Recombinant humanized III-type collagen with high activity as well as preparation method and application of recombinant humanized III-type collagen

Abstract

The invention belongs to the technical field of biomedicine, and particularly discloses recombinant humanized III-type collagen with high activity as well as a preparation method and application of the recombinant humanized III-type collagen. The recombinant humanized III-type collagen comprises a first basic repetitive unit, the amino acid sequence of the first basic repetitive unit is shown as SEQ ID NO: 1, and the recombinant humanized III-type collagen is formed by repeating the first basic repetitive unit for 1-10 times. The recombinant humanized III-type collagen provided by the invention is formed by repeating 51 amino acids derived from a human III-type collagen alpha1 chain functional region for different times, and compared with the reported recombinant III-type collagen, the recombinant humanized III-type collagen provided by the invention has the advantages that the molecular weight is small; the stable triple-helix structure is realized, and the biological activity and the stability are relatively high. The recombinant humanized III-type collagen has outstanding performance in the aspects of promoting cell proliferation, adhesion, migration and the like, is good in biocompatibility and high in safety, and is suitable for the fields of cosmetics, tissue engineering, medical instruments and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly active recombinant humanized type III collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is the most abundant structural protein in the extracellular matrix of mammals, playing a crucial role in the human body. It accounts for 25%-30% of the total protein content and is widely distributed in numerous tissues such as skin, bones, tendons, and blood vessels. It is essential for maintaining tissue integrity, promoting cell adhesion and migration, and regulating physiological processes such as wound healing. Among the 28 known types of collagen, type I and type III collagen are particularly abundant in the skin. Type III collagen, as a core component of early development and soft tissue repair, is significant for maintaining tissue flexibility and tensile strength. Due to its high hydrophilicity, it binds a large number of water molecules, maintaining skin hydration. As a key part of the skin's extracellular matrix, it also promotes cell adhesion, proliferation, and migration, acting as a "scaffold" in wound healing, being synthesized first to build the basic framework for tissue repair. The receptors for type III collagen mainly include integrins (such as α1β1 and α2β1), DDR1, and GPVI. Among them, integrins mediate cell adhesion by recognizing GFOGER-like motifs (such as GLOGER) in collagen; DDR1 regulates cell proliferation, migration, and matrix remodeling by relying on GVMGFO-like sequences in the collagen triple helix structure; and the platelet receptor GPVI participates in hemostasis by binding to poly-GPO repeat sequences to trigger platelet activation and aggregation. Therefore, type III collagen has become a highly promising and ideal candidate material in the fields of biomedicine, high-end cosmetics, and tissue engineering.

[0003] Currently, the main methods for obtaining type III collagen are animal extraction, chemical synthesis, and genetic engineering recombination. However, both animal extraction and chemical synthesis methods have significant drawbacks. Animal-derived collagen may carry pathogens such as viruses and bacteria, posing a serious risk of immune reactions and disease transmission to users. While chemical synthesis can precisely control the amino acid sequence of collagen, the process requires large amounts of toxic and harmful chemical reagents, resulting in high costs. The synthesized collagen also has a small molecular weight, making it difficult to form a complete triple helix structure, which greatly limits its application in the medical and cosmetic fields.

[0004] The function of collagen is closely related to its amino acid sequence length. In existing technologies, long-sequence collagen, while containing complete triple-helix domains and multiple functional fragments, can better mimic the biological functions of natural collagen, such as promoting cell proliferation and tissue regeneration. However, due to their excessive length (usually exceeding 1000 amino acids), they often contain multiple enzyme cleavage sites, making their structure inherently unstable. They are prone to degradation or aggregation during in vitro preparation and storage, exhibiting poor stability, especially under conditions of high temperature and extreme pH, leading to functional failure. While short-sequence collagen (e.g., 30-100 amino acids) improves stability, the lack of complete functional domains significantly reduces its biological activity. Its cell adhesion ability and tissue repair efficiency are far lower than those of natural collagen, making it difficult to meet practical application needs. Furthermore, existing research on the "sequence length versus stability and activity balance" focuses on single-dimensional optimization, such as simply shortening the sequence or replacing individual amino acids, without developing a systematic sequence design strategy, thus failing to simultaneously address both stability and biological function. It is evident that existing collagen sequences present a contradiction between sequence length and stability, and there is a lack of targeted sequence design strategies. Developing a collagen sequence of moderate length (e.g., 100-500 amino acids), good stability, and excellent biological function has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned issues, this invention proposes a novel, fully-sequenced recombinant humanized type III collagen based on genetic engineering and synthetic biology techniques. Through core functional region screening, domain recombination, and expression system optimization, it overcomes existing technological bottlenecks and provides a revolutionary solution for regenerative medicine and functional skincare.

