Fibronecked-(X VII) type collagen fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a fibronectin-XVII type collagen fusion protein as well as a preparation method and application of the fibronectin-XVII type collagen fusion protein. The fusion protein comprises at least one XVII type collagen functional domain fragment and at least one fibronectin cell binding domain fragment, the novel COLXVII-FN fusion protein is obtained by carrying out recombinant expression on an XVII type collagen helical region functional fragment and a fibronectin structural domain fragment by utilizing a genetic engineering technology. Through in-vitro cell verification and efficacy test, the fusion protein shows the dual efficacy of collagen and fibronectin, and has obvious cell adhesion promotion and anti-inflammatory activity, the cell adhesion promotion can enhance the intercellular connection, and the anti-inflammatory activity of the fusion protein is helpful to alleviate skin inflammation response. The compound also shows the activity of promoting cell proliferation and adhesion, and accelerates the renewal of cells; in conclusion, the fusion protein has wider application potential in the fields of skin care products, cosmetics, medicines, biomedical materials and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fibronectin-type XVII collagen fusion protein, a preparation method and an application thereof. Background Art

[0002] Type XVII collagen, also known as transmembrane collagen, is primarily distributed within the basement membranes of various tissues and plays a key role in cell attachment and signal transduction. Unlike most collagens, type XVII collagen, also known as anchoring fibers, is the primary component of anchoring fibers in the epidermal-dermal basement membrane zone attachment structure. XVII (encoded by the COL17A1 gene) is a transmembrane protein composed of 1,497 amino acids. It forms a structural component of hemidesmosomes and plays a crucial role in the interaction between epithelial cells and the basement membrane. It regulates epithelial cell adhesion, separation, development and differentiation, and has a significant impact on keratinocyte differentiation and regeneration. The N-terminus of type XVII collagen is located outside the cell, interacting with other components of the basement membrane, such as laminin and fibronectin, while the C-terminus extends into the cell interior. This unique structure confers on type XVII collagen its key function in providing support and stability for cell attachment.

[0003] Currently, relevant literature reports that recombinant human type XVII collagen has excellent cell adhesion and cell proliferation promoting effects, and can be used in skin care products and skin repair medical materials. However, the cell adhesion and cell proliferation promoting effects of currently commercialized recombinant human type XVII collagen are still relatively low, and its functions are relatively limited. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention lies in a fibronectin-type XVII collagen fusion protein, a preparation method and a use thereof.

[0005] To this end, the present invention provides the following technical solutions:

[0006] An embodiment of the present invention provides a fusion protein comprising: at least one type XVII collagen functional domain fragment and at least one cell binding domain fragment of fibronectin.

[0007] The fusion protein of the present invention is a bioactive molecule with potential applications, which may promote tissue repair and regeneration by enhancing cell adhesion and promoting cell migration. Type XVII collagen is an important basement membrane component that is involved in cell attachment and signal transduction, and the binding of fibronectin to sample fibers and microfibrils is also crucial to tissue structure and function. Therefore, by combining fibronectin with the functional domain of type XVII collagen, the fusion protein possesses the efficacy of both fibronectin and type XVII collagen, helping to enhance the adhesion between cells and basement membranes mediated by type XVII collagen, promote cell proliferation and differentiation, and thus achieve tissue repair and regeneration. In short, the fusion protein has a wide range of application potential, especially in the fields of skin care, skin problem treatment and tissue engineering, and has important research value.

[0008] In some embodiments, the type VII collagen functional region fragment is selected from any one of the following amino acid sequences (1)-(2):

[0009] (1) having an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.2;

[0010] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.2 described in (1).

[0011] In some embodiments, the cell-binding region fragment of fibronectin is selected from any one of the following amino acid sequences (1)-(2):

[0012] (1) having the amino acid sequence shown in SEQ ID NO. 3;

[0013] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in SEQ ID NO. 3 described in (1).

[0014] In some embodiments, the C-terminus of the type XVII collagen functional domain fragment is fused to a cell-binding domain fragment of fibronectin. Research conducted by the present invention has found that collagen is more readily expressed in Pichia pastoris, and the expression level is relatively high. Therefore, fusion of the C-terminus of the type XVII collagen functional domain fragment to a cell-binding domain fragment of fibronectin makes it easier to achieve higher expression levels of the fusion protein.

[0015] In some embodiments, the type XVII collagen functional domain fragment and the cell-binding domain fragment of the fused fibronectin are connected by at least one linker; preferably, the number of linkers is 1-3. Furthermore, the number of linkers is 1, 2, or 3.

[0016] In some embodiments, the amino acid sequence of the linker is GGGGS.

[0017] In some embodiments, the fusion protein is selected from any one of the following amino acid sequences (1)-(2):

[0018] (1) having the amino acid sequence shown in SEQ ID NO. 4;

[0019] (2) An amino acid sequence having an identity of 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the amino acid sequence shown in SEQ ID NO. 4 described in (1).

[0020] An embodiment of the present invention provides a biomaterial, including any one of the following:

[0021] 1) A nucleic acid molecule encoding the aforementioned fusion protein; optionally, the nucleic acid molecule is DNA or RNA;

[0022] 2) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the aforementioned fusion protein;

[0023] 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1);

[0024] 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3);

[0025] 5) A host cell containing the recombinant vector described in 2) or 3) or 4).

[0026] In some embodiments, the original plasmid vector of the recombinant vector is a pPIC9K plasmid.

[0027] In some embodiments, the host cell comprises a yeast. Further, the yeast is selected from Pichia pastoris.

[0028] In some embodiments, the method for constructing the recombinant vector comprises: inserting a nucleic acid molecule encoding the fusion protein into a plasmid vector through restriction enzyme cleavage sites to obtain a recombinant vector. Furthermore, synthesizing a gene fragment based on the gene sequence encoding the fusion protein, and inserting the synthesized gene fragment into the Pichia pastoris expression plasmid pPIC9K through restriction enzyme cleavage sites EcoRI and NotI to obtain the recombinant plasmid pPIC9K-C1701-FN.

