A fibronectin and collagen fusion protein and a method for preparing the same

Abstract

The application discloses a fibronectin and collagen fusion protein and a preparation method thereof, the amino acid sequence of the fusion protein is shown as SEQ.ID.NO.4, the fused collagen can effectively reduce the cytotoxicity of the fibronectin and improve the biological safety of raw materials under the premise of not affecting the efficacy of the fibronectin; compared with the original single sequence, the fusion protein can effectively improve the cell proliferation rate of the fibronectin; the fusion with the collagen also obviously increases the expression amount of the fibronectin, and the expression amount can reach 2g / L, which is much higher than the expression amount of the original single sequence; at the same time, the stability of the fibronectin at-20 DEG C, 4 DEG C and 40 DEG C is much higher than the stability of the original single sequence.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a fusion protein of fibronectin and collagen and its preparation method. Background Technology

[0002] Fibronectin (Fn) is a large glycoprotein with a molecular weight of approximately 440 kDa. It is a dimer composed of two 220 kDa subunits linked by disulfide bonds. Its molecular shape is V-shaped, consisting of two similar A and B chains. Each Fn subunit contains six dense globular regions, each with a specific function, binding to specific ligands and exhibiting a variety of biological functions. Fn is widely involved in cell migration, adhesion, proliferation, hemostasis, and tissue repair, mobilizing the mononuclear phagocyte system to clear harmful substances from damaged tissues, and also functions as a growth factor.

[0003] Studies on the role of fibrinogen (Fn) in wound healing have revealed that during the initial coagulation phase of wound healing, Fn is released from platelets and deposits on damaged collagen and fibrin, enhancing platelet adhesion, phagocyte and fibroblast migration, and cell proliferation. Fn also activates neutrophils, enhancing their adhesion and accelerating their aggregation towards the wound site. In the mid-stage of wound healing, Fn is an important chemokine for fibroblasts, enhancing cell adhesion and chemotaxis by helping to maintain cytoskeleton structure.

[0004] When fibrin cross-links with fibrin, it adheres to the blood clot, facilitating the movement of fibroblasts and their rapid entry into the wound area. Once inside the wound, fibroblasts secrete large amounts of fibrin, which, along with type III collagen, deposits in the intercellular matrix. The actual healing process begins when fibroblasts spin a fibrin network. Fibronectin also enhances angiogenesis by stimulating endothelial cell migration.

[0005] Collagen, a fibrous protein, is basically composed of procollagen molecules. Procollagen molecules are fibrous proteins formed by three strands of α-helical peptide chains, approximately 300 nm in length, 1.5 nm in diameter, and 300 kDa in molecular weight. Collagen contains a large amount of glycine, accounting for about one-third of the total amino acid residues, and its primary structure (peptide chain) consists entirely of (GLy-XY)n repeating sequences. Proline and hydroxyproline are the next most abundant, accounting for 15% to 30% of the total content. These two amino acids are unique to collagen, but the content of essential amino acids such as tryptophan, tyrosine, and methionine is low.

[0006] Collagen protects the skin and improves its elasticity. Because collagen and its hydrolysates have a similar structure to human skin collagen, they are highly compatible and can diffuse into the deeper layers of the skin, providing excellent nutrition. Furthermore, the outer surface of collagen molecules contains numerous carboxyl and hydroxyl groups, as well as abundant natural moisturizing factors such as glycine, which enhance the water-retention capacity of tissue cells, thus keeping the skin moisturized. Many collagen peptides are involved in the growth, differentiation, division, proliferation, and migration of skin cells, providing nutrients to the skin, delaying skin aging, and promoting wound repair. Collagen solutions also have strong anti-radiation properties, making them widely used in the cosmetics industry.

[0007] Currently, in the preparation of fibrinogen (Fn), cryoprecipitates obtained from processed plasma are rich in Fn. Initially, a series of steps were used to prepare Fn, including heated defibrination, glycine defibrination, polyethylene glycol (PEG) precipitation, Al(OH)3 adsorption, citric acid precipitation, and DEAE cellulose ion exchange. However, this method is complex and has a low Fn recovery rate. Another approach involves direct adsorption of plasma Fn using an anti-Fn affinity chromatography column followed by elution with glycine hydrochloride. However, this method requires specific anti-Fn antibodies, resulting in extremely high costs. In subsequent production, a gelatin Sepharose 4B affinity chromatography method using urea as the eluent was developed for Fn preparation. However, the urea eluent obtained by this method contains a high amount of fibrinogen. During purification, fibronectinogen precipitates as fibrin, clogging the chromatography column and affecting Fn preparation. Furthermore, Fn is a cold-insoluble protein, and Fn solutions are prone to degradation and instability when stored at 4°C.

[0008] In summary, existing Fn production methods are difficult to scale up, and the poor stability of single-sequence fibronectin limits its application conditions and effects. The current production volume of Fn and the unstable nature of the protein make it difficult to meet the increasingly strong market demand. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, the present invention aims to provide a fibronectin and collagen fusion protein, its preparation method, and its application.

[0010] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0011] In a first aspect, a fibronectin-collagen fusion protein is provided, the amino acid sequence of which is shown in SEQ.ID.NO.4.

[0012] Preferably, the fusion protein is composed of the following amino acid peptides fused together:

[0013] Fibronectin peptide, amino acid sequence as shown in SEQ.ID.NO.1;

[0014] Collagen peptides, with amino acid sequences as shown in SEQ.ID.NO.2;

[0015] The linking peptide segments have the amino acid sequences shown in SEQ.ID.NO.3.

[0016] This invention selected a fibronectin polypeptide sequence and a collagen sequence to attempt a fusion protein, aiming to leverage the high expression capacity of collagen in Pichia pastoris to promote the expression of fibronectin in the same yeast. This invention significantly promoted fibronectin expression through fusion with collagen, achieving an expression level of 2 g / L, far exceeding the expression level of the original single sequence. Simultaneously, the fusion protein exhibited significantly higher stability at -20℃, 4℃, and 40℃ compared to the original single sequence.