[0006] The first aspect of the present invention provides a highly active recombinant humanized type III collagen, its preparation method and application, wherein the recombinant humanized type III collagen comprises a first basic repeating unit, the amino acid sequence of which is shown in SEQ ID NO:1, and the recombinant humanized type III collagen is formed by repeating the first basic repeating unit 1-10 times.

[0007] When the number of repetitions exceeds 2, each first basic repeating unit is directly connected.

[0008] SEQ ID NO:1: GRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFP.

[0009] Preferably, the number of the first basic repeating units is 3-5.

[0010] Preferably, the number of the first basic repeating units is 3.

[0011] Preferably, the number of the first basic repeating units is 4.

[0012] Preferably, the number of the first basic repeating units is 5.

[0013] The inventors have discovered that the recombinant humanized type III collagen provided by this invention has good biocompatibility and excellent biological activity, including promoting cell proliferation, adhesion, and migration, thereby achieving the effect of promoting tissue regeneration and repair. The recombinant humanized type III collagen has high stability, is not easily degraded, and is safe to use. It can be applied in industries such as skin care products, tissue engineering materials, and medical devices.

[0014] The collagen of this invention is a recombinant humanized type III collagen constructed through short amino acid sequence repetitions. It is generally believed that the fewer the number of repetitions of the basic repeating unit, the smaller the molecular weight, and the more difficult it is to form a stable triple helix structure, leading to weakened mechanical properties of collagen fibers and susceptibility to protease degradation, making it difficult to reach a stable equilibrium state. However, the recombinant humanized type III collagen of this invention uses 51 amino acids as repeating units, repeated 3-5 times, and has been verified to possess a triple helix structure. Furthermore, biological function evaluation has shown that this protein has significant cell proliferation, migration, and adhesion promotion functions, exhibiting good biological activity and stability.

[0015] A second aspect of the present invention also provides a nucleic acid molecule encoding the above-mentioned recombinant humanized type III collagen.

[0016] Nucleic acid molecules are a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), biological macromolecules composed of many nucleotide monomers, and are one of the most fundamental substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA, and in some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0017] The aforementioned nucleic acid molecules can be optimized based on the codon preferences of the host cell, and then gene fragments can be artificially synthesized. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0018] Furthermore, the nucleic acid molecule includes a second basic repeat unit, the nucleotide sequence of which is shown in SEQ ID NO:2. The nucleic acid molecule is formed by repeating the second basic repeat unit 1-10 times. When the number of repetitions exceeds 2, the second basic repeat units are directly linked together.

[0019] SEQ ID NO:2: GGGAGGCCCGGAGAAAGAGGACTAAAAGGTGAGAACGGCCTGCCGGGTGAAAACGGCGCACCGGGCGAGCGCGGTTTTCGTGGTCCGGCTGGCCCAAATGGCATCCCGGGTGAGAAGGGTCCGGCGGGTGAACGTGGCGTTATGGGTTTCCCG.

[0020] Furthermore, the nucleotide sequence was optimized for codon preference in E. coli, with a codon fitness index (CAI) ≥ 0.85.

[0021] Thirdly, the present invention also provides an expression vector that carries any of the above-mentioned nucleic acid molecules.

[0022] Furthermore, the vector comprises an expression control element operatively linked to the nucleic acid, a purification tag nucleotide, and / or a leader sequence nucleotide, wherein the purification tag is selected from one or both of His tags and Sumo tags.

[0023] Preferably, the carrier is pET-30a(+).

[0024] Fourthly, the present invention also provides a cell that carries any of the above-mentioned nucleic acid molecules, or contains any of the above-mentioned carriers, or expresses the above-mentioned recombinant humanized type III collagen.

