Recombinant human FGF4 protein and its application in skin wound repair

Abstract

The present invention discloses a recombinant human FGF4 protein, which is an amino acid sequence as shown in SEQ ID NO: 1, 2, 3 or 4, or a protein derived from the above amino acid sequence by substitution, deletion or addition of one or several amino acids and having the activity of a recombinant human FGF4 protein; the activity of the protein provides a function capable of promoting keratinocyte migration. The present invention also discloses the use of the recombinant human FGF4 protein or a composition thereof in the preparation of a drug or kit capable of promoting keratinocyte migration and promoting skin wound recovery. The present invention discovered through in vivo and in vitro pharmacological experiments that the recombinant human FGF4 protein (rFGF4) can promote keratinocyte migration, significantly promote the healing of acute wounds and chronic diabetic wounds, and improve the quality of wound healing. Therefore, FGF4 can be prepared for use in skin wounds, especially chronic, refractory wounds.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant human FGF4 protein and an application thereof in skin wound repair. Background Art

[0002] As the largest organ in the human body, the skin is an important barrier between internal organs and the external environment, maintaining the homeostasis of the body's internal environment. The skin is in long-term contact with the external environment and is inevitably exposed to various damaging stimuli. If the damaged skin is not repaired in time, the body is extremely vulnerable to pathogens, causing secondary infections and even death. With the increasing aging of the population, the incidence of skin wounds, especially chronic and difficult-to-heal wounds such as diabetic foot, pressure sores, and venous ulcers, is increasing. The disease has a complex pathogenesis, a long course of disease, and a high disability rate. There is currently no effective treatment option, which is a difficult problem facing clinical work.

[0003] The fibroblast growth factor (FGF) family is one of the largest growth factor families in living organisms, comprising 22 members. It holds broad promise for application in tissue repair and regeneration. Paracrine FGF2 has been found to be induced in wounded skin and to have a significant role in promoting wound healing. While the FGF family is extensive, the role of other FGFs in skin wound repair is limited. Currently, there are no reports of FGF4 protein being used for wound repair. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a recombinant human FGF4 protein.

[0006] Another object of the present invention is to provide a method for producing recombinant human FGF4 protein.

[0007] Another object of the present invention is to provide the use of the recombinant human FGF4 protein or the composition in the preparation of a drug or kit capable of promoting keratinocyte migration and promoting skin wound recovery.

[0008] To this end, the technical solution provided by the present invention is:

[0009] A recombinant human FGF4 protein, which is a protein of a) or b) or c) or d) or e) as follows:

[0010] a) the amino acid sequence shown in SEQ ID NO: 1,

[0011] b) the amino acid sequence shown in SEQ ID NO: 2,

[0012] c) the amino acid sequence shown in SEQ ID NO: 3,

[0013] d) the amino acid sequence shown in SEQ ID NO: 4,

[0014] e) a protein derived from a) or b) or c) or d) having the activity of a recombinant human FGF4 protein, wherein the amino acid sequence in a) or b) or c) or d) is substituted, deleted or added with one or more amino acids;

[0015] The activity of the recombinant human FGF4 protein provides the function of promoting keratinocyte migration.

[0016] A composition containing one or more of the recombinant human FGF4 proteins.

[0017] A DNA molecule encoding the recombinant human FGF4 protein.

[0018] A recombinant vector contains the DNA molecule and a regulatory sequence for expression that is operably linked to the DNA molecule.

[0019] A host cell containing the DNA molecule or the recombinant vector.

[0020] The method for producing recombinant human FGF4 protein comprises the following steps: culturing the host cells in a culture medium, and producing the recombinant human FGF4 protein encoded by the recombinant human FGF4 protein gene contained in the recombinant vector in the host cells.

[0021] Preferably, the method for producing the recombinant human FGF4 protein further comprises the following step: purifying the produced recombinant human FGF4 protein.

[0022] The use of the recombinant human FGF4 protein or the composition in the preparation of a drug or a kit capable of promoting keratinocyte migration and promoting skin wound recovery.

[0023] Preferably, in the application, the skin wounds include acute skin wounds and chronic diabetic wounds, the acute skin wounds include mechanical injuries, traffic injuries, surgical wounds and burn wounds, and the chronic diabetic wounds include diabetic foot, pressure sores and venous ulcers.

[0024] Preferably, the application also includes the aspect of plastic surgery wound repair.

[0025] The present invention has at least the following beneficial effects:

[0026] Through in vitro and in vivo pharmacological experiments, the present invention discovered that recombinant human FGF4 protein (rFGF4) can promote keratinocyte migration, significantly accelerate the healing of acute wounds and chronic diabetic wounds, and improve the quality of wound healing. Therefore, FGF4 can be prepared for use on skin wounds, especially chronic, refractory wounds.