[0029] In some embodiments, the method for constructing a host cell containing the recombinant vector comprises: enzymatically linearizing the recombinant vector and then transferring it into the host cell; further, the enzymatically linearizing the recombinant vector comprises: enzymatically linearizing the recombinant plasmid pPIC9K-C17-FN using QuickCut SacⅠ at a digestion temperature of 37°C for 5 hours, adding 3M NaAc and anhydrous ethanol, placing the mixture at -20°C overnight, centrifuging at 4°C and 13,000 rpm for 20 minutes, discarding the supernatant, collecting the precipitate, rinsing it with 75% ethanol, centrifuging it again, discarding the supernatant, removing water and residual ethanol from the collected precipitate, and dissolving it with ddH2O to obtain the linearized plasmid pPIC9K-C17-FN. Furthermore, the step of transferring the enzyme-linearized recombinant vector into the host cell includes: mixing the linearized recombinant vector pPIC9K-C17-FN with Pichia pastoris GS115 competent cells, ice bathing, electroporating, adding sorbitol solution, mixing, transferring to a sterile EP tube, incubating at 30° C. for 1 h-2 h, spreading on an MD plate, standing at room temperature for 10 min, and then inverting and culturing at 30° C. for 2 d-5 d until a single colony grows;

[0030] Single colonies from the MD plate were picked and transferred to 96-well plates containing YPD liquid medium supplemented with G418. After further incubation at 30°C for 24 hours, the bacterial solution in a third 96-well plate was aspirated and 1 μL was spotted onto YPD plates supplemented with 1 mg / mL, 2 mg / mL, and 4 mg / mL G418, respectively, to screen for recombinant humanized collagen-producing yeast strains.

[0031] An embodiment of the present invention provides a method for preparing the aforementioned fusion protein, comprising:

[0032] Synthesizing the gene encoding the aforementioned fusion protein;

[0033] constructing a recombinant vector containing the encoding gene;

[0034] The recombinant vector is transformed into host cells and fermented and cultured.

[0035] In some embodiments, the original plasmid vector of the recombinant vector is a pPIC9K plasmid.

[0036] In some embodiments, the host cell comprises yeast; further, the yeast is selected from Pichia pastoris.

[0037] In some embodiments, the method for constructing the recombinant vector comprises: inserting a nucleic acid molecule encoding the fusion protein into a plasmid vector through restriction enzyme cleavage sites to obtain a recombinant vector. Furthermore, synthesizing a gene fragment based on the gene sequence encoding the fusion protein, and inserting the synthesized gene fragment into the Pichia pastoris expression plasmid pPIC9K through restriction enzyme cleavage sites EcoRI and NotI to obtain the recombinant plasmid pPIC9K-C1701-FN.

[0038] In some embodiments, the method for constructing a host cell containing the recombinant vector comprises: enzymatically linearizing the recombinant vector and then transferring it into the host cell; further, the enzymatically linearizing the recombinant vector comprises: enzymatically linearizing the recombinant plasmid pPIC9K-C17-FN using QuickCut SacⅠ at a digestion temperature of 37°C for 5 hours, adding 3M NaAc and anhydrous ethanol, placing the mixture at -20°C overnight, centrifuging at 4°C and 13,000 rpm for 20 minutes, discarding the supernatant, collecting the precipitate, rinsing it with 75% ethanol, centrifuging it again, discarding the supernatant, removing water and residual ethanol from the collected precipitate, and dissolving it with ddH2O to obtain the linearized plasmid pPIC9K-C17-FN. Furthermore, the step of transferring the enzyme-linearized recombinant vector into the host cell includes: mixing the linearized recombinant vector pPIC9K-C17-FN with Pichia pastoris GS115 competent cells, ice bathing, electroporating, adding sorbitol solution, mixing, transferring to a sterile EP tube, incubating at 30° C. for 1 h-2 h, spreading on an MD plate, standing at room temperature for 10 min, and then inverting and culturing at 30° C. for 2 d-5 d until a single colony grows;

[0039] Single colonies from the MD plate were picked and transferred to 96-well plates containing YPD liquid medium supplemented with G418. After further incubation at 30°C for 24 hours, the bacterial solution in a third 96-well plate was aspirated and 1 μL was spotted onto YPD plates supplemented with 1 mg / mL, 2 mg / mL, and 4 mg / mL G418, respectively, to screen for recombinant engineered yeast.

[0040] In some embodiments, the fermentation conditions include any one of the following:

[0041] The inoculation amount is 8% to 10%; further, the inoculation amount can be any one of 8%, 9%, 10% or a range value between any two values.

[0042] The stirring speed is 200 to 400 rpm; further, the stirring speed can be any one of 200, 250, 300, 350, 400 rpm or a range between any two values.

[0043] The tank pressure is 0.03 to 0.05 MPa; further, the tank pressure can be any one of 0.03, 0.04, and 0.05 MPa or a range between any two values.

[0044] The pH is controlled to be 5.0-6.0; further, the pH can be any one of 5, 5.5, 6 or a range between any two values.

[0045] The temperature is 25-30°C; further, the temperature can be any one of 25, 28, 30°C or a range between any two values.

[0046] The dissolved oxygen is controlled at 25-35%; further, the dissolved oxygen can be any one of 25%, 28%, 30%, 33%, 35% or a range between any two values.

[0047] After the fermentation reaches the set value, methanol is added for induction culture, the pH is adjusted to 5.0-6.0, the dissolved oxygen is controlled at 25-35%, and the induction is carried out for 48-72 hours, preferably 48 hours.

