A recombinant fibronectin fusion protein and its application

Abstract

The present invention discloses a recombinant fibronectin fusion protein and its application. The recombinant fibronectin fusion protein includes a recombinant fibronectin mutant (FN1) and platelet factor 4 (PF4); the amino acid sequence of the recombinant fibronectin fusion protein is shown in SEQ ID NO.3 or SEQ ID NO.4. The recombinant fibronectin fusion protein of the present invention has a molecular weight of less than 90KDa, good solubility, a simple preparation process, and convenient purification; the recombinant fibronectin fusion protein provided by the present invention has a novel recombinant fibronectin, which can inhibit inflammation during wound healing, thereby efficiently promoting the healing of mouse skin injuries, and can provide new ideas for the development of wound healing drugs. Moreover, when the present invention creatively fuses platelet factor 4 to the N-terminus of the fibronectin mutant, it can significantly and effectively promote cell proliferation activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a recombinant fibronectin fusion protein and applications thereof. Background Art

[0002] As the body's first line of defense against the outside world, the skin is vulnerable to environmental damage. Skin injuries are often accompanied by inflammation and infection, and in severe cases, can be life-threatening. Skin injury healing is generally divided into inflammation, proliferation, and remodeling. To address skin injuries, a variety of drugs and wound dressings have been developed. Among them, human fibronectin (FN) plays a role in all stages of wound healing, making it an ideal material for wound healing.

[0003] Fibronectin, also known as adhesion protein, is a large glycoprotein produced by the liver and vascular tissue with a molecular weight of approximately 440 kDa. Fibronectin is an extracellular matrix protein involved in numerous physiological processes, including cell adhesion, spreading, migration, proliferation, and mobilization. Fibronectin molecules consist of three types of repeating subunits: FNI and FNII are disulfide-stabilized β-sheets, while FNIII is a tubular structure composed of seven β-sheets susceptible to mechanical deformation. Fibronectin promotes cell growth and proliferation, improves cell attachment, and mediates a wide range of wound and tissue healing, as well as peripheral neurite outgrowth. It also has a significant market as a matrix in the medical aesthetics industry. Fibronectin is primarily derived from animal tissue and blood, resulting in limited production and high costs, which severely restrict its industrial use and market value. With the advancement of genetic engineering technology, small molecule fibronectins with similar functions to natural fibronectin can be expressed through recombinant techniques. However, these proteins still suffer from large molecular weights, low recombinant expression efficiency, poor solubility, and low activity. Summary of the Invention

[0004] To address the above-mentioned problems in the prior art, the present invention provides a highly active, low-molecular-weight recombinant fibronectin fusion protein and its application. The recombinant fibronectin fusion protein provided by the present invention has a simple purification process, can effectively promote cell proliferation and adhesion, and exhibits a better promoting effect on wound healing.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In a first aspect, a recombinant fibronectin fusion protein comprises a recombinant fibronectin mutant (FN1) and platelet factor 4 (PF4); the amino acid sequence of the recombinant fibronectin fusion protein is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0007] The amino acid sequence of the recombinant fibronectin mutant is shown in SEQ ID NO. 1. To enhance the function of the recombinant fibronectin mutant, platelet factor 4 is fused to FN1. The amino acid sequence of platelet factor 4 is shown in SEQ ID NO. 2. In one embodiment, platelet factor 4 is fused to the N-terminus of FN1 to construct the recombinant fibronectin fusion protein PF4-FN1, whose amino acid sequence is shown in SEQ ID NO. 3. In another embodiment, platelet factor 4 is fused to the C-terminus of FN1 to construct the recombinant fibronectin fusion protein FN1-PF4, whose amino acid sequence is shown in SEQ ID NO. 4.

[0008] In a second aspect, a method for preparing a recombinant fibronectin fusion protein comprises the following steps:

[0009] Codon optimization was performed using Escherichia coli as a host to obtain a gene sequence of a recombinant fibronectin fusion protein with an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4, which was cloned into a pET30a vector to obtain a recombinant plasmid PF4-FN1-pET30a or FN1-PF4-pET30a. The recombinant plasmid was transformed into BL21 (DE3) competent cells, and a positive single clone was picked and inoculated into LB medium and cultured overnight to an OD value of 0.6-0.8. An inducer was added to induce expression for 12-15 hours, and the cells were collected by centrifugation.

[0010] The cells were resuspended, crushed, and centrifuged to collect the supernatant; the supernatant was filtered through a filter membrane as a crude protein sample, and the target protein was gradient eluted with an eluent containing 50-500mM imidazole. The content and purity of the target protein in each eluent were detected by SDS-PAGE gel electrophoresis; the imidazole in the buffer was replaced, and the target protein was concentrated.

[0011] In a third aspect, a use of the recombinant fibronectin fusion protein described in the first aspect in the preparation of a drug for treating skin injury healing and repair.