[0017] The efficacy advantages of the fusion protein of this invention

[0018] Firstly, collagen promotes skin growth and renewal, stimulates the production of collagen and elastin, maintains and repairs the skin's barrier function, and enhances the skin's ability to cope with environmental changes. Fibronectin effectively promotes wound healing by activating neutrophils, enhancing their adhesion, accelerating their aggregation in the wound area, and reducing the wound surface area. In skin wound repair and healing, fibronectin can shorten healing time and reduce scarring, making it widely used in regenerative medicine. In skincare, fibronectin (Fn) has multiple uses, specifically: Anti-wrinkle effects: Wrinkles are mainly caused by the loss of collagen, elastin, and reticular fibers that support the skin. Collagen is the most important component of the extracellular matrix, maintaining skin elasticity, resilience, and radiance along with other extracellular matrix components. Fibronectin (Fn), as an important signaling molecule, induces the expression and secretion of collagen in the extracellular matrix, promotes the secretion of hyaluronic acid and other glycoprotein molecules, and affects the directional alignment of the ECM. Therefore, fibronectin (Fn) has a good effect on smoothing wrinkles. Skin whitening and spot-fading effects: Skin needs to maintain normal physiological metabolism, such as the shedding and regeneration of keratinocytes and the regulation of melanocyte contents. It has been found that abnormalities in normal physiological metabolism not only lead to scarring but also to disordered melanocyte regulation, resulting in age spots. Age spots involve abnormalities in the proportion and structure / function of skin cells. Fn promotes normal skin physiological metabolism and also has the ability to prevent abnormal melanocyte secretion. This invention also conducted cell proliferation experiments on the fusion protein, using the MTT assay to detect its effects on HFF-1 human fibroblasts, mainly including its effects on cell survival, growth status, and cell growth rate. This fusion protein can improve various aspects of skin condition, including elasticity, wound healing, acne scars, and firmness, making it a promising new skincare ingredient.

[0019] Secondly, a process for preparing the fibronectin and collagen fusion protein as described in the first aspect is provided, comprising the following steps:

[0020] 1) Gene acquisition

[0021] The nucleic acid sequence corresponding to the fusion protein was artificially designed and synthesized, as shown in SEQ.ID.NO.5, and restriction enzyme sites were directly added to both ends of the sequence;

[0022] 2) Construction of pPIC9k-Fn-HLC recombinant plasmid

[0023] The artificially synthesized sequence and the Pichia pastoris universal vector were double-digested using XhoI and NotI restriction endonucleases. After digestion, the corresponding target gene and vector were recovered, and the Pichia pastoris was transformed into E. coli using T4 DNA ligase. The correct pPIC9k-Fn-HLC recombinant plasmid was obtained by sequencing and identification.

[0024] 3) pPIC9k-Fn-HLC recombinant plasmid was transformed into GS115 yeast competent cells

[0025] The pPIC9k-Fn-HLC recombinant plasmid was linearized with SalI, purified, and then 200 μL of the recovered plasmid was dried and concentrated to 10-20 μL in a 45°C oven. The pPIC9k-Fn-HLC recombinant plasmid was then transformed into Pichia pastoris GS115 using an electroporator, and high-copy transformants were screened using G418.

[0026] 4) Identification of recombinant yeast transformants

[0027] Genomic DNA was extracted from transformants using the freeze-thaw method and identified by PCR.

[0028] 5) Expression of GS115 / pPIC9k-Fn-HLC yeast transformants in shake flasks

[0029] Transformants correctly identified by PCR were expressed via shake-flask fermentation using BMGY and BMMY media. Fermentation was induced with methanol, and the expression results were detected by SDS-PAGE electrophoresis. Expression strains were then screened and obtained.

[0030] 6) Fusion protein fermentation process

[0031] The fermentation mode of induced fermentation using a shake flask as primary seed, a 30L seed tank as secondary seed, and a 100L fermenter was adopted. Methanol was used to induce the expression of the fusion protein. After the wet bacterial weight was induced to be greater than 300g / L, the increase in the target protein no longer increased significantly, the cells aged, and the fermentation was confirmed to have terminated.

[0032] 7) Fusion protein purification process

[0033] The crude fusion protein was prepared by centrifugation, microfiltration, and ultrafiltration. The fermentation broth of the fusion protein was then purified using a composite weak cation exchange chromatography column. The elution was carried out on the column, and the eluent was collected. Finally, the eluent was dialyzed, desalted, and freeze-dried to obtain the fusion protein with a purity of up to 95% (detected by electrophoresis).

[0034] Thirdly, a nucleic acid molecule is provided, the nucleic acid molecule comprising a nucleotide sequence or its complementary sequence encoding the fibronectin-collagen fusion protein described in the first aspect. Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ.ID.NO.5.

[0035] Fourthly, a carrier is provided that contains the nucleic acid molecule described in the third aspect.

[0036] Fifthly, the application of the fusion protein of fibronectin and collagen as described in the first aspect, the nucleic acid sequence as described in the third aspect, and the carrier as described in the fourth aspect in cosmetics or medical aesthetic products is provided.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The fusion protein of this invention has been proven to effectively reduce fibronectin cytotoxicity and improve the biosafety of raw materials by fusing with collagen without affecting the efficacy of fibronectin. Compared with the original single sequence, it can effectively increase the cell proliferation rate of fibronectin. The fusion with collagen also significantly increases the expression level of fibronectin, reaching 2 g / L, which is much higher than the expression level of the original single sequence. On the other hand, the fusion protein molecular sequence provided by this invention selects highly stable and highly hydrophilic collagen peptides for fusion expression with fibronectin, which can effectively increase the water solubility and stability of fibronectin. The stability of the fusion protein at -20℃, 4℃, and 40℃ is much higher than that of the original single sequence, thereby solving the problems of instability and easy degradation of fibronectin, making its application conditions more extensive and stable.

[0039] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of the invention. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 13D structure modeled for fibronectin and collagen fusion protein molecule SWISS-MODEL;

[0042] Figure 2 PCR identification results of GS115 / 9K-FN-HLC recombinant transformants;

[0043] Figure 3 The results of shake-flask induced expression identification of GS115 / 9K-FN-HLC strain;

[0044] Figure 4 Fermentation results for the GS115 / 9K-FN-HLC fusion protein;

[0045] Figure 5 The purification results of the GS115 / 9K-FN-HLC fusion protein;

[0046] Figure 6 The results show a comparison of the expression levels of the fusion protein and the Fn protein.