[0048] In a preferred embodiment, the seed liquid of the recombinant yeast engineered bacteria is added to a 30L fermentation tank containing 10-15L fermentation medium (BSM) at an inoculum size of 8% to 10%. The initial stirring speed is 200-400 rpm, the tank pressure is 0.03-0.05 MPa, the pH is controlled at 5.0-6.0, and the temperature is 25-30°C. The air flow and speed are adjusted to control the dissolved oxygen at about 25-35%. When the carbon source is exhausted, the dissolved oxygen rises sharply, and 50% glycerol (50% glycerol aqueous solution by mass) is started to be fed to supplement the carbon source; when the wet weight increases to about 200-220 g / L, the glycerol feeding is stopped, and the starvation culture is carried out for 1-2 hours. After the glycerol is exhausted, methanol is started to be fed, and the methanol induction culture stage is entered. Ammonia water is used to adjust the pH to 5.0-6.0, and the speed, air flow and methanol addition rate are adjusted to control the dissolved oxygen at about 25-35%. The fermentation is terminated after 48 hours of induction.

[0049] In some embodiments, the fermentation medium can be a conventional medium. Furthermore, the fermentation medium is selected from BSM medium. Furthermore, the fermentation medium has a formula of: 80% to 85% H3PO4 30 mL / L; CaSO4·2H2O 0.93 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 16 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L; and biotin (500×) 6.35 mL / L.

[0050] The PTM1 solution was prepared according to the Invitrogen operating manual. The specific formula was as follows: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02 g / L; CoCl2 0.5 g / L; ZnCl2 20.0 g / L; FeSO4·7H2O 65.0 g / L; biotin 0.2 g / L; H2SO4 5.0 mL / L. The solution was sterilized by filtration through a 0.22 μm filter membrane and stored at room temperature.

[0051] The embodiments of the present invention provide a use of the aforementioned fusion protein, the aforementioned biomaterial, or the fusion protein prepared by the aforementioned preparation method, including any one of the following:

[0052] (1) Promoting cell proliferation or preparing products that promote cell proliferation;

[0053] (2) promoting cell adhesion or preparing products that promote cell adhesion;

[0054] (3) Use in the preparation of products for inhibiting inflammation;

[0055] (4) Preparation of skin care products or cosmetics;

[0056] (5) Preparation of biomedical materials;

[0057] Optionally, the product includes skin care products, cosmetics, medicines or biomedical materials;

[0058] Optionally, the inhibition of inflammation comprises inhibiting TNF-α and / or IL-6.

[0059] The technical solution of the present invention has the following advantages:

[0060] 1. The present invention provides a fusion protein comprising: at least one type XVII collagen functional domain fragment and at least one fibronectin cell-binding domain fragment. Using genetic engineering techniques, the present invention recombinantly expresses the type XVII collagen helical domain fragment and the fibronectin structural domain fragment to produce a novel COL XVII-FN fusion protein. In vitro cell validation and efficacy testing have demonstrated that this fusion protein exhibits the dual efficacy of collagen and fibronectin, exhibiting significant cell adhesion and anti-inflammatory activities. Promoting cell adhesion can enhance intercellular connectivity, while its anti-inflammatory activity helps alleviate skin inflammatory responses. It also exhibits activity in promoting cell proliferation and adhesion, accelerating cell renewal. In summary, this fusion protein has broad application potential in skincare products, cosmetics, pharmaceuticals, and biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0062] Figure 1 is a map of the recombinant XVII collagen fused to fibronectin plasmid in Example 1 of the present invention;

[0063] Figure 2 This is the process of screening the recombinant yeast strain capable of high-efficiency expression in Example 1 of the present invention;

[0064] Figure 3 This is a graph showing the SDS-PAGE electrophoresis results of the shake flask fermentation supernatant in Example 1 of the present invention; Lane M: Marker; Lanes 1-10: represent the results of 10 positive transformants GS115 / pPIC9K-C1701-FN;

[0065] Figure 4 This is the electrophoresis result of samples taken from the fermentation tank culture of the recombinant bacteria pPIC9K-C1701-FN in Example 1 of the present invention;

[0066] Figure 5 This is the result of the purified recombinant fibronectin-type XVII collagen fusion protein in Example 1 of the present invention;

[0067] Figure 6 This is the result of the cell proliferation promotion test in Experimental Example 1 of the present invention;

[0068] Figure 7 This is the result of the cell adhesion promotion test in Experimental Example 2 of the present invention;

[0069] Figure 8 is the relative cell viability curve in Experimental Example 3 of the present invention;

[0070] Figure 9 The results of negative control and blank control group detection of TNF-α and IL-6 in Experimental Example 3 of the present invention are as follows:

[0071] Figure 10 These are the results of detecting TNF-α and IL-6 in the experimental group and the negative control group in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0072] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0073] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0074] The gene sequences involved in the following examples were all synthesized by GenScript Biotech Co., Ltd.

[0075] Example 1 Fusion Protein

[0076] This embodiment provides a method for preparing a fusion protein, comprising the following steps:

[0077] 1. Obtaining fusion protein recombinant plasmid

[0078] In this example, the amino acid sequence of the type XVII collagen functional domain fragment selected is shown in SEQ ID NO. 1 or SEQ ID NO. 2. The amino acid sequence of the cell-binding domain fragment of fibronectin selected is shown in SEQ ID NO. 3. The cell-binding domain fragment of fibronectin is fused to the C-terminus of the type XVII collagen functional domain fragment via a linker. Studies have found that when expressing collagen in Pichia pastoris, the expression level of the type XVII collagen functional domain fragment selected as the fusion protein is relatively low. Therefore, in this example, the type XVII collagen functional domain fragment selected as SEQ ID NO. 1 and the cell-binding domain fragment of fibronectin selected as SEQ ID NO. 3 are fused. The target fusion protein has the amino acid sequence shown in SEQ ID NO. 5. A terminator is added to the C-terminus of the gene encoding the amino acid sequence of the target fusion protein, and the DNA sequence encoding the protein is optimized for Pichia pastoris expression, making the target fusion protein more suitable for expression in Pichia pastoris. The optimized base sequence of the target fusion protein was commissioned to GenScript Biotech Co., Ltd. for gene fragment synthesis. The coding gene of the synthesized target fusion protein is shown in SEQ ID NO. 5. The synthesized gene fragment was inserted into the pPIC9K plasmid through the EcoRI and NotI restriction sites to obtain the recombinant XVII collagen fusion fibronectin plasmid pPIC9K-C1701-FN. The plasmid map is shown in Figure 1 The nucleotide sequence is shown in SEQ ID NO.8.

