Recombinant fibronectin and application thereof

Abstract

The invention relates to recombinant fibronectin and application thereof, and the recombinant fibronectin comprises an amino acid fragment as shown in SEQ ID No.4. The recombinant fibronectin disclosed by the invention has the capability of promoting cell adhesion, extension and growth, and also has a relatively good inhibiting and killing effect on common pathogenic bacteria (such as staphylococcus aureus causing acne and other skin problems) causing skin infection.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant fibronectin and an application thereof. Background Art

[0002] Antimicrobial peptides are a class of small molecule polypeptides that exist in organisms and can resist the invasion of external microorganisms. They are an important part of the innate immune system. Most antimicrobial peptides have antimicrobial activity and have inhibitory and killing effects on Gram-positive and Gram-negative bacteria, fungi and enveloped viruses. Plectasin is the first fungal defensin isolated from Pseudopleceania nigrella. It inhibits the synthesis of cell walls by interacting with peptidoglycan precursor lipid II to achieve the purpose of sterilization. Studies have found that Plectasin has antibacterial activity against Gram-positive bacteria, especially Streptococcus and Staphylococcus. Staphylococcus aureus often attacks human skin, soft tissues, blood and other tissues and organs, colonizes in many parts of the body, and causes host infection. Atopic dermatitis caused by Staphylococcus aureus is one of the most common skin diseases in children.

[0003] Fibronectin, also known as fibronectin (FN), is a high molecular weight (about 440 kDa) glycoprotein of the extracellular matrix that binds to integrin transmembrane receptor proteins. Similar to integrins, fibronectin binds to extracellular matrix components such as collagen, fibrin and heparin sulfate proteoglycans (such as syndecan). Fibronectin is a protein dimer composed of two almost identical monomers connected by a pair of disulfide bonds at the C-terminus. Each fibronectin subunit has a molecular weight of 230-250 kDa and contains three types of motifs: type I, type II and type III. These motifs are arranged into several functional and protein-binding domains along the length of the fibronectin monomer. Fibronectin has four protein domains that allow fibronectin to bind to other fibronectin molecules. One of the fibronectin domains, I1-5, is called the "assembly domain", which is necessary for the establishment of fibronectin matrix assembly. Motif III9-10 corresponds to the "cell binding domain" of fibronectin. The RGD sequence (Arg-Gly-Asp) is located in III10, which is the cell attachment site on the cell surface through α5β1 and αVβ3 integrins. This "synergistic site" is in the III9 motif, which can mediate the synergistic effect of fibronectin and α5β1 integrin (also known as integrin).

[0004] Fibronectin has many biological functions, such as being essential for wound healing and embryonic development. Cellular fibronectin aggregates in the extracellular matrix, an insoluble network that separates and supports the organs and tissues of an organism. Fibronectin and fibrin are deposited at the site of injury to form blood clots, stop bleeding and protect subcutaneous tissue. Secondly, FN plays an important role in cell adhesion, extension, growth, migration and differentiation. Fibronectin has a wide range of applications and a huge market in medicine, cosmetics and scientific research, but the production of natural fibronectin extracted from human or animal blood and tissues is extremely limited and expensive. And because the fibronectin molecule is too large, it is difficult to prepare fibronectin using DNA recombinant technology, and the resulting fibronectin has low biological activity. Therefore, the production of FN and its application in many aspects are limited. Summary of the invention

[0005] Based on this, one embodiment of the present application requires a recombinant fibronectin that has strong biological activity and antibacterial function.

[0006] The technical solutions include the following:

[0007] The recombinant fibronectin comprises the amino acid fragment shown in SEQ ID No.4.

[0008] A nucleic acid molecule encoding the recombinant fibronectin.

[0009] In one embodiment, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.8.

[0010] An expression vector comprising the nucleic acid molecule.

[0011] In one embodiment, the expression vector comprises a plasmid.

[0012] A host cell, which expresses the recombinant fibronectin, comprises the nucleic acid molecule or the expression vector.

