Recombinant IV type collagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of protein engineering and genetic engineering, and particularly relates to recombinant IV-type collagen as well as a preparation method and application thereof. According to the invention, three high-activity amino acid sequences are screened by taking a human-derived IV-type collagen alpha1 chain as a template, and the nucleotide sequences of the three amino acid sequences are optimized by pichia pastoris codon preference and then are respectively integrated onto a pichia pastoris genome to obtain three recombinant IV-type collagen engineering yeast strains; through shake-flask culture, two engineering yeast strains can be successfully fermented to prepare the recombinant IV type collagen, and are respectively named as IV1-2 and IV1-3. Compared with a commercially available collagen product, the recombinant IV-type collagen prepared by the technical scheme has better oxidation resistance and barrier repair capability, the effect of IV1-3 is better than that of IV1-2, and the recombinant IV-type collagen has good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and genetic engineering technology, specifically relating to a recombinant type IV collagen, its preparation method, and its application. Background Technology

[0002] Collagen is the most abundant protein in the human body, accounting for about 30% of total protein. It is a major component of the extracellular matrix and plays an important role in maintaining tissue integrity, promoting cell adhesion and proliferation, and wound healing. Based on its structure and function, collagen can be divided into several types. Type IV collagen is a triple helix molecule composed of three α chains, each containing approximately 1700 amino acids, forming a long rod-like structure. There are six types of α chains (α1-α6) in the human body, encoded by different genes (COL4A1 to COL4A6). The most common combination is α1α1α2, which is mainly distributed in the basement membrane, forming the reticular scaffold of the basement membrane, maintaining tissue mechanical stability, constituting selective barriers (such as the glomerular basement membrane), regulating substance exchange, and mediating cell adhesion, migration, and differentiation through receptors such as integrins. It also binds to growth factors (such as VEGF and TGF-β) and matrix components (laminarin), regulating microenvironment homeostasis.

[0003] Traditional collagen is mainly derived from animal tissues, such as cowhide and pigskin. However, these collagens suffer from high immunogenicity, potential pathogen carriers, and significant batch-to-batch variability. With the development of genetic engineering technology, the research and application of recombinant collagen has gradually become a hot topic. Animal and plant expression systems are costly and time-consuming, making them difficult to meet industrialization needs. In contrast, microbial fermentation for recombinant collagen production is low-cost, short-cycle, and easier to cultivate, making it more suitable for commercial production. However, bacterial expression systems are prone to pyrogen residues, resulting in impure products that are difficult to apply clinically. Furthermore, the target protein is usually expressed in inclusion body form, making product purification complex. Additionally, prokaryotic expression systems have imperfect post-translational modification systems, leading to lower biological activity of the expressed products, which negatively impacts product quality. Moreover, the imperfect post-translational modification systems of prokaryotic expression systems result in lower biological activity of the expressed products, thus adversely affecting the overall product quality. Simultaneously, the prepared collagen products also suffer from poor biological activity. Summary of the Invention

[0004] In response to the aforementioned existing technologies, the inventors, through long-term technical and practical exploration, have provided a recombinant type IV collagen, its preparation method, and its applications. Experiments have demonstrated that the recombinant type IV collagen produced by this invention possesses excellent antioxidant and skin barrier repair properties, and is highly suitable for large-scale industrial production. Based on the above research results, this invention has been completed.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a recombinant type IV collagen, wherein the recombinant type IV collagen is selected from: (a1) A protein consisting of the amino acid sequence shown in either SEQ ID NO.2 or SEQ ID NO.3; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) is a protein whose amino acid sequence composition is 80% or more identical to that of (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

[0006] In a second aspect, the present invention provides a nucleic acid molecule capable of encoding the aforementioned recombinant type IV collagen.

[0007] Specifically, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) A nucleotide sequence as shown in SEQ ID NO.5 or SEQ ID NO.6; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the recombinant type IV collagen; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode recombinant type IV collagen with the same or similar function.

[0008] A third aspect of the present invention provides a carrier comprising at least the aforementioned nucleic acid molecules.