[0025] Fifthly, the present invention also provides an engineered Escherichia coli strain for producing the above-mentioned recombinant humanized type III collagen, comprising any one of the above-mentioned vectors, wherein the Escherichia coli is strain BL21.

[0026] Sixthly, the present invention also provides a method for preparing recombinant humanized type III collagen, comprising the following steps: 1) Pre-culture engineered Escherichia coli in LB medium at 37°C until OD. 600 ≥0.6; 2) Cool to 16-30℃ and add 0.5-2 mM IPTG to induce expression for 10-20 hours; 3) After lysing the bacterial cells, the target protein was captured by Ni-NTA affinity chromatography; 4) After purification, the target collagen with a purity of ≥95% is obtained, which is the recombinant humanized type III collagen.

[0027] Furthermore, purification may employ one or a combination of methods, including but not limited to salting out, chromatographic chromatography, acid-base precipitation, and membrane separation.

[0028] Preferably, purification is achieved using a combination of chromatographic chromatography and membrane separation.

[0029] Preferably, purification employs a combination of ion exchange chromatography and membrane separation.

[0030] In a seventh aspect, the present invention also provides a solution for promoting wound repair, comprising recombinant humanized type III collagen and sodium hyaluronate, wherein the concentration of recombinant humanized type III collagen is 0.1-2% w / v, the concentration of sodium hyaluronate is 0.5-2% w / v, and the recombinant humanized type III collagen and the sodium hyaluronate form a clear and transparent aqueous solution under pH 6.5-7.5 conditions.

[0031] Furthermore, the aqueous solution comprises 0.5% (w / w) recombinant human type III collagen, 1% sodium hyaluronate, 0.1% panthenol, and 3% glycerol, with deionized water as the solvent.

[0032] Eighthly, the present invention also provides the use of collagen in the manufacture of skin care products, tissue engineering materials or medical devices.

[0033] In summary, the present invention includes at least one of the following beneficial technical effects: 1) The recombinant humanized type III collagen provided by the present invention is composed of 51 amino acids from the α1 chain functional region of human type III collagen repeated a different number of times, and has 100% sequence identity with human collagen. Compared with previously reported type III recombinant collagen, this recombinant humanized type III collagen has a smaller molecular weight, is easier to separate and purify, and can be prepared with a purity of over 95% through simple chromatographic separation. 2) This recombinant humanized type III collagen has a stable triple helix structure, high biological activity, and stability. 3) In terms of application, the recombinant humanized type III collagen provided by the present invention shows outstanding performance in promoting cell proliferation, adhesion, and migration, has good biocompatibility, and is more suitable for the fields of cosmetics and medical devices. 4) The recombinant humanized type III collagen provided by the present invention has good biological activity and stability, no introduction of foreign amino acid residues, no risk of rejection or sensitization, no cytotoxicity was observed in the detection, and has high safety, and can be widely used in the fields of skin care products, tissue engineering materials, or medical devices. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram of a recombinant humanized type III collagen expression plasmid. Figure 2 The results of whole bacterial culture and ultrasonically disrupted supernatant electrophoresis of recombinant humanized type III collagen; Figure 3 The results are obtained by electrophoresis of purified recombinant humanized type III collagen. Figure 4 The circular dichroism (CD) spectrum of recombinant humanized type III collagen; Figure 5 The results of the cell proliferation rate detection for recombinant humanized type III collagen; Figure 6 The image shows the cell proliferation effect of recombinant humanized type III collagen. Figure 7 The adhesion effect of recombinant humanized type III collagen; Figure 8 The effect of recombinant humanized type III collagen on promoting cell adhesion is illustrated in the image. Figure 9 Results of the assay for the cell migration-promoting ability of recombinant humanized type III collagen; Figure 10 The results of the cytotoxicity test for recombinant humanized type III collagen; Figure 11 The results of stability testing for recombinant humanized type III collagen; Figure 12 The results of an experiment on the treatment of pruritus in mice with recombinant humanized type III collagen; Figure 13 Experimental results showing that recombinant humanized type III collagen can increase the skin hydration of mice; Figure 14 The results of experiments to increase the content of type I collagen in mouse skin using recombinant humanized type III collagen. Detailed Implementation