[0047] Figure 7 The results show the temperature stability comparison between the fusion protein and the Fn protein;

[0048] Figure 8 To compare the growth curves of HFF-1 cells in vitro culture at different concentrations of fusion protein samples;

[0049] Figure 9 The results of the fusion protein promoting HFF-1 cell proliferation;

[0050] Figure 10 Comparison of cell scratches after HFF-1 cells were treated with the fusion protein and Fn protein;

[0051] Figure 11 The results show the comparison between the effects of fusion protein and Fn protein samples on cell migration rate. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0054] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0055] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0056] Example

[0057] 1. Fusion protein molecular design

[0058] 1.1 The protein and gene sequences of collagen (type III) and fibronectin were obtained by searching on the NCBI website.

[0059] The amino acid sequence of fibronectin is shown in SEQ ID NO.1.

[0060] The amino acid sequence of collagen is shown in SEQ.ID.NO.2.

[0061] The amino acid sequence of the linker peptide is shown in SEQ ID NO.3.

[0062] The amino acid sequence of the fusion protein is shown in SEQ ID NO.4.

[0063] 1.2 Based on the codon preference of Pichia pastoris, the nucleic acid sequence of the fusion protein was artificially designed, and restriction enzyme sites were directly added to both ends of the sequence. After chemical synthesis, it could be directly inserted into the vector. The final determined nucleotide sequence is shown in SEQ.ID.NO.5.

[0064] 1.3 Primer Design:

[0065] Fn-HLC-XF:5'-TATCTCGAGAAAAGAGAGGAATTCGGTCCTCCCGGCGAACCAGGTAAT-3'

[0066] Fn-HLC-NR:5'-TATGCGGCCGCTTCTGGACCTGGTGGACCTTGTGGACCTTG-3'

[0067] 1.4 Fusion protein structure and theoretical properties:

[0068] See the 3D model of the fusion protein simulation. Figure 1

[0069] The molecular weight of the protein is 25.24 kDa.

[0070] Construction of the pPIC9k-Fn-HLC recombinant plasmid with an isoelectric point of 8.772 p.

[0071] 2.1 Materials:

[0072] 2×TaqPCRMastermix (Tiangen Biochemical), T4DNALigase (Thermo), DH5α competent cells (Tiangen Biochemical), restriction endonuclease NotIFastDigest (Thermo), restriction endonuclease XhoIFastDigest (Thermo), agarose gel DNA recovery kit (Tiangen Biochemical), LB medium.

[0073] 2.2 Equipment:

[0074] PCR instrument (Bio-RAD), horizontal electrophoresis apparatus, centrifuge, shaker.

[0075] 2.3 Methods:

[0076] 2.3.1 Double enzyme digestion of target gene and vector

[0077] To construct sticky ends suitable for ligation, this experiment used XhoI and NotI restriction endonucleases to double-digest the whole-gene synthesis plasmid T-Fn-HLC and the pPIC9K vector. The digestion systems are shown in Tables 2-1 and 2-2, respectively. The specific digestion steps are as follows:

[0078] (1) Prepare the enzyme digestion reaction system in clean PCR tubes, as shown in Table 2-1 and Table 2-2 respectively:

[0079] Table 2-1: Restriction enzyme digestion system of the whole-genome synthetic plasmid T-Fn-HLC

[0080]

[0081]

[0082] Table 2-2: pPIC9k restriction enzyme digestion system

[0083] Components Volume (μL) pPIC9K carrier 5.0 10×Buffer 2.0 NotI 1.0 XhoI 1.0 <![CDATA[ddH2O]]> 11.0 20μL in total

[0084] (2) Gently tap the tube wall, mix the solution with a suspension apparatus, and then centrifuge briefly to concentrate the solution at the bottom of the tube;

[0085] (3) Place the tube in a 37°C water bath and digest for 17 min.

[0086] (4) After the enzyme digestion step, the enzyme digestion product is detected by 1% agarose gel electrophoresis, and the gel is cut and recovered according to the requirements of the Tiangen agarose gel DNA recovery kit.

[0087] (5) The recovered product must be the same size as the expected fragment before it can be used directly for the ligation reaction to construct the expression vector.

[0088] 2.3.2 Ligation of enzyme digestion products

[0089] To construct the expression vector pPIC9K-Fn-HLC, Thermo's T4 DNA ligase was used to ligate the digested products and the pPIC9K vector digested products. The ligation system is shown in Table 2-3, and the specific ligation steps are as follows:

[0090] (1) Prepare the ligation reaction system in clean PCR tubes, as shown in Table 2-3:

[0091] Table 2-3: T4 ligase ligation system

[0092]

[0093]

[0094] (2) Gently tap the tube wall, mix the solution with a suspension apparatus, and then centrifuge briefly to concentrate the solution at the bottom of the tube;

[0095] (3) Place the EP tube of the T4 connection system in a 22℃ constant temperature connection instrument and connect for 4 hours;

[0096] (4) After completing the above ligation steps, the ligation product can be directly used for the transformation of competent bacteria.

[0097] 2.3.3 Conversion of Linkage Products

[0098] The experiment used E. coLiDH5α competent cells for plasmid transformation. The specific experimental steps are as follows:

[0099] (1) Take out 50 μL of DH5α competent cells stored in a -80℃ freezer and quickly insert it into an ice box. After the frozen bacterial block thaws, add 5 μL of the two aforementioned ligation products to the ultra-clean workbench. After gently stirring the bottom of the tube with your finger, place the EP tube on the ice box and let it stand for 30 min.

[0100] (2) Place the EP tube in a 42℃ water bath for 90 seconds to heat shock, and then quickly place it in an ice box for 3 minutes. This process should be handled carefully to avoid violent shaking, otherwise the conversion efficiency will be affected.

[0101] (3) Add 600 μL of antibiotic-free sterile LB liquid culture medium to each EP tube, shake to mix, and incubate at 180 rpm for 45 minutes in a shaker at 37°C.

[0102] (4) Take out about 200 μL of incubation solution from the clean bench and spread it evenly on LB agar plates containing 50 ug / mL ampicillin antibiotic.