[0009] In a fourth aspect, the present invention provides a host cell containing the aforementioned nucleic acid molecule and the aforementioned carrier; or, the host cell is capable of expressing the aforementioned recombinant type IV collagen.

[0010] A fifth aspect of the present invention provides a method for preparing recombinant type IV collagen, comprising the steps of: The recombinant type IV collagen was expressed using the aforementioned host cells, and then separated and purified to obtain the final product.

[0011] A sixth aspect of the present invention provides the use of the above-described recombinant type IV collagen in the preparation of health foods, cosmetics, medical aesthetic products, or pharmaceuticals.

[0012] A seventh aspect of the present invention provides a cosmetic product comprising at least the above-described recombinant type IV collagen.

[0013] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects: The above technical solution uses the human type IV collagen α1 chain as a template to screen for three highly active amino acid sequences. The nucleotide sequences of these three amino acid sequences are then optimized using Pichia pastoris codon preference and integrated into the Pichia pastoris genome, resulting in three engineered yeast strains producing recombinant type IV collagen. Shake-flask culture revealed that two of these engineered yeast strains could be successfully fermented to produce recombinant type IV collagen, named IV1-2 and IV1-3, respectively. Compared to commercially available collagen products, the recombinant type IV collagen prepared using this technical solution exhibits superior antioxidant and barrier repair capabilities, with IV1-3 showing better efficacy than IV1-2, demonstrating significant practical application value. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0015] Figure 1 This is an SDS-PAGE protein electrophoresis result of the fermentation supernatant of recombinant type IV collagen in Example 3 of the present invention; where lane M represents the standard protein with a molecular weight of 180 kDa; lane 1 represents the control bacteria. P. pastoris GS115 / pPIC9K fermentation supernatant; lane 2 represents recombinant bacteria. P. pastoris GS115 / pPIC9K-IV1-1 fermentation supernatant, lane 3 represents recombinant bacteria. P. pastoris GS115 / pPIC9K-IV1-2 fermentation supernatant, lane 4 represents recombinant bacteria. P .pastoris GS115 / pPIC9K-IV1-3 fermentation supernatant.

[0016] Figure 2 This is a graph showing the DPPH free radical scavenging rate of the sample in Example 5 of the present invention.

[0017] Figure 3This is a comparison of the results of different collagen proteins promoting FLG mRNA expression in HaCaT cells in Example 6 of the present invention. In the figure, control group 1 represents the blank control group; control group 2 represents the comparative experimental group of commercially available recombinant collagen; experimental group 1 represents the recombinant type IV collagen IV1-2 group prepared in this invention, and experimental group 2 represents the recombinant type IV collagen IV1-3 group prepared in this invention. In the figure, compared with control group 1, P < 0.0001 (****), which is considered to be a significant difference. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] In a typical embodiment of the present invention, a recombinant type IV collagen is provided, wherein the recombinant type IV collagen is selected from: (a1) A protein consisting of the amino acid sequence shown in either SEQ ID NO.2 or SEQ ID NO.3; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) is a protein whose amino acid sequence composition is 80% or more identical to that of (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

[0021] In (a2), the substitution, deletion and / or addition of one or more amino acid residues generally refers to the substitution and / or deletion and / or addition of no more than 15 amino acid residues.

[0022] The proteins shown in (a1)–(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0023] In this invention, amino acid substitution refers to the replacement of an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be conserved amino acid substitution, meaning that compared to the original amino acid sequence, several amino acids are replaced by amino acids with similar or related properties to form a peptide.

[0024] In this invention, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0025] In this invention, amino acid addition can refer to adding 1, 2 or 3 or more amino acids at any position at the C-terminus, N-terminus or between the C-terminus and N-terminus of the amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0026] In another specific embodiment of the present invention, a nucleic acid molecule is provided, which is capable of encoding the above-mentioned recombinant type IV collagen.

