A binding peptide capable of binding to multiple fibroblast growth factors and use thereof

Abstract

The present application relates to a binding peptide and its application in purifying fibroblast growth factor. The binding peptide of the present application can bind to fibroblast growth factor, in particular to fibroblast growth factor-1, and thus can be used for purifying fibroblast growth factor or detecting fibroblast growth factor.

Description

Technical Field

[0001] This invention relates to the field of polypeptides, and particularly to a binding peptide. Background Technology

[0002] Fibroblast growth factors (FGFs) are a large family of more than 20 structure-related signaling proteins. They activate downstream signaling pathways by binding to fibroblast growth factor receptors on the cell surface, thereby broadly regulating cell proliferation, differentiation, migration, and survival. During embryonic development, fibroblast growth factors are crucial for organogenesis and limb development; in adults, they participate in tissue damage repair, angiogenesis, and the maintenance of glucose and lipid metabolism homeostasis. Based on their important biological functions, members of the fibroblast growth factor family show promising applications in wound healing, metabolic diseases (such as diabetes and fatty liver), and skeletal developmental abnormalities.

[0003] However, very few fibroblast growth factors (GF) are marketed as drugs. Besides limitations imposed by the protein's inherent physicochemical properties and clinical factors, protein purification processes are also crucial. For example, the purity of GF is essential for its activity detection, molecular crystal structure analysis, and clinical application. While GF family proteins can be purified using heparin, pharmacopoeias impose stringent quality standards and testing requirements on protein drugs, particularly regarding purity. Although most GF molecules possess a heparin-binding domain, theoretically allowing for separation and purification using heparin affinity chromatography, in practice, heparin affinity chromatography cannot meet the high-purity requirements for all GFs. Furthermore, detection methods and intracellular activity analysis of GF need further development. Summary of the Invention

[0004] One aspect of the present invention provides a binding peptide having the amino acid sequence shown in SEQ ID No. 1.

[0005] The second invention provides a nucleic acid encoding a binding peptide as described in the first invention.

[0006] In one specific embodiment, the sequence of the nucleic acid is shown in SEQ ID No. 2.

[0007] The third invention provides the use of the binding peptide according to the first invention in binding with fibroblast growth factor.

[0008] In one specific embodiment, the binding peptide is used for the purification or detection of the fibroblast growth factor. For example, when it is necessary to isolate fibroblast growth factor from a sample or when a large amount of pure fibroblast growth factor is required, the binding peptide can be used to isolate or purify the fibroblast growth factor; when it is necessary to perform molecular detection of the fibroblast growth factor, the binding peptide can be visualized (e.g., modified with a fluorescent group) and then the fibroblast growth factor can be detected or quantitatively analyzed; when it is necessary to inactivate the fibroblast growth factor in vivo or in vitro, the binding peptide can be used to bind to the fibroblast growth factor to achieve the purpose of inactivating the fibroblast growth factor, in which case the binding peptide acts as a protein inactivating agent.

[0009] In one specific embodiment, the binding peptide is used as an affinity chromatography packing material to purify the purified fibroblast growth factor via affinity chromatography.

[0010] In one specific embodiment, the fibroblast growth factor is detected by binding the binding peptide to the fibroblast growth factor to inactivate the fibroblast growth factor.

[0011] In one specific embodiment, the binding peptide is modified with a fluorescent group to detect the fibroblast growth factor. For example, a fluorescent group is modified on the carboxyl group at the C-terminus or the amino group at the N-terminus of the binding peptide.

[0012] In one specific embodiment, the fibroblast growth factor is at least one of fibroblast growth factor-1, fibroblast growth factor-14, and fibroblast growth factor-19. The amino acid sequences of fibroblast growth factor-1, fibroblast growth factor-14, and fibroblast growth factor-19 can be the amino acid sequences of the corresponding fibroblast growth factors listed in CN2021107794524, i.e., the amino acid sequences of the corresponding human fibroblast growth factors.