[0036] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0037] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted nucleotides, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0038] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0039] In this invention, the protein sequence screened was derived from human type III collagen COL3A1, whose NCBI reference sequence number is NP_000081. For details, please refer to: https: / / www.ncbi.nlm.nih.gov / protein / 1780266031. The specific sequence of NP_000081 is shown below:

[0040] 1 mmsfvqkgsw lllallhpti ilaqqeaveg gcshlgqsya drdvwkpepc qicvcdsgsv 61 lcddiicddq eldcpnpeip fgeccavcpq pptaptrppn gqgpqgpkgd pgppgipgrn 121 gdpgipgqpg spgspgppgi cescptgpqn yspqydsydv ksgvavggla gypgpagppg 181 ppgppgtsgh pgspgspgyq gppgepgqag psgppgppga igpsgpagkd gesgrpgrpg 241 erglpgppgi kgpagipgfp gmkghrgfdg rngekgetga pglkgenglp gengapgpmg 301 prgapgergr pglpgaagar gndgargsdg qpgppgppgt agfpgspgak gevgpagspg 361 sngapgqrge pgpqghagaq gppgppging spggkgemgp agipgapglm gargppgpag 421 angapglrgg agepgkngak gepgprgerg eagipgvpga kgedgkdgsp gepganglpg 481 aagergapgf rgpagpngip gekgpagerg apgpagprga agepgrdgvp ggpgmrgmpg 541 spggpgsdgk pgppgsqges grpgppgpsg prgqpgvmgf pgpkgndgap gkngerggpg 601 gpgpqgppgk ngetgpqgpp gptgpggdkg dtgppgpqgl qglpgtggpp gengkpgepg 661 pkgdagapga pggkgdagap gergppglag apglrggagp pgpeggkgaa gppgppgaag 721 tpglqgmpge rgglgspgpk gdkgepggpg adgvpgkdgp rgptgpigpp gpagqpgdkg 781 eggapglpgi agprgspger getgppgpag fpgapgqnge pggkgergap gekgeggppg 841 vagppggsgp agppgpqgvk gergspggpg aagfpgargl pgppgsngnp gppgpsgspg 901 kdgppgpagn tgapgspgvs gpkgdagqpg ekgspgaqgp pgapgplgia gitgarglag 961 ppgmpgprgs pgpqgvkges gkpganglsg ergppgpqgl pglagtagep grdgnpgsdg 1021 lpgrdgspgg kgdrgengsp gapgapghpg ppgpvgpagk sgdrgesgpagpagapgpag 1081 srgapgpqgp rgdkgetger gaagikghrg fpgnpgapgs pgpagqqgaigspgpagprg 1141 pvgpsgppgk dgtsghpgpi gppgprgnrg ergsegspgh pgqpgppgppgapgpccggv 1201 gaaaiagigg ekaggfapyy gdepmdfkin tdeimtslks vngqieslispdgsrknpar 1261 ncrdlkfchp elksgeywvd pnqgckldai kvfcnmetge tcisanplnvprkhwwtdss 1321 aekkhvwfge smdggfqfsy gnpelpedvl dvhlaflrll ssrasqnityhcknsiaymd 1381 qasgnvkkal klmgsnegef kaegnskfty tvledgctkh tgewsktvfeyrtrkavrlp 1441 ivdiapydig gpdqefgvdv gpvcfl The amino acid sequence selected in this invention is extracted from the above sequence.

[0041] Example 1: Preparation of recombinant humanized type III collagen in an Escherichia coli expression system 1. Plasmid construction According to the amino acid sequence shown in SEQ ID No. 1, codon optimization was performed on the E. coli expression system. The 5' end sequence was CATATG, the 3' end sequence was AAGCTT, the start codon was CATATG, and a 6His tag was carried to obtain the target gene sequence, i.e., the first basic repeat unit, as follows: GRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFP.

[0042] The protein whose first basic repeat unit is repeated three times is named SYJY-PRO3-3, and its sequence is as follows: GRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFP.

[0043] The protein whose first basic repeat unit is repeated 4 times is named SYJY-PRO3-4, and its sequence is as follows: GRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFP.