[0013] In one embodiment, the host cell comprises a eukaryotic cell.

[0014] In one embodiment, the eukaryotic cell comprises Pichia pastoris.

[0015] The method for preparing the recombinant fibronectin comprises culturing the host cells, and separating and purifying the recombinant fibronectin expressed by the host cells.

[0016] The use of the recombinant fibronectin in one or more of the following:

[0017] (1) Application in the preparation of products that promote cell adhesion, extension and growth;

[0018] (2) Application in the preparation of drugs for promoting transdermal absorption; and

[0019] (3) Application in the preparation of products for inhibiting bacterial growth.

[0020] A cosmetic product comprising the recombinant fibronectin.

[0021] Compared with the traditional technology, this application has the following beneficial effects:

[0022] The present application intercepts a part of the most important functional region of fibronectin, and connects the transdermal enhancement peptide TD-1, the antimicrobial peptide Plectasin and the fibronectin FN through a connecting peptide, and the connection sequence is TD-1-FN-Plectasin. The obtained recombinant fibronectin with antimicrobial function has the biological function and activity of fibronectin and a small molecular weight, which can effectively solve the difficulties in preparing fibronectin by DNA recombinant technology and the technical defects of low biological activity of the prepared fibronectin.

[0023] The recombinant fibronectin of the present application has the ability to promote cell adhesion, extension and growth, and at the same time has a good inhibitory and killing effect on common pathogenic bacteria that cause skin infections (such as Staphylococcus aureus that causes skin problems such as acne). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0025] Figure 1 A schematic diagram of the design concept provided for Example 1;

[0026] Figure 2 The purification status of recombinant fibronectin; M stands for Maker; 1 is the supernatant before induction of expression; 2 is the supernatant after 72h of induction of expression; 3 is the protein sample after affinity chromatography;

[0027] Figure 3 To detect the cell adhesion activity of recombinant fibronectin;

[0028] Figure 4 The antibacterial effect of recombinant fibronectin, Figure A shows Staphylococcus aureus (1-4 are repeated in parallel), and Figure B shows methicillin-resistant Staphylococcus aureus (1-6 are repeated in parallel);

[0029] Figure 5The figures are a comparison of the antibacterial activity of recombinant fibronectin constructed in different sequence connection orders. Figures AE are the antibacterial results of recombinant fibronectin rFN-1 to rFN-5, and Figure F is the antibacterial result of the recombinant fibronectin TD-1-FN-Plectasin in the present invention. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0032] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] In the present application, “preferred” is only used to describe an implementation method or example with better effects. It should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0034] The present application study found that the fusion protein (SEQ ID No. 4) formed by sequentially connecting a transdermal functional short peptide (TD-1, SEQ ID No. 1), a fibronectin domain (FN, SEQ ID No. 2), and Plectasin (SEQ ID No. 3) through a connecting peptide (GGGGS) can significantly enhance the transdermal effect of the fusion protein, enhance the antibacterial ability, and promote cell adhesion, extension and growth ability.

[0035] One embodiment of the present application provides a recombinant fibronectin with antibacterial function, wherein the recombinant fibronectin includes an amino acid fragment shown in SEQ ID No.4.

[0036] The fusion protein (SEQ ID No.4) formed by sequentially connecting SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 through a connecting peptide has better antibacterial ability and the ability to promote cell adhesion, extension and growth than fusion proteins connected in other ways.

[0037] The transdermal fibronectin TD-1-FN-Plectasin of the present application has the ability to promote cell adhesion, extension and growth. Due to the addition of the transdermal peptide TD-1 sequence and the antimicrobial peptide Plectasin sequence, the recombinant fibronectin also has better transdermal absorption effect and the ability to inhibit and kill common skin pathogens such as Staphylococcus aureus, and has multiple functions of promoting skin repair and maintaining skin health.

[0038] In a specific example, the amino acid fragment is at least 90%, at least 95%, at least 98%, at least 99% or 100% homologous to the amino acid fragment shown in SEQ ID No. 4 after substitution, deletion or addition of one or more amino acids, and has the same or similar functions.