[0012] In a fourth aspect, a use of the recombinant fibronectin fusion protein described in the first aspect in the preparation of a drug for promoting cell adhesion and growth.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The recombinant fibronectin fusion protein of the present invention has a molecular weight below 90 kDa, good solubility, a simple preparation process, and convenient purification. The recombinant fibronectin fusion protein provided by the present invention is a novel recombinant fibronectin that can inhibit inflammation during wound healing, thereby effectively promoting the healing of mouse skin injuries and providing new insights into the development of wound healing drugs. Furthermore, the present invention innovatively fuses platelet factor 4 to the N-terminus of a fibronectin mutant, significantly and effectively promoting cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 Schematic diagram for the construction of recombinant fibronectin fusion protein;

[0017] Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified recombinant fibronectin fusion protein;

[0018] Figure 3 This is a diagram showing the effect of recombinant fibronectin on promoting cell adhesion;

[0019] Figure 4 This is a diagram showing the effect of recombinant fibronectin on crude cell proliferation;

[0020] Figure 5 Schematic diagram of recombinant fibronectin fusion protein promoting mouse skin wound healing. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0022] The term "about" as used herein refers to a range of ±20% of the value thereafter. In some embodiments, the term "about" refers to a range of ±10% of the value thereafter. In some embodiments, the term "about" refers to a range of ±5% of the value thereafter.

[0023] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of".

[0024] As used herein, the terms "optionally," "either," "arbitrarily," or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but need not be present.

[0025] The term "about" as used herein refers to a range of ±20% of the value thereafter. In some embodiments, the term "about" refers to a range of ±10% of the value thereafter. In some embodiments, the term "about" refers to a range of ±5% of the value thereafter.

[0026] The term "and / or" used in the present invention should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0027] Example 1 Expression and identification of recombinant fibronectin fusion protein

[0028] Through functional domain and efficacy analysis, specific functional domains of fibronectin (III1H, III2-4, III8-10, III13) were selected and assembled into a mutant (FN1). Platelet factor 4 (PF4, amino acid sequence as shown in SEQ ID NO. 2) was fused to its N-terminus or C-terminus to construct mutants PF4-FN1 and FN1-PF4. The fusion protein construction process is as follows Figure 1 shown.

[0029] Codon optimization was performed using Escherichia coli as the host, resulting in the gene sequences of FN1 (amino acid sequence shown in SEQ ID NO. 1), PF4-FN1 (amino acid sequence shown in SEQ ID NO. 3), and FN1-PF4 (amino acid sequence shown in SEQ ID NO. 4). These sequences were then cloned into the pET30a vector to generate recombinant plasmids FN1-pET30a, PF4-FN1-pET30a, and FN1-PF4-pET30a, respectively. The recombinant plasmids were transformed into BL21(DE3) competent cells, and positive single colonies were selected and inoculated into 10 mL of LB medium (containing kanamycin at a final concentration of 50 μg / mL). The cells were cultured overnight at 37°C and then transferred to fresh LB medium at a 1% inoculum. The cells were cultured to an OD (absorbance) of 0.6-0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was then added to induce expression at 23°C for 12-15 hours. The cells were collected by centrifugation at 6000 rpm for 10 min and then processed in the next step or temporarily stored at -20°C.

[0030] The amino acid sequence of the recombinant fibronectin fusion protein is:

[0031] MIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDAST (SEQ ID NO.1).

[0032] The amino acid sequence of platelet factor 4 is:

[0033] EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO.2).

[0034] The amino acid sequence of the recombinant fibronectin fusion protein PF4-FN1 is:

[0035] MEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLESGGGGSGGGGSIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDAST (SEQ ID NO.3).

[0036] The amino acid sequence of the recombinant fibronectin fusion protein FN1-PF4 is as follows:

[0037] MIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTGGGGSGGGGSEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO.4).

[0038] Example 2 Purification of Recombinant Fibronectin Fusion Protein

[0039] Buffer A: 20 mM PBS, 150 mM NaCl, pH 7.5.

[0040] Buffer B: 20 mM PBS, 150 mM NaCl, 500 mM imidazole, pH 7.5.

[0041] Purification was performed using an AKTA purifier connected to a Ni NTA column and a step-elution method.

[0042] The bacteria obtained in Example 1 were resuspended with PBS at a ratio of 1:10, and after resuspension, the bacteria were broken using a high-pressure homogenizer. Centrifuge at 8000rpm for 30min and collect the supernatant. The supernatant was filtered with a 0.22μm filter membrane and used as a crude protein sample, which was purified using an AKTA system and a Ni-NTA column. The target protein was gradient eluted with an eluent containing 50-500mM imidazole, and the content and purity of the target protein in each eluent were detected by SDS-PAGE gel electrophoresis. The 50KDa ultrafiltration system replaced the imidazole in the buffer with PBS and concentrated the target protein. The purified target protein was detected by SDS-PAGE, and the results were as follows: Figure 2 As shown, after purification of FN1, PF4-FN1 and FN1-PF4, the target protein bands were obvious, the molecular weight was normal, and there were no significant miscellaneous bands.

