Recombinant fibronectin fusion protein and application thereof

Abstract

The invention discloses a recombinant fibronectin fusion protein and application thereof. The recombinant fibronectin fusion protein comprises a recombinant fibronectin mutant (FN1) and a platelet factor 4 (PF4), the amino acid sequence of the recombinant fibronectin is as shown in SEQ ID NO. 3 or SEQ ID NO. 4. The molecular weight of the recombinant fibronectin fusion protein is less than 90KDa, and the recombinant fibronectin fusion protein is good in solubility, simple in preparation process and convenient to purify; the recombinant fibronectin provided by the invention has novel recombinant fibronectin, and can inhibit inflammation in a wound healing process, so that mouse skin injury healing is efficiently promoted, and a new thought can be provided for development of wound healing drugs. In addition, when the platelet factor 4 is creatively fused to the N end of the fibronectin mutant, the cell proliferation activity can be obviously and effectively promoted.

Description

Technical Field

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

[0002] As the first line of defense between the human body and the outside world, the skin is easily damaged by the external environment. Skin damage is often accompanied by inflammation and infection, and in severe cases may be life-threatening. Skin injury healing is generally divided into inflammation, proliferation and remodeling. In order to treat skin injuries, a variety of drugs and wound dressings have been developed. Among them, human fibronectin (Fibronectin, FN) plays a role in all stages of wound healing and is an ideal material for wound healing.

[0003] Fibronectin, also known as adhesion protein, is a macromolecular glycoprotein produced by the liver and vascular tissue, with a molecular weight of about 440KDa. Fibronectin is an extracellular matrix protein that participates in a variety of physiological processes, including cell adhesion, diffusion, migration, proliferation and transfer. Fibronectin molecules include three types of repeating subunits, FNI and FNII are disulfide-stabilized β-folds, while FNIII is a tubular structure of 7 strands of β-folds susceptible to mechanical deformation. Fibronectin can promote cell growth and proliferation, increase cell adhesion rate, mediate a large number of wounds and tissue healing and peripheral neurite growth, and also has a very large market as a matrix in the medical beauty industry. Fibronectin is mostly derived from animal tissues and blood, with limited production and high prices, which severely limits the use of fibronectin in the industry and its market value. With the development of genetic engineering technology, some small molecule fibronectins with similar functions to natural fibronectin can be expressed through recombinant technology, but they are still accompanied by problems such as large molecular weight, low efficiency of recombinant expression, poor solubility and poor activity. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides a highly active and 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 in the wound healing process.

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

[0006] Wherein, the amino acid sequence of the recombinant fibronectin mutant is shown in SEQ ID NO.1. In order to improve the function of the recombinant fibronectin mutant, platelet factor 4 is fused with FN1. The amino acid sequence of the 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 a recombinant fibronectin fusion protein PF4-FN1, and its 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 a recombinant fibronectin fusion protein FN1-PF4, and its amino acid sequence is shown in SEQ ID NO.4.

[0007] In a second aspect, a method for preparing a recombinant fibronectin fusion protein comprises the following steps: Using Escherichia coli as a host, codon optimization was performed 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, and the gene sequence 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, cultured overnight until the OD value was 0.6-0.8, an inducer was added to induce expression for 12-15 hours, and the bacteria were collected by centrifugation; The cells were resuspended, crushed and centrifuged to collect the supernatant; the supernatant was filtered through a filter membrane and used as a crude protein sample, and the target protein was gradient eluted with an eluent containing 50-500 mM 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.

[0008] 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.

[0009] 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.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The recombinant fibronectin fusion protein of the present invention has a molecular weight of less than 90KDa, good solubility, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is 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 paying any creative work.

[0012] Figure 1 Schematic diagram for constructing recombinant fibronectin fusion protein; Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified recombinant fibronectin fusion protein; Figure 3 This is a diagram showing the effect of recombinant fibronectin on promoting cell adhesion; Figure 4 This is a rough cell proliferation effect diagram of recombinant fibronectin fusion protein; Figure 5 Schematic diagram of recombinant fibronectin fusion protein promoting mouse skin wound healing. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0014] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0015] The term "comprises" or "comprising" as used in the present invention 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 exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0016] The terms "optionally", "any", "arbitrarily" or "any" used in the present invention mean that the event or environment described later may but need not occur, and the description includes occasions where the event or environment 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 does not have to be present.

[0017] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0018] 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.

[0019] Example 1 Expression and identification of recombinant fibronectin fusion protein Through functional domain and efficacy analysis, specific functional domains of fibronectin (III1H, III2-4, III8-10, III13) were selected to assemble into a mutant (FN1), and 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 construction process of fusion protein is as follows Figure 1 shown.