[0027] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is one of the gel images showing the protein purity detected by SDS-PAGE electrophoresis after purification of the protein with the amino acid sequence shown in SEQ ID NO: 4 in one embodiment of the present invention.

[0029] Figure 2 This is a graph showing the results of the recombinant human FGF4 protein (SEQ. ID NO: 1) in one embodiment of the present invention promoting keratinocyte migration.

[0030] Figure 3 This is a graph showing the results of promoting keratinocyte migration by the recombinant human FGF4 protein (SEQ.ID NO: 2) in one embodiment of the present invention.

[0031] Figure 4 This is a graph showing the results of promoting keratinocyte migration by the recombinant human FGF4 protein (SEQ.ID NO: 3) in one embodiment of the present invention.

[0032] Figure 5 This is a graph showing the results of the recombinant human FGF4 protein (SEQ.ID NO: 4) in one embodiment of the present invention promoting keratinocyte migration.

[0033] Figure 6 This is a diagram showing the effect of the recombinant human FGF4 protein (SEQ.ID NO: 4) in one embodiment of the present invention on promoting acute wound repair.

[0034] Figure 7 This is a diagram showing the effect of the recombinant human FGF4 protein (SEQ.ID NO: 4) in one embodiment of the present invention on promoting the repair of refractory diabetic wounds. DETAILED DESCRIPTION

[0035] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0036] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0037] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0038] The present invention provides a recombinant human FGF4 protein, which is a protein of the following a) or b) or c) or d) or e):

[0039] a) the amino acid sequence shown in SEQ ID NO: 1,

[0040] b) the amino acid sequence shown in SEQ ID NO: 2,

[0041] c) the amino acid sequence shown in SEQ ID NO: 3,

[0042] d) the amino acid sequence shown in SEQ ID NO: 4,

[0043] e) A protein derived from a) or b) or c) or d) having the activity of a recombinant human FGF4 protein, wherein the amino acid sequence in a) or b) or c) or d) is substituted, deleted or added with one or more amino acids; such as multiple N-terminally truncated recombinant human rFGF4 proteins and mutants thereof, or a protein having more than 70% similarity to the amino acid sequence of the present invention.

[0044] The activity of the recombinant human FGF4 protein provides the function of promoting keratinocyte migration.

[0045] The present invention also provides a composition containing one or more of the aforementioned recombinant human FGF4 proteins. Preferably, a protein comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:4 can be used alone. Alternatively, a protein comprising the amino acid sequence set forth in SEQ ID NO:1 and derivatives thereof can be used, or a protein comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence set forth in SEQ ID NO:2, and derivatives thereof can be used. Alternatively, a protein comprising the amino acid sequence set forth in SEQ ID NO:2 and the amino acid sequence set forth in SEQ ID NO:3 can be used. These are not listed here one by one; one or more of the aforementioned recombinant human FGF4 proteins can be selected for use according to actual needs.

[0046] The present invention also provides a DNA molecule encoding the recombinant human FGF4 protein. This DNA molecule, also known as a "gene," is the primary agent of genetic variation and the fundamental genetic unit controlling biological traits. The base sequence within a gene that encodes RNA or protein is called a structural gene. The DNA molecule referred to in the present invention is a structural gene.

[0047] The present invention also provides a recombinant vector comprising the DNA molecule and a regulatory sequence for expression operably linked to the DNA molecule. A "recombinant vector" refers to an expression vector to which a gene has been linked and which can replicate in a host cell.

[0048] The present invention also provides a host cell containing the DNA molecule or the recombinant vector. The host cell refers to a cell in which a target gene is cloned or a protein is expressed.

[0049] The present invention also provides a method for producing a recombinant human FGF4 protein, comprising the steps of culturing the host cells in a culture medium, and producing the recombinant human FGF4 protein encoded by the recombinant human FGF4 protein gene contained in a recombinant vector in the host cells. Preferably, the cells expressing the recombinant human FGF4 protein are collected, lysed, and the supernatant containing the recombinant human FGF4 protein is collected.

[0050] In the above scheme, preferably, the following step is further included: purifying the produced recombinant human FGF4 protein. Preferably, the collected supernatant containing the recombinant human FGF4 protein can be purified using a heparin affinity chromatography column.

[0051] The present invention also provides use of the recombinant human FGF4 protein or the composition in preparing a drug or a kit capable of promoting keratinocyte migration and promoting skin wound recovery.

[0052] In the above scheme, preferably, the skin wounds include acute skin wounds and chronic diabetic wounds. The acute skin wounds include mechanical injuries, traffic injuries, surgical wounds and burn wounds. The chronic diabetic wounds include diabetic foot, pressure sores and venous ulcers.