[0044] The protein whose first basic repeat unit is repeated 5 times is named SYJY-PRO3-5, and its sequence is as follows: GRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGF RGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFPGRPGERGLKGENGLPGENGAPGERGFRGPAGPNGIPGEKGPAGERGVMGFP.

[0045] Each basic repeating unit was directly linked, and the resulting target gene sequences were synthesized and transformed into pET-30a(+) plasmids. The nucleotide sequences were optimized for E. coli codon bias, with codon fitness indices (CAI) of 0.90, 0.91, and 0.90, respectively. A schematic diagram of a recombinant humanized type III collagen expression plasmid is shown below. Figure 1 As shown.

[0046] 2. Construction of Escherichia coli expression strains The plasmid containing the target gene was transformed into competent E. coli cells BL21(DE3). The specific steps were as follows: BL21(DE3) competent cells were removed from the ultra-low temperature freezer and thawed on ice. 100 μl of plasmid (100 ng) was added to 100 μl of competent cells and gently pipetted to mix thoroughly. The cells were placed on ice for 30 min and then heat-shocked in a water bath at 42°C for 90 s. The cells were then placed on ice for another 3 min, and 100 μl of room temperature LB medium was added. The cells were then cultured in a shaker at 37°C and 200 rpm for 60 min. The bacterial culture was mixed and spread onto TSA plates containing kanamycin. The plates were inverted and incubated overnight at 37°C. Transformants were screened, and the colonies that grew were the candidate strains of E. coli expressing recombinant humanized type III collagen.

[0047] 3. Target protein expression induction Single colonies of the constructed Escherichia coli expression strain were picked and added to 10 ml of LB medium containing 50 μg / ml kanamycin (1% peptone, 0.5% yeast extract and 1% sodium chloride). The culture was activated at 37°C and 220 rpm for 7 h. After activation, the strain could be frozen at -80°C after adding glycerol to obtain glycerol strain.

[0048] Transfer 100 μL of glycerol bacteria at a 0.1% inoculum to a 250 mL culture flask (containing 100 mL of LB medium containing 50 μg / mL kanamycin), and incubate at 37 °C and 220 rpm for 7 h, until OD reaches the target value. 600When the concentration reaches ≥0.6, transfer the solution to a 2L Erlenmeyer flask (containing 1000ml of LB medium with 50μg / ml kanamycin), lower the temperature to 25℃, and add IPTG at a final concentration of 1mM at 220rpm for 16h induction culture.

[0049] After fermentation, 1 ml of bacterial culture was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the precipitate was resuspended in water, and the expression effect of the target protein was detected by SDS-PAGE. The detection results of SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 are as follows. Figure 2 As shown, there are obvious protein expression bands at molecular weights of approximately 17kDa, 22kDa, and 27kDa, indicating that the engineered Escherichia coli strain capable of highly expressing SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 has been successfully constructed.

[0050] Example 2. Collagen Purification Centrifuge the bacterial culture at 10,000 rpm for 30 min, discard the supernatant, add water to the precipitate at a ratio of 1:20, sonicate for 30 min to break it up, centrifuge at 10,000 rpm for 30 min, discard the precipitate, and obtain the protein supernatant.

[0051] First, equilibrate the column with 20 mM PBS, 0.5 M NaCl, 20 mM imidazole, and pH 7.4 buffer. Then, pass the protein supernatant obtained in the previous steps through a nickel affinity chromatography column at a controlled rate, loading 5 times the column volume. Next, elute with 20 mM PBS, 0.5 M NaCl, 40 mM imidazole, and pH 7.4 buffer to remove contaminating proteins. Finally, elute with 20 mM PBS, 0.5 M NaCl, 200 mM imidazole, and pH 7.4 buffer to collect the target protein, recombinant humanized type III collagen.

[0052] The collected recombinant humanized type III collagen was further purified using Q Sepharose Fast Flow ion exchange chromatography. The loading buffer was 0.02 mol / L Tris-HCl buffer (pH 9.0 ± 0.1). The loading volume was calculated based on the dynamic loading capacity of the Q Sepharose Fast Flow medium. After loading, the sample was washed with the loading buffer until the absorbance returned to baseline. Elution was then performed with 0.02 mol / L Tris-HCl elution buffer, and the resulting recombinant humanized type III collagen was collected.