[0027] Specifically, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) A nucleotide sequence as shown in SEQ ID NO.5 or SEQ ID NO.6; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the recombinant type IV collagen; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode recombinant type IV collagen with the same or similar function.

[0028] In this invention, the stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA. Hybridization can be performed in a mixed solution of EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 65°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by washing once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0029] It should be noted that the term "identity" refers to sequence similarity to an amino acid / nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0030] The aforementioned 80% or more identity can be 80%, 85%, 90%, 95%, or 99% or more identity.

[0031] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., without specific limitations.

[0032] In another specific embodiment of the present invention, a carrier is provided, the carrier comprising at least the above-mentioned nucleic acid molecules.

[0033] In this invention, a "vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) to a recipient cell. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends; nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid" (specifically pPIC9K), which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of a target gene. Such vectors are referred to herein as "expression vectors." Recombinant expression vectors may contain a form suitable for expressing nucleic acids in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0034] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned nucleic acid molecule and the above-mentioned carrier; or, the host cell is capable of expressing the above-mentioned recombinant type IV collagen.

[0035] The host cell includes bacterial cells or fungal cells; The bacteria can be any one or more of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0036] In one or more specific embodiments of the present invention, the bacteria are Escherichia coli (e.g., Escherichia coli). E. coil TOP10), Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0037] In one or more specific embodiments of the present invention, the fungus includes yeast, and more specifically, Pichia pastoris (including Pichia pastoris GS115, KM71, and SMD1168). Pichia pastoris is one of the most common exogenous protein expression systems, exhibiting rapid growth, a well-defined genetic background, and simple operation, making it an ideal host for producing large quantities of protein in a short time. Furthermore, Pichia pastoris possesses a systematic gene editing system, a complete protein secretion and expression mechanism, and the ability to modify exogenous proteins after translation, making it a widely used heterologous host in the food and pharmaceutical fields. Therefore, the present invention preferentially selects Pichia pastoris.

[0038] In another specific embodiment of the present invention, a method for preparing recombinant type IV collagen is provided, comprising the steps of: The recombinant type IV human collagen was expressed using the aforementioned host cells, and then separated and purified to obtain the final product.

[0039] The separation and purification steps include removing impurities from proteins, centrifugation, hollow fiber membrane clarification, ultrafiltration membrane concentration, ion exchange chromatography, etc., which are not specifically limited here.

[0040] In another specific embodiment of the present invention, the application of the above-mentioned recombinant type IV collagen in the preparation of health foods, cosmetics, medical aesthetic products or pharmaceuticals is provided.

[0041] The aforementioned health food is a type of food that shares common characteristics with general foods. It can regulate the body's functions and is suitable for consumption by specific groups of people, but it is not intended to treat diseases.

[0042] The cosmetics mentioned refer to chemical industrial products or fine chemical products that are applied to any part of the human body surface, such as skin, hair, nails, lips, and teeth, by means of smearing, spraying, or other similar methods, to achieve the purpose of cleaning, maintaining, beautifying, modifying appearance, or correcting body odor and maintaining a good condition. In this invention, the cosmetics can be applied to the skin to achieve antioxidant and skin barrier repair effects; therefore, the cosmetics mentioned in this invention can be skin care products.

[0043] Meanwhile, in this invention, the cosmetic dosage form can be an aqueous solution, emulsion, ointment, etc. Furthermore, other cosmetic categories derived and prepared based on the above-mentioned basic cosmetic categories, such as facial cleanser, sunscreen, toner, face cream, eye cream, essence water (lotion), hand cream (ointment), body lotion, and face mask, are also within the scope of protection of this application.

[0044] In this invention, the medical aesthetic products refer to products used in the field of medical aesthetics, which are different from ordinary daily skincare products and beauty salon skincare products. Some commonly used medical aesthetic products include medical aesthetic masks and dressings. The use of these products requires professional medical personnel or relevant guidance. Medical aesthetic products, such as dressings, can be applied to those with impaired skin barrier function after procedures such as dermatitis, eczema, sensitive skin, and post-procedure treatments such as photofacial rejuvenation, laser beauty treatments, and chemical peels.