[0020] Escherichia coli was used as the host for codon optimization, and the gene sequences of FN1 (amino acid sequence as shown in SEQ ID NO.1), PF4-FN1 (amino acid sequence as shown in SEQ ID NO.3) and FN1-PF4 (amino acid sequence as shown in SEQ ID NO.4) were obtained respectively; they were cloned into the pET30a vector to obtain recombinant plasmids FN1-pET30a, PF4-FN1-pET30a, and FN1-PF4-pET30a. The recombinant plasmids were transformed into BL21 (DE3) competent cells, and the positive single clones were picked and inoculated into 10mL LB medium (containing kanamycin with a final concentration of 50μg / mL), cultured at 37℃ overnight, and transferred to fresh LB medium according to 1% inoculum, cultured to OD value (absorbance) of 0.6-0.8, and IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 1mM was added, and induced expression at 23℃ for 12-15h. The cells were collected by centrifugation at 6000 rpm for 10 min and then processed in the next step or temporarily stored in a freezer at -20 °C.

[0021] The amino acid sequence of the recombinant fibronectin fusion protein is: MIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDAST (SEQ ID NO.1).

[0022] The amino acid sequence of platelet factor 4 is: EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO.2).

[0023] The amino acid sequence of the recombinant fibronectin fusion protein PF4-FN1 is: MEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLESGGGGSGGGGSIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDAST (SEQ ID NO.3).

[0024] The amino acid sequence of the recombinant fibronectin fusion protein FN1-PF4 is as follows: MIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPVTSNTVTGETTPFSPLVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYIVNVYQISEDGEQSLILSTSQTTAPDAPPDTTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVVIQQETTGTPRSDTVPSPRDLQFVEVTDVKVTIMWTPPESAVTGYRVDVIPVNLPGEHGQRLPISRNTFAEVTGLSPGVTYYFKVFAVSHGRESKPLTAQQTTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTNVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTGGGGSGGGGSEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO.4).

[0025] Example 2 Purification of Recombinant Fibronectin Fusion Protein Buffer A: 20 mM PBS, 150 mM NaCl, pH 7.5.

[0026] Buffer B: 20 mM PBS, 150 mM NaCl, 500 mM imidazole, pH 7.5.

[0027] Purification was performed using an AKTA purifier connected to a Ni NTA column and a step-elution method.

[0028] The bacteria obtained in Example 1 were resuspended with PBS at a ratio of 1:10, and after resuspension, the bacteria were broken with 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 was 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.

[0029] Example 3 Test of the cell adhesion promoting activity of recombinant fibronectin fusion protein 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 duplicate 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 recombinant proteins 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 with culture medium, and 20,000 cells were plated in each well of the 96-well plate. After culturing 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 state. After culturing for 12 hours, 10 μL of CCK8 reagent was added to each well. After incubation for 2 hours, the A450 absorbance was read with an enzyme 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.

[0030] Example 4 Test of the cell proliferation promoting activity of recombinant fibronectin fusion protein Approximately 4-7 × 10 4 HaCat cells were cultured at 37℃ for 24h, and treated with 200μg / ml FN1, PF4-FN1 and FN1-PF4 respectively. PBS buffer was used as negative control and cultured at 37℃ for 48h. 10μL CCK-8 reagent was added to each well, and the cells were cultured at 37℃ for 2h before being taken out. The A450 absorbance was read by an ELISA reader to determine the cell proliferation activity. The results are shown in Figure 4 As shown, compared with PBS, FN1, PF4-FN1 and FN1-PF4 all showed good activity in promoting cell proliferation, among which the cell-promoting activity of PF4-FN1 was higher than that of FN1 and FN1-PF4.

[0031] Example 5 Schematic diagram of recombinant fibronectin promoting wound healing in mice skin A mouse full-thickness skin wound model was constructed to evaluate the effect of the protein solution on promoting wound healing in vivo. All mice were housed in an SPF-grade facility, managed according to a 12-hour light / 12-hour dark cycle, and had free access to food and water. The experiment was conducted after 7 days of adaptive feeding. The mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, fixed in a prone position, shaved on the back, 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 removed to make a circular full-thickness skin defect wound, which was pressed to stop bleeding for later use.

[0032] After surgery, all mice received local administration (application) 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 allow it to fully absorb the protein solution, and then applied to the wound. Once a day for 7 days, photos were taken to record the wound healing conditions 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 the figure. Figure 5 As shown, the healing speed of all experimental groups was significantly better than that of the experimental group. For the same protein, the wound healing speed was faster when treated with high concentration. Among the three proteins, the wound healing effect of PF4-FN1 was significantly better than that of FN1 and FN1-PF4. It was basically completely healed on the 7th day, indicating that the fusion of PF4 was helpful for the promotion of FN1's wound healing activity. The promotion effect of PF4-FN1 was better than that of FN1-PF4, which means that the position of PF4 fusion has a certain influence on the function of the fusion protein. When fused to the N-terminus of FN1, it has the highest biological activity.

[0033] Obviously, the embodiments described above are only some embodiments of the present application, rather than all 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 is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this 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 treating skin injury healing and repair.

5. Use of the recombinant fibronectin fusion protein as claimed in claim 1 in the preparation of a drug for promoting cell adhesion and growth.

Images