[0013] The beneficial effects of the present invention are as follows: The binding peptide of the present invention has a high affinity for fibroblast growth factors, especially fibroblast growth factor-1, fibroblast growth factor-14 and fibroblast growth factor-19. Therefore, it can be used to purify fibroblast growth factors. In addition, it can also be used to detect or inactivate fibroblast growth factors. Attached Figure Description

[0014] Figure 1The SDS-PAGE electrophoresis images of recombinant bacteria pGS-B176 / BL21(DE3) before and after induction are shown. Lane 1: bacterial suspension sample after induction; Lane 2: bacterial suspension sample before induction; M: protein molecular weight marker. The arrow with a black border indicates the induced expression of the fusion protein GFP-SUMO-B176.

[0015] Figure 2 The SDS-PAGE electrophoresis images of each sample during the purification of the binding peptide B176 are shown. Lane 1: pGS-B176 / BL21(DE3) supernatant; Lane 2: elution buffer of the fusion protein GFP-SUMO-B176; Lane 3: enzymatic digestion buffer; Lane 4: elution buffer after enzymatic digestion; Lane 5: elution buffer of other proteins; Lane 6: concentrated binding peptide B176; M: protein molecular weight label. The black arrow indicates the binding peptide B176; the black-bordered arrow indicates the fusion protein GFP-SUMO-B176.

[0016] Figure 3 The results of the surface plasmon resonance (SPR) method are shown, which is used to analyze the affinity fit between the binding peptide B176 and human fibroblast growth factor-1.

[0017] Figure 4 The activity analysis of the binding peptide B176 in inhibiting fibroblast growth factor-1-promoted fibroblast proliferation is shown.

[0018] Figure 5 The following are SDS-PAGE electrophoresis images of samples from the gel separation and purification of fibroblast growth factor-19 (hFGF-19) using the conjugated peptide B176. M: Protein molecular weight marker; Lanes 1-3: pET-GSFGF19-CsPA-U1p1 / BL21 supernatant; Lane 4: Supernatant eluent; Lane 5: Elution buffer for other proteins; Lane 6: hFGF-19 elution buffer. Arrows indicate human fibroblast growth factor-19. Detailed Implementation

[0019] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0020] Unless otherwise specified, the strains, plasmids, proteases, and biochemical reagents used in the embodiments of this invention can all be purchased commercially.

[0021] Unless otherwise specified, the molecular biology manipulation methods used in the embodiments of the present invention are based on "Molecular Cloning: A Laboratory Manual" (Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0022] The Escherichia coli pET-21a expression vector (catalog number: 69740) and Escherichia coli BL21(DE3) (catalog number: 69450) were purchased from Sigma-Aldrich.

[0023] The nickel-nitrotriacetic acid affinity chromatography gel (NI-NTA) (catalog number: 17371203) and SP cation exchange chromatography gel (catalog number: 17072901) used for protein purification were purchased from Cytiva Biotechnology (Hangzhou) Co., Ltd.

[0024] The CM5 chip (item number: BR100399) was purchased from Cytiva Biotechnology (Hangzhou) Co., Ltd.

[0025] Restriction endonucleases BamH Product I (item number: D6053) was purchased from Beyotime Biotechnology Co., Ltd.

[0026] LB liquid medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 ml distilled water, sterilized at 121°C for 20 minutes.

[0027] Phosphate buffer: 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L sodium dihydrogen phosphate, 2 mmol / L potassium dihydrogen phosphate, 0.2% surfactant NP-40, pH=7.0.

[0028] Basic phosphate buffer: 5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, pH=7.4. Example 1: De novo design of binding peptides

[0029] De novo design of fibroblast growth factor binding peptides: 1) A preliminary model of the peptide backbone was designed using the AI ​​(Artificial Intelligence) software RFDiffusion (version 1.1.0). Then, the synthetic direction and local structure of the peptide were gradually adjusted to obtain the final binding peptide backbone model; 2) Based on the binding peptide backbone model, various amino acids were randomly filled using the software ProteinMPNN (version 1.0.1) to obtain no less than 2000 first candidate sequences of binding peptides; 3) The first candidate sequences of each binding peptide were input into the AlphaFold 3 analysis platform for evaluation, and second candidate sequences of binding peptides were screened; 4) Molecular dynamics simulation analysis of each second candidate sequence of binding peptides was performed using the GROMACS (version 2020.6) software to screen third candidate sequences of binding peptides. One of the third candidate sequences of a binding peptide is shown in SEQ ID No. 1, and the binding peptide is numbered B176. Example 2: Construction and expression of peptide-binding vectors

