A nucleotide encoding highly hydrophilic recombinant human type IV collagen YS-hydro-col4 and its applications

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a nucleotide encoding a highly hydrophilic recombinant human type IV collagen YS-hydro-col4 and its application. The encoded highly hydrophilic recombinant human type IV collagen YS-hydro-col4 contains the amino acid sequence shown in SEQ ID NO:1. In the hydrophilicity test, its contact angle is about 24°, indicating good hydrophilicity. Compared with the unmodified recombinant human type IV collagen (about 47°), the contact angle is significantly reduced, and the hydrophilicity is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nucleotide encoding a highly hydrophilic recombinant human type IV collagen YS-hydro-col4 and its applications. Background Technology

[0002] Collagen is a general term for a class of biological macromolecules. 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 total protein content. For example, type IV collagen, a type of collagen, is an important component of the basement membrane, located near the membrane at the boundary between the epidermis and dermis. Type IV collagen provides a stable epidermal-dermal junction, ensuring the mechanical integrity of the skin. It also regulates and acts as a ligand signaling mediator, binding to cell receptors such as integrins, thereby affecting cell proliferation, migration, and differentiation. In the same way, it influences the polar upward migration and differentiation of keratinocytes in the basal layer of the epidermis. Therefore, it plays a crucial role in the body. For example, the invasion and metastasis of skin tumors first require breaching the basement membrane. In liver disease, with the development of inflammation, fibrous tissue proliferates actively, and a large amount of collagen is deposited during fibrous tissue formation. Various types of collagen increase, but the most important increase is in type IV collagen, which constitutes the basement membrane.

[0003] The unique biological structure of collagen, especially type IV collagen, along with its excellent biocompatibility, biodegradability, and low antigenicity, has led to its widespread application in various fields of production and daily life, including biomedicine, cosmetics, health products, and food. For example, type IV collagen is currently used as a membrane material for immobilizing enzymes and in meat product labels; as a food adhesive for synthetic fiber membranes and food protective layers; and as an antioxidant to maintain the bright color of food. It can also improve the tenderness, water content, and softness of meat products. Furthermore, it may have potential applications in the preparation of artificial basement membranes.

[0004] Therefore, it is of great significance to discover or design a nucleotide that can encode highly hydrophilic type IV collagen-like proteins. Summary of the Invention

[0005] To address the above problems, one objective of this invention is to provide a nucleotide that can encode a highly hydrophilic recombinant human type IV collagen YS-hydro-col4. The highly hydrophilic recombinant human type IV collagen YS-hydro-col4 encoded by this nucleotide is obtained by modifying recombinant human type IV collagen. The resulting highly hydrophilic recombinant human type IV collagen YS-hydro-col4 can achieve a contact angle as low as approximately 24° in hydrophilicity tests, exhibiting good hydrophilicity. This is significantly lower than the contact angle (approximately 47°) of unmodified recombinant human type IV collagen, indicating a significant improvement in hydrophilicity.

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

[0007] In one aspect, the present invention provides a nucleotide encoding a highly hydrophilic recombinant human type IV collagen YS-hydro-col4, wherein the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 comprises the amino acid sequence shown in SEQ ID NO:1.

[0008] Another aspect of the present invention provides a recombinant expression vector or gene expression kit comprising the above-described nucleotides.

[0009] In another aspect, the present invention provides an engineered bacterium comprising the above-described nucleotides or the above-described recombinant expression vector.

[0010] In another aspect, the present invention provides the application of the above-mentioned recombinant expression or the above-mentioned engineered bacteria in the preparation of highly hydrophilic recombinant human type IV collagen YS-hydro-col4.

[0011] In another aspect, the present invention provides a highly hydrophilic recombinant human type IV collagen YS-hydro-col4, comprising the amino acid sequence shown in SEQ ID NO:1.

[0012] In another aspect, the present invention provides a composition comprising highly hydrophilic recombinant human type IV collagen YS-hydro-col4, wherein the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 comprises the amino acid sequence shown in SEQ ID NO:1.

[0013] In another aspect, the present invention provides a formulation comprising the above-described highly hydrophilic recombinant human type IV collagen YS-hydro-col4 or the above-described composition.

[0014] In another aspect, the present invention provides the application of the above-mentioned highly hydrophilic recombinant human type IV collagen YS-hydro-col4, the above-mentioned nucleotides, the above-mentioned recombinant expression vector, the above-mentioned engineered bacteria, or the above-mentioned composition in the preparation of formulations, the formulations including pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, or health products.

