration: Dehydrate the sections in 50%, 70%, 80%, 90%, 95% and 100% ethanol for 2 min each.

[0058] (13) Transparency: Place the sections in xylene I and xylene II for 5 min each.

[0059] (14) Mounting: After wiping off the surrounding liquid, add 1-2 drops of neutral resin (purchased from Soleris, G8590), and cover with a cover glass.

[0060] (15) Observation: The sections were observed and photographed under an inverted microscope.

[0061] II. IHC staining of mouse skin tissue

[0062] (1) De-waxing: The sections were sequentially placed in xylene I and xylene II for 5 min each.

[0063] (2) Hydrating: The sections were then sequentially placed in 100%, 95%, 80%, 70%, 60%, and 50% ethanol for 5 min each. The sections were gently washed with ultrapure water.

[0064] (3) Antigen retrieval: The sections were placed in citrate buffer (purchased from Merck, C9999-100ML) and then treated with a microwave oven for 3 times, 3 min each time.

[0065] (4) Permeabilization: The sections were washed with PBS for 3 times, 3 min each time, and then placed in permeabilization solution (Triton X-100 (purchased from Biyun Tian, P0096-100ml)) for incubation at room temperature for 30 min.

[0066] (5) Blocking endogenous peroxidase: The sections were washed with PBS for 3 times, 3 min each time, and then placed in 3% v / v hydrogen peroxide for blocking at room temperature for 20 min.

[0067] (6) Blocking: The sections were washed with PBS for 3 times, 3 min each time, and then incubated with 5% w / v BSA at room temperature for 30 min.

[0068] (7) Incubation of primary antibody: The corresponding primary antibody (Anti-Ki67 Rabbit pAb (purchased from Servicebio, GB111141), Anti-CD31 Rabbit pAb (purchased from Servicebio, GB11063-2)) was added dropwise on the tissue, and incubated overnight at 4°C.

[0069] (8) Washing: The sections were washed with PBS for 3 times, 3 min each time.

[0070] (9) Incubation of secondary antibody: The corresponding secondary antibody (HRP-labeled rabbit anti-goat IgG (purchased from Servicebio, GB23204)) was added dropwise, and incubated at room temperature for 30 min on a shaking bed.

[0071] (10) Washing: The sections were washed with PBS for 3 times, 3 min each time.

[0072] (11) DAB color development: 50 μL of freshly prepared DAB color developing solution was added to each section, and observed under a microscope.

[0073] (12) Counterstaining: After repeated washing with ultrapure water, add hematoxylin for counterstaining, then differentiate with 1% hydrochloric acid ethanol, and then repeatedly wash with ultrapure water.

[0074] (13) Dehydration: Dehydrate the section with 50%, 70%, 80%, 90%, 95% and 100% ethanol for 2 min each. Then put it into xylene I and xylene II for 5 min each.

[0075] (14) Mounting: After wiping off the surrounding liquid, add 1-2 drops of neutral resin, and cover with a cover glass.

[0076] (15) Observation: Observe and take pictures of the section under an inverted microscope.

[0077] After the mice were sacrificed at 7 days, we further analyzed the changes in the mouse skin tissue by H&E staining, Ki67 and CD31 immunohistochemical staining. As shown in Figure 4 the IMQ+PBS group was significantly thicker (p<0.001) than the blank control group, reaching 144.04±7.35 μm, while M-F3 (5 mg / kg), M-F3 (20 mg / kg) and calcipotriol could significantly inhibit the thickening of the skin (p<0.001), and the effect of the M-F3 (20 mg / kg) group was better than that of the M-F3 (5 mg / kg), with thicknesses of 84.12±8.80 μm, 50.24±5.21 μm and 60.58±5.91 μm, respectively. Ki67 is a marker protein used to evaluate cell proliferation in tissues, and CD31 is a platelet endothelial cell mucosal molecule that can be used to reflect the angiogenesis status in tissues. M-F3 (5 mg / kg), M-F3 (20 mg / kg) and calcipotriol can significantly down-regulate the up-regulation of Ki67 and CD31 in the skin of IMQ-induced mice. This indicates that M-F3 can effectively inhibit the abnormal proliferation of the epidermis and angiogenesis in psoriasis mice.