[0045] In this invention, the medicine can be a pharmaceutical-medical device product, specifically a product composed of both a medicine and a medical device, and manufactured as a single entity. Specifically, the pharmaceutical-medical device product can be a topical dressing containing the aforementioned recombinant type IV collagen.

[0046] In another specific embodiment of the present invention, a cosmetic product is provided, the cosmetic product comprising at least the above-mentioned recombinant type IV collagen.

[0047] Furthermore, the cosmetics mentioned are specifically skincare products.

[0048] Of course, the cosmetics may also contain any other ingredients permitted in the cosmetic field, including but not limited to dispersants, suspending agents, emulsifiers, emollients, humectants, preservatives, pH adjusters, thickeners, and fragrances. Those skilled in the art can choose to add these ingredients according to the actual situation, and no specific limitations are made here.

[0049] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, all experimental methods used are conventional methods. Unless otherwise specified, all strains, cells, materials, and reagents used are commercially available.

[0050] Example 1: Sequence Selection of Recombinant Type IV Collagen The amino acid sequence of natural human type IV collagen α1 chain protein was obtained from the NCBI website (GenBank: AAA53098.1). Based on the characteristics of natural collagen as an insoluble fibrous protein with highly repetitive amino acid sequences, and using various techniques such as bioinformatics, three highly active amino acid sequences were successfully screened. The three recombinant type IV collagen gene sequences were named IV1-1 (amino acid sequence as shown in SEQ ID NO.1), IV1-2 (amino acid sequence as shown in SEQ ID NO.2), and IV1-3 (amino acid sequence as shown in SEQ ID NO.3), respectively.

[0051] Example 2 Construction of an expression system containing recombinant type IV collagen gene sequence Based on the codon bias of Pichia pastoris, the amino acid sequences of the three recombinant type IV collagens in Example 1 were codon optimized. The optimized nucleotide sequences encoding gene IV1-1 are shown in SEQ ID NO.4, gene IV1-2 in SEQ ID NO.5, and gene IV1-3 in SEQ ID NO.6.

[0052] The above three nucleotide sequences were synthesized by Nanjing GenScript Biotech Co., Ltd., and cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K, respectively, and transformed into... E. coilAmong the TOP10, recombinant expression vectors pPIC9K-IV1-1, pPIC9K-IV1-2, and pPIC9K-IV1-3 were obtained while ensuring that the reading frame did not shift. After DNA sequencing and alignment, all three recombinant expression vectors were successfully constructed.

[0053] The three recombinant expression vectors were linearized with SalI rapid digestion enzyme and then electroporated into... P. pastoris In host cells expressing GS115, three high-copy-count Pichia pastoris strains expressing type IV collagen were obtained by screening recombinant transformants with genistein G418. These strains are: P. pastoris GS115 / pPIC9K-IV1-1, P. pastoris GS115 / pPIC9K-IV1-2 and P. pastoris GS115 / pPIC9K-IV1-3.

[0054] Example 3: Shake-flask fermentation of recombinant type IV collagen-expressing Pichia pastoris strain The obtained recombinant type IV collagen Pichia pastoris expression strain P. pastoris GS115 / pPIC9K-IV1-1, P. pastoris GS115 / pPIC9K-IV1-2 and P. pastoris GS115 / pPIC9K-IV1-3 were cultured in shake flasks.

[0055] The fermentation steps are as follows: Single colonies were picked and inoculated into 40 mL of YPD medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) and cultured at 30℃ and 200 rpm for 24 h. A 10% inoculum was then transferred to 40 mL of the initial expression medium BMGY (yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶). -4 Incubate in 30℃, 200 rpm for 24 h at 30℃ and 10 g / L glycerol (yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ g / L). Collect bacterial cells by centrifugation, wash with physiological saline, and then transfer to 40 mL of BMMY induction expression medium (yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ g / L). -4 The medium was incubated at 30°C and 200 rpm with pure methanol added every 24 h until the final concentration was 1.0% (v / v) for 96 h.