[0015] The beneficial effects of this invention include: the nucleotide provided by this invention can encode a recombinant human type IV collagen YS-hydro-col4 with relatively high hydrophilicity. The contact angle of the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 encoded by this nucleotide can be as low as about 24° in the hydrophilicity test, which is good hydrophilicity. Compared with the contact angle of unmodified recombinant human type IV collagen (about 47°), the hydrophilicity is significantly reduced and the hydrophilicity is significantly improved. Attached Figure Description

[0016] Figure 1 The results of SDS-PAGE analysis of highly hydrophilic recombinant human type IV collagen YS-hydro-col4 are shown; M is the marker.

[0017] Figure 2 The graph shows the contact angle test results between the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 and the recombinant human type IV collagen standard; the horizontal axis represents the group and the vertical axis represents the contact angle value. Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0020] This invention provides a nucleotide encoding the aforementioned highly hydrophilic recombinant human type IV collagen YS-hydro-col4, which comprises the sequence shown in SEQ ID NO:1. It should be noted that all nucleotides capable of encoding type IV highly hydrophilic collagen comprising the sequence shown in SEQ ID NO:1 are nucleotides of this invention.

[0021] In some specific embodiments, the nucleotides encoding the aforementioned highly hydrophilic recombinant human type IV collagen YS-hydro-col4 may include the sequence shown in SEQ ID NO:2, or may include nucleotides that can still encode the aforementioned highly hydrophilic type IV collagen after modifying SEQ ID NO:2.

[0022] Another embodiment of the present invention provides a recombinant expression vector or gene expression kit containing the above-described nucleotides. It should be noted that the nucleotides encoding the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 can be loaded onto an expression vector to form a recombinant expression vector for the expression of the above-described nucleotides. The expression vector is known in the art, such as the pPIC9K vector; or a gene expression kit known in the art can be used for expression.

[0023] In another embodiment of the present invention, an engineered bacterium is provided, comprising the above-described nucleotides or the above-described recombinant expression vector. It should be noted that the above-described recombinant expression vector can be expressed by an engineered bacterium, which 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. Furthermore, the method for 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.

[0024] In another embodiment of the present invention, the above-mentioned recombinant expression or gene expression kit and the above-mentioned engineered bacteria are used in the preparation of type I high hydrophilic collagen. It should be noted that the high hydrophilic recombinant human type IV collagen YS-hydro-col4 can be prepared using a recombinant expression or gene expression kit and engineered bacteria. In some specific embodiments, the method for preparing type IV high hydrophilic collagen may include the following steps: introducing the encoding gene of the high hydrophilic recombinant human type IV collagen YS-hydro-col4 into engineered bacteria to obtain recombinant bacteria, culturing the recombinant bacteria, inducing protein expression, and extracting and purifying the protein. In other specific embodiments, a recombinant vector pPIC9K-YS-hydro-col4 (highly hydrophilic recombinant human type IV collagen YS-hydro-col4) can be constructed and then introduced into Pichia GS115 to obtain recombinant Pichia-pPIC9K-YS-hydro-col4; Pichia-pPIC9K-YS-hydro-col4 is cultured, and 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 to obtain highly hydrophilic recombinant human type IV collagen YS-hydro-col4.

[0025] In another embodiment of the present invention, a highly hydrophilic recombinant human type IV collagen YS-hydro-col4 is provided, comprising the amino acid sequence shown in SEQ ID NO:1. It should be noted that this highly hydrophilic recombinant human type IV collagen YS-hydro-col4 is obtained by modifying recombinant human type IV collagen. Its contact angle in hydrophilicity testing can be as low as approximately 24°, exhibiting good hydrophilicity, which is significantly lower than the contact angle (approximately 47°) of unmodified recombinant human type IV collagen, indicating a significant improvement in hydrophilicity.

[0026] Another embodiment of the present invention provides a composition comprising at least a highly hydrophilic recombinant human type IV collagen YS-hydro-col4 with an amino acid sequence as shown in SEQ ID NO:1. It should be noted that the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 of the present invention can be used in combination with other collagen or other pharmaceutical active ingredients to form a composition that synergistically enhances efficacy.

[0027] Another embodiment of the present invention provides a formulation comprising the above-described highly hydrophilic recombinant human type IV collagen YS-hydro-col4 or the above-described composition. It should be noted that the above-described highly hydrophilic recombinant human type IV collagen YS-hydro-col4 or the above-described composition can be prepared into a formulation by adding excipients.

[0028] In some specific embodiments, the formulation includes, but is not limited to, drugs, medical devices, biomaterials, tissue-engineered products, cosmetics, or health products.

[0029] It should be noted that the dosage form of the above-mentioned preparations may be powder, tablet, granule, pill, emulsion, ointment, solution, or gel. The administration or application method may be oral, injection, infusion, or topical application.