[0030] The nucleotide coding sequence of the binding peptide B176 is shown in SEQ ID No. 2 and was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0031] Using genetic engineering methods, a nucleic acid fragment encoding a binding peptide was cloned downstream of the GFP-SUMO nucleic acid in the *E. coli* expression vector pET-GFP-SUMO (see Example 3 in CN2021107794524) to form a recombinant expression vector pGS-B176. This recombinant plasmid was transformed into the *E. coli* host BL21(DE3), and positive recombinant bacteria were screened using ampicillin resistance (final concentration 100 μg / mL) and named pGS-B176 / BL21(DE3). The fusion protein encoded by the GFP-SUMO-B176 nucleic acid is a GFP-SUMO binding peptide fused to its N-terminus; this fusion protein is abbreviated as GFP-SUMO-B176.

[0032] The pGS-B176 / BL21(DE3) strain was inoculated into a test tube containing 5 mL of ampicillin-containing liquid LB medium and activated by incubation at 37°C and 220 rpm for 12 hours. The activated bacterial culture was then inoculated at 1% (v / v) into 50 mL of fresh ampicillin-containing liquid LB medium and cultured at 37°C and 220 rpm with shaking until OD reached. 600=1.0 (approximately 2 to 3 hours), after sampling (pre-induction bacterial culture), add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol / L, and continue induction culture at 37°C for 4 hours to obtain the post-induction bacterial culture. Centrifuge the pre-induction and post-induction bacterial cultures at 12000 rpm respectively, collect the bacterial cells, and resuspend the bacterial cells in phosphate buffer to obtain the pre-induction and post-induction bacterial suspensions. Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on the pre-induction and post-induction bacterial suspensions. The results are shown in the figure. Figure 1 . Figure 1 The results showed that the fusion protein GFP-SUMO-B176 was successfully expressed in Escherichia coli BL21(DE3). Example 3: Isolation and purification of binding peptide B176

[0033] Cryopreserved glycerol culture of pGS-B176 / BL21(DE3) was inoculated at a ratio of 1:100 (volume / volume) into a 3.7 L Erlenmeyer flask containing 100 mg / L ampicillin in liquid LB medium. The culture was incubated at 37°C and 220 rpm for 4 hours with shaking to obtain activated bacterial culture. Subsequently, the activated bacterial culture was inoculated at 10% (volume / volume) into a 200 L fermenter containing 5% (mass / volume) glucose and a final concentration of 100 μg / mL ampicillin in liquid LB medium. Fermentation parameters were set as follows: pH = 7.0-7.2; dissolved oxygen >30%; stirring speed increased from 250 rpm to 650 rpm as fermentation progressed; and cultured at 37°C. When the glucose content in the medium decreased to 0.1% (mass / volume), a fed-batch culture was started, consisting of 20% (mass / volume) glucose and a final concentration of 100 μg / mL ampicillin in liquid LB medium. When the wet bacterial weight reached 35±1 g / L, IPTG with a final concentration of 1 mmol / L was added for induction. After 4 hours of induction, the bacterial cells were collected by centrifugation to obtain pGS-B176 / BL21(DE3) cells.

[0034] Weigh 20 g of pGS-B176 / BL21(DE3) bacterial cells and resuspend the cells in 200 mL of basal phosphate buffer to prepare a bacterial suspension. Place the bacterial suspension in an autoclave and homogenize twice consecutively at 4°C and pressures of 800 and 1200 bar. Collect the lysis buffer and place it in a sterile centrifuge tube. Centrifuge at 12000 rpm for 30 minutes and collect the supernatant of pGS-B176 / BL21(DE3).