[0079] 2. Construction of expression plasmid

[0080] 2.1 Plasmid linearization

[0081] The plasmid was linearized using QuickCut SacⅠ at 37°C for 5 h. The enzyme digestion system was as follows:

[0082] Table 1. Enzyme digestion system

[0083]

[0084]

[0085] After enzyme digestion, add 0.1 volumes of 3M NaAc (pH 5.2) and 2.5 volumes of anhydrous ethanol, and incubate at -20°C overnight. Centrifuge at 13,000 rpm for 20 minutes at 4°C, discard the supernatant. Rinse with 700 μl of 75% ethanol, centrifuge at 13,000 rpm for 20 minutes, and discard the supernatant. Repeat this process. Invert the EP tube onto absorbent paper on a clean bench for approximately 10 minutes to remove as much water and residual ethanol as possible. Redissolve the plasmid in 20 μl of ddH2O, dilute 1 μl 10-fold, and assay the nucleic acid concentration using a one-drop assay.

[0086] 2.2 Preparation of competent yeast cells

[0087] After streaking the Pichia pastoris host strain GS115, a single colony was picked and inoculated into 20 ml of YPD and cultured at 30°C and 225 rpm for 24 h; the colony was transferred to 50 ml of YPD liquid medium at a ratio of 1:1000 and cultured at 30°C and 225 rpm until the OD 600nm =1.3-1.5; transfer the bacterial solution to a sterile 50 ml centrifuge tube, centrifuge at 4°C, 1500 rpm for 5 min, discard the supernatant and collect the bacteria; resuspend the bacterial pellet with 50 ml of pre-cooled sterile ultrapure water, centrifuge at 4°C, 1500 rpm for 5 min; discard the supernatant, and resuspend the bacterial pellet with 50 ml of pre-cooled sterile ultrapure water; centrifuge at 4°C, 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 40 ml of pre-cooled sterile 1 M (mol / L) sorbitol; centrifuge at 4°C, 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 100-150 μl of pre-cooled sterile 1 M sorbitol, gently rotate to mix, and place on ice for use.

[0088] 2.3 Electrotransformation into Pichia pastoris GS115 competent cells

[0089] Take 100μl of Pichia pastoris GS115 competent cells obtained in step 2.2 and mix with 10μl of linearized plasmid obtained in step 2.1, transfer to a pre-cooled electroporation cuvette, and immediately place on ice for 5 minutes. Select the yeast mode of the electroporator and perform electroporation. Then immediately add 1mL of pre-cooled 1M sorbitol to the electroporation cuvette, mix well, transfer the mixture to a sterile EP tube, and incubate in a 30℃ incubator for 1-2 hours. Take 100μl~200μl of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 10 minutes, and incubate it upside down in a 30℃ incubator for about 2~5 days until a single colony appears.

[0090] 2.4 Screening of positive transformants

[0091] Add 2 mL of sterile double-distilled water to the surface of the MD plate, and then gently scrape off the His on the surface of the plate with a sterile triangular spreader. +The transformants were transferred to 50 mL centrifuge tubes. The colonies were counted and diluted to 10 with sterile water. 5 Spread a cell suspension at a concentration of 10 cells / ml onto a YPD plate containing 0.5 mg / mL G418 (Geneticin 418), invert, and incubate at 30°C for 3-4 days until a single colony appears. Pick a single colony from the YPD plate and transfer it to a 96-well plate containing 200 μl of YPD medium. Continue incubating at 30°C. After 48 hours, homogenize the bacterial suspension by aeration, transfer 10 μl per well to a new 96-well plate containing 190 μl YPD, and continue incubating for 24 hours before repeating the above steps. After 24 hours, homogenize the bacterial suspension in a third 96-well plate by aeration, and transfer 1 μl to YPD plates containing 1 mg / mL, 2 mg / mL, and 4 mg / mL G418, respectively, and continue incubating. If the transformant can grow on the plate containing the high concentration of 4 mg / mL G418, it indicates that the transformant contains multiple copies of the target gene. After this step of screening, recombinant yeast strains with high expression efficiency can be obtained. The screening process is as follows: Figure 2 shown.

[0092] 2.5 Identification of positive transformants

[0093] Take 50 μl of the remaining bacterial solution from the first 96-well plate in step 2.4 above, boil it in a boiling water bath for 10 min, freeze it in liquid nitrogen for 30 min, boil it in a boiling water bath for 10 min, repeat freeze-thaw cycles, centrifuge it at 12,000 rpm for 5 min, and use the supernatant as a PCR template to determine whether the target gene has been integrated into the yeast chromosome.

[0094] Upstream primer: 5'AOX1 (5'-GACTGGTTCCAATTGACAAGC-3') (SEQ ID NO. 6);

[0095] Downstream primer: 3'AOX1 (5'-GCAAATGGCATTCTGACATCC-3') (SEQ ID NO. 7);

[0096] PCR amplification system:

[0097] Table 2. PCR amplification system

[0098] PCR system Volume (μl) Upstream primer (10 μmol / L) 0.5 Downstream primer (10 μmol / L) 0.5 Taq DNA polymerase 2.5 Sterile double-distilled water 20 DNA template 1.5 Total volume 25

[0099] PCR amplification conditions: pre-denaturation at 98°C for 5 min, thermal denaturation at 98°C for 50 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 35 cycles; annealing at 72°C for 10 min.