[0039] In a specific example, a nucleic acid molecule encodes the above-mentioned recombinant fibronectin with antibacterial function.

[0040] In a preferred specific example, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.4.

[0041] One embodiment of the present application also provides an expression vector, comprising the above-mentioned nucleic acid molecule, in which the nucleic acid molecule is operably connected to one or more regulatory nucleotide sequences.

[0042] The term "expression vector" refers to a nucleic acid carrier into which a nucleic acid molecule can be inserted. When an expression vector can express the protein encoded by the inserted nucleic acid molecule, it is called an expression plasmid. The expression plasmid can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Plasmids are generally well known to those skilled in the art. In some embodiments, the recombinant plasmid in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0043] In a specific example, in addition to the above nucleic acid molecules, the expression vector may also include nucleotide sequences such as promoters, enhancers, non-coding regions, etc., to achieve the purpose of improving the expression level, expression efficiency, product activity and other aspects of the gene performance.

[0044] In a specific example, the expression vector includes one or more of a virus and a plasmid.

[0045] In one specific example, the plasmid includes pPIC9K.

[0046] One embodiment of the present application further provides a host cell, which expresses the above-mentioned recombinant fibronectin, including the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0047] The term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into a host cell to construct a recombinant cell, which is then used to express the recombinant fibronectin provided in the present application. The recombinant fibronectin can be obtained by culturing the recombinant cell.

[0048] In one embodiment, the host cell includes a eukaryotic cell.

[0049] In a specific example, the eukaryotic cell includes a yeast cell, an insect cell, or a mammalian cell.

[0050] Optionally, the yeast cell comprises Pichia pastoris.

[0051] It is understood that, in other examples, the host cell may also be other eukaryotic cells other than those listed above.

[0052] One embodiment of the present application also provides a method for preparing the above-mentioned recombinant fibronectin, comprising culturing the host cells, and isolating and purifying the recombinant fibronectin expressed by the host cells.

[0053] In one specific example, purification is performed by phenyl gel column and / or anion exchange chromatography.

[0054] One embodiment of the present application also provides the use of the above-mentioned recombinant fibronectin with antibacterial function in one or more of the following:

[0055] (1) Application in the preparation of products that promote cell adhesion, extension and growth;

[0056] (2) Application in the preparation of drugs that promote transdermal absorption;

[0057] (3) Application in the preparation of products for inhibiting bacterial growth.

[0058] One embodiment of the present application also provides a cosmetic product, comprising the above-mentioned recombinant fibronectin.

[0059] This product has the function of repairing the skin, killing harmful bacteria in the skin and regulating the balance of skin flora. It can be used in repairing and anti-inflammatory beauty.

[0060] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0061] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0062] Example 1

[0063] 1. Obtaining the target fragment and expression vector of recombinant fibronectin with antibacterial function (the expression vector is labeled as: TD-1-FN-Plectasin)

[0064] Figure 1 This is a schematic diagram of the design ideas provided in the embodiment. The commercial vector pPIC9K is used (including the location of the functional region of pPIC9K; the sequence map of pPIC9K, the restriction sites are designed according to the position of the pPIC9K related sequence; the structure of the target fragment of the recombinant fibronectin with antibacterial function). TD-1 is connected to the C-terminus of fibronectin and connected by the connecting peptide GGGGS.

[0065] The amino acid sequence of TD-1 is SEQ ID No. 1: ACSSSPSKHCG, and the nucleotide sequence thereof is SEQ ID No. 5: GCGTGTTCTAGCAGTCCTAGTAAACACTGCGGC.

