A highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation and its application. The highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation comprises an amino acid sequence as shown in SEQ ID NO: 1. Based on its good hydrophilicity, a highly hydrophilic type V collagen preparation can be obtained for use in the fields of cosmetics, biomedicine, health products or food.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation and application thereof. 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 tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of the total protein content. Type V collagen is one type of collagen, which is usually distributed around cells and around type I collagen. It may play a bridging role between the basement membrane and connective tissue, and is also called pericellular collagen.

[0003] In addition to the unique biological structure of collagen and its good biocompatibility, degradability and low antigenicity, current research has found that type V collagen can regulate the size of cardiac scars after heart injury, and that it helps regulate the fibrous cartilage and hyaline cartilage layers in the condyle of the temporomandibular joint, indicating that it has certain potential in application fields such as biomedicine, cosmetics, health products or food.

[0004] Therefore, it is necessary to design a highly hydrophilic type V collagen preparation for use in the fields of cosmetics, biomedicine, health products or food. Summary of the invention

[0005] In response to the above problems, one of the objects of the present invention is to provide a recombinant human type V collagen YS-hydro-col5 preparation with relatively high hydrophilicity. The highly hydrophilic recombinant human type V collagen YS-hydro-col5 in the preparation is obtained by modifying recombinant human type V collagen, and its hydrophilicity is significantly improved compared with that of unmodified recombinant human type V collagen.

[0006] In order to achieve the above purpose, the following technical solutions can be adopted:

[0007] On the one hand, the present invention provides a highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation, which comprises highly hydrophilic recombinant human type V collagen YS-hydro-col5, and the highly hydrophilic recombinant human type V collagen YS-hydro-col5 comprises the amino acid sequence shown in SEQ ID NO:1.

[0008] Another aspect of the present invention provides a highly hydrophilic recombinant human type V collagen YS-hydro-col5, which comprises the amino acid sequence shown in SEQ ID NO:1.

[0009] In another aspect, the present invention provides a composition comprising highly hydrophilic recombinant human type V collagen YS-hydro-col5, wherein the highly hydrophilic recombinant human type V collagen YS-hydro-col5 comprises the amino acid sequence shown in SEQ ID NO:1.

[0010] In another aspect, the present invention provides a nucleotide encoding a highly hydrophilic recombinant human type V collagen protein YS-hydro-col5. The encoded highly hydrophilic recombinant human type V collagen protein YS-hydro-col5 comprises an amino acid sequence as shown in SEQ ID NO:1.

[0011] In another aspect, the present invention provides a recombinant expression vector or a gene expression kit comprising the above-mentioned nucleotides.

[0012] In another aspect, the present invention provides an engineered bacterium comprising the above-mentioned nucleotide or the above-mentioned recombinant expression vector.

[0013] In another aspect, the present invention provides a use of the above-mentioned recombinant expression or the above-mentioned engineering bacteria in the preparation of highly hydrophilic recombinant human type V collagen YS-hydro-col5.

[0014] In another aspect, the present invention provides a use of the highly hydrophilic recombinant human type V collagen YS-hydro-col5, the composition, the nucleotide, the recombinant expression vector or the gene expression kit or the engineered bacteria in the preparation of preparations, including drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0015] The beneficial effects of the present invention include: in a hydrophilicity test, the highly hydrophilic recombinant human type V collagen YS-hydro-col5 provided by the present invention has a contact angle of about 24°, and has good hydrophilicity; the contact angle is significantly lower than that of the unmodified recombinant human type V collagen (about 44°), and the hydrophilicity is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The SDS-PAGE test results of highly hydrophilic recombinant human type V collagen YS-hydro-col5; M is a marker;

[0017] Figure 2 This is a contact angle test result diagram of highly hydrophilic recombinant human type V collagen YS-hydro-col5 and recombinant human type V collagen standard; the horizontal axis is the group; the vertical axis is the contact angle value. DETAILED DESCRIPTION

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

[0019] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence 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 combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0020] One embodiment of the present invention provides a highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 preparation, which comprises highly hydrophilic recombinant human-like type V collagen YS-hydro-col5, and the highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 comprises the amino acid sequence shown in SEQ ID NO: 1. It should be noted that the highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 or the above-mentioned composition can be added with auxiliary materials to prepare products in the fields of cosmetics, biomedicine, health products or food.