[0078] Example 6: Q-PCR detection of mouse skin tissue factors

[0079] (1) Take out the tissue stored at -80°C, cut it into small pieces with scissors, add 200 μL RNA iso, and grind it with a handheld electric grinding rod until there are no obvious fragments. All operations are performed on ice. Add 800 μL RNA iso and stand at room temperature for 5 min. Centrifuge at 4°C, 12000g for 5 min.

[0080] (2) Transfer the supernatant to a new 1.5 mL EP tube, add 200 μL chloroform, mix well by inverting, and stand for 5 min.

[0081] (3) 4℃, 12000g centrifuge 15min. The liquid is divided into three layers, and the uppermost clear liquid is taken to a new 1.5mL EP tube, and 600mL of isopropanol is added. After mixing, centrifuge (4℃, 2000g, 10min).

[0082] (4) Discard the supernatant, add 1mL of 75% v / v ethanol to resuspend the white precipitate at the bottom. 4℃, 8000g centrifuge for 5min.

[0083] (5) Discard the supernatant, resuspend with 20μL DEPC water, and measure the RNA concentration and purity with Nano.

[0084] (6) Remove gDNA, and store the remaining RNA at -80℃.

[0085] Reaction system: 5×gDNA Eraser Buffer 2.0μL, gDNA Eraser 1.0μL, total RNA 1.0μg, ddH2O to 10μL.

[0086] Reaction conditions: 42℃ 2min; 4℃ 10min.

[0087] (7) Reverse transcription

[0088] Reaction system: 10μL of reaction solution from the previous step, PrimerScript Enzyme Mix I 1.0μL, 5×primerScript Buffer 2 4.0μL, RT primer Mix 1.0μL, ddH2O to 20μL.

[0089] Reaction conditions: 37℃ 15min, 85℃ 5s, 4℃ ∞.

[0090] (8) The primers are as follows:

[0091]

[0092] (9) Q-PCR reaction

[0093] Reaction system: upstream primer (10μM) 0.4μL, downstream primer (10μM) 0.4μL, cDNA 1.0μg, 2×ChamQSYBR qPCR Master Mix 10μL, ddH2O to 20μL.

[0094] Reaction conditions: 95℃ 3min; 95℃ 10s, 60℃ 30s, 40 cycles; 65℃ ∞.

[0095] Using GAPDH as the internal reference, the relative expression of the gene was analyzed by CFX Maestro.

[0096] To further investigate the effect of M-F3 on the inflammation of IMQ-induced psoriasis in mice, we detected the mRNA levels of related inflammatory cytokines and chemokines in the skin tissues by Q-PCR. As shown in Figure 5 Figure 6, IMQ stimulation up-regulated the mRNA levels of inflammatory cytokines IL-1β, IL-6, IL-17 and IL-23 (p<0.001), while M-F3 and calcipotriol treatment down-regulated the mRNA levels of these related inflammatory cytokines to different extents, and the therapeutic effect of M-F3 showed dose dependence. These data indicated that M-F3 could effectively alleviate the inflammation of IMQ-induced psoriasis in mice.

[0097] Example 7 Western blot detection of protein levels in skin tissues

[0098] (1) The tissue stored at -80℃ was taken out, cut into small pieces with scissors, and added with 200 μL RIPA lysis buffer, and then lysed in ice bath for 30 min, during which the vortex was constantly used. The lysis buffer was centrifuged (4℃, 12000g, 45 min), and the supernatant was taken into a new 1.5 mL Ep tube. The protein concentration of the supernatant was determined by BCA kit (purchased from Thermo Scientific TM , 23235).