[0056] The theoretical molecular weight of protein IV1-1 is 9.8 kDa, V1-2 is 12.2 kDa, and V1-3 is 14.8 kDa. SDS-PAGE protein electrophoresis was performed on the fermentation supernatant of the three recombinant type IV collagen-expressing Pichia pastoris strains and the fermentation supernatant of the control strain GS115 / pPIC9K. The electrophoresis results are as follows: Figure 1 As shown. Recombinant engineered bacteria P. pastoris GS115 / pPIC9K-IV1-2 (lane 3) and P. pastoris The fermentation supernatant of GS115 / pPIC9K-IV1-3 (lane 4) showed an extra protein band near the theoretical size of the recombinant protein, while the engineered bacteria... P. pastoris The fermentation supernatant of GS115 / pPIC9K-IV1-1 (lane 2) did not show a clear protein band at the theoretical size of recombinant protein IV1-1, indicating that recombinant type IV collagen IV1-2 and IV1-3 were successfully expressed, while IV1-1 was not successfully expressed.

[0057] Example 4 Preparation of recombinant type IV collagen IV1-2 and IV1-3 Recombinant bacteria P. pastoris GS115 / pPIC9K-IV1-2 and P. pastoris GS115 / pPIC9K-IV1-2 was cultured in shake flasks according to Example 3, and the fermentation supernatant was purified by the following steps to obtain pure recombinant collagen.

[0058] ① Hollow fiber clarification: The fermentation supernatant above is filtered through a 250kDa hollow fiber membrane to remove bacteria and large molecular impurities; ② Ultrafiltration membrane concentration and replacement: The feed solution is concentrated using a 3kDa ultrafiltration membrane pack (manufacturer: Sartorius) and simultaneously replaced with ion solution A (20mM Tris, pH 7.5) until the conductivity at the permeate end is equivalent to that of ion solution A. ③ Ion exchange chromatography: Cation exchange chromatography was used, with 200 mM sodium chloride (pH 7.5) for elution, and the elution peaks were collected.

[0059] ④ Ultrafiltration membrane desalination: The feed solution was desalinated using a 3kDa ultrafiltration membrane until the conductivity at the permeate end dropped to 70μS / cm; ⑤ Freeze-drying: The ultrafiltration concentrate obtained above was filtered through a 0.45μm microfiltration membrane and then placed in a freeze-drying tray for freeze-drying.

[0060] The proteins IV1-2 and IV1-3 purified by the above steps had a purity greater than 98% and a yield greater than 50%.

[0061] Example 5: Antioxidant experiment of recombinant type IV collagen IV1-2 and IV1-3 Appropriate amounts of recombinant type IV collagen IV1-2 (experimental group 1) and IV1-3 (experimental group 2) were placed in separate test tubes. 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol was added. The mixture was vigorously shaken for 10 s and then allowed to react at room temperature for 30 min. After the reaction, the absorbance of the reaction mixture was measured at 517 nm. Distilled water was used as a blank control (control group 1) instead of the sample solution, and an equal amount of commercially available recombinant collagen was used as a comparative experiment (control group 2). The DPPH free radical scavenging activity of the sample solution = (absorbance of blank control - absorbance of sample) / absorbance of blank control.

[0062] The results are as follows Figure 2 As shown, samples IV1-2 and IV1-3 exhibited significant DPPH free radical scavenging ability compared to the blank control, and their free radical scavenging rate was higher than that of commercially available collagen. Among them, the free radical scavenging rate of recombinant type IV collagen IV1-3 was slightly higher than that of IV1-2. Therefore, the recombinant type IV collagen IV1-2 and IV1-3 prepared in this invention both have higher antioxidant effects than commercially available collagen.

[0063] Example 6: Barrier repair performance test of recombinant type IV collagen IV1-2 and IV1-3 Filagrin (FLG) is an important component of the keratinized capsule, ensuring the integrity of the skin barrier. FLG deficiency leads to disordered lipid bilayer structure and delayed maturation, while also causing reduced keratinocyte density, increased intercellular permeability, and decreased photoprotective function, ultimately resulting in skin barrier damage.