[0021] In some specific embodiments, the highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation includes but is not limited to drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0022] It should be noted that the highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation can be in the form of powder, tablet, granule, pill, emulsion, paste, solution or gel, and can be administered orally, injected, dripped or applied externally.

[0023] It should also be noted that the above-mentioned highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation can be a moisturizing, anti-wrinkle or tissue repair cosmetic. Based on the hydrophilicity of the highly hydrophilic recombinant human type V collagen YS-hydro-col5, the cosmetic can have a good moisturizing effect, keep the skin hydrophilic, increase skin elasticity, that is, it can improve the moisturizing and anti-wrinkle beauty effects; the above-mentioned highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation can also be a drug for tissue repair, which can better retain the advantages of type V collagen skin scaffold, repair skin barrier, etc., while helping to enhance the ability of wound healing, that is, it can enhance the liquid absorption, liquid retention and moisture permeability functions, 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] The embodiment of the present invention provides a highly hydrophilic recombinant human-like type V collagen YS-hydro-col5, which comprises an amino acid sequence as shown in SEQ ID NO: 1. It should be noted that the highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 is obtained by modifying recombinant human type V collagen, and its contact angle in the hydrophilicity test can be as low as about 24°, with good hydrophilicity, which is significantly lower than the contact angle of unmodified recombinant human type V collagen (about 44°), and the hydrophilicity is significantly improved.

[0025] Another embodiment of the present invention provides a composition, comprising highly hydrophilic recombinant human-like type V collagen YS-hydro-col5, wherein the highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 comprises an amino acid sequence as shown in SEQ ID NO: 1. It should be noted that the highly hydrophilic recombinant human-like type V collagen YS-hydro-col5 can be combined with other collagens or other active pharmaceutical ingredients to form a composition to synergistically enhance the efficacy.

[0026] Another embodiment of the present invention provides a nucleotide encoding the highly hydrophilic recombinant human type V collagen YS-hydro-col5 having an amino acid sequence as shown in SEQ ID NO: 1. It should be noted that all nucleotides that can encode the highly hydrophilic recombinant human type V collagen YS-hydro-col5 are nucleotides in the present invention.

[0027] In some specific embodiments, the nucleotide encoding the highly hydrophilic recombinant human type V collagen YS-hydro-col5 may include the sequence shown in SEQ ID NO:2, or may include nucleotides that are modified from SEQ ID NO:2 and can still encode the highly hydrophilic recombinant human type V collagen YS-hydro-col5.

[0028] Another embodiment of the present invention provides a recombinant expression vector or a gene expression kit, comprising the above-mentioned nucleotide. It should be noted that the above-mentioned nucleotide encoding the highly hydrophilic recombinant human type V collagen YS-hydro-col5 can be loaded on an expression vector to form a recombinant expression vector for the expression of the above-mentioned nucleotide, and the expression vector is known in the art, such as a pPIC9K vector; or a gene expression kit known in the art can be used for expression.

[0029] Another embodiment of the present invention provides an 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 an engineered bacterium, and the engineered bacterium can be a cloning host bacterium or an expression host bacterium; the cloning host bacterium can be an Escherichia coli DH5α strain; the expression host bacterium can be Pichia pastoris GS115. In addition, the method of introducing the recombinant expression vector into the engineered bacterium is known in the art, for example, it can be introduced into the engineered bacterium by electroporation transformation.

