A highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition and its applications

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to a highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition and its applications. The composition comprises highly hydrophilic recombinant human type III collagen YS-hydro-col3 having the amino acid sequence shown in SEQ ID NO:1. Due to the good hydrophilicity of the highly hydrophilic recombinant human type III collagen YS-hydro-col3, the highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition in the present invention can be well applied in application fields such as biomedicine, cosmetics, health products or food.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition and its application. Background Art

[0002] Collagen is a general term for a class of biological macromolecular proteins. It is one of the main components of animal connective tissues and is also the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of the total protein content. With its unique biological structure, as well as good biocompatibility, degradability, and low antigenicity, collagen has been widely used in many fields of production and life such as biomedicine, cosmetics, health products, or food in recent years.

[0003] As one type of collagen, type III collagen is the most abundant collagen in infant skin and is also called "infant collagen". It is mostly located in the superficial dermis, vascular intima, intestine, etc. and will be lost with age and various factors. It has a delicate reticular structure, controls surrounding cells and moisture, and determines the elasticity and smoothness of the skin. Type III collagen can repair aging fibers, supply nutrition to the skin, enhance skin metabolism, thereby improving various skin aging problems such as dry fine lines, roughness, pigmentation, dryness, and water shortage, and making the skin recover to be delicate, smooth, moist, and bright. And it can restore the damaged skin mucosal barrier, repair damaged elastic fibers, fill and repair the damaged and aging skin, promote blood coagulation during wound repair, and avoid the formation of scars. It also has the function of reconstructing the reticular structure, enhancing the dilatability of damaged skin, and restoring skin elasticity. Its macromolecular active collagen can form a collagen film on the stratum corneum, prevent the evaporation of internal moisture to the outside, lock water and moisturize, and improve the water content of the stratum corneum. By increasing the hydrophilicity of type III collagen, its effects of shrinking pores and tightening the skin may be exerted to a greater extent; it may also increase cell adhesion activity and water solubility, and improve its tissue repair ability; that is, improving the hydrophilicity of type III collagen can optimize the efficacy of collagen in application fields such as biomedicine, cosmetics, health products, or food.

[0004] Therefore, it is of great significance to explore or design a product of highly hydrophilic type III-like collagen. Summary of the Invention

[0005] In view of the above problems, one of the objectives of the present invention is to provide a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 that can be applied in the fields of biomedicine, cosmetics, health products, or food. The composition includes the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, which is obtained by modifying recombinant human type III collagen. The contact angle in its hydrophilicity test can be as low as about 23°, indicating good hydrophilicity and significantly improved hydrophilicity compared to unmodified recombinant human type III collagen.

[0006] To achieve the above objective, the following technical solutions can be adopted:

[0007] On the one hand, the present invention provides a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition, which contains the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, and the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 contains the amino acid sequence shown in SEQ ID NO:1.

[0008] On the other hand, the present invention provides a preparation, which contains the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition or the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, and the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 contains the amino acid sequence shown in SEQ ID NO:1.

[0009] On yet another aspect, the present invention provides a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, which contains the amino acid sequence shown in SEQ ID NO:1.

[0010] On yet another aspect, the present invention provides a nucleotide that encodes the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, and the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 contains the amino acid sequence shown in SEQ ID NO:1.

[0011] On yet another aspect, the present invention provides a recombinant expression vector or a gene expression kit, which contains the above-mentioned nucleotide.

[0012] On yet another aspect, the present invention provides an engineered bacterium, which contains the above-mentioned nucleotide or the above-mentioned recombinant expression vector.

[0013] In another aspect, the present invention provides an application of the above-mentioned recombinant expression or the above-mentioned engineered bacterium in the preparation of highly hydrophilic recombinant human type III collagen YS-hydro-col3.