[0053] In the above scheme, preferably, the application also includes (protein derivative field) plastic surgery wound repair.

[0054] In the above scheme, as a preference, it also includes applications in protein derivative fields such as burns and scalds.

[0055] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following examples are provided for illustration: Example 1

[0056] Genetic engineering expression and purification of multiple recombinant human FGF4 proteins (SEQ.ID NO: 1, SEQ.ID NO: 2, SEQ.ID NO: 3 and SEQ.ID NO: 4)

[0057] A cDNA fragment encoding human FGF4 (Met1-Leu206, Ser54-Leu206, Ala31-Leu206, and Ala67-Leu206) was amplified by PCR and cloned into an E. coli expression vector using the NcoI and XhoI restriction endonuclease sites. The FGF4 expression vector was added to competent E. coli BL-21 (DE3) cells in LB liquid medium containing 2% glucose and 30 mg / mL kanamycin and incubated at 37°C, 200 rpm for amplification. When the OD at 600 nm was 0.8-1.0, 1 ml of the culture was aspirated to prepare a pre-induction sample with a final concentration of 1 mM. IPTG (1 M IPTG, used at a ratio of 1:1000) was added to induce recombinant protein expression. The cells were incubated at 37°C for 4 hours to prepare a post-induction sample.

[0058] The cells were harvested using a large-capacity homogenizer and lysed in 25 mM NaCl containing 300 mM NaCl. + / K + Phosphate buffer (pH 7.5) was used at a ratio of 40 ml phosphate buffer per 800 ml bacterial culture. Cells were disrupted using a cell sonicator, and the lysate was centrifuged at 4°C, 15,000 rpm for 30 minutes to obtain the supernatant, and the precipitate was discarded.

[0059] Heparin affinity chromatography column (HiTrap TM Heparin HP, GE Healthcare) was pre-equilibrated in buffer A (150 mM NaCl, 25 mM HEPES, pH 7.5), and the recombinant protein was then purified using a heparin affinity chromatography column. The bound rFGF4 was eluted using a linear gradient of 0-2 M NaCl in 25 mM HEPES (pH 7.5). In this example, a linear gradient of 25 mM HEPES (pH 7.5) containing 2 M NaCl was used for the eluent, and the proportion of the eluent in buffer A gradually increased from 0% to 100% within 40 min. The purity of the purified protein was detected by SDS-PAGE electrophoresis, and the test results are shown in FIG. Figure 1 The protein was concentrated using a gel filtration column (SuperdexTM-75GE Healthcare, Piscataway, NJ) in buffer C (1 M NaCl, 25 mM Tris-HCl, pH 8.0) to obtain recombinant human FGF4 protein with a purity of >98%.

[0060] >SEQ.ID NO:1(Met1-Leu206)

[0061] MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESLVALSLARLPVAAQPCEAAVQSGAGDYLLGIKRRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIALSKNGKTKKNRVSPTMKVTHFLPRPL

[0062] >SEQ.ID NO:2(Ser54-Leu206)

[0063] SLARLPVAAQPCEAAVQSGAGDYLLGIKRRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIALSKNGKTKKGNRVSPTMKVTHPLPRL

[0064] >SEQ.ID NO:3(Ala31-Leu206)

[0065] AAPTAPNGTLEAELERRWESLVALSLARLPVAAQPCEAAVQSGAGDYLLGIKRRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIALSKNGKTKKGNRVSPTMKVTHPLPRL

[0066] >SEQ.ID NO:4(Ala67-Leu206)

[0067] MAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIALSKNGKTKKGNRVSPTMKVTHLFLPRL

[0068] Example 2

[0069] Keratinocyte migration assay simulates the effect of rFGF4 (SEQ.ID NO:1) on wound healing in vitro

[0070] Human keratinocytes (HaCaT) were counted and seeded in equal numbers in 24-well plates. They were cultured in a cell culture incubator until the cells adhered and confluent into a monolayer. A straight line was drawn through the confluent monolayer using a P200 micropipette tip to create a cellular "wound." Culture medium (Minimum Essential Medium (MEM) basal culture medium + 1% FBS + 1% penicillin and streptomycin) containing 50 ng / mL rFGF4 was added to the HaCaT cells. A PBS vehicle was used as a control. Cell migration was observed and imaged using an inverted microscope on the day of culture (0 h) and 48 h later.

[0071] The experimental results are as follows Figure 2 As shown, after 48 hours of treatment with 50 ng / ml rFGF4, the healing rate of the "scratch wound" of HaCaT cells was significantly faster than that of the PBS control group, and the statistical results of the migration area showed statistically significant differences, indicating that rFGF4 (SEQ.ID NO: 1) can promote the migration of keratinocytes.