[0030] Another embodiment of the present invention provides the above-mentioned recombinant expression or gene expression kit and the above-mentioned engineering bacteria for use in preparing type I highly hydrophilic collagen. It should be noted that type V highly hydrophilic collagen can be prepared by recombinant expression or gene expression kit and engineering bacteria. In some specific embodiments, the method for preparing type V highly hydrophilic collagen may include the following steps: introducing the coding gene of type V highly hydrophilic collagen into the engineering bacteria, obtaining recombinant bacteria, culturing the recombinant bacteria, inducing protein expression, and extracting and purifying the protein. In some other specific embodiments, a recombinant vector pPIC9K-YS-hydro-col5 (highly hydrophilic recombinant human type V collagen YS-hydro-col5) can be constructed and then introduced into Pichia pastoris GS115 to obtain recombinant Pichia pastoris Pichia-pPIC9K-Col5A1; Pichia-pPIC9K-Col5A1 is cultured, methanol is added to the culture medium to induce protein expression, and then the supernatant is collected; the protein in the supernatant can be purified by trichloroacetic acid precipitation method to obtain highly hydrophilic recombinant human type V collagen YS-hydro-col5.

[0031] Another embodiment of the present invention provides an application of the above-mentioned type V highly hydrophilic collagen, the above-mentioned composition, the above-mentioned nucleotide, the above-mentioned recombinant expression vector or gene expression kit or the above-mentioned engineered bacteria in the preparation of preparations, and the preparations include drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products. It should be noted that, as mentioned above, based on the hydrophilicity of the highly hydrophilic recombinant human type V collagen YS-hydro-col5, it and its nucleic acid nucleotides and expression vectors and engineered bacteria can be used in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products to improve the quality of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0032] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0033] In the following examples, Escherichia coli DH5α competent cells were purchased from Beijing Solebow Technology Co., Ltd.; Pichia pastoris GS115 competent cells were purchased from Invitrogen.

[0034] In the following examples, the pPIC9K plasmid is a Pichia pastoris expression vector purchased from Invitrogen, the promoter of the plasmid is AOX1, the vector size is 9276 bp, and the vector resistance is Ampicillin and Kanamycin. The plasmid uses the alpha factor to secrete a signal peptide to secrete the expression protein gene.

[0035] 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 agar powder and dissolve it in 860 ml distilled water, sterilize it at 121 degrees for 20 minutes, cool it to about 60 degrees, and add 10×YNB 100ml, 500× biotin 2ml and 50% glucose 40ml, mix well and pour into a sterilized culture dish quickly; YPD solid medium: weigh 20g peptone, 10g yeast extract and 15g agar powder, dissolve in distilled water and make up to 960ml, autoclave at 121 degrees for 20min, cool to about 60 degrees, add 50% glucose 40ml, mix well and pour into a sterilized culture dish quickly; BMGY medium: 20g peptone and 10g yeast extract are dissolved in 780ml distilled water, autoclave at 121 degrees for 20min, after cooling to room temperature, add 100ml 1M potassium phosphate buffer (PH6.0), 100ml 10×YNB, 2ml 500× biotin and 20ml 50% glycerol; BMMY medium: the glycerol in the BMGY medium is replaced with methanol, and the rest is the same as the BMGY medium.

[0036] In the following examples, G418 (Geneticin) is an aminoglycoside antibiotic purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0037] In the following examples, all experimental reagents not otherwise specified are conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; all experimental methods not otherwise specified are conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0038] In the following examples, recombinant human type V collagen standard was purchased from Abcam

[0039] Example 1 Design and expression of type V highly hydrophilic collagen-like protein

[0040] (1) Protein design

[0041] In the embodiment of the present invention, the recombinant human type V collagen standard is modified and designed to obtain the highly hydrophilic recombinant human type V collagen YS-hydro-col5 (hereinafter also represented by YS-hydro-col5). The amino acid sequence of YS-hydro-col5 is shown in SEQ ID NO: 1, and its full length is 1364 amino acids.