[0014] In another aspect, the present invention provides an application of a reagent in the preparation of a preparation, and the reagent is selected from the above-mentioned highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition, the above-mentioned highly hydrophilic recombinant human type III collagen YS-hydro-col3, the above-mentioned nucleotide, the above-mentioned recombinant expression vector or gene expression kit, or the above-mentioned engineered bacterium; the preparation includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0015] The beneficial effects of the present invention include: in the hydrophilicity test, the contact angle of the highly hydrophilic recombinant human type III collagen YS-hydro-col3 provided by the present invention is about 23°, and the hydrophilicity is good; compared with the contact angle of the unmodified recombinant human type III collagen (about 45°), it is significantly reduced, and the hydrophilicity is significantly improved. Description of the Drawings

[0016] Figure 1 It is the SDS-PAGE detection result of highly hydrophilic recombinant human type III collagen YS-hydro-col3; M is the marker;

[0017] Figure 2 It is the contact angle experiment result diagram of highly hydrophilic recombinant human type III collagen YS-hydro-col3 and the recombinant human type III collagen reference product; the abscissa is the group; the ordinate is the contact angle value. Detailed Embodiments

[0018] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned invention content still fall within the protection scope of the present invention.

[0019] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is an obvious different meaning in the context, the singular form of the expression includes the plural form of the expression. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or their combinations may exist or can be added. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

[0020] An embodiment of the present invention provides a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition, which at least contains highly hydrophilic recombinant human-like type III collagen YS-hydro-col3. The highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 contains the amino acid sequence shown in SEQ ID NO:1. It should be noted that the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 can be combined with other collagens or other pharmaceutically active ingredients to form a composition, synergistically enhancing the efficacy. Moreover, based on the high hydrophilicity of the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 in the present invention, the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition of the present invention has strong hydrophilicity.

[0021] It should also be noted that the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 in the present invention is obtained by modifying recombinant human-derived type III collagen. The contact angle in its hydrophilicity test can be as low as about 23°, with good hydrophilicity. Compared with the contact angle of unmodified recombinant human-derived type III collagen (about 45°), it is significantly reduced, and the hydrophilicity is significantly improved.

[0022] Another embodiment of the present invention provides a preparation, which contains the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 or the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition. It should be noted that the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 or the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 composition can be added with excipients to prepare a preparation. It should be noted that the preparation includes but is not limited to drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products. In addition, the dosage form of the above-mentioned preparation can be powder, tablet, granule, pill, emulsion, ointment or solution, gel. The administration or usage method can be oral, injection, infusion or external application.

[0023] It should also be noted that the above-mentioned preparation can be a cosmetic for moisturizing, anti-wrinkle or tissue repair. Based on the hydrophilicity of the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, this cosmetic can have a good moisturizing effect, keep the skin in good hydrophilicity, and increase skin elasticity, that is, it can improve the functions of moisturizing and anti-wrinkle beauty; the above-mentioned preparation can also be a drug for tissue repair, which can better retain the type III collagen skin scaffold and has the advantages of repairing the skin barrier, etc., and at the same time helps to enhance the ability of wound healing, that is, it can enhance the functions of liquid absorption, liquid retention and moisture permeability, and promote wound healing; the above-mentioned preparation can also be used as an additive in food to improve the tenderness, water content and softness of meat products.

[0024] An embodiment of the present invention provides a highly hydrophilic recombinant human-like type III collagen YS-hydro-col3, which contains the amino acid sequence shown in SEQ ID NO:1. As described above, this highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 has good hydrophilicity and stronger hydrophilicity than the unmodified recombinant human type III collagen.

[0025] Another embodiment of the present invention provides a nucleotide, which encodes the nucleotide of the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3. The highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 contains the amino acid sequence shown in SEQ ID NO:1. It should be noted that all nucleotides that can encode the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 containing the sequence shown in SEQ ID NO:1 are the nucleotides in the present invention.

[0026] In some specific embodiments, the above-mentioned nucleotide may include the sequence shown in SEQ ID NO:2, or may also be a nucleotide obtained by modifying SEQ ID NO:2 and still capable of encoding the above-mentioned highly hydrophilic recombinant human-like type III collagen YS-hydro-col3.

[0027] Another embodiment of the present invention provides a recombinant expression vector or a gene expression kit, which contains the above-mentioned nucleotide. It should be noted that the above-mentioned nucleotide encoding the highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 can be loaded on an expression vector to form a recombinant expression vector for the expression of the above-mentioned nucleotide. The expression vector is well-known in the art, such as the pPIC9K vector; or expression can be carried out using a gene expression kit well-known in the art.