[0072] Example 3

[0073] Keratinocyte migration assay simulates the effect of rFGF4 (SEQ.ID NO: 2) on wound healing in vitro

[0074] The specific experimental method is the same as in Example 2. The experimental results are as follows Figure 3 As shown, after 48 hours of treatment with 50 ng / ml rFGF4, the healing rate of the "scratch wound" of HaCaT cells was significantly faster than that of the PBS control group, and the statistical results of the migration area showed statistically significant differences, indicating that rFGF4 (SEQ.ID NO: 2) can promote the migration of keratinocytes.

[0075] Example 4

[0076] Keratinocyte migration assay simulates the effect of rFGF4 (SEQ.ID NO:3) on wound healing in vitro

[0077] The specific experimental method is the same as in Example 2. The experimental results are as follows Figure 4 As shown, after 48 hours of treatment with 50 ng / ml rFGF4, the healing rate of the "scratch wound" of HaCaT cells was significantly faster than that of the PBS control group, and the statistical results of the migration area showed statistically significant differences, indicating that rFGF4 (SEQ.ID NO: 3) can promote the migration of keratinocytes.

[0078] Example 5

[0079] Keratinocyte migration assay simulates the effect of rFGF4 (SEQ.ID NO:4) on wound healing in vitro

[0080] The specific experimental method is the same as in Example 2. The experimental results are as follows Figure 5 As shown, after 48 hours of treatment with 50 ng / ml rFGF4, the healing rate of the "scratch wound" of HaCaT cells was significantly faster than that of the PBS control group, and the statistical results of the migration area showed statistically significant differences, indicating that rFGF4 (SEQ.ID NO: 4) can promote the migration of keratinocytes.

[0081] Example 6

[0082] Study on the effect of rFGF4 (SEQ.ID NO:4) on the repair of acute skin wounds

[0083] Male C57BL / 6 mice (8 weeks old) were randomly divided into two groups: a control group (PBS vehicle) and a rFGF4-treated group (300 ng), with 10 mice in each group. The dorsal hair of the mice was shaved using a shaver and depilatory cream to expose the dorsal skin. A full-thickness incision was then made in the dorsal skin using a sterile 8 mm diameter skin biopsy punch. The drug was administered once daily for 11 days using a wound application.

[0084] The experimental results are as follows Figure 6 As shown, the healing rate of skin wounds in the rFGF4-treated group was significantly accelerated compared to the PBS control group (83% vs 97%). In addition, H&E and Masson staining of wound tissue sections showed that the skin wounds of mice in the rFGF4-treated group showed shorter scar width and higher collagen deposition levels, indicating that rFGF4 can significantly promote acute wound healing.

[0085] Example 7

[0086] Study on the effect of rFGF4 (SEQ.ID NO:4) on the repair of diabetic refractory wounds

[0087] Male db / db diabetic mouse model: 1. Genetic Background: C57BL / 6J mice with Leptin receptor gene knockout, resulting in homozygous infertility; 2. Characteristics: Susceptible to obesity, rapid weight gain, decreased activity, and high blood lipids; 3. Application: This model is used for T2D diabetes drug screening, physiological and biochemical indicators of obesity, and drug treatment research.

[0088] Male db / db diabetic mice (8 weeks old) were randomly divided into two groups: a control group (PBS vehicle) and a rFGF4-treated group (300 ng per wound site), with 10 mice in each group. The dorsal hair of the mice was shaved using a shaver and depilatory cream to expose the dorsal skin. A full-thickness incision was then made in the dorsal skin using a sterile 6 mm diameter skin biopsy punch. The drug was administered once daily for 15 days using a wound application.

[0089] The experimental results are as follows Figure 7 As shown in the results, compared with the PBS control group, rFGF4 can significantly promote the wound healing rate of diabetic mice. The wound healing rate of the rFGF4-treated group was about 93%, while the wound healing rate of the control group was about 75%. Figure 7 In addition, H&E staining of wound tissue on day 15 showed that the wound surface of the treatment group had a higher level of re-epithelialization. More importantly, multiple new hair follicles and other skin appendages appeared around the wound (arrows, Figure 7 At the same time, Masson staining results showed that rFGF4 could promote collagen deposition near the wound ( Figure 7 These experimental results indicate that rFGF4 can significantly improve the speed and quality of diabetic wound healing.

[0090] The number of modules and processing scales described herein are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

[0091] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Use of recombinant human FGF4 protein in the preparation of a drug or kit for promoting the recovery of acute skin wounds and chronic diabetic wounds, wherein the amino acid sequence of the recombinant human FGF4 protein is shown in SEQ ID NO: 4.

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