[0066] The amino acid sequence of fibronectin is SEQ ID No. 2: IQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTGSAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT, and its nucleotide sequence is SEQ ID No. 6: ATCCAGTGGAACGCGCCACAGCCGAGCCACATATCAAAATATATTCTTAGGTGGCGTCCCAAAAATAGTGTTGGCAGATGGAAAGAAGCTACAATCCCTGGACATCTAAACAGCTACACCATCAAGGGATTGAAACCTGGCGTCGTGTATGAGGGGCAACTTATCTCTATACAGCAATATGGTCACCAAGAAGTCACGAGGTTCGACTTTACCACCACGAGTACTAGCACTGGCTCCGCTGTGCCTCCGCCCACCGACTTACGGTTTACAAACATAGGACCAGATACCATGAGAGTAACTTGGGCACCCCCCCCGTCCATCGATTTGACGAACTTCCTGGTGCGTTACTCCCCAGTCAAGAACGAAGAAGACGTAGCCGAGCTTTCAATTTCGCCCTCAGATAATGCGGTAGTGTTGACAAATCTACTCGTCTCGGACGTACCTCGCGACTTAGAGGTTGTTGCAGCAACTCCAACCTCTCTCCTGATATCGTGGGATGCTCCAGCCGTCACGGTTCGATACTATCGTATAACGTACGGCGAGACGGGTGGAAACTCCCCGGTACAAGAATTTACTGTACCGGGGTCCAAATCTACCGCAACTATCAGCGGCCTAAAACCTGGGGTGGATTATACAATTACAGTTTACGCCGTCACTGGACGAGGTGACTCTCCCGCGTCGTCAAAACCGATTTCTATTAATTACAGGACT。

[0067] The amino acid sequence of the antimicrobial peptide Plectasin is SEQ ID No.3: GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY, and the nucleotide sequence thereof is SEQ ID No.7: GGCTTTGGTTGTAATGGGCCCTGGGATGAAGACGATATGCAGTGTCACAATCATTGTAAGAGTATTAAGGGGTACAAGGGTGGGTACTGCGCTAAGGGTGGATTCGTTTGCAAGTGCTAT.

[0068] Therefore, the amino acid sequence of the target recombinant fibronectin fragment with antibacterial function is SEQ ID No.4: ACSSSPSKHCGGGGGSIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTGSAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGGGSGFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY, and its nucleotide sequence is SEQ ID No.8: GCGTGTTCTAGCAGTCCTAGTAAACACTGCGGCGGTGGGGGGGGATCAATCCAGTGGAACGCGCCACAGCCGAGCCACATATCAAAATATATTCTTAGGTGGCGTCCCAAAAATAGTGTTGGCAGATGGAAAGAAGCTACAATCCCTGGACATCTAAACAGCTACACCATCAAGGGATTGAAACCTGGCGTCGTGTATGAGGGGCAACTTATCTCTATACAGCAATATGGTCACCAAGAAGTCACGAGGTTCGACTTTACCACCACGAGTACTAGCACTGGCTCCGCTGTGCCTCCGCCCACCGACTTACGGTTTACAAACATAGGACCAGATACCATGAGAGTAACTTGGGCACCCCCCCCGTCCATCGATTTGACGAACTTCCTGGTGCGTTACTCCCCAGTCAAGAACGAAGAAGACGTAGCCGAGCTTTCAATTTCGCCCTCAGATAATGCGGTAGTGTTGACAAATCTACTCGTCTCGGACGTACCTCGCGACTTAGAGGTTGTTGCAGCAACTCCAACCTCTCTCCTGATATCGTGGGATGCTCCAGCCGTCACGGTTCGATACTATCGTATAACGTACGGCGAGACGGGTGGAAACTCCCCGGTACAAGAATTTACTGTACCGGGGTCCAAATCTACCGCAACTATCAGCGGCCTAAAACCTGGGGTGGATTATACAATTACAGTTTACGCCGTCACTGGACGAGGTGACTCTCCCGCGTCGTCAAAACCGATTTCTATTAATTACAGGACTGGTGGGGGGGGATCAGGCTTTGGTTGTAATGGGCCCTGGGATGAAGACGATATGCAGTGTCACAATCATTGTAAGAGTATTAAGGGGTACAAGGGTGGGTACTGCGCTAAGGGTGGATTCGTTTGCAAGTGCTAT。.