[0035] The pGS-B176 / BL21(DE3) supernatant was loaded onto a nickel-nitrotriacetic acid affinity chromatography gel column (NI-NTA column). The NI-NTA column was then washed with 600 mL of elution buffer 1 (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 50 mmol / L imidazole, pH=7.4) containing a low concentration of imidazole to remove unbound proteins. The eluent was then used as the protein eluent. Elution was then performed with 200 mL of elution buffer 2 (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 150 mmol / L sodium chloride, 300 mmol / L imidazole, pH=7.4) containing a high concentration of imidazole to obtain the fusion protein GFP-SUMO-B176 eluent. The basal phosphate buffer and the elution buffer of the fusion protein GFP-SUMO-B176 were mixed at a volume ratio of 5:1, and then 200 IU of SUMO protease (Bestpsys Biotechnology Co., Ltd., catalog number: SUE-S5127-1000U) were added. The mixture was incubated at 4°C for 60 minutes to obtain the enzymatic hydrolysate. The hydrolysate was loaded onto an NI-NTA column, and the elution buffer (the elution buffer after enzymatic hydrolysis) was collected. The elution buffer was then loaded onto an SP cation exchange chromatography gel column and eluted with elution buffer 3 containing sodium chloride (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 600 mmol / L sodium chloride, pH=7.4). The solution was then concentrated using an ultrafiltration tube to obtain the conjugated peptide B176 concentrate, i.e., a conjugated peptide B176 solution with a concentration of 0.5 mg / mL.

[0036] Samples were taken at various stages, including pGS-B176 / BL21(DE3) supernatant, elution buffer for other proteins, elution buffer for the fusion protein GFP-SUMO-B176, enzyme digestion buffer, digested buffer, and concentrated B176 binding peptide solution, and analyzed by SDS-PAGE electrophoresis. The results are shown in [Figure number missing]. Figure 2 . Example 4: Detection of the affinity of binding peptide B176 for fibroblast growth factor 1

[0037] For details on the preparation of human fibroblast growth factor-1, please refer to CN2021107794524. Human fibroblast growth factor-1 eluted with sodium chloride-containing elution buffer was prepared, i.e., human fibroblast growth factor-1 eluent. The human fibroblast growth factor-1 eluent was loaded onto a Sephadex G-25 filter chromatography gel. The sodium chloride-containing elution buffer was replaced with basal phosphate buffer to remove sodium chloride. The protein was concentrated using an ultrafiltration centrifuge tube to obtain a human fibroblast growth factor-1 solution with a concentration of 2 mg / mL.

[0038] Human fibroblast growth factor-1 solution (2 mg / mL human fibroblast growth factor-1, 5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, pH=7.4) was diluted to 10 μg / mL with 20 mmol / L sodium acetate aqueous solution at pH 5.0 to obtain human fibroblast growth factor-1 diluted solution.

[0039] The affinity of binding peptide B176 for human fibroblast growth factor-1 was detected using the surface plasmon resonance (SPR) method with a Biacore T200 instrument.

[0040] Following the operating instructions in the Biacore T200 instrument manual, human fibroblast growth factor-1 in the diluted solution was coupled to the CM5 chip using the Biacore T200 instrument to obtain a ligand-coupled chip.

[0041] The binding peptide B176 solution with a final concentration of 50 μmol / mL (0.98 mg / mL) prepared in accordance with Example 3 was diluted to 2.5 μmol / mL, 5 μmol / mL, 7.5 μmol / mL, 10 μmol / mL and 34.8 μmol / mL using a dilution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.15 mmol / L sodium chloride, pH=7.4) to obtain different concentrations of binding peptide B176 dilutions.

[0042] The ligand-coupled chip was placed into the Biacore T200 instrument. Then, different concentrations of the bound peptide B176 dilutions were placed as test solutions on sample tube racks, which were then placed into the Biacore T200 instrument. Measurements were performed according to the instrument's operating instructions. The parameters for the test solutions entering the ligand-coupled chip were set as follows: flow rate 30 μL / min, sample binding time 180 s, and dissociation time 300 s. Results are shown below. Figure 3 .

[0043] Figure 3 The results showed that the affinity of binding peptide B176 for fibroblast growth factor-1 reached 6 × 10⁻⁶. -6 Moles per liter. Example 5: Reliability analysis of the complex formed by binding peptide B176 and members of the fibroblast growth factor family.