[0103] (5) Place the LB plate upside down in a 37°C constant temperature incubator and incubate overnight.

[0104] 2.3.4 Identification of recombinant positive transformants

[0105] Select the positive transformants that have grown, perform bacterial culture PCR identification, and sequence the strains that are correctly identified by PCR. The specific experimental steps are as follows:

[0106] (1) Pick the positive transformants that grow from the LB plate containing 50ug / mL ampicillin antibiotic, number the strains, and inoculate them into 5mL of LB medium containing 50ug / mL ampicillin antibiotic.

[0107] (2) Place on a shaker and incubate overnight at 200 rpm and 37°C.

[0108] (3) On the second day, using the culture medium as a template, primers Fn-HLC-XF and Fn-HLC-NR were designed and prepared according to the reaction system in Table 2-4 for PCR.

[0109] Table 2-4: Bacterial PCR System

[0110] Components Volume (μL) bacterial solution 2.0 Fn-HLC-XF 1.0 Fn-HLC-NR 1.0 2XTaqPCRMix 10.0 <![CDATA[ddH2O]]> 6.0 20μL in total

[0111] (4) PCR program settings: Set the following program in the PCR instrument.

[0112] 1) 94℃, 5min

[0113] 2) 94℃, 30s (decontaminate)

[0114] 3) Annealing at 60℃ for 30 seconds.

[0115] 4) 72℃, 1 min (extended)

[0116] 5) Repeat steps 2 to 4 for 30 cycles.

[0117] 6) 72℃, 5min

[0118] 7) 4℃, Forever

[0119] (5) Perform PCR products on 1% agarose gel electrophoresis. Based on the electrophoresis results, preliminarily determine the positive transformants and prepare glycerol tubes with the corresponding bacterial solutions in a 1:1 ratio of bacterial solutions to 50% glycerol. Store at -20℃.

[0120] (6) 100 μL of bacterial culture was aliquoted from the glycerol tube seed and sequenced. The sequencing results were compared with the designed sequence, and the correct positive transformants were stored.

[0121] 3. pPIC9k-Fn-HLC recombinant plasmid was transformed into GS115 yeast competent cells

[0122] 3.1 Materials:

[0123] Plasmid mini-extraction kit (Tiangen Biochemical), general DNA purification and recovery kit (Tiangen Biochemical), restriction endonuclease SalIFastDigest (Thermo).

[0124] 3.2 Equipment:

[0125] PCR instrument (Bio-RAD), horizontal electrophoresis apparatus, centrifuge, shaker.

[0126] 3.3 Method:

[0127] 3.3.1 Medium-scale extraction of recombinant plasmids

[0128] (1) Take 10 μL of the bacterial culture that needs to be electroporated and inoculate it into 20 mL of LB medium containing 50 μg / mL ampicillin.

[0129] (2) Take 15 mL of bacterial solution, centrifuge at 12000 rpm for 1 min at room temperature to collect the bacterial cells.

[0130] (3) Extract pPIC9K-Fn-HLC plasmid according to the Tiangen plasmid small-scale extraction kit requirements. Finally, elute with 300 μL of sterile ddH2O. Store at -20℃.

[0131] 3.3.2 Linearization of Recombinant Plasmids

[0132] The pPIC9k-Fn-HLC plasmid extracted in medium quantities was linearized using SalI. The linearization reaction system is shown in Table 3-1 below:

[0133] Table 3-1: Linearization reaction system of pPIC9k-Fn-HLC plasmid

[0134] Components Volume (μL) pPIC9k-Fn-HLC plasmid 300 10×FastDigestbuffer 200 SaLI 20 <![CDATA[ddH2O]]> 1480 2000μL in total

[0135] (1) Take a sterilized 2mL centrifuge tube, prepare the reaction system according to Table 2-1, mix well, and centrifuge briefly.

[0136] (2) Dispense the solution into 4 tubes at 400 μL / tube and digest them in a water bath at 37°C for 30 minutes.

[0137] (3) Linearization of plasmids by 1% agarose gel electrophoresis.

[0138] (4) When using the Tiangen Universal DNA Purification and Recovery Kit (DP214-02), use 50 μL ddH2O per tube to recover the linearized plasmid during elution.

[0139] (5) Combine the 4 tubes of plasmids, totaling 200 μL, bake at 45°C to dry and concentrate the plasmids to 10-20 μL. Store at -20°C.

[0140] 3.3.3 Preparation of Pichia pastoris competent cells GS115

[0141] (1) Pick a single colony of Pichia pastoris strain GS115 and inoculate it into 5 mL of sterile YPD medium. Incubate at 29°C and 200 rpm for 8 hours.

[0142] (2) After 8 hours, 400uL of culture medium was taken and inoculated into 100mL of sterile YPD medium. The medium was then cultured overnight at 29℃ and 200rpm on a shaker.

[0143] (3) The next day, take 1 mL of culture medium, dilute it to 10 mL, zero the instrument with ddH2O as a control, and measure the OD using a spectrophotometer. 600 OD 600 When the value is between 1.1 and 1.7, it can be used for competent state preparation.

[0144] (4) Take 50 mL of culture medium, centrifuge at 4℃, 3000 rpm for 5 min and discard the supernatant.

[0145] (5) Resuspend the cells in 30 mL of sterile ice-cooled ddH2O, centrifuge at 4°C, 3000 rpm for 5 min, and discard the supernatant.

[0146] (6) Repeat step (5) twice.

[0147] (7) Resuspend the cells in 30 mL of sterile, ice-cold 1MD-sorbitol, centrifuge at 4°C, 3000 rpm for 5 min, and discard the supernatant.

[0148] (8) Repeat step (7) twice.

[0149] (9) Take 400uL of sterilized ice-cold 1MD-sorbitol to resuspend the cells, which are the competent cells.

[0150] (10) Dispense competent cells into 90μL / EP tubes.

[0151] 3.3.4 Electroconversion and Screening of Linearized Plasmids

[0152] (1) Soak a 2mm electric shock cup in 75% ethanol for 30 minutes.

[0153] (2) Rinse three times with anhydrous ethanol and air dry on a clean bench.