[0030] It should also be noted that the above-mentioned preparations can be cosmetics for moisturizing, anti-wrinkle, or tissue repair. Based on the hydrophilicity of highly hydrophilic recombinant human type IV collagen YS-hydro-col4, these cosmetics can have good moisturizing effects, maintain good skin hydrophilicity, and increase skin elasticity, thus enhancing moisturizing and anti-wrinkle effects. The above-mentioned preparations can also be drugs for tissue repair, which can better retain the skin scaffold of type IV collagen, repair the skin barrier, and help enhance the ability to heal wounds, that is, enhance the functions of fluid absorption, fluid retention, and moisture permeability, and promote wound healing. The above-mentioned preparations can also be used as additives in food to improve the tenderness, water content, and softness of meat products.

[0031] Another embodiment of the present invention provides the application of the above-mentioned highly hydrophilic recombinant human type IV collagen YS-hydro-col4, the above-mentioned nucleotides, the above-mentioned recombinant expression vector, the above-mentioned engineered bacteria, or the above-mentioned composition in the preparation of formulations, including pharmaceuticals, medical devices, biomaterials, tissue-engineered products, cosmetics, or health products. It should be noted that, as described above, based on the hydrophilicity of the highly hydrophilic recombinant human type IV collagen YS-hydro-col4, it, along with its nucleotides, expression vector, and engineered bacteria, can be used in the preparation of pharmaceuticals, medical devices, biomaterials, tissue-engineered products, cosmetics, or health products to improve the quality of these products.

[0032] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0033] 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.

[0034] 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 is resistant to ampicillin and kanamycin. This plasmid utilizes the alpha factor to secrete a signal peptide, thereby secreting the expressed protein gene.

[0035] In the following examples, the MD solid culture medium, YPD solid culture medium, BMGY culture medium, and BMMY culture medium used were all prepared in the laboratory, and their formulas are as follows: MD solid culture medium: Weigh 15g of agar powder into 860ml of distilled water, autoclave at 121 degrees Celsius for 20 minutes, cool to about 60 degrees Celsius, and then add 10×YNB. 100ml of peptone, 2ml of 500× biotin, and 40ml of 50% glucose were mixed and quickly poured into sterilized petri dishes; YPD solid medium: 20g of peptone, 10g of yeast extract, and 15g of agar powder were dissolved in distilled water and brought to a final volume of 960ml. The mixture was autoclaved at 121°C for 20min, cooled to about 60°C, and 40ml of 50% glucose were added. The mixture was then quickly poured into sterilized petri dishes; BMGY medium: 20g of peptone and 10g of yeast extract were dissolved in 780ml of distilled water. The mixture was autoclaved at 121°C for 20min, cooled to room temperature, and 100ml of 1M potassium phosphate buffer (pH 6.0), 100ml of 10× YNB, 2ml of 500× biotin, and 20ml of 50% glycerol were added; BMMY medium: glycerol in BMGY medium was replaced with methanol, otherwise the same as 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, unless otherwise specified, all experimental reagents are conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; unless otherwise specified, all experimental methods are conventional methods in the art, and relevant experimental manuals, such as the Molecular Cloning Experimental Manual or the instructions of relevant reagent manufacturers, can be consulted.

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

[0039] Example 1: Design and Expression of Highly Hydrophilic Recombinant Human Type IV Collagen YS-hydro-col4

[0040] (1) Design of YS-hydro-col4 protein

[0041] In this embodiment of the invention, a highly hydrophilic recombinant human type IV collagen standard was modified and designed to obtain a highly hydrophilic recombinant human type IV collagen YS-hydro-col4 (hereinafter also referred to as YS-hydro-col4). The amino acid sequence of YS-hydro-col4 is shown in SEQ ID NO:1, and its full length is 1395 amino acids.

[0042] (2) Construction of expression vector

[0043] (a) The amino acid sequence of YS-hydro-col4 was translated into the corresponding DNA sequence. The coding gene sequence of Col4A1 is shown in SEQ ID NO:2, with a full length of 4185 bases. The recognition sequence of restriction endonuclease SnaB I (TACGTA) was added to the 5' end of the coding gene sequence of YS-hydro-col4, 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-col4. The whole gene of the target gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0044] (b) The pPIC9K plasmid was used as the expression vector, and the pPIC9K plasmid and the synthesized target gene were double-digested using restriction endonucleases SnaB I (Thermo Fisher Scientific) and Avr II (Thermo Fisher Scientific), respectively. The digestion system (20 μl) consisted of: 16 μl ddH2O, 2 μl 10×Buffer, 1 μl DNA, 0.5 μl SnaB I, and 0.5 μl Avr II; the digestion conditions were: 37℃ for 3 h, followed by inactivation at 80℃ for 20 min.