[0028] Another embodiment of the present invention provides a genetically engineered bacterium comprising the above-mentioned nucleotide or the above-mentioned recombinant expression vector. It should be noted that the above-mentioned recombinant expression vector can be expressed by a genetically engineered bacterium, and the genetically engineered bacterium can be a cloning host bacterium or an expression host bacterium; the cloning host bacterium can be Escherichia coli DH5α strain; the expression host bacterium can be Pichia pastoris GS115. In addition, the method for introducing the recombinant expression vector into the genetically engineered bacterium is known in the art, for example, it can be introduced into the genetically engineered bacterium by electroporation.

[0029] Another embodiment of the present invention provides the use of the above-mentioned recombinant expression or gene expression kit and the above-mentioned genetically engineered bacterium in the preparation of highly hydrophilic recombinant human type III collagen YS-hydro-col3. It should be noted that highly hydrophilic recombinant human type III collagen YS-hydro-col3 can be prepared by a recombinant expression or gene expression kit and a genetically engineered bacterium. In some specific embodiments, the method for preparing highly hydrophilic recombinant human type III collagen YS-hydro-col3 may include the following steps: introducing the coding gene of highly hydrophilic recombinant human type III collagen YS-hydro-col3 into a genetically engineered bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium, inducing protein expression, and extracting and purifying the protein. In some other specific embodiments, a recombinant vector pPIC9K-YS-hydro-col3 (highly hydrophilic recombinant human type III collagen YS-hydro-col3) can be constructed and then introduced into Pichia pastoris GS115 respectively to obtain recombinant Pichia pastoris Pichia-pPIC9K-YS-hydro-col3; culturing Pichia-pPIC9K-YS-hydro-col3, adding methanol to the culture medium to induce protein expression, and then collecting the supernatant; the protein in the supernatant can be purified by trichloroacetic acid precipitation method to obtain type III highly hydrophilic collagen.

[0030] Another embodiment of the present invention provides the use of a reagent in the preparation of a preparation, wherein the reagent is selected from the above-mentioned highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition, the above-mentioned highly hydrophilic recombinant human type III collagen YS-hydro-col3, the above-mentioned nucleotide, the above-mentioned recombinant expression vector or gene expression kit, or the above-mentioned genetically engineered bacterium; the preparation includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0031] To better understand the present invention, the content of the present invention will be further clarified below with specific examples, but the content of the present invention is not limited to the following examples.

[0032] In the following examples, Escherichia coli DH5α competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Pichia pastoris GS115 competent cells were purchased from Invitrogen.

[0033] In the following examples, the pPIC9K plasmid is a Pichia pastoris expression vector purchased from Invitrogen. The promoter of this plasmid is AOX1, the vector size is 9276 bp, and the vector resistance is Ampicillin and Kanamycin. This plasmid utilizes the alpha factor secretion signal peptide to secrete and express the protein gene.

[0034] In the following examples, the MD solid medium, YPD solid medium, BMGY medium, and BMMY medium used were all prepared in the laboratory, and their formulas are as follows: MD solid medium: Weigh 15 g of agar powder into 860 ml of distilled water, autoclave at 121 °C for 20 min, cool to about 60 °C, and successively add 100 ml of 10×YNB, 2 ml of 500× biotin, and 40 ml of 50% glucose. After mixing, quickly pour it into a sterilized petri dish; YPD solid medium: Weigh 20 g of peptone, 10 g of yeast extract, and 15 g of agar powder, dissolve in distilled water and make up the volume to 960 ml, autoclave at 121 °C for 20 min, cool to about 60 °C, add 40 ml of 50% glucose, and quickly pour it into a sterilized petri dish after mixing; BMGY medium: 20 g of peptone and 10 g of yeast extract are dissolved in 780 ml of distilled water, autoclave at 121 °C for 20 min. After cooling to room temperature, add 100 ml of 1 M potassium phosphate buffer (pH 6.0), 100 ml of 10×YNB, 2 ml of 500× biotin, and 20 ml of 50% glycerol; BMMY medium: The glycerol in the BMGY medium is replaced with methanol, and the others are the same as the BMGY medium.

[0035] In the following examples, G418 (Geneticin) is an aminoglycoside antibiotic purchased from Shanghai Macklin Biochemical Co., Ltd.

[0036] In the following examples, the experimental reagents not specifically described are all conventional reagents in the art, which can be prepared according to the conventional methods in the art or obtained from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and relevant experimental manuals can be referred to, such as the Molecular Cloning Experiment Manual or the instructions of relevant reagent manufacturers.