[0154] (3) Add 10 μL of linearized plasmid to 90 μL of competent cells, mix well, and then transfer 100 μL of the mixture to a 2 mm electrocautery cup and pre-cool on ice for 10 min.

[0155] (4) Set the electric shock parameters. The yeast parameters are: voltage 2000V, capacitance 25uF, resistance 200Ω.

[0156] (5) Quickly dry the external moisture of the electric shock cup, put it into the electric shock tank, and perform electric shock.

[0157] (6) After the electrocution is complete, immediately add 1.5 mL of 1 M ice-cooled D-sorbitol, mix well, and then aspirate the bacterial solution into a 1.5 mL sterile centrifuge tube.

[0158] (7) Screening on MD plates: 200uL of bacterial solution per plate, spread gently with a sterile spreader, incubate at 29℃ upside down for 3-4 days.

[0159] (8) Prepare a 2 mg / mL G418 plate. Pick all the single colonies grown in the MD plate in step (7) onto the G418 plate with a sterile toothpick and incubate at 29°C upside down for 4-5 days.

[0160] (9) Prepare YPD plates. Pick all the single colonies grown in the G418 plate in step (8) onto the YPD plates with a sterile toothpick and incubate them upside down at 29°C for 1-2 days.

[0161] 4. Identification of recombinant yeast transformants

[0162] 4.1 Materials:

[0163] 2×TaqPCRMastermix (Tiangen Biotech).

[0164] 4.2 Equipment

[0165] PCR instrument (Bio-RAD), centrifuge, microwave oven, -80℃ freezer.

[0166] 4.3 Method:

[0167] 4.3.1 Genomic DNA extraction from transformants

[0168] (1) Based on the number of transformants grown in the YPD plate, take an EP tube and place it on a centrifuge rack, then add 50uL of double-distilled water.

[0169] (2) Pick up the colonies growing in the YPD plate with a toothpick and put them into an EP tube. Mix well and centrifuge at 4000 rpm for 1 min at room temperature. Discard the supernatant.

[0170] (3) Microwave oven, medium heat, heat for 5 minutes.

[0171] (4) Freeze in a refrigerator at -80℃ for 20 minutes.

[0172] (5) Microwave oven, medium heat, heat for 5 minutes.

[0173] (6) Freeze in a refrigerator at -80℃ for 20 minutes.

[0174] (7) Microwave oven, medium heat, heat for 5 minutes.

[0175] (8) Add 50uL of double-distilled water, mix well, and centrifuge at 4000rpm for 1min at room temperature.

[0176] (9) Transfer the supernatant (genomic DNA) from the centrifugation into a sterile centrifuge tube and store at -20°C.

[0177] 4.3.2 Genomic PCR identification of transformants

[0178] Using extracted genomic DNA as a template, PCR identification was performed with primers for the inserted target gene fragment. Strains that amplified the target fragment were considered positive clones. The PCR reaction system is shown in Table 4-1 below:

[0179] Table 4-1: GS115 / pPIC9k-Fn-HLC Genomic DNA PCR Reaction System

[0180] Components Volume (μL) Genomic DNA 4.0 Fn-HLC-XF 2.0 Fn-HLC-NR 2.0 2XTaqPCRMix 20.0 <![CDATA[ddH2O]]> 12.0 40μL in total

[0181] (1) Take a sterilized EP tube, prepare the reaction system according to Table 4-1, mix well, and centrifuge briefly.

[0182] (2) PCR program settings: Set the following program in the PCR instrument.

[0183] 1) 94℃, 5min

[0184] 2) 94℃, 30s (decontaminate)

[0185] 3) Annealing at 60℃ for 30 seconds.

[0186] 4) 72℃, 1 min (extended)

[0187] 5) Repeat steps 2 to 4 for 30 cycles.

[0188] 6) 72℃, 5min

[0189] 7) 4℃, Forever

[0190] (3) Based on the PCR results, see details below. Figure 2 Positive transformants were initially identified, and their corresponding strains were used for shake-flask expression. Plates were stored at -4°C.

[0191] 5. Expression of GS115 / pPIC9k-Fn-HLC yeast transformants in shake flasks

[0192] 5.1 Materials:

[0193] BMGY medium, BMMY medium

[0194] 5.2 Equipment: Shaking table

[0195] 5.3 Method:

[0196] (1) Pick a single positive clone and inoculate it into a 250mL Erlenmeyer flask containing 30mL BMGY medium. Incubate at 29℃ and 225rpm for 60 hours on a shaker.

[0197] (2) Number the 50mL centrifuge tubes, pour the BMGY culture medium into the centrifuge tubes, centrifuge at 3000rpm for 5min at room temperature to collect the bacterial cells in the shake flask.

[0198] (3) Add 30 mL of sterile double-distilled water and resuspend the bacterial cells. Centrifuge at 3000 rpm for 5 min at room temperature to collect the bacterial cells.

[0199] (4) Repeat step (3) twice.

[0200] (5) Collect the cells at room temperature, centrifuge at 3000 rpm for 5 min, add 30 mL of BMMY medium to resuspend the cells, pour 30 mL of the mixed BMMY culture into a 250 mL Erlenmeyer flask, and culture at 29 °C and 225 rpm on a shaker.

[0201] (6) Add 300 μL of methanol every 24 hours.

[0202] (7) Add 300 μL of methanol after 48 hours.

[0203] (8) Add 300 μL of methanol after 72 hours.

[0204] (9) After 96 hours, collect the supernatant from the shake flask and store it at -20°C.

[0205] (10) SDS-PAGE electrophoresis was used to detect the shake-flask results, preliminarily confirming the expression of the target protein. The shake-flask induction expression identification results of the GS115 / 9K-FN-HLC strain were also detailed. Figure 3 .

[0206] 6. Fermentation of fusion proteins

[0207] 6.1 Materials:

[0208] Yeast powder, peptone, glycerin, etc. (see ingredient list for details) are all from Sinopharm Reagent.

[0209] 6.2 Equipment:

[0210] Shaking incubator, sterilizer, clean bench, 100L fermenter system.