[0045] (c) The digested pPIC9K plasmid and the target gene were recovered separately, and the target gene was ligated into the pPIC9K vector using T4 DNA ligase (New England Biolabs). The ligation system (20 μl) consisted of: 2 μl of 10× buffer, 0.2 μl of T4 DNA ligase, 3 μl of pPIC9K, 1 μl of the target gene, and ddH2O to a final volume of 20 μl. The ligation conditions were: overnight at 16°C.

[0046] (d) The ligation product was transformed into *E. coli* DH5α clones using the heat shock method. The transformation method was as follows: *E. coli* DH5α competent cells were removed from a -80°C freezer and incubated on ice for 5 min. After the glycerol in the competent cells thawed, the competent cells were added to the ligation product, mixed by pipetting 3-4 times, and incubated on ice for 30 min. The tube walls were quickly dried with absorbent paper, and the tubes were heat-shocked at 42°C for 90 s, followed immediately by an ice bath for 2 min. Under aseptic conditions, 800 μl of LB liquid medium was added, and the tubes were incubated at 37°C and 150 rpm for 45 min. The cells were collected by centrifugation at 8000 rpm for 5 min, and a portion of the supernatant was discarded. The remaining approximately 100 μl of supernatant was used to resuspend the *E. coli* cells, which were then evenly spread onto LB solid medium containing 100 μg / ml ampicillin. The tubes were then incubated upside down at 37°C for 10-16 h. Single clones were picked and inoculated into liquid LB medium containing 100 μg / ml ampicillin. After incubation at 37°C for 10-16 h, the bacterial culture was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to identify positive clones.

[0047] (e) Using the plasmid extraction kit (Omega, D6943-01), the plasmids of the positive clones with the correct sequence were extracted according to the kit instructions to obtain the recombinant plasmid pPIC9K-YS-hydro-col4.

[0048] (3) Pichia pastoris transformation

[0049] (a) Take 20 μg of recombinant plasmid (pPIC9K-YS-hydro-col4) and digest it with restriction endonuclease Sac I (Promega); the digestion system (20 μl): 16 μl ddH2O, 1 μl recombinant plasmid, 2 μl 10×buffer, 1 μl Sac I; digestion conditions: digest at 37℃ for 2 h; after inactivation at 65℃ for 20 min, use a plasmid extraction kit (Omega, D6943-01) to recover the linearized recombinant plasmid 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 the mixture to a 0.2 cm electroporation cuvette, place it on ice for 10 min, and then place it in an electroporator for electroporation transformation. The electroporation conditions are: voltage 1.5 kV, capacitance 25 μF, resistance 200 Ω, and electroporation time 10 ms.

[0051] (c) After the electroshock is completed, remove the electroshock conversion cup, add 1 ml of 1 M sorbitol solution that has been pre-cooled on ice to the electroshock conversion cup, and gently mix it by blowing with a pipette tip.

[0052] (d) Transfer the liquid in the electroporation cup to a 2ml EP tube and shake it on a shaker at 30℃ for 40min; spread all the liquid onto MD solid medium and incubate at 30℃ for 2d.

[0053] (e) Use an inoculation loop to pick a single colony growing on MD solid medium and inoculate it onto YPD solid medium containing 4.0 mg / ml G418. Incubate overnight at 30°C and screen for positive clones to obtain recombinant Pichia-pPIC9K-YS-hydro-col4.

[0054] (4) Protein expression and purification

[0055] (a) Pick single colonies of recombinant Pichia-pPIC9K-YS-hydro-col4 and inoculate them into Erlenmeyer flasks containing 50 ml of BMGY medium. Then place them in a shaker and incubate overnight at 30°C and 220 rpm until OD600 = 1-2.

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

[0057] (c) Resuspend the cells in BMMY medium until OD600 = 0.2-0.6; transfer the bacterial culture to a 500ml Erlenmeyer flask and induce expression at 30℃ and 200rpm. Add methanol to the medium every 24h to a final concentration of 1% (v / v).

[0058] (d) After the induction expression was completed, the supernatant was collected after centrifugation at 3000g for 20 min at 4℃ and used to purify the protein.

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

[0060] (f) Centrifuge at 12000 rpm for 10 min, discard the supernatant and collect the precipitate; invert the EP tube onto absorbent paper and place it in a 37℃ oven for 10 min-20 min to ensure that there is no obvious liquid residue on the tube wall.