[0042] (2) Expression vector construction

[0043] (a) The amino acid sequence of YS-hydro-col5 was translated into the corresponding DNA sequence. The coding gene sequence of YS-hydro-col5 is shown in SEQ ID NO: 2, and its total length is 4092 bases. The recognition sequence (TACGTA) of restriction endonuclease SnaB I was added to the 5' end of the coding gene sequence of Col5A1, and the recognition sequence (CCTAGG) of restriction endonuclease Avr II was added to the 3' end to obtain the target gene sequence of YS-hydro-col5, and the target gene sequence was commissioned to be fully synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0044] (b) pPIC9K plasmid was used as an expression vector, and restriction endonucleases SnaB I (Thermo Fisher Scientific) and Avr II (Thermo Fisher Scientific) were used to perform double enzyme digestion on pPIC9K plasmid and the synthesized target gene, respectively. The enzyme digestion system (20 μl) included 16 μl of ddH2O, 2 μl of 10×Buffer, 1 μl of DNA, 0.5 μl of SnaB I, and 0.5 μl of Avr II; the enzyme digestion conditions were: 37°C for 3 h, and inactivation at 80°C for 20 min.

[0045] (c) Recover the pPIC9K plasmid and target gene after restriction digestion, and connect the target gene to the pPIC9K vector by ligation reaction using T4 DNA ligase (New England Biolabs). The ligation system (20 μl) includes: 10× buffer 2 μl, T4 DNA ligase 0.2 μl, pPIC9K 3 μl, target gene 1 μl, ddH2O to 20 μl; ligation conditions: 16°C overnight.

[0046] (d) Use the heat shock method to transform the ligation product into the E. coli DH5α cloning strain. The transformation method is as follows: Take out the E. coli DH5α competent cells from the -80℃ refrigerator and place them on ice for 5 minutes. After the glycerol of the competent cells melts, add the competent cells to the ligation product, mix them by pipetting 3-4 times with the pipette tip, and place them on ice for 30 minutes. Quickly wipe the water off the tube wall with absorbent paper, place it at 42℃ for 90 seconds, and then immediately place it on ice for 2 minutes. Add 800μl of LB liquid culture medium under sterile conditions and culture it at 37℃, 150rpm for 45 minutes. Collect the cells by centrifugation at 8000rpm for 5 minutes, discard part of the supernatant, resuspend the E. coli with the remaining 100μl of supernatant, and then evenly spread it on the LB solid culture medium containing 100μg / ml ampicillin, place it in a 37℃ incubator, and culture it upside down for 10h-16h. A single clone was picked and inoculated into liquid LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 10 h-16 h, the bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to identify positive clones.

[0047] (e) A plasmid extraction kit (Omega, D6943-01) was used to extract the plasmid of the positive clone with the correct sequence according to the kit instructions to obtain the recombinant plasmid pPIC9K-YS-hydro-col5.

[0048] (3) Pichia pastoris transformation

[0049] (a) 20 μg of recombinant plasmid (pPIC9K-YS-hydro-col5) was taken and digested with restriction endonuclease Sac I (Promega); digestion system (20 μl): 16 μl ddH2O, 1 μl recombinant plasmid, 2 μl 10× buffer, 1 μl Sac I; digestion conditions: digestion at 37°C for 2 h; after inactivation at 65°C for 20 min, the linearized recombinant plasmid was recovered using a plasmid extraction kit (Omega, D6943-01) according to the kit instructions.

[0050] (b) Take 15 μl of the recovered linearized recombinant plasmid and mix it with 100 μl of Pichia pastoris GS115 competent cells in a 1.5 ml EP tube, then transfer it to a 0.2 cm electroporation cup, 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.

[0051] (c) After the electroporation is completed, remove the electroporation cup and add 1 ml of 1 M sorbitol solution pre-cooled on ice into the electroporation cup. Mix by gently pipetting with a pipette.