[0100] The amplified product was subjected to 1.5% agarose gel electrophoresis to identify whether a gene fragment of the expected size was amplified. A positive transformant with a gene fragment of the expected size was recorded as GS115 / pPIC9K-C1701-FN.

[0101] 3. Induced Expression of Recombinant Yeast

[0102] The culture medium formula used is as follows:

[0103] 1) YPD complete medium:

[0104] Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L (solid medium containing 2% agar);

[0105] 2)MD solid medium:

[0106] YNB without amino acids, nitrogen source 13.4 g / L; 0.4 mg / L biotin; 20 g / L glucose (solid medium containing 2% agar);

[0107] 3) BMGY yeast growth medium:

[0108] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L KH2PO4, add water to 890mL, sterilize at 121℃ for 20 minutes, then add 100mL of 10×YNB (13.4g / L), 1mL of 500×biotin (4×10 -4 g / L), glycerol 10mL.

[0109] 4) BMMY yeast induction medium:

[0110] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L KH2PO4, add water to 895mL, sterilize at 121℃ for 20 minutes, then add 100×YNB 100mL (13.4g / L), 500×biotin 1mL (4×10 -4 g / L), methanol 5mL.

[0111] The specific operation is as follows: pick a single colony of the positive transformant identified in 2.4 and inoculate it into BMGY medium, and culture it at 30℃ and 220rpm for 24h until the OD 600nm =2-6. According to the measured OD 600nm Adjust the volume of BMGY culture solution to the value, collect the cells at 3000rpm for 10min, and resuspend the cells with BMMY medium of the same volume as BMGY to make the initial OD 600nThe m value was 2.0. The culture was continued at 30°C and 220 rpm. 0.5 v / v% methanol was added to the culture medium every 24 h. The bacterial culture samples were taken 24 h, 48 h, 72 h, and 96 h after induction, and the expression supernatant was collected by centrifugation. The expression level of the target protein was analyzed by SDS-PAGE electrophoresis ( Figure 3 ).

[0112] Result analysis: SDS-PAGE electrophoresis showed that C1701-FN (SEQ ID No.5) was expressed in the yeast engineering strain ( Figure 3 ) was successfully expressed.

[0113] 4. Fermentation in fermenter

[0114] Seed medium: YPD (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L);

[0115] Basal fermentation medium (BSM): 85% H3PO4 10-30mL / L; CaSO4·2H2O 0.93g / L; K2SO4 18.2g / L;

[0116] MgSO4·7H2O 16 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L; biotin (500×) 6.35 mL / L.

[0117] The PTM1 solution was prepared according to the Invitrogen operating manual. The specific formula was as follows: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02 g / L; CoCl2 0.5 g / L; ZnCl2 20.0 g / L; FeSO4·7H2O 65.0 g / L; biotin 0.2 g / L; H2SO4 5.0 mL / L. The solution was sterilized by filtration through a 0.22 μm filter membrane and stored at room temperature.

[0118] Specific implementation methods:

[0119] (1) taking the recombinant bacteria, activating them, and inoculating them into YPD medium for amplification to obtain seed liquid;

[0120] (2) The seed liquid was added to a 30 L fermentation tank containing 10 to 15 L of fermentation medium (BSM) at an inoculum rate of 8% to 10%, with an initial stirring speed of 200 to 400 rpm and a tank pressure of 0.03 to 0.05 MPa. The pH was controlled at 5.0 to 6.0 and the temperature was 25 to 30°C. The air flow rate and speed were adjusted to control the dissolved oxygen at about 25 to 35%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol (50% glycerol aqueous solution by mass) was added to supplement the carbon source.

[0121] (3) When the wet weight increases to about 200-220 g / L, stop feeding glycerol, starve the culture for 1-2 hours, and start feeding methanol after the glycerol is exhausted, entering the methanol induction stage. Use ammonia water to adjust the pH to 5.0-6.0, adjust the rotation speed, air flow rate and feeding rate of methanol to control the dissolved oxygen at about 25-35%, and end the fermentation after 48 hours of induction;

[0122] (4) The fermentation product was centrifuged at 9000 rpm for 30 min to separate the solid and liquid to obtain the fermentation supernatant. The supernatant was ultrafiltered and concentrated using a hollow fiber ultrafiltration system with a molecular weight cutoff of 10.0 KD. The retentate was collected to obtain the crude extract of the fusion protein. The yield of the protein solution was about 0.8-1.0 g / L. The electrophoresis results of the samples taken during the fermentation process were as follows: Figure 4 shown.

[0123] 6 Purification

[0124] The culture supernatant was collected by centrifugation.

[0125] Using a cation exchange medium (chromatographic filler is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system), the chromatography column was equilibrated with PB buffer (20mM, pH 5.0) until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 5mL / min, and the UV A280 absorbance value was detected. When it increased, the sample was loaded. After the loading was completed, the cationic chromatography medium was equilibrated with 20mM PB buffer until the UV and conductivity dropped to the minimum and no longer changed, and the sample loading was stopped. Then, 20mM PB buffer containing NaCl (concentration of 1M) was used to elute and collect the corresponding protein. The eluate containing the target protein was harvested from the column chromatography and dialyzed to obtain a preliminary purified protein semi-finished product, which was subjected to electrophoresis. At the same time, the supernatant containing the target protein before column chromatography and the flow-through obtained from the column chromatography were subjected to electrophoresis to obtain the recombinant fibronectin-type XVII collagen fusion protein ( Figure 5 ).