[0099] (2) SDS-PAGE electrophoresis and membrane transfer: The installation sequence of the transfer module clamp is black base plate, sponge, filter paper, gel, NC membrane, filter paper, sponge, and white base plate. Pay attention to air bubbles and installation sequence. After installation, load into the membrane transfer tank. Place an ice bag in the tank for cooling. Turn on the power and adjust the current to 300 mA for 90 min. After membrane transfer, remove the NC membrane and wash the membrane with TBST for 3 times, 3 min each time. Place the NC membrane in 5% skim milk for room temperature blocking for 2 h. Discard the skim milk and wash the membrane with TBST for 3 times, 3 min each time. Discard TBST and add the corresponding primary antibody (Cytokeratin 6A Polyclonal Antibody (purchased from Proteintech, 10590-1-AP), Cytokeratin 17-Specific Polyclonal Antibody (purchased from Proteintech, 17516-1-AP), FGF2 Antibody (purchased from Signalway Antibody, 38109-1), AKT Monoclonal Antibody (purchased from Proteintech, 60203-2-Ig), Phospho-AKT (Ser473) Monoclonal Antibody (purchased from Proteintech, CL488-66444), PI3 Kinase P85 Alpha Monoclonal Antibody (purchased from Proteintech, 60225-1-Ig), PI3-kinase p85-alpha (Phospho-Tyr607) Antibody (purchased from Signalway Antibody, 12057)), and incubate on a shaker at 4°C overnight. Recover the primary antibody and wash the membrane with TBST for 3 times, 3 min each time. Discard TBST and add the corresponding secondary antibody (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (purchased from Proteintech, SA00001-2), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (purchased from Proteintech, SA00001-1)), and incubate on a shaker at room temperature for 2 h. Discard the secondary antibody and wash the membrane with TBST for 3 times, 3 min each time. Develop with a gel imager (purchased from Biorad), evenly drop the developing solution on the NC membrane, adjust the exposure time according to the signal strength, and take a picture for record.

[0100] The above results show that M-F3 can effectively alleviate IMQ-induced inflammation, and then we further explore the effects of M-F3 on the expression of key proteins CK6, CK17 and FGF-2 in the process of psoriasis. CK6 and CK7 are the marker proteins of keratinocyte proliferation and differentiation, and FGF-2 is a well-known growth factor that plays a key role in cell proliferation and vascular differentiation, and they are all highly expressed in the skin of psoriasis patients. We extracted the proteins of the back skin tissue and performed Western blot detection, and the results are shown in Figure 6 FGF-2 can significantly up-regulate the expression of CK6, CK17 and FGF-2 (p<0.001), and M-F3 and calcipotriol can significantly inhibit the up-regulation of CK6, CK17 and FGF-2 induced by IMQ, and the inhibitory effect of M-F3 is concentration-dependent. This shows that M-F3 can inhibit the abnormal proliferation of keratinocytes and the symptoms of angiogenesis induced by IMQ.

[0101] Through the above research, we prove that M-F3 can effectively alleviate IMQ-induced psoriasis in mice, but the specific mechanism is still unclear. In order to explore the mechanism of M-F3, we detected the related signal pathways by Western blot, and the results are shown in Figure 7 The total protein levels of AKT and PI3K p85α do not change after IMQ, M-F3 and calcipotriol treatment, but IMQ can up-regulate the phosphorylation levels of AKT (Ser473) and PI3K p85α (p<0.001), and the phosphorylation levels of AKT (Ser473) and PI3K p85α significantly decrease after treatment with M-F3 and calcipotriol. This means that M-F3 plays a role in alleviating IMQ-induced psoriasis in mice by inhibiting the phosphorylation levels of AKT (Ser473) / PI3K p85α.

[0102] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. Application of FGF-2 nanobody in the preparation of drugs for treating psoriasis: The amino acid sequence of the FGF-2 nanobody is shown in SEQ ID NO.

1.

2. The application of the FGF-2 nanobody according to claim 1 in the preparation of drugs for treating psoriasis, characterized in that: The sequence of the nucleic acid encoding the FGF-2 nanobody is shown in SEQ ID NO. 2.

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