[0069] In this example, the commercial vector pPIC9K ( Figure 1 ) was used, which was purchased from Wuhan Miaoling Biotechnology Co., Ltd. According to Figure 1The restriction sites EcoR I and Not I were selected for the design of the relevant sequence positions. The gene sequences encoding the recombinant fibronectin are all artificially optimized Pichia pastoris preferred codons and are obtained by artificial synthesis. The 5' and 3' ends of the synthesized recombinant fibronectin full-length DNA fragment each have a restriction endonuclease site, corresponding to EcoR I and Not I, respectively. The target fragment of the recombinant fibronectin will be inserted between these two restriction sites to obtain an expression plasmid encoding the recombinant fibronectin. The recombinant DNA fragment was commissioned to Guangzhou Ruibo Biotechnology Co., Ltd. to synthesize and construct a recombinant expression plasmid.

[0070] 2. Expression, purification and electrophoresis identification of recombinant fibronectin

[0071] 1. Preparation of Pichia pastoris competent cells

[0072] (1) Take the original strain of Pichia pastoris stored at -80℃ and activate it by streaking on YPD medium. Pick two monoclonal strains from the newly activated Pichia pastoris plate and inoculate them into a 250mL conical flask containing 40mL YPD liquid medium. Cultivate overnight at 29℃ at 230rpm with shaking. Pay attention to monitor the OD600 value. When the OD600 value reaches 1.2±0.1, take out the culture flask containing the bacterial solution and place it on ice for precooling.

[0073] (2) Transfer the pre-cooled bacterial suspension on ice to a 50 mL centrifuge tube, centrifuge at 4°C, 4000 rpm for 5 min (the centrifuge needs to be pre-cooled in advance), discard the supernatant after centrifugation, and collect the cells. Resuspend the cells in 40 mL of pre-cooled sterile water, centrifuge at 4°C, 4000 rpm for 5 min, discard the supernatant after centrifugation, and collect the cells. Resuspend the cells again in 40 mL of pre-cooled sterile water, centrifuge at 4°C, 4000 rpm for 5 min, discard the supernatant after centrifugation, and collect the cells. Then resuspend the cells in 5 mL of pre-cooled 1M D-sorbitol, centrifuge at 4°C, 4000 rpm for 5 min, discard the supernatant after centrifugation, and collect the cells. Finally, resuspend the cells in 1 mL of pre-cooled 1M D-sorbitol, gently pipette to mix, and place on ice for later use.

[0074] 2. Competent Cell Transformation

[0075] (1) Drain 75% alcohol from the electrophoresis cup in a clean bench and add 1 mL of 1M D-sorbitol to rinse. After rinsing, drain the sorbitol solution and invert the electrophoresis cup to drain the sorbitol solution. Then, pre-cool it on ice for later use.

[0076] (2) Take 5-10 μg (10 μL) of the linearized recombinant expression plasmid and 80 μL of the competent cells mentioned above and add them to a 0.2 cm pre-cooled electroporation cuvette (add DNA first and then yeast competent cells), mix gently and let stand for 5 min.

[0077] (3) Wipe off the condensed water on the outside of the electroporation cup, place it on the electroporator (BIO-RAD), and perform electroporation at 2000 V, 200 Ω, and 25 μF.

[0078] (4) Immediately after electroporation, add 1 mL of pre-cooled 1 M D-sorbitol to the electroporation cup, transfer the electroporation product to a sterile 10 mL centrifuge tube, and place it in a 29°C incubator for 1 h.

[0079] (5) After 1 h, place the 10 mL centrifuge tube in a clean bench, add 1 mL of YPD liquid medium, and shake in a constant temperature shaker at 230 rpm and 29°C for 1-2 h to recover the strain.