[0037] In the following examples, the recombinant human type III collagen reference standard was purchased from Abcam.

[0038] Example 1 Design and Expression of Highly Hydrophilic Recombinant Human-like Type III Collagen YS-hydro-col3

[0039] (1) Protein Design

[0040] In this example of the present invention, the recombinant human type III collagen reference product was modified and designed to obtain highly hydrophilic recombinant human-like type III collagen YS-hydro-col3 (hereinafter also referred to as YS-hydro-col3). The amino acid sequence of YS-hydro-col3 is shown in SEQ ID NO:1, with a total length of 1068 amino acids.

[0041] (2) Construction of Expression Vector

[0042] (a) The amino acid sequence of YS-hydro-col3 was translated into the corresponding DNA sequence respectively. The coding gene sequence of YS-hydro-col3 is shown in SEQ ID NO:2, with a total length of 3204 bases. The recognition sequence of restriction endonuclease SnaB I (TACGTA) was added to the 5' end of the coding gene sequence of YS-hydro-col3, and the recognition sequence of restriction endonuclease Avr II (CCTAGG) was added to the 3' end to obtain the target gene sequence of YS-hydro-col3. The whole gene synthesis of the target gene sequence was entrusted to Sangon Biotech (Shanghai) Co., Ltd.

[0043] (b) The pPIC9K plasmid was used as the expression vector, and the pPIC9K plasmid and the synthesized target gene were double digested with restriction endonucleases SnaB I (Thermo Fisher Scientific) and Avr II (Thermo Fisher Scientific) respectively. Among them, the digestion system (20 μl): ddH2O 16 μl, 10× Buffer 2 μl, DNA 1 μl, SnaB I 0.5 μl, Avr II 0.5 μl; digestion conditions: digest at 37°C for 3 h and inactivate at 80°C for 20 min.

[0044] (c) The digested pPIC9K plasmid and the target gene were recovered respectively, and the target gene was ligated into the pPIC9K vector through a ligation reaction using T4 DNA ligase (New England Biolabs). Among them, the ligation system (20 μl): 10× buffer 2 μl, T4 DNA ligase 0.2 μl, pPIC9K 3 μl, target gene 1 μl, supplemented with ddH2O to 20 μl; ligation conditions: ligate overnight at 16°C.

[0045] (d) The ligation product was transformed into Escherichia coli DH5α cloning strain by heat shock method. The transformation method is as follows: Take out the Escherichia coli DH5α competent cells from the -80°C refrigerator and place them on ice for 5 min. After the glycerol of the competent cells melts, add the competent cells to the ligation product, pipette up and down 3 - 4 times to mix evenly, and let it stand on ice for 30 min. Quickly dry the water on the tube wall with absorbent paper, place it at 42°C for heat shock for 90 s, and then immediately place it on ice for 2 min. Under sterile conditions, add 800 μl of LB liquid medium, culture at 37°C and 150 rpm for 45 min. Centrifuge at 8000 rpm for 5 min to collect the bacteria, discard part of the supernatant, resuspend the Escherichia coli with the remaining about 100 μl of supernatant, and then evenly coat it on the LB solid medium containing 100 μg / ml ampicillin, put it into a 37°C incubator, and incubate it upside down for 10 - 16 h. Pick a single clone and inoculate it into the liquid LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 10 - 16 h, send the bacterial solution to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to identify the positive clone.

[0046] (e) The plasmid of the positive clone with the correct sequence was extracted using a plasmid extraction kit (Omega, D6943 - 01) according to the kit instructions to obtain the recombinant plasmid pPIC9K - YS - hydro - col3.

[0047] (3) Pichia pastoris transformation

[0048] (a) Take 20 μg of the recombinant plasmid (pPIC9K - YS - hydro - col3), and use the restriction endonuclease Sac I (Promega) to digest the recombinant plasmid; the digestion system (20 μl): 16 μl of ddH2O, 1 μl of the recombinant plasmid, 2 μl of 10× buffer, 1 μl of Sac I; digestion condition: digest at 37°C for 2 h; after inactivating at 65°C for 20 min, use a plasmid extraction kit (Omega, D6943 - 01) to recover the linearized recombinant plasmid according to the kit instructions.