[0126] Experimental Example 1 Cell proliferation test

[0127] Refer to Appendix 3528 of the 2020 edition (Part III) of the Chinese Pharmacopoeia. This assay is based on the finding that recombinant proteins stimulate the growth of mouse embryonic fibroblasts (BALB / c 3T3 cells). The growth of BALB / c 3T3 cells varies depending on the biological activity of different recombinant proteins, thereby detecting the biological activity of promoting cell proliferation in vitro.

[0128] Test materials:

[0129] Complete cell culture medium: Add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C.

[0130] Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C.

[0131] Digestion solution: 0.25% trypsin.

[0132] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0133] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder and dissolve in 20 ml of PBS. Sterilize by filtering through a 0.22 μm filter. Store at 4°C in the dark.

[0134] Maintenance medium: serum-free medium containing 0.4%;

[0135] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0136] Samples: Recombinant fibronectin-type XVII collagen fusion protein COLXVII-FN (C1701-FN prepared in Example 1), diluted to 1 mg / mL; recombinant humanized type XVII collagen (COLXVII, purchased from Aladdin, product number rp212899), dissolved and diluted to 1 mg / mL; recombinant fibronectin (FN) was provided by Wuhu Interfil Biological Products Industry Research Institute Co., Ltd., with a protein content of 1 mg / mL.

[0137] Specific implementation method: BALB / c 3T3 cell line was cultured in complete culture medium at 37°C and 5% carbon dioxide, and the cell density was controlled to be 2.0×10 5 24 to 36 hours after passage, the cells were used for biological activity assay. The culture medium in the culture flask was discarded, the cells were digested and collected, and 5.0×10 5A cell suspension of 100 μl cells / ml was plated in a 96-well cell culture plate, with 100 μl per well, and cultured at 37°C and 5% CO2. After 24 hours, the plate was replaced with maintenance medium and incubated at 37°C and 5% CO2 for another 24 hours. The maintenance medium was discarded from the prepared cell culture plate, and 100 μl of the standard solution (refer to Chinese Pharmacopoeia, Part III, 3528; the standard is human epidermal growth factor) and the test sample (the aforementioned sample) solution were added to each well, with duplicate wells for each sample. The plates were incubated at 37°C and 5% CO2 for 72 hours. Twenty μl of MTT solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 5 hours. All procedures were performed under sterile conditions. After discarding the liquid from the plate, 100 μl of DMSO was added to each well, mixed, and the absorbance was measured at 570 nm using a microplate reader with a reference wavelength of 630 nm. The results were recorded.

[0138] Data processing: The test data are processed using a computer program or four-parameter regression method, and the results are calculated according to the following formula:

[0139] Corrected titer (U / ml) = Pr × Ds × Es / Dr × Er;

[0140] Where Pr is the biological activity of the standard, U / ml;

[0141] Ds is the pre-dilution multiple of the test sample;

[0142] Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample;

[0143] Dr is the pre-dilution multiple of the standard;

[0144] Er is the dilution multiple of the half-effective dose of the standard;

[0145] The results are as follows Figure 6 As shown in the figure, according to the specific activity results, the activity of recombinant fibronectin-type XVII collagen in promoting BALB / c 3T3 cell proliferation is 8585 U / mg, which is significantly higher than that of commercial recombinant humanized type XVII collagen (about 200 U / mg) and recombinant fibronectin (no obvious activity). In the figure, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0146] Experimental Example 2 Cell Adhesion Promotion Test

[0147] Test materials:

[0148] Complete cell culture medium: Add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C.

[0149] Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C.

[0150] Digestion solution: 0.25% trypsin.

[0151] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0152] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0153] BSA (purchased from Sigma).

[0154] Test sample: recombinant fibronectin-type XVII collagen fusion protein (COLXVII-FN, C1701-FN prepared in Example 1), diluted to 1 mg / mL; recombinant humanized type XVII collagen (COLXVII, purchased from Aladdin, product number rp212899), dissolved and diluted to 1 mg / mL; recombinant fibronectin (FN) was provided by Wuhu Interfil Biological Products Industry Research Institute Co., Ltd., with a protein content of 1 mg / mL.

[0155] Specific implementation method: The test sample was pre-diluted to 0.5 μg / ml with PBS. After pre-dilution, a 2-fold serial dilution was performed in a 96-well plate, making a total of 8 dilutions. 50 μl of the sample of different dilutions was added to each well. Three replicates were made for each concentration. A negative control (no sample) was set up, and 50 μl of PBS was added as a control. The plates were incubated at 4°C overnight. After incubation, the liquid in the plate was discarded, and 100 μl of PBS was added to each well for washing three times. After washing, 100 μl of 30 μg / μL BSA was added to each well for blocking, and the plates were incubated at 37°C for 1 hour. After incubation, the liquid in the plate was discarded, and 100 μl of PBS was added to each well for washing three times. Then, the fibroblast suspension was added, and the cell seeding density was 5.0×10 5 cells / ml, inoculate 100 μl per well, and incubate in an incubator for 5 h. Wash the incubated cell plate three times with PBS, observe the cell adhesion under a microscope, and count the number of adherent cells at five points excluding the edge under a 200x microscope. The titer is calculated based on the curve fitted based on the counting results.

[0156] Data processing: The experimental data were processed using a computer program or four-parameter regression method to obtain the formula: Y = (ad) / [1+(x / c)b]+d. Data points of X and Y were collected or generated, where X is the gradient dilution factor (1, 2, 4, 8, 16, 32, 64, 128) or the corresponding protein concentration (mg / mL or μg / mL), and Y is the average number of cells per well corresponding to X. 2 Statistical indicators such as σ2 and σ3 values ​​were used to evaluate the fitting effect and ensure the goodness of model fitting.