[0052] (d) Transfer the liquid in the electroporation transformation cup to a 2 ml EP tube and shake it at 30°C for 40 min. Spread all the liquid on MD solid culture medium and culture it at 30°C for 2 days.

[0053] (e) A single colony grown on the MD solid medium was picked up with an inoculation loop and inoculated onto a YPD solid medium containing 4.0 mg / ml G418. The culture was incubated at 30°C overnight, and positive clones were screened to obtain recombinant Pichia pastoris Pichia-pPIC9K-YS-hydro-col5.

[0054] (4) Protein expression and purification

[0055] (a) Single colonies of the recombinant Pichia pastoris (Pichia-pPIC9K-YS-hydro-col5) were picked and inoculated into conical flasks containing 50 ml of BMGY medium. The flasks were then placed in a shaker and cultured overnight at 30°C and 220 rpm until OD600 = 1-2.

[0056] (b) Transfer the bacterial solution to a centrifuge tube and centrifuge at 3000 g for 5 min at room temperature. Remove the supernatant and collect the cells.

[0057] (c) Resuspend the cells in BMMY culture medium to OD600 = 0.2-0.6; transfer the culture medium to a 500 ml conical flask and induce expression at 30°C, 200 rpm, and add methanol to the culture medium every 24 hours to a final concentration of 1% (v / v).

[0058] (d) After induction of expression, the cells were centrifuged at 3000 g for 20 min at 4°C and the supernatant was collected for protein purification.

[0059] (e) Take 100% trichloroacetic acid (TCA) equivalent to 1 / 9 of the volume of the supernatant and add it to the centrifuge tube containing the supernatant, shake to mix, and precipitate at 4°C overnight.

[0060] (f) Centrifuge at 12,000 rpm for 10 min, discard the supernatant, and collect the precipitate; invert the EP tube onto absorbent paper and place it in a 37°C oven for 10-20 min to ensure that there is no obvious liquid residue on the tube wall.

[0061] (g) Add 200 μl of cold propanol, vortex to mix, and let stand at room temperature for 10 min to wash away the TCA remaining on the tube wall and bottom.

[0062] (h) Repeat steps (6) and (7) 2-3 times to obtain the purified protein YS-hydro-col5.

[0063] (5) SDS-PAGE detection of protein YS-hydro-col5

[0064] (a) Prepare SDS-PAGE protein electrophoresis gel, wherein the concentration of separation gel is 10% and the concentration of stacking gel is 5%, and the formula is as follows: separation gel 10%: 30% acrylamide solution 3.3 ml, ddH2O 4 ml, gel buffer 2.5 ml, 10% SDS 0.1 ml, 10% AP solution 0.1 ml, TEMED 0.004 ml; stacking gel 5%: 30% acrylamide solution 0.67 ml, ddH2O 2.7 ml, gel buffer 0.5 ml, 10% SDS 0.04 ml, 10% AP solution 0.04 ml, TEMED 0.04 ml.

[0065] (b) Sample treatment: Take an appropriate amount of the protein to be tested, add Loading Buffer, shake and mix, boil in boiling water for 5min-10min to denature the protein, centrifuge at 12000rpm for 10min, take the supernatant, and load 10μl. The electrophoresis parameters are: constant voltage 80V, 120V after entering the separation gel.

[0066] SDS-PAGE results are as follows Figure 1 As shown, the protein molecular weight of YS-hydro-col5 is about 95 kDa, which is consistent with the expected protein molecular weight.