[0049] (b) Take 15 μl of the recovered linearized recombinant plasmid, mix it with 100 μl of Pichia pastoris GS115 competent cells in a 1.5 ml EP tube, and then transfer it to a 0.2 cm electroporation cuvette, place it on ice for 10 min, and put it into an electroporator for electroporation; the electroporation conditions are: voltage 1.5 kv, capacitance 25 μF, resistance 200 Ω, and electroporation time 10 ms.

[0050] (c) After the electroporation is completed, take out the electroporation cuvette, add 1 ml of ice - pre - cooled 1 M sorbitol solution to the electroporation cuvette, and gently pipette to mix evenly.

[0051] (d) Transfer the liquid in the electroshock conversion cup to a 2 ml EP tube, and shake it on a shaker at 30 °C for 40 min; spread all the liquid on an MD solid medium and incubate it at 30 °C for 2 days.

[0052] (e) Use an inoculation loop to pick a single colony grown on the MD solid medium and inoculate it onto a YPD solid medium containing 4.0 mg / ml G418, and incubate it overnight at 30 °C to screen for positive clones, obtaining the recombinant Pichia pastoris Pichia-pPIC9K-YS-hydro-col3.

[0053] (4) Protein expression and purification

[0054] (a) Pick a single colony of the recombinant Pichia pastoris (Pichia-pPIC9K-YS-hydro-col3) and inoculate it into a conical flask containing 50 ml of BMGY medium, then place it in a shaker and incubate it overnight at 30 °C and 220 rpm until OD600 = 1 - 2.

[0055] (b) Transfer the bacterial liquid to a centrifuge tube, centrifuge it at 3000 g for 5 min at room temperature, remove the supernatant, and collect the cells.

[0056] (c) Resuspend the cells with BMMY medium to OD600 = 0.2 - 0.6; transfer the bacterial liquid to a 500 ml conical flask and perform induction expression at 30 °C and 200 rpm, and add methanol to the medium every 24 h to a final concentration of 1% (v / v).

[0057] (d) After the induction expression is completed, centrifuge at 4 °C and 3000 g for 20 min, collect the supernatant for protein purification.

[0058] (e) Take trichloroacetic acid (TCA) with a concentration of 100% equivalent to 1 / 9 of the volume of the supernatant and add it to the centrifuge tube containing the supernatant, shake well, and precipitate overnight at 4 °C.

[0059] (f) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and collect the precipitate; invert the EP tube on absorbent paper and place it in an oven at 37 °C for 10 min - 20 min to make the tube wall have no obvious liquid residue.

[0060] (g) Add 200 μl of cold propanol, shake well, and let it stand at room temperature for 10 min to wash away the residual TCA on the tube wall and the bottom of the tube.

[0061] (h) Repeat steps (f) and (g) 2 - 3 times to obtain the purified protein YS-hydro-col3.

[0062] (5) SDS-PAGE detection of protein YS-hydro-col3

[0063] (a) Prepare an SDS-PAGE protein electrophoresis gel. The concentration of the separating gel is 10%, and the concentration of the stacking gel is 5%. The formula is as follows: Separating gel 10%: 3.3 ml of 30% acrylamide solution, 4 ml of ddH2O, 2.5 ml of gel buffer, 0.1 ml of 10% SDS, 0.1 ml of 10% AP solution, 0.004 ml of TEMED; Stacking gel 5%: 0.67 ml of 30% acrylamide solution, 2.7 ml of ddH2O, 0.5 ml of gel buffer, 0.04 ml of 10% SDS, 0.04 ml of 10% AP solution, 0.04 ml of TEMED.

[0064] (b) Sample treatment: Take an appropriate amount of the protein to be tested, add Loading Buffer, mix well by shaking, boil in boiling water for 5 - 10 minutes to denature the protein, centrifuge at 12000 rpm for 10 minutes, take the supernatant, and load 10 μl. Among them, the electrophoresis parameters are: constant voltage 80V, and 120V after entering the separating gel.

[0065] The results of SDS-PAGE are as Figure 1 shown. The molecular weight of the protein YS-hydro-col3 is about 95 kDa, which is consistent with the expected protein molecular weight.