[0044] AlphaFold3 can evaluate the conformational interaction of two proteins. The software manual and related application guidelines include: focusing on combining predicted aligned error (PAE), predicted local distance difference test (pLDDT) confidence score, interface predicted TM-score (iPTM), and predicted template modeling score (pTM) to comprehensively evaluate the quality of the complex. Among them, PAE is used to characterize the certainty of the relative spatial arrangement between the binding peptide and the target protein, and is one of the key parameters for evaluating the reliability of interfacial interactions. The lower the PAE value, the more stable the relative positioning of the two in the complex, and the more reliable the predicted binding mode. PAE < 5 often indicates that the relative positioning is relatively reliable. pLDDT is used to measure the confidence level of the model in predicting the local atomic coordinates of the binding peptide and the target protein. It is usually scored from 0 to 1. A score higher than 70 indicates that the local structure prediction has high reliability. pTM mainly reflects the prediction accuracy of the overall fold topology and the relative arrangement of domains. Its value range is generally 0 to 1. A value > 0.6 indicates that the overall conformation of the model is reliable. iPTM is an important indicator for evaluating the reliability of the prediction of the interface and relative spatial arrangement of different intermolecular interactions in the complex. Its value range is generally 0 to 1. A value > 0.6 usually indicates that the interface binding mode and intermolecular orientation results are reliable.

[0045] To systematically evaluate the ability of binding peptide B176 to form complexes with members of the fibroblast growth factor family, AlphaFold3 was used to assess the conformational interaction between binding peptide B176 and FGF family members (the amino acid sequence can be the amino acid sequence of human fibroblast growth factor in CN2021107794524). Based on the software instructions and relevant application guidelines, the obtained model was comprehensively evaluated using multiple indicators.

[0046] The results are shown in Table 1. The results indicate that the binding peptide B176 has a good binding ability to fibroblast growth factor-1, fibroblast growth factor-14, and fibroblast growth factor-19.

[0047] Table 1 Example 6: Effects of binding peptide B176 on the function of human fibroblast growth factor-1

[0048] Binding peptide treatment group: A 0.5 mg / mL solution of binding peptide B176 prepared according to Example 3 was added to Dulbecco's Modified Eagle Medium / F-12 (DMEM / F-12, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 41420ES76) without serum to obtain a medium containing binding peptide B176.

[0049] FGF-1 treatment group: Add the 2 mg / mL human fibroblast growth factor-1 solution prepared in Example 4 to DMEM / F-12 to make the final concentration of human fibroblast growth factor-1 200 ng / mL, and prepare a culture medium containing fibroblast growth factor-1.

[0050] Mixed factor treatment group 1: Add 0.5 mg / mL of binding peptide B176 solution prepared according to Example 3 and 2 mg / mL of human fibroblast growth factor-1 prepared in Example 4 to DMEM / F-12, so that the final concentration of binding peptide B176 is 50 ng / mL and the final concentration of human fibroblast growth factor-1 is 200 ng / mL, to prepare a culture medium containing mixed factor 1.

[0051] Mixed factor treatment group 2: A 0.5 mg / mL solution of binding peptide B176 prepared according to Example 3 and a 2 mg / mL human fibroblast growth factor-1 prepared in Example 4 were added to DMEM / F-12 to make the final concentration of binding peptide B176 200 ng / mL and the final concentration of human fibroblast growth factor-1 200 ng / mL, thus preparing a culture medium containing mixed factor 2.

[0052] The above culture media should be placed at 4 degrees Celsius and allowed to stand for 30 minutes after preparation before use.