[0211] 6.3 Methods

[0212] 6.3.1 Preparation of Seeds in Shake Flasks

[0213] Table 6-1: Seed Culture Medium Formulation

[0214]

[0215]

[0216] Prepare the culture medium and dispense 200ml into 500mL Erlenmeyer flasks. Wrap each flask in kraft paper and sterilize in a sterilizer at 121–125℃ and 0.10–0.13MPa for 25 minutes. After cooling, ensure the medium passes a sterility test before inoculation. Take 200µL of the frozen seed culture and inoculate it into a sterile blank seed shake flask (add one extra flask for sterility testing). Incubate at 29±0.5℃ on a shaker (220±10 rpm) for 20–28 hours. Randomly select one flask from the cultured seed culture. The flask should be brownish-yellow in appearance, with a pH of 6.0–6.5. Microscopic examination should show uniform cell morphology, good growth, and no contaminating bacteria. Under flame protection, ignite an alcohol lamp and combine 500–800mL of the seed culture from the shake flask into a sterile inoculation bottle, then inoculate into the seed tank.

[0217] 6.3.2 Secondary Seed Culture

[0218] 6.3.2.1 Secondary seed culture medium formulation

[0219] Table 6-2: Seed Culture Medium Formulation

[0220] Culture medium formulation Mixing ratio (g / L) Phosphoric acid 26.7 Anhydrous calcium sulfate 0.93 potassium sulfate 18.2 Magnesium sulfate heptahydrate 14.9 potassium hydroxide 4.13 glycerin 20 Bubble Enemy 0.1 PTM1 0.5

[0221] Table 6-3: PTM1 Formulation

[0222] Culture medium formulation Mixing ratio (g / L) Anhydrous copper sulfate 6.0 Sodium iodide 0.08 Manganese sulfate monohydrate 0.3 Sodium molybdate dihydrate 0.2 Boric acid 0.02 cobalt chloride 0.5 Zinc chloride 20 Ferrous sulfate heptahydrate 65 sulfuric acid 5 Biotin 0.2

[0223] 6.3.2.2 Operation

[0224] After sterilizing the prepared culture medium in the seed tank at 121–122℃ for 25±2 minutes, sterile air is introduced to maintain pressure for later use. Before inoculation, the tank temperature is adjusted to 29.0±0.5℃ and stirring is started. During inoculation, the inoculation port is protected by an alcohol swab flame using the flame ring inoculation method, ensuring that all the seed liquid from the shake flask is transferred into the tank. The liquid level inside the tank must be observed simultaneously to prevent liquid escape. After inoculation, the air inlet and outlet valves are adjusted to maintain the tank pressure at 0.030–0.050 MPa, and primary seed culture begins.

[0225] 6.3.2.3 Process Requirements

[0226] Table 6-4: Requirements for Secondary Seed Processing Parameters

[0227]

[0228]

[0229] 6.3.3 100L fermenter fermentation culture

[0230] 6.3.3.1 Fermentation medium formulation

[0231] Table 6-5: Fermentation medium formulation

[0232] Culture medium formulation Mixing ratio (g / L) Phosphoric acid 26.7 Anhydrous calcium sulfate 0.93 potassium sulfate 18.2 Magnesium sulfate heptahydrate 14.9 potassium hydroxide 4.13 glycerin 15 Bubble Enemy 0.1 <![CDATA[PTM1]]> 0.5

[0233] 6.3.3.2 Operation

[0234] The prepared culture medium was poured into the fermenter and sterilized at 121–122℃ for 30 ± 2 minutes. Sterile air was then introduced, and the mixture was cooled and pressurized for later use. The inoculation pipeline from the seed tank to the fermenter was sterilized by high-pressure steam according to the sterilization procedure. Before inoculation, the tank temperature was adjusted to 28.0 ± 2.0℃, and the flow rate was adjusted to the maximum value (3–5 m³ / h). 3 / h), pressurize the tank to 0.030-0.050 MPa, stir to 500 rpm, and calibrate the dissolved oxygen electrode to (95-100%). When transferring the seed solution, adjust the fermenter stirring, stop the seed tank stirring, increase the seed tank pressure, decrease the fermenter pressure, and transfer the seed solution from the seed tank to the fermenter. Once all the seed solution has been transferred to the fermenter, and the transfer is complete, close the inoculation valve, adjust the tank pressure to 0.030-0.050 MPa, and adjust the fermenter air flow rate to 2-5 m³ / h. 3 Stir at 100 rpm for 1 hour and then ferment.

[0235] After dissolved oxygen levels recover, fermentation enters the glycerol batch growth stage, supplemented with 50% glycerol at a rate of 10-15 mL / L / h. When the wet cell weight reaches 200 g / L, methanol induction is performed using 2.0% methanol, induced 10 times. Induction continues until the wet cell weight exceeds 300 g / L, at which point the increase in target protein no longer increases significantly, indicating cell aging, and fermentation can be confirmed as terminated.

[0236] 6.3.3.3 Process Requirements

[0237] Table 6-6: Requirements for Secondary Seed Processing Parameters

[0238]

[0239] 6.3.4 Can Release Operation

[0240] Upon receiving notification that the tank can be released, open the discharge valve and begin releasing the contents of the tank.

[0241] 6.4 Fermentation Results

[0242] from Figure 4 It can be seen that the FN-HLC fusion protein can be fully expressed by methanol induction, with an expression level greater than 2 mg / mL.

[0243] 7. Purification process of fusion proteins

[0244] 7.1 Materials:

[0245] Citric acid monohydrate (Sinopharm), trisodium citrate dihydrate (Sinopharm), sodium chloride (Sinopharm), CM-SepharoseFF

[0246] 7.2 Equipment:

[0247] Centrifuge, 5kD hollow fiber ultrafiltration system, chromatography purification system.

[0248] 7.3 Method

[0249] 7.3.1. Preparation of crude fermentation broth:

[0250] The fermentation broth was subjected to solid-liquid separation using a large-capacity refrigerated centrifuge at 4000 rpm (6260×g) for 15 min, and the fermentation broth was collected.

[0251] The fermentation broth was filtered through a 0.22µm microfiltration membrane system to remove a small amount of bacterial cells and other large molecular proteins, and the filtrate was collected.