[0080] 3. Transformant coating screening

[0081] The cultured bacterial suspension was centrifuged at 25°C and 4000 rpm for 5 min. After centrifugation, the excess supernatant was poured out in the clean bench, and 200 μL of supernatant was retained. After mixing, 50 μL was spread on the MD plate and cultured at 29°C for 3-4 days to observe the growth of the transformed Pichia pastoris transformants.

[0082] 4. Expression and Purification of Recombinant Fibronectin

[0083] (1) Pick a single clone and inoculate it into a 250 ml shake flask containing 25 ml BMGY medium (Buffered Glycerol-complex Medium). Shake at 29°C and 220 rpm until OD600 = 4.0. Centrifuge at 25°C and 3000g for 5 min. Remove the supernatant, collect the cells, resuspend the cells in BMMY medium to OD600 = 1.0, add methanol to a final concentration of 0.5%, and induce expression in a 1 L shake flask. Every 24 hours, add methanol to a final concentration of 0.5% to continue induction. At multiple time points such as 24, 48, 72 and 96 h, take 1 ml of culture medium into a 1.5 ml centrifuge tube, and centrifuge at 25°C and 12000g for 5 min to collect the bacteria. These samples are used to analyze the expression level and determine the best time to collect cells after induction. It was tested that the expression level of the recombinant fibronectin yeast clone changed with time. After 4 days of induction, the protein yield reached 2 g / L. Figure 2 Conclusion: The Pichia pastoris expression system can be used to obtain high-yield yeast fermentation of small molecule recombinant fibronectin.

[0084] (2) Centrifuge the fermented bacterial solution at 3000g for 5 minutes, collect the supernatant and discard the precipitate. Allow the protein in the supernatant to bind to the phenyl gel column and elute with 20mM phosphate buffer (pH=7.5). Bind the washed eluate to the anion exchange resin and elute with 150mM NaCl, 20mM phosphate, and 0.5M urea solution. The purity of the obtained protein was determined by SDS-PAGE electrophoresis to have a single protein band, no degradation bands, and a purity greater than 95%.

[0085] Example 2 Experiment of promoting cell adhesion and growth with recombinant fibronectin

[0086] The purified recombinant fibronectin in Example 1 was used to promote cell adhesion, extension and growth. The specific steps are as follows: Mouse embryonic fibroblasts NIH / 3T3 were used for the experiment.

[0087] (1) Coating preparation

[0088] 100 μL of the recombinant fibronectin purified in Example 1 and a D-PBS blank control were added to the 96-well plate. Four wells were prepared for each sample coating and incubated in a 37°C, 5% CO2 incubator for 1 h to 4 h. Excess coating solution was removed from the wells, 100 μL of 1% BSA-PBS solution was added, and incubated in a 37°C, 5% CO2 incubator for 1 h. After removing the liquid in the wells, wash three times with D-PBS, discard the washing solution, seal with a sealing film, and place at 4°C for use.

[0089] (2) Cell preparation

[0090] NIH / 3T3 cells were cultured in a 37°C, 5% CO2 cell culture incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. The cells were diluted to 5 × 10 using complete culture medium premixed with Hoechst 33342 fluorescent stain (10%). 4 / mL. 100 μL of cells were added to the wells, covered with aluminum foil, and incubated for 1 h at 37 °C, 5% CO2. Three replicates were measured, and the fourth well was used to adjust the microscope parameters and its measurement value was not used.

[0091] (3) Detection

[0092] An inverted microscope was used to capture fluorescent tile images of each of the three wells. Each well was filled with D-PBS to form a "reverse meniscus", bubbles were blown out and covered with sealing film. Centrifuged (inverted) at 22 °C for 5 min at optimal relative centrifugal force. After centrifugation, the sealing film was discarded and the supernatant was removed from the wells. After washing once with D-PBS, 100 μL of D-PBS was added. Each of the three wells was photographed to verify the cell adhesion, extension and growth promoting activity of recombinant fibronectin. The experimental results of fibroblasts NIH / 3T3 are shown in Figure 2. Figure 3 shown.