[0053] Human dermal fibroblasts (Wuhan Pronosei Biotechnology Co., Ltd., catalog number: CP-H103) were seeded into DMEM / F-12 medium containing 10% fetal bovine serum and cultured at 37°C with 5% CO2 for 24 hours. The original medium was discarded, and DMEM / F-12 medium containing 0.5% fetal bovine serum was added, followed by 12 hours of further culture. The medium was then discarded, and the appropriate medium was added according to the designated groups. DMEM / F-12 medium without fetal bovine serum was used as a negative control group (labeled as the control group), and the cells were cultured in an incubator for 36 hours. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT, Shanghai Beyotime Biotechnology Co., Ltd., catalog number: ST316) was added to each well, and after incubation for 4 hours, 100 μL of dimethyl sulfoxide (DMSO, Shanghai Beyotime Biotechnology Co., Ltd., catalog number: ST038) was added to each well, and the cells were thoroughly mixed with a shaker. The absorbance, or OD, was measured using an ELISA reader at a wavelength of 450 nm. 450 The results are shown below. Figure 4 . Figure 4 The results showed that, compared with the negative control group, the FGF-1 treatment group (labeled as FGF-1) had a significant ability to promote dermal cell proliferation, while the binding peptide treatment group (labeled as binding peptide B176) lacked the ability to promote cell proliferation. The mixed factor treatment group 1 (labeled as FGF-1+B176L) and the mixed factor treatment group 2 (labeled as FGF-1+B176H) showed a significant decrease in promoting dermal cell proliferation compared with the FGF-1 treatment group, and the degree of decrease increased with the increase of the binding peptide dose, indicating that the binding peptide can inactivate human fibroblast growth factor-1. Example 7: Separation of fibroblast growth factor-19 using gel chromatography with conjugated peptide B176

[0054] The recombinant strain expressing human fibroblast growth factor-19 (hFGF-19) was pET-GSFGF19-CsPA-U1p1 / BL21 (CN2021107794524). pET-GSFGF19-CsPA-U1p1 / BL21 was fermented using the same procedures as in Example 9 of CN2021107794524, and the cells were then suspended in Tris-HCl (50 mmol / L Tris-HCl buffer, 0.1 mol / L sodium chloride, pH=8.0), lysed, and centrifuged to obtain the supernatant of pET-GSFGF19-CsPA-U1p1 / BL21.

[0055] The bound peptide B176 solution was prepared according to Example 3, and then sent to Nanjing Dulai Biotechnology Co., Ltd. for small-batch preparation of gel chromatography material conjugated with bound peptide B176 for the separation and purification of fibroblast growth factor-19.

[0056] The supernatant of pET-GSFGF19-CsPA-U1p1 / BL21 was loaded onto a chromatography gel containing the conjugated peptide B176, and the elution was collected as the supernatant elution. Then, the impurities were removed with washing buffer (50 mmol / L Tris-HCl, 0.3 mol / L sodium chloride, pH=8.0), and the elution was the impurity elution buffer. The elution was then carried out with elution buffer (50 mmol / L Tris-HCl, 1.0 mol / L sodium chloride, pH=8.0) to obtain the hFGF-19 elution buffer containing the target protein.

[0057] Samples were taken at various stages of the eluent process, including the supernatant of pET-GSFGF19-CsPA-U1p1 / BL21, the supernatant eluent, the eluent containing other proteins, and the hFGF-19 eluent. SDS-PAGE electrophoresis was performed, and the results are shown below. Figure 5 . Figure 5 The results indicate that the binding peptide B176 of the present invention can be used to purify fibroblast growth factors with which it has an affinity.

Claims

1. A binding peptide having the amino acid sequence shown in SEQ ID No.

1.

2. A nucleic acid encoding the binding peptide as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The sequence of the nucleic acid is shown in SEQ ID No.

2.

4. The use of the binding peptide according to claim 1 in binding with fibroblast growth factor.

5. The application according to claim 4, characterized in that, The binding peptide is used for the purification or detection of the fibroblast growth factor.

6. The application according to claim 5, characterized in that, The binding peptide is used as an affinity chromatography packing material to purify the purified fibroblast growth factor via affinity chromatography.

7. The application according to claim 5, characterized in that, The fibroblast growth factor is detected by binding the binding peptide to the fibroblast growth factor to inactivate the fibroblast growth factor.

8. The application according to claim 5, characterized in that, The binding peptide was modified with a fluorescent group to detect the fibroblast growth factor.

9. The application according to any one of claims 4 to 8, characterized in that, The fibroblast growth factor is at least one of fibroblast growth factor-1, fibroblast growth factor-14, and fibroblast growth factor-19.

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