[0064] Immortalized human keratinocytes (HaCaT cells) were collected, and a cell suspension was prepared using high-glucose DMEM cell culture medium. 2 mL of the cell suspension was added to each well of a 6-well plate, resulting in a cell count of 2.6 × 10⁶ cells. 5 / well. The experiment included a blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), recombinant type IV collagen IV1-2 prepared according to this invention (experimental group 1), and recombinant type IV collagen IV1-3 prepared according to this invention (experimental group 2), with 3 replicates for each group. The 6-well plate was incubated in a cell culture incubator (5% CO2, 37℃) for 24 hours. When the cell confluence rate reached 50%~60%, the culture medium was discarded. 2 mL of high-glucose DMEM cell culture medium was added to each well of the blank control group; 2 mL of high-glucose DMEM cell culture medium containing the corresponding collagen was added to each of the other four groups. The concentration of collagen in the high-glucose DMEM cell culture medium was 1 mg / mL. After incubating the 6-well plates in an incubator (37℃, 5% CO2) for 24 hours, the cells in each well were washed twice with 2 mL of PBS buffer. Following the Total RNA Extraction Kit, 1 mL of RNAiso Plus was added, and the cells were lysed by pipetting. RNA extraction, reverse transcription, and quantitative real-time PCR were performed according to the kit instructions to detect the relative mRNA expression level of the barrier-related protein FLG. A 2... -△△CT The method is used for calculation.

[0065] Different collagen proteins promote the expression of FLG mRNA in HaCaT cells, such as Figure 3 As shown, compared with the blank control group (control group 1), commercially available recombinant collagen (control group 2), recombinant type IV collagen IV1-2 prepared in this invention (experimental group 1), and recombinant type IV collagen IV1-3 prepared in this invention (experimental group 2) can all significantly increase the relative expression level of FLG mRNA. Among them, the recombinant type IV collagen IV1-2 and IV1-3 prepared in this invention have stronger ability to promote FLG mRNA expression than commercially available recombinant collagen. Therefore, the recombinant type IV collagen IV1-2 and IV1-3 of this invention have obvious skin barrier repair function.

[0066] Matters not covered in this invention are common knowledge.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant type IV collagen, characterized in that, The recombinant type IV collagen was selected from: (a1) A protein consisting of the amino acid sequence shown in either SEQ ID NO.2 or SEQ ID NO.3; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) is a protein whose amino acid sequence composition is 80% or more identical to that of (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is capable of encoding the recombinant type IV collagen of claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) A nucleotide sequence as shown in SEQ ID NO.5 or SEQ ID NO.6; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the recombinant type IV collagen; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode recombinant type IV collagen with the same or similar function.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2 or 3.

5. The carrier as described in claim 4, characterized in that, The vector is obtained by effectively linking the nucleic acid molecule described in any one of claims 2-3 to the expression vector, wherein the expression vector is any one or more of a viral vector, plasmid, bacteriophage, kinase, or artificial chromosome; further, the expression vector is a plasmid.

6. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in any one of claims 2-3 and the vector as described in any one of claims 4-5; or, the host cell is capable of expressing the recombinant type IV collagen as described in claim 1.

7. The host cell as described in claim 6, characterized in that, The host cells include bacterial cells and fungal cells; further, the fungal cells are Pichia pastoris.

8. A method for preparing recombinant type IV collagen, characterized in that, Including the following steps: The recombinant type IV human collagen is expressed using the host cell described in claim 6 or 7, and then separated and purified to obtain the final product.

9. The use of the recombinant type IV collagen as described in claim 1 or 2 in the preparation of health foods, cosmetics, medical aesthetic products or pharmaceuticals.

10. A cosmetic product, characterized in that, The cosmetic product contains the recombinant type IV collagen as described in claim 1 or 2; further, the cosmetic product is a skin care product.

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