[0093] From the results, it can be seen that D-PBS has no function of promoting cell adhesion, and recombinant fibronectin has biological activity of promoting cell adhesion. In cells without serum, recombinant fibronectin can effectively promote cell adhesion, extension and growth within 1 hour, proving that the recombinant fibronectin obtained by connecting the transdermal enhancement peptide TD-1, fibronectin and antimicrobial peptide through the connecting peptide retains the biological activity of fibronectin in promoting cell adhesion, extension and growth.

[0094] Example 3 Antibacterial experiment of recombinant fibronectin

[0095] 1. Plate antibacterial assay of recombinant fibronectin

[0096] (1) Select monoclonal strains of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), incubate at 37°C and 180 rpm for 5-6 h, and measure the OD 600 Value, control pathogenic bacteria liquid OD 600 The value was 0.4±0.02, and the antibacterial activity of recombinant fibronectin was determined by the double-layer Oxford cup inhibition zone method.

[0097] (2) In a clean bench, take a sterile culture dish and pour 10 mL of sterile LB medium into it. After it solidifies, take out the sterile Oxford cup with sterile tweezers, quickly burn it under the outer flame of an alcohol lamp, and place it vertically on the surface of the culture medium.

[0098] (3) Use a pipette to draw up the OD 600 Add the pathogen suspension of 0.4 to the sterilized LB medium cooled to about 50℃ at a ratio of 1:1000 and shake well to make the LB medium mixed with the test bacteria, and take about 15 mL and pour it into the culture dish with LB bottom plate.

[0099] (4) After it solidifies, remove the Oxford cup with tweezers and use a pipette to draw 100 μL of recombinant fibronectin solution into the well.

[0100] (5) After culturing in a 37°C incubator for 16-17 hours, observe the antibacterial activity of the recombinant fibronectin and measure the diameter of the inhibition zone. Take photos of the inhibition plate and keep them. Figure 4 .

[0101] 2. Liquid antibacterial assay of recombinant fibronectin

[0102] (1) After the recombinant fibronectin sample obtained in Example 1 was sterilized by filtration, a protein concentration standard curve was drawn using the Bradford method with BSA as the standard, and the concentration of the recombinant fibronectin was calculated according to the formula.

[0103] (2) Dilute the protein solution in multiple ratios to 1-32 μg / mL.

[0104] (3) Performing MIC (Minimum Inhibitory Concentration) determination in a 96-well cell culture plate. The specific operation is as follows:

[0105] a) Preparation of four bacterial suspensions of Gram-positive bacteria such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria such as Pseudomonas aeruginosa and Aeromonas hydrophila: Streak the bacteria to be tested on MH plates and culture overnight;

[0106] b) Adjust the bacterial concentration to 6×10 5 CFU / ml for future use.

[0107] c) For each tested bacterium, set up a blank control group, a positive control group and a test sample experimental group according to the following operation, set up 2 parallel samples for each group, and repeat 3 times for each bacterium:

[0108] I. Positive control group: add 50 μl sodium phosphate buffer and 50 μl bacterial suspension;

[0109] II. Blank control group: add 50 μl of the protein sample to be tested and 50 μl of sodium phosphate buffer;

[0110] III. Sample experimental group: Add 50 μl of the protein sample to be tested and 50 μl of bacterial suspension.

[0111] d) After 24-48 hours of bacterial culture, observe the MIC results.

[0112] The results of the antibacterial experiment are shown in Table 1. Recombinant fibronectin has a broad-spectrum antibacterial activity and can effectively inhibit Gram-positive bacteria such as Staphylococcus aureus (MIC 2~4μg / mL) and methicillin-resistant Staphylococcus aureus (MIC 4~8μg / mL), but has a poor growth inhibition effect on Gram-negative bacteria Pseudomonas aeruginosa (MIC>100μg / mL) and Aeromonas hydrophila (MIC>100 μg / mL).