[0043] Example 3 Test of cell adhesion promoting activity of recombinant fibronectin fusion protein

[0044] The purified target proteins FN1, PF4-FN1 and FN1-PF4 were plated on cell culture plates at 20 μg / mL, with PBS as the control. Each group had 8 replicate wells and 2 non-wash control wells to observe the effect of each target protein on cell adhesion activity. Experimental method: 96-well plates were coated with 20 μg / ml of recombinant protein FN1 / PF4-FN1 / FN1-PF4 and treated at 37°C or room temperature for 1 hour. After absorbing the protein solution, the plates were blocked with 1% sterile BSA at room temperature for half an hour. HaCat cells were resuspended in culture medium, 20,000 cells were plated on each well of the 96-well plate, and cultured at 37°C for 1.5 hours. Each well was washed 3 times with PBS, and complete culture medium was added to observe the cell adhesion status. After 12 hours of culture, 10 μL of CCK8 reagent was added to each well. After incubation for 2 hours, the A450 absorbance was read with a microplate reader to determine the cell adhesion activity. The results are as follows Figure 3 As shown, compared with PBS, FN1, PF4-FN1 and FN1-PF4 all showed good cell adhesion promoting activity, among which the activity of FN1 was slightly higher than that of PF4-FN1 and FN1-PF4.

[0045] Example 4 Test of the cell proliferation promoting activity of recombinant fibronectin fusion protein

[0046] Add approximately 4-7 × 10 4HaCat cells were cultured at 37°C for 24 hours and treated with 200 μg / ml of FN1, PF4-FN1, and FN1-PF4, respectively. PBS buffer was used as a negative control and the cells were cultured at 37°C for another 48 hours. 10 μL of CCK-8 reagent was added to each well and the cells were cultured at 37°C for 2 hours. The A450 absorbance was read on a microplate reader to determine cell proliferation activity. The results are shown in Figure 2. Figure 4 As shown, compared with PBS, FN1, PF4-FN1 and FN1-PF4 all showed better cell proliferation promoting activity, among which PF4-FN1 had higher cell proliferation promoting activity than FN1 and FN1-PF4.

[0047] Example 5 Schematic diagram of recombinant fibronectin promoting wound healing in mice

[0048] A full-thickness skin wound model was constructed in mice to evaluate the effect of the protein solution on promoting wound healing in vivo. All mice were housed in an SPF-grade facility, maintained on a 12-hour light / 12-hour dark cycle, and had free access to food and water. Experiments were conducted after 7 days of adaptive feeding. Mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital. The mice were fixed in a prone position, the back was shaved, the skin was disinfected with iodine and wiped three times with 70% ethanol. A circular hole with a diameter of 8 mm was made on the back with a punch, and the full-thickness skin was excised to create a circular full-thickness skin defect wound. Pressure was applied to stop bleeding and the wound was then used for further treatment.

[0049] After surgery, all mice received local administration (smearing) of the corresponding drugs. A pipette was used to draw 0.25 mL of protein solution into a sterile 1.5 mL ep tube. A sterile cotton swab was soaked to fully absorb the protein solution, and then the drug was applied to the wound. Once a day for 7 days, the wound healing was recorded on day 0, day 3, day 5 and day 7. Among them, the control group was treated with PBS, and the experimental groups were treated with FN1 (1 μM), FN1 (10 μM), PF4-FN1 (1 μM), PF4-FN1 (10 μM), FN1-PF4 (1 μM) and FN1-PF4 (10 μM), respectively. The wound healing conditions are shown in Figure 2. Figure 5 As shown, all experimental groups showed significantly better healing rates than the experimental group. For the same protein, high concentrations resulted in faster wound healing. Of the three proteins, PF4-FN1 significantly enhanced wound healing compared to FN1 and FN1-PF4, achieving near-complete healing by day 7. This suggests that the PF4 fusion enhances FN1's wound healing activity. The greater PF4-FN1-promoting effect compared to FN1-PF4 suggests that the location of the PF4 fusion protein influences its function, with the highest biological activity observed when fused to the N-terminus of FN1.

[0050] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. A recombinant fibronectin fusion protein, characterized in that: The recombinant fibronectin fusion protein includes a recombinant fibronectin mutant and platelet factor 4; the amino acid sequence of the recombinant fibronectin fusion protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. The recombinant fibronectin fusion protein according to claim 1, characterized in that: The amino acid sequence of the recombinant fibronectin mutant is shown in SEQ ID NO.

1.

3. The recombinant fibronectin fusion protein according to claim 1, characterized in that: The amino acid sequence of the platelet factor 4 is shown in SEQ ID NO.

2.

4. Use of the recombinant fibronectin fusion protein as claimed in claim 1 in the preparation of a drug for healing and repairing skin injuries.

Images