[0252] The filtrate was ultrafiltered through a 5kD pre-ultrafiltration system until the conductivity of the retentate was less than 5 mS / cm. The retentate volume was then concentrated to less than 30 L. The retentate was released and the system was flushed with 20 L of purified water to recover residual protein. The retentate and the recovered solution were thoroughly mixed. The pH was adjusted to 5.0 ± 0.2 with citric acid monohydrate, and the conductivity was adjusted to 4.5–6.0 mS / cm with purified water and sodium chloride for later use.

[0253] 7.3.2 Chromatographic purification of crude product solution:

[0254] Column packing: 5L of BorgRon composite weak cation exchange packing material MMC was packed into a Huideyi EC-0140-0500-G chromatography column;

[0255] Chromatography buffer formulation:

[0256] Buffer A: Prepare 100L of purified water, add 3.44g / L citric acid monohydrate and 6.95g / L trisodium citrate dihydrate, and after complete dissolution, the pH of the solution should be 5.0±0.2 and the conductivity should be 4.5~5.0mS / cm;

[0257] Buffer B: Prepare 60L of purified water, add 1.95g / L citric acid monohydrate and 9.53g / L trisodium citrate dihydrate, and after complete dissolution, the pH of the solution should be 5.9±0.2 and the conductivity should be 6.0~6.5mS / cm;

[0258] Buffer C: Prepare 15 L of 2 Molar sodium chloride solution using purified water.

[0259] Chromatographic purification process (flow rate 0.4 L / min)

[0260] A solution is fed into the column for equilibration—sample loading—A solution is fed into the column for reequilibration—B solution is used to elute the target protein—C solution is used to regenerate the chromatography column;

[0261] Collect the target protein peak eluted by solution B (pure solution).

[0262] 7.3.3 Ultrafiltration Concentration

[0263] The eluent was ultrafiltered using a 5kD pre-ultrafiltration system until the conductivity of the retentate was less than 4 mS / cm, and then concentrated until the protein content was higher than 1 mg / mL. The ultrafiltration process was then complete.

[0264] 7.3.4 Freeze-drying

[0265] The concentrated solution obtained by ultrafiltration is placed in a freeze dryer and freeze-dried.

[0266] 7.4 Results

[0267] Finally, 220g of lyophilized fusion protein powder was obtained with a purity of up to 95%. Details of the electrophoresis results for the lyophilized powder and purification process can be found in [link to documentation]. Figure 5 .

[0268] 8. Comparison experiment between fusion protein and Fn protein

[0269] 8.1 Materials:

[0270] BMGY medium, BMMY medium

[0271] 8.2 Equipment: Shaking table, electrophoresis system

[0272] 8.3.1 Method for comparing the expression levels of fusion proteins and Fn proteins:

[0273] (1) Select GS115 / 9K-Fn-HLC and GS115 / 9K-Fn (laboratory prepared strain) strains, and inoculate them into 250mL Erlenmeyer flasks containing 30mL BMGY medium, and culture them in a shaker at 29℃ and 225rpm for 60 hours.

[0274] (2) Number the 50mL centrifuge tubes, pour the BMGY culture medium into the centrifuge tubes, centrifuge at 3000rpm for 5min at room temperature to collect the bacterial cells in the shake flask.

[0275] (3) Add 30 mL of sterile double-distilled water and resuspend the bacterial cells. Centrifuge at 3000 rpm for 5 min at room temperature to collect the bacterial cells.

[0276] (4) Repeat step (3) twice.

[0277] (5) Collect the cells at room temperature, centrifuge at 3000 rpm for 5 min, add 30 mL of BMMY medium to resuspend the cells, pour 30 mL of the mixed BMMY culture into a 250 mL Erlenmeyer flask, and culture at 29 °C and 225 rpm on a shaker.

[0278] (6) Add 300 μL of methanol every 24 hours.

[0279] (7) Add 300 μL of methanol after 48 hours.

[0280] (8) Add 300 μL of methanol after 72 hours.

[0281] (9) After 96 hours, collect the supernatant from the shake flask and store it at -20°C.

[0282] (10) SDS-PAGE electrophoresis was used to detect the shake-flask results. The expression results of the target protein and the expression of the fusion protein and Fn protein were compared. For details, please refer to [link to relevant documentation]. Figure 6 .

[0283] 8.3.2 Method for comparing the stability of fusion proteins and Fn proteins:

[0284] (1) Select Fn-HLC fusion protein and Fn protein lyophilized powder, and prepare 9% NaCl aqueous solution with a protein content of 0.5g / mL respectively, and dispense them.

[0285] (2) The proteins were placed at -20℃, 4℃, 40℃, and 30 days respectively, and their protein stability was detected by electrophoresis. For the comparison of temperature stability between the fusion protein and the Fn protein, please refer to [link to relevant documentation]. Figure 7 .

[0286] 8.4 Results

[0287] 8.4.1 Comparison of expression levels between fusion protein and Fn protein

[0288] Table 8-1: Figure 6 protein electrophoresis grayscale value

[0289] swimming lane Sample Name target strip grayscale value 2 Fn 1125.22 3 Fn-HLC 12858.81

[0290] from Figure 6Electrophoresis grayscale scan results showed that, at the shake flask level, the fusion of collagen and fibronectin significantly increased the expression level of the target protein by up to 10 times. The fusion of collagen significantly increased the yield of fibronectin. Fermentation tank expression confirmed that the expression level could reach 2 g / L, which is much higher than the original single sequence expression level.

[0291] 8.4.2 Comparison of temperature stability between fusion protein and Fn protein

[0292] Table 8-2: Figure 7 protein electrophoresis grayscale value

[0293]

[0294]

[0295] from Figure 7 Electrophoretic grayscale scanning results showed that the single-sequence Fn sample was relatively stable after 30 days of storage at -20℃, with fewer degradation bands and the target band exhibiting the highest grayscale value. After 30 days of storage at 4℃, the band value decreased slightly, showing obvious degradation bands. After 30 days of storage at 40℃, it was completely degraded, and the target band was no longer visible. In contrast, the fusion protein was stable after 30 days of storage at -20℃, 4℃, and 40℃, with no obvious degradation bands. Grayscale analysis showed only minor degradation after 30 days of storage at 4℃ and 40℃, demonstrating a stability far superior to that of the original single sequence.