[0067] Example 2 Hydrophilicity Test

[0068] In the embodiment of the present invention, the contact angle of the collagen fiber membrane is tested as follows:

[0069] Using acetic acid aqueous solution as solvent, protein YS-hydro-col5 and PEO (polyethylene oxide) in Example 1 as solutes to prepare a collagen solution with a total mass concentration of 7% (wherein the mass ratio of protein YS-hydro-col5 and PEO is 90:10), and stirring at room temperature for 2 hours; pour the collagen solution into a 10mL syringe with a metal nozzle, fix the syringe on a spiral propeller and set the parameters, connect the positive electrode of the high-voltage generator to the syringe needle, and connect the negative electrode to a bracket with aluminum foil; under room temperature conditions, the electrospinning process parameters are: spinning voltage 25Kv, receiving distance 20cm, propulsion rate 0.8mL / h, temperature 20℃, and relative air humidity below 50%; after spinning is completed, collect the aluminum foil and obtain a collagen fiber membrane. The collagen fiber membrane was cut into rectangles, and the contact angles of different films were detected by a video contact angle meter (the contact angle test is an experiment to evaluate the hydrophilicity and hydrophobicity of a substance, and the contact angle is regarded as an indicator for evaluating the degree of hydrophilicity and hydrophobicity; if θ<90°, the solid surface is hydrophilic, that is, the liquid is easier 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). The distilled water that was boiled and cooled was automatically added to the sample surface at a rate of 5 μl each time through the instrument. The built-in digital camera recorded the shape of the water droplets and recorded and calculated the surface contact angle. Three parallel samples were measured for each sample, and the average value was calculated.

[0070] The recombinant human type V collagen standard was also tested for hydrophilicity according to the above method.

[0071] The hydrophilicity test results of YS-hydro-col5 and recombinant human type V collagen standard are as follows Figure 2 As shown, the contact angle of the recombinant human type V collagen standard (before modification) is about 44°, while that of YS-hydro-col5 (after modification) is about 24°. Compared with the recombinant human type V collagen standard, the contact angle of YS-hydro-col5 is reduced, indicating that the hydrophilicity of the highly hydrophilic recombinant human type V collagen YS-hydro-col5 of the present invention is significantly improved compared with the recombinant human type V collagen standard.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A highly hydrophilic recombinant human type V collagen YS-hydro-col5 preparation, comprising highly hydrophilic recombinant human type V collagen YS-hydro-col5, wherein the amino acid sequence of the highly hydrophilic recombinant human type V collagen YS-hydro-col5 is as shown in SEQ ID NO: 1; the preparation is a drug, a medical device, a biomaterial, a tissue engineering product or a cosmetic.

2. Highly hydrophilic recombinant human type V collagen YS-hydro-col5, with an amino acid sequence as shown in SEQ ID NO:

1.

3. A composition comprising highly hydrophilic recombinant human type V collagen YS-hydro-col5, wherein the amino acid sequence of the highly hydrophilic recombinant human type V collagen YS-hydro-col5 is shown in SEQ ID NO:

1.

4. Nucleotide, encoding highly hydrophilic recombinant human type V collagen YS-hydro-col5, the amino acid sequence of the encoded highly hydrophilic recombinant human type V collagen YS-hydro-col5 is shown in SEQ ID NO:

1.

5. The nucleotide according to claim 4, characterized in that The nucleotide sequence is as shown in SEQ ID NO:

2.

6. A recombinant expression vector or a gene expression kit comprising the nucleotide according to claim 4 or 5.

7. An engineered bacterium comprising the nucleotide according to claim 4 or 5 or the recombinant expression vector or gene expression kit according to claim 6.

8. Use of the recombinant expression or gene expression kit according to claim 6 or the engineering bacteria according to claim 7 in the preparation of highly hydrophilic recombinant human type V collagen YS-hydro-col5.

9. Use of the highly hydrophilic recombinant human type V collagen YS-hydro-col5 according to claim 2, the composition according to claim 6, the nucleotide according to claim 4 or 5, the recombinant expression vector or gene expression kit according to claim 6, or the engineered bacteria according to claim 7 in the preparation of a preparation, wherein the preparation is a drug, a medical device, a biomaterial, a tissue engineering product or a cosmetic.

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