[0157] Where: a is the estimated value of the asymptote on the curve;

[0158] b is the slope of the curve;

[0159] c is the dose corresponding to half of the maximum binding (i.e., half-effective concentration);

[0160] d is the estimated value of the asymptote under the curve;

[0161] Then, the dilution factor is raised to the power of c (i.e. 2 c ) value was calculated to obtain the titer U / ml, which was then divided by the corresponding protein concentration to obtain the specific activity U / mg.

[0162] The results are as follows Figure 7 As shown in the results, recombinant fibronectin-type XVII collagen has significant cell adhesion promoting activity, with a specific activity of approximately 8230 U / mg. However, commercial type 17 collagen (COL XVII) had no significant activity, and the specific activity of single fibronectin (FN) was 4684 U / mg, lower than that of recombinant fibronectin-type XVII collagen fusion protein. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0163] Experimental Example 3 Anti-inflammatory and soothing efficacy test - in vitro TNF-α and IL-6 content determination

[0164] 1. Toxicity test based on mouse macrophage RAW264.7

[0165] Refer to Medical Device Biological Evaluation Part 5: In Vitro Cytotoxicity Tests (GB / T 16886.5-2017). Cytotoxicity is measured by observing cell morphology and the MTT assay, and by quantifying the number of live and dead cells in the sample and measuring cell metabolic activity.

[0166] Test materials:

[0167] Complete cell culture medium: DMEM culture medium (containing double antibodies) with 10% fetal bovine serum added and stored at 4°C.

[0168] Serum-free culture medium: DMEM culture medium (containing double antibodies), stored at 4°C.

[0169] Digestion solution: 0.25% trypsin.

[0170] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up to 1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

[0171] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder and dissolve in 20 ml of PBS. Sterilize by filtering through a 0.22 μm filter. Store at 4°C in the dark.

[0172] Mouse macrophage RAW264.7 cells were purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences.

[0173] Sample: recombinant fibronectin-type XVII collagen fusion protein (COLXVII-FN, C1701-FN prepared in Example 1), diluted to 1 mg / mL.

[0174] Specific implementation method: Mouse macrophage RAW264.7 cells were cultured in complete culture medium at 37°C and 5% CO2, and the cell density was controlled at 1.0×10 5 cells / mL, discard the culture medium in the culture flask 24 to 36 hours after passage. After digestion, collect the cells with complete culture medium and make 5.0×10 5 cells / mL cell suspension. 100 μL of the cell suspension was inoculated into each well of a 96-well plate and cultured for 24 hours. After the cells had grown into a monolayer, the original culture medium was aspirated. 100 μL of the test sample dilutions (samples were serially diluted 10-fold in complete cell culture medium) at different concentrations, a blank control (complete cell culture medium), and a negative control (PBS) were added to the experimental groups. Six replicate wells were prepared for each group except the experimental groups. Within the experimental groups, three replicate wells were prepared for each concentration of each sample. The plates were incubated at 37°C, 5% CO2. After 24 hours, the 96-well plates were removed and cell morphology was observed under a microscope. The liquid was then aspirated and 50 μL of MTT (1 mg / mL) was added to each well. The plates were incubated in a CO2 incubator for 2 hours. The MTT solution was discarded and 100 μL of DMSO solution was added to each well. The plates were shaken and the absorbance was measured at 570 nm (reference wavelength 650 nm) on a microplate reader. Calculate cell survival rate. When the cell survival rate is lower than that of the blank control group, the sample is cytotoxic. Otherwise, it is non-cytotoxic. Statistical method: mean ± standard deviation (`x ± s); survival rate (%) = (OD 570 Mean / blank control group OD 570 mean)×100%.

[0175] Results: As Figure 8 As shown, the relative cell survival rate was >90% compared to the blank control group, with no significant difference (P>0.05). Cell morphology showed no significant changes compared to the blank control group. The highest dose group, 1-10 μg / ml, was selected, with an average cell survival rate exceeding 90%.

[0176] 2. Detection of TNF-α and IL-6 levels by enzyme-linked immunosorbent assay (ELISA)

[0177] Studies have shown that lipopolysaccharide (LPS) can trigger a strong inflammatory response in RAW264.7 macrophages, manifested by upregulation of TNF-α and IL-6 mRNA levels and increased cytokine secretion. By establishing an LPS-induced pro-inflammatory differentiation model of RAW264.7 macrophages, the efficacy of the recombinant protein in inhibiting inflammation in vitro was investigated.

[0178] Test materials:

[0179] Cells: Mouse macrophage RAW264.7 cells were purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences.

[0180] Reagents: lipopolysaccharide (Sigma), DMEM medium (high glucose type), 0.25% trypsin, FBS, double antibody, PBS, MTT, DMSO, dexamethasone (Sinopharm), TNF-α and IL-6 ELISA detection kits (purchased from R&D).

[0181] Samples: recombinant fibronectin-type XVII collagen fusion protein stock solution (COLXVII-FN, C1701-FN prepared in Example 1), protein concentration approximately 1.0 mg / mL; commercial type XVII collagen (COLXVII, Jiangsu Chuangshi, C170801), formulated at 1 mg / mL; recombinant fibronectin (FN) provided by Wuhu Interfil Biological Products Industry Research Institute Co., Ltd., protein content 1 mg / mL; corporate reference product: recombinant serum protein peptide (Wuhu Interfil), 1 mg / mL.

[0182] Specific implementation method: RAW264.7 cells in the logarithmic growth phase were resuspended and diluted to 1×10 5 / ml, add 100μl of cell suspension to each well of the 96-well plate, add 200μl of PBS to each well around the plate as a moisturizing well, and place it in a 37°C, 5% CO2 incubator for 24 hours. Observe the morphology, density and uniform distribution of RAW264.7 cells to determine whether they meet the needs of subsequent experiments. Then, aspirate and discard all the culture medium, and the subsequent culture medium used is DMEM basal medium without FBS. Specific grouping is shown in Table 1. For different groups, 0.9ml of DMEM culture medium (pretreatment solution) containing different components + 0.1ml of LPS solution are added.