[0296] 9 Fusion Protein Promotes HFF-1 Cell Proliferation Experiment

[0297] 9.1 Materials:

[0298] DMEM medium (containing 15% fetal bovine serum & 5% fetal bovine serum), PBS, 75% ethanol, cell culture flasks, 96-well plates, pipettes, centrifuge tubes, alcohol lamp, 1mL syringe, 0.22μL filter membrane, steel ruler, marker pen

[0299] 9.2 Equipment:

[0300] Liquid nitrogen tank, clean bench, cell culture incubator, inverted microscope, centrifuge, ELISA reader

[0301] 9.3 Method:

[0302] 9.3.1 Plating: 10^3 cells / well were inoculated into 7 wells of a 96-well plate. After 24 hours (adjustable), different concentration gradients of the fusion protein diluted with 5% serum (40 μg / ml, 80 μg / ml, 120 μg / ml, 160 μg / ml, 200 μg / ml, 240 μg / ml, 280 μg / ml, 320 μg / ml, 360 μg / ml, 400 μg / ml) were added for further culture. The blank control group received only 5% serum. The plates were then incubated, and one plate was sampled every 24 hours for MTT assay.

[0303] 9.3.2 Record cell state using an inverted imaging microscope, then remove the culture medium and add 50 μL of MTT solution to each well. Incubate at 37°C with 5% CO2 for 2 h.

[0304] The MTT solution was then removed, and 100 μL of isopropanol was added to each well. The mixture was then shaken at room temperature for 30 min. The absorbance of each well was measured at 570 nm using an ELISA reader.

[0305] 9.3.3 Data Recording and Calculation: Analyze the absorbance values ​​of the fusion protein (calculate the average OD value for each concentration), perform data analysis using Prism8, and plot line graphs of OD values ​​at each concentration and time. The higher the OD value, the greater the number of cells, indicating a more significant cell proliferation-promoting effect of the fusion protein.

[0306] 9.4 Results

[0307] Cells are in the logarithmic growth phase from day 4 to 6, and reach maximum viable cell density on day 6. As nutrients and living space decrease, cells undergo apoptosis due to nutrient depletion on day 7 to 8.

[0308] Depend on Figure 8-9 It was found that the fusion protein was non-toxic to cell growth and effectively promoted the growth and proliferation of HFF-1 cells. Furthermore, the proliferative effect of different concentrations of the fusion protein was superior to that of 5% fetal bovine serum, indicating a positive role in cell growth. After 6 days of culture, the cell number in the sample group containing 200 μg / ml of fusion protein was greater than that in the blank control group. The fusion protein significantly promoted fibroblast adhesion and growth.

[0309] 10. Fusion protein cell scratch assay

[0310] 10.1 Materials:

[0311] DMEM medium (containing 15% fetal bovine serum & 2% fetal bovine serum), PBS, 75% ethanol, cell culture flasks, 6-well plates, pipettes, centrifuge tubes, alcohol lamp, 1mL syringe, 0.22μL filter membrane, steel ruler, marker pen

[0312] 10.2 Equipment:

[0313] Liquid nitrogen tank, clean bench, cell culture incubator, inverted microscope, centrifuge, ELISA reader

[0314] 10.3 Method:

[0315] Plating: Inoculate 2×10^6 cells into 6-well plates, culture for 24 hours (adjustable), streak with a pipette tip, and wash the cells 3 times with PBS.

[0316] Subsequently, serum-free culture medium containing the same concentrations of single-sequence protein and fusion protein were added for culture, specifically Fn-1: 50 μg / mL and Fn-HLC-1: 50 μg / mL, respectively. A blank control containing only serum-free culture medium was also set up. Images were taken under a microscope at 0, 6, 20, and 28 hours of culture.

[0317] In the cell scratch assay, the images were analyzed using ImageJ and Prism8 to create bar charts showing cell migration rate and time. A higher cell migration rate indicated a more significant effect of the fusion protein in promoting cell proliferation at that concentration.

[0318] 10.4 Results:

[0319] Depend on Figures 10-11 It was found that both Fn-HLC-1 concentrations of 50 μg / ml and Fn-1 concentrations of 50 μg / ml promoted cell proliferation from 0 h to 28 h, with Fn-HLC-1 showing a significant effect from 6 h to 28 h (P < 0.05). Among them, the Fn-HLC-1 concentration of 50 μg / ml showed the best proliferative effect.

[0320] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A fusion protein of fibronectin and collagen, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ.ID.NO.

4.

2. A method for preparing the fusion protein of fibronectin and collagen as described in claim 1, characterized in that, Includes the following steps: A. Design the target gene; B. Construct the pPIC9k-Fn-HLC recombinant plasmid; C.pPIC9k-Fn-HLC recombinant plasmid was transformed into GS115 yeast competent cells; Identification and expression of D.GS115 / pPIC9k-Fn-HLC yeast transformant; E. Fermentation and purification of fusion proteins; The target gene is the nucleic acid sequence shown in SEQ ID NO.

5.

3. The method for preparing the fusion protein of fibronectin and collagen according to claim 2, characterized in that: In step B, the artificially synthesized sequence and the Pichia pastoris universal vector are double-digested using Xho I and Not I restriction endonucleases. After digestion, the corresponding target gene and vector are recovered, and the gene is ligated and transformed into E. coli using T4 DNA ligase. After sequencing, the pPIC9k-Fn-HLC recombinant plasmid is obtained.

4. The method for preparing the fusion protein of fibronectin and collagen according to claim 2, characterized in that: In step C, the pPIC9k-Fn-HLC recombinant plasmid was linearized using SalI and then purified and recovered. The pPIC9k-Fn-HLC recombinant plasmid was electroporated into Pichia pastoris GS115, and high-copy transformants were screened using G418.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule sequence is a nucleotide sequence or its complementary sequence encoding the fusion protein of fibronectin and collagen as described in claim 1.

6. A carrier, characterized in that: It contains the nucleic acid molecule as described in claim 5.

7. The application of the fusion protein of fibronectin and collagen as described in claim 1 in the preparation of cosmetics or medical aesthetic products.

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