[0066] Example 2 Hydrophilicity test

[0067] In the embodiment of the present invention, the contact angle of the collagen fiber membrane is tested as follows:

[0068] Using an acetic acid aqueous solution as the solvent, prepare a collagen solution with a total mass concentration of 7% with the protein YS-hydro-col3 and PEO (polyethylene oxide) in Example 1 as solutes (where the mass ratio of the protein YS-hydro-col3 and PEO is 90:10), and stir at room temperature for 2 h; Pour the collagen solution into a 10 mL syringe with a metal nozzle, fix the syringe on a screw propeller and set the parameters, connect the positive pole of the high-voltage generator to the syringe needle, and the negative pole to the bracket with aluminum foil; Under room temperature conditions, the electrospinning process parameters are: spinning voltage 25 Kv, receiving distance 20 cm, propulsion rate 0.8 mL / h, temperature 20 °C, and air relative humidity below 50%; After electrospinning, collect the aluminum foil to obtain the collagen fiber membrane.

[0069] The collagen fiber membrane was cut into rectangles, and a video contact angle measuring instrument was used to detect the contact angles of different membranes (contact angle measurement is an experiment to evaluate the hydrophilicity and hydrophobicity of substances, and the contact angle size is regarded as an index to evaluate the degree of hydrophilicity and hydrophobicity; if θ < 90°, the solid surface is hydrophilic, that is, the liquid is more likely to wet the solid, and the smaller the angle, the better the wettability; if θ > 90°, the solid surface is hydrophobic, that is, the liquid is not easy to wet the solid and is easy to move on the surface). After boiling and cooling, 5 μl of distilled water was automatically dropped onto the sample surface through the instrument each time. The built-in digital camera recorded the shape of the water droplets and recorded and calculated the surface contact angle size. Three parallel samples were measured for each sample, and the average value was calculated.

[0070] In addition, the recombinant human type III collagen reference product was also tested for hydrophilicity according to the above method.

[0071] The above test results are as Figure 2 shown. The contact angle of the recombinant human type III collagen reference product (before modification) was about 45°, while that of YS-hydro-col3 (after modification) was about 23°. Compared with the recombinant human type III collagen reference product, the contact angle of YS-hydro-col3 decreased, indicating that the hydrophilic recombinant human-like type III collagen YS-hydro-col3 of the present invention significantly improved compared with the recombinant human type III collagen reference product.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A highly hydrophilic recombinant human type III collagen YS-hydro-col3 composition, characterized in that, The high hydrophilic recombinant human type III collagen YS-hydro-col3 composition contains high hydrophilic recombinant human type III collagen YS-hydro-col3, and the amino acid sequence of the high hydrophilic recombinant human type III collagen YS-hydro-col3 is shown as SEQ ID NO:

1.

2. A preparation, characterized in that, The preparation contains the high hydrophilic recombinant human type III collagen YS-hydro-col3 composition or high hydrophilic recombinant human type III collagen YS-hydro-col3 described in claim 1, and the amino acid sequence of the high hydrophilic recombinant human type III collagen YS-hydro-col3 is shown as SEQ ID NO:

1.

3. The preparation according to claim 2, characterized in that, The preparation includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

4. Highly hydrophilic recombinant human type III collagen YS-hydro-col3, characterized in that, The amino acid sequence of the high hydrophilic recombinant human type III collagen YS-hydro-col3 is shown as SEQ ID NO:

1.

5. A nucleic acid molecule encoding highly hydrophilic recombinant human type III collagen YS-hydro-col3, characterized in that, The amino acid sequence of the high hydrophilic recombinant human type III collagen YS-hydro-col3 is shown as SEQ ID NO:

1.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown as SEQ IDNO:

2.

7. A recombinant expression vector comprising the nucleic acid molecule according to claim 5 or 6.

8. An engineered bacterium, characterized in that, Contains the nucleic acid molecule described in claim 5 or 6 or the recombinant expression vector of claim 7.

9. Use of the recombinant expression vector according to claim 7 or the engineered bacterium according to claim 8 in the preparation of highly hydrophilic recombinant human type III collagen YS-hydro-col3.

10. Use of a reagent in the preparation of a preparation, characterized in that, The reagent is selected from the high hydrophilic recombinant human type III collagen YS-hydro-col3 composition described in claim 1, the high hydrophilic recombinant human type III collagen YS-hydro-col3 described in claim 4, the nucleic acid molecule described in claim 5 or 6, the recombinant expression vector described in claim 7 or the engineered bacterium described in claim 8; the preparation includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

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