[0183] Table 1 Experimental groups for the effect of LPS on the RAW264.7 cell inflammation model

[0184]

[0185]

[0186] Each group was treated with triplicate wells and incubated at 37°C, 5% CO₂ for 6 hours. After pretreatment, 0.1 ml of DMEM medium was added to the blank control group, while 0.1 ml of DMEM medium containing 10 μg / ml LPS was added to the LPS model group and each drug group. The cells were incubated at 37°C, 5% CO₂ for 24 hours. Cell culture supernatants were collected 24 hours after LPS stimulation. Levels of the cytokines TNF-α and IL-6 were measured using ELISA kits. The procedures were performed according to the kit manufacturer's instructions.

[0187] Data Processing and Analysis: All experiments were repeated three times and expressed as mean ± SD. One-way analysis of variance (SNK assay) and multiple comparisons were performed using SPSS 22.0. TNF-α and IL-6 inhibition rates were calculated as follows: Inhibition rate (%) = (1-T / C) × 100%, where: T: mean TNF-α or IL-6 content of the test substance; C: mean TNF-α or IL-6 content of the negative control.

[0188] Results: As Figure 9 and Figure 10 As shown in the test results, it can be seen that Figure 9 As shown, the negative control significantly upregulated expression compared with the blank control, indicating that the cell inflammation model was established. Figure 10 As shown in the results, the positive control (dexamethasone) had a 49.3% inhibition rate for TNFα and a 38.8% inhibition rate for IL-6 compared to the negative control (LPS). The COLⅩⅦ-FN fusion protein had a 41.37% inhibition rate for TNFα and a 77.31% inhibition rate for IL-6, demonstrating significant anti-inflammatory and soothing effects. In contrast, the control group, commercialized collagen XVII (COLⅩⅦ), had a 0 inhibition rate for TNFα and a 8.19% inhibition rate for IL-6, demonstrating no significant anti-inflammatory activity. Meanwhile, recombinant fibronectin (FN) had a 0 inhibition rate for both TNFα and IL-6, exhibiting no significant inhibitory effect on their secretion and failing to exert its anti-inflammatory effect.

[0189] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that include: At least one type XVII collagen functional domain fragment, at least one fibronectin cell binding domain fragment.

2. The fusion protein according to claim 1, characterized in that The type VII collagen functional region fragment is selected from any one of the following amino acid sequences (1)-(2): (1) having an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.2; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence represented by any one of SEQ ID NO. 1 to SEQ ID NO. 2 described in (1).

3. The fusion protein according to claim 1 or 2, characterized in that The cell binding region fragment of fibronectin is selected from any one of the following amino acid sequences (1)-(2): (1) having the amino acid sequence shown in SEQ ID NO. 3; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence shown in SEQ ID NO. 3 described in (1).

4. The fusion protein according to any one of claims 1 to 3, characterized in that The C-terminus of the type XVII collagen functional region fragment is fused with the cell binding region fragment of fibronectin; And / or, the type XVII collagen functional region fragment and the cell binding region fragment of the fused fibronectin are connected by at least one linker; preferably, the number of linkers is 1-3; And / or, the linker is GGGGS.

5. The fusion protein according to claim 4, characterized in that The fusion protein is selected from any one of the following amino acid sequences (1)-(2): (1) having the amino acid sequence shown in SEQ ID NO. 4; (2) An amino acid sequence having an identity of 75% or more with the amino acid sequence shown in SEQ ID NO. 4 described in (1).

6. A biomaterial, characterized in that Includes any of the following: 1) A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5; optionally, the nucleic acid molecule is DNA or RNA; 2) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the fusion protein according to any one of claims 1 to 5; 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1); 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3); 5) A host cell containing the recombinant vector described in 2) or 3) or 4).

7. The biomaterial according to claim 6, characterized in that Including: the initial plasmid vector of the recombinant vector is pPIC9K plasmid; and / or, the host cell comprises yeast; And / or, the method for constructing the recombinant vector comprises: inserting the nucleic acid molecule encoding the fusion protein into a plasmid vector through an enzyme cleavage site to obtain the recombinant vector; And / or, the method for constructing the host cell containing the recombinant vector comprises: linearizing the recombinant vector by enzyme digestion, then transferring the recombinant vector into competent host cells, culturing, and screening positive transformants.

8. A method for preparing the fusion protein according to any one of claims 1 to 5, characterized in that: include: Synthesizing a gene encoding the fusion protein according to any one of claims 1 to 5; constructing a recombinant vector containing the encoding gene; The recombinant vector is transformed into host cells and fermented and cultured.

9. The preparation method according to claim 8, characterized in that The initial plasmid vector of the recombinant vector is pPIC9K plasmid; and / or, the host cell comprises yeast; And / or, the fermentation culture conditions include any one of the following: The inoculation rate is 8% to 10%; Stirring speed 200-400 rpm; Tank pressure 0.03~0.05MPa; Control pH 5.0-6.0; Temperature 25-30℃; Dissolved oxygen is controlled at 25-35%; And / or, after the fermentation culture reaches a set value, methanol is added for induction culture, the pH is adjusted to 5.0-6.0, the dissolved oxygen is controlled at 25-35%, and the induction is carried out for 48-72 hours.

10. A use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to claim 6 or 7, or the fusion protein prepared by the preparation method according to claim 8 or 9, comprising any of the following: (1) Promoting cell proliferation or preparing products that promote cell proliferation; (2) promoting cell adhesion or preparing products that promote cell adhesion; (3) Use in the preparation of products for inhibiting inflammation; (4) Preparation of skin care products or cosmetics; (5) Preparation of biomedical materials; Optionally, the product includes skin care products, cosmetics, medicines or biomedical materials; Optionally, the inhibition of inflammation comprises inhibiting TNF-α and / or IL-6.

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