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

[0062] (h) Repeat steps (6) and (7) 2-3 times to obtain purified protein YS-hydro-col4.

[0063] (5) SDS-PAGE detection of proteins

[0064] (a) Prepare SDS-PAGE protein electrophoresis gels with a separating gel concentration of 10% and a stacking gel concentration of 5%. The formulations are 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, and 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, and 0.04 ml of TEMED.

[0065] (b) Sample preparation: Take an appropriate amount of the protein to be tested, add Loading Buffer, vortex to mix, boil in water for 5-10 minutes to denature the protein, centrifuge at 12000 rpm for 10 minutes, take the supernatant, and load 10 μl of the sample. Electrophoresis parameters: constant voltage 80V, 120V after entering the separating gel.

[0066] SDS-PAGE results are as follows Figure 1 As shown, the molecular weight of YS-hydro-col4 is approximately 95 kDa, which is consistent with the expected molecular weight.

[0067] Example 2 Hydrophilicity Test

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

[0069] Using acetic acid aqueous solution as solvent, a collagen solution with a total mass concentration of 7% was prepared using protein Col4A1 and PEO (polyethylene oxide) from Example 1 as solutes (wherein the mass ratio of protein YS-hydro-col4 to PEO was 90:10). The solution was stirred at room temperature for 2 hours. The collagen solution was poured into a 10 mL syringe with a metal nozzle. The syringe was fixed on a screw propeller and the parameters were set. The positive terminal of the high-voltage generator was connected to the syringe needle, and the negative terminal was connected to a support with aluminum foil. The electrospinning process parameters were as follows: spinning voltage 25 kV, receiving distance 20 cm, propulsion rate 0.8 mL / h, temperature 20 °C, and relative humidity below 50%. After spinning, the aluminum foil was collected, and a collagen fiber membrane was obtained.

[0070] Collagen fiber membranes were cut into rectangles, and the contact angles of different films were measured using a video contact angle meter (contact angle testing is an experiment to evaluate the hydrophilicity and hydrophobicity of a substance; the contact angle is considered an indicator of the degree of hydrophilicity and hydrophobicity; if θ < 90°, the solid surface is hydrophilic, meaning the liquid easily wets the solid, and the smaller the angle, the better the wettability; if θ > 90°, the solid surface is hydrophobic, meaning the liquid does not easily wet the solid and easily moves on the surface). Boiled and cooled distilled water was automatically added in 5 μl drops to the sample surface each time, and the built-in digital camera recorded the water droplet morphology and calculated the surface contact angle. Three parallel samples were measured for each type of sample, and the average value was calculated.

[0071] The hydrophilicity test of the recombinant human type IV collagen standard was also performed using the above method.

[0072] The hydrophilicity test results of protein YS-hydro-col4 and recombinant human type IV collagen standards are as follows: Figure 2 As shown, the contact angle of the recombinant human type IV collagen standard (before modification) is approximately 47°, while that of YS-hydro-col4 (after modification) is approximately 24°. Compared with the recombinant human type IV collagen standard, the contact angle of YS-hydro-col4 is reduced, indicating that the hydrophilicity of the highly hydrophilic recombinant human type IV collagen YS-hydro-col4 of this invention is significantly improved compared with the recombinant human type IV collagen standard.

[0073] 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 it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nucleotide encoding a high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4, wherein an amino acid sequence of the high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4 encoded by the nucleotide is shown as SEQ ID NO:

1.

2. The nucleotide of claim 1, wherein, consisting of a sequence shown as SEQ ID NO:

2. 3.A recombinant expression vector comprising the nucleotide of claim 1 or 2. 4.An engineered bacterium comprising the nucleotide of claim 1 or 2 or the recombinant expression vector of claim 3. 5.Use of the recombinant expression vector of claim 3 or the engineered bacterium of claim 4 in the preparation of a high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4. 6.A high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4, wherein an amino acid sequence of the high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4 is shown as SEQ ID NO:

1. 7.A composition comprising a high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4, wherein an amino acid sequence of the high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4 is shown as SEQ ID NO:

1. 8.A preparation comprising the high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4 of claim 6 or the composition of claim 7, wherein the preparation is selected from a drug, a medical device, a biomaterial, a tissue engineering product, a cosmetic, or a health product. 9.Use of the nucleotide of claim 1 or 2, the recombinant expression vector of claim 3, the engineered bacterium of claim 4, the high-hydrophilicity recombinant human-like type IV collagen YS-hydro-col4 of claim 6, or the composition of claim 7 in the preparation of a preparation, wherein the preparation is selected from a drug, a medical device, a biomaterial, a tissue engineering product, a cosmetic, or a health product.

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