[0113] Table 1

[0114]

[0115] Note: CGMCC No.: China Microbiological Culture Collection Number;

[0116] MIC: Minimum inhibitory concentration, represented by ab, where a represents the highest concentration at which bacterial growth is visible to the naked eye, and b represents the minimum concentration at which no bacterial growth is seen.

[0117] Example 4 Comparison of antibacterial activity of recombinant fibronectin composed of different sequence connection orders

[0118] 1. According to the steps in Example 1, recombinant fibronectins with amino acid sequences of rFN-1 to rFN-5 were obtained, and the obtained recombinant fibronectins rFN-1 to rFN-5 were purified and endotoxins were removed.

[0119] The three sequences of SEQ ID No.1 (TD-1, ACSSSPSKHCG), SEQ ID No.2 (FN, IQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTGSAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT) and SEQ ID No.3 (Plectasin, GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY) were connected in other order to form a recombinant fibronectin with a sequence different from SEQ ID No.4:

[0120] (1) connecting SEQ ID No.1-SEQ ID No.3-SEQ ID No.2 in the order of connection, forming SEQ ID No.9, thereby forming recombinant fibronectin rFN-1;

[0121] (2) connecting SEQ ID No.2-SEQ ID No.1-SEQ ID No.3 in the order of connection to form SEQ ID No.10, thereby forming recombinant fibronectin rFN-2;

[0122] (3) forming recombinant fibronectin rFN-3 according to the connection order of SEQ ID No.2-SEQ ID No.3-SEQ ID No.1, SEQ ID No.11;

[0123] (4) connecting SEQ ID No.3-SEQ ID No.1-SEQ ID No.2 in the order of connection to form SEQ ID No.12, thereby forming recombinant fibronectin rFN-4;

[0124] (5) The sequence of connection is SEQ ID No.3-SEQ ID No.2-SEQ ID No.1, and SEQ ID No.13 forms recombinant fibronectin rFN-5.

[0125] 2. The recombinant fibronectin TD1-FN-Plectasin obtained after purification and removal of endotoxin was used as a control, and rFN-1 to rFN-5 were used to detect the antibacterial effects of the six proteins on bacteria, and the antibacterial activities of recombinant fibronectin with different sequence connection orders were compared and analyzed.

[0126] Figure 5 Middle A- Figure 5 Figure E shows the antibacterial activity of recombinant fibronectin with different sequence connection orders (rFN-1 to rFN-5). Compared with the recombinant fibronectin obtained by connecting the other five sequences, the recombinant fibronectin of the present application (SEQ ID No. 4) ( Figure 5 Middle F) has relatively more significant antibacterial activity against Staphylococcus aureus, proving that the recombinant fibronectin formed according to the sequence connection order of the present application can better inhibit the growth of bacteria.

[0127] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. Recombinant fibronectin, characterized in that The recombinant fibronectin includes the amino acid fragment shown in SEQ ID No.

4.

2. A nucleic acid molecule, characterized in that It encodes the recombinant fibronectin according to claim 1.

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

8.

4. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 2 or 3.

5. The expression vector according to claim 4, characterized in that The expression vector includes a plasmid.

6. A host cell, characterized in that It expresses the recombinant fibronectin described in claim 1, comprises the nucleic acid molecule described in claim 2 or 3, or the expression vector described in claim 4 or 5.

7. The host cell according to claim 6, characterized in that The host cell includes a eukaryotic cell, and optionally, the eukaryotic cell includes Pichia pastoris.

8. The method for preparing the recombinant fibronectin according to claim 1, characterized in that: The method comprises culturing the host cell according to claim 7, and separating and purifying the recombinant fibronectin expressed by the host cell.

9. Use of the recombinant fibronectin according to claim 1 in one or more of the following: (1) Application in the preparation of products that promote cell adhesion, extension and growth; (2) Application in the preparation of drugs for promoting transdermal absorption; and (3) Application in the preparation of products for inhibiting bacterial growth.

10. A beauty product, characterized in that: The cosmetic product comprises the recombinant fibronectin according to claim 1.

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