[0053] SDS-PAGE assay: Take SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5, centrifuge to collect the precipitate, resuspend, add loading buffer, denature in boiling water bath, and then centrifuge to collect the supernatant.

[0054] Place the polymerized gel plate into the electrophoresis tank, add buffer, spot the sample and marker, connect the electrodes, first perform electrophoresis on the stacking gel at a low voltage of 80V, then perform electrophoresis on the separating gel at a high voltage of 120V, and stop when the indicator band is close to the bottom of the gel.

[0055] The protein bands were stained with Coomassie Brilliant Blue staining solution and then destained with destaining solution. The protein bands were observed and compared with the marker to analyze protein expression.

[0056] The electrophoretic results of recombinant humanized type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 are as follows: Figure 3 As shown in the figure. SDS-PAGE analysis results showed that the purity of recombinant humanized type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 was ≥95%.

[0057] Example 3. Spatial structure detection of the target protein SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 solutions were prepared at 1 mg / mL using 10 mM, pH 7.4 PBS buffer. Before testing, the SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 solutions were filtered through a 0.22 μm filter membrane to remove particulate impurities, followed by degassing to prevent air bubbles from affecting the detection results. A 1 mm thick cuvette was used, and the wavelength scanning range was 190-250 nm with 2.5 nm intervals between each step to obtain circular dichroism chromatograms. The tertiary structure of the protein was identified by analyzing the characteristic peaks of the spectrum.

[0058] Experimental results are as follows Figure 4 As shown: Referring to the recombinant collagen standard YY / T 1849-2022, the circular dichroism (CD) spectrum of collagen shows a negative peak at a wavelength near 195 nm and a positive peak at a wavelength near 221 nm, indicating that SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 all have a triple helix structure.

[0059] Example 4. Detection of the cell proliferation-promoting ability of recombinant humanized type III collagen Cell culture and absorbance detection: a) Dilute the recombinant humanized type III collagen test samples (including SYJY-PRO3-3, SYJY-PRO3-4, SYJY-PRO3-5, and commercially available human type III collagen (abcam, ab7535)) to 1 mg / ml using D-PBS, and then perform 5-fold serial dilutions to obtain 4 concentrations; b) Add 100 μL of different concentrations of recombinant humanized type III collagen, commercially available human type III collagen, and D-PBS control to each well. Coat 3 wells with each concentration and incubate at 37°C for 2 hours. c) After incubation, remove the plate, discard the excess coating solution in the wells, add 100 μL / well of 1% BSA-PBS solution, and incubate at 37℃ in a 5% CO2 incubator for 1-2 h. d) After removing the liquid from the wells, wash three times with D-PBS (200 μL / well). Discard the washing solution, seal with sealing film, and store at 4°C for later use. e) Cell processing: Remove the cell culture flasks, discard the culture medium, wash twice with 5 ml D-PBS, add 3 ml trypsin to each flask, incubate at 37°C for 2 minutes, observe the cell digestion status, and terminate digestion with complete culture medium. After thorough mixing, transfer to centrifuge tubes. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in 2 ml DMEM medium, mix 50 μl of the cell suspension with 50 μl of trypan blue staining solution, and count the cells. Adjust the cell density to 1.5 × 10⁻⁶ cells using assay medium. 4 pcs / ml; f) Allow the well plates coated with recombinant humanized type III collagen to reach room temperature beforehand. Seed the diluted cells at 200 μl per well into each 96-well plate. Set up a blank control group with 200 μl / well of the corresponding detection medium. Incubate the cells at 37°C in a 5% CO2 incubator for 48 h. g) After culturing for 48 hours, remove the plate and discard the culture supernatant. Dilute the CCK8 stock solution with DMEM medium at a ratio of 1:10, mix thoroughly, add 100 μl to each well, and continue culturing the cell culture plate. h) After culturing for 2 hours, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader; i) Results Analysis: Cell proliferation rate was calculated using the OD450 of each well. The absorbance value of the recombinant humanized collagen group was At, the absorbance value of the control group was Ac, and the absorbance value of the blank group was A0. Proliferation rate (%) = (At - A0) / (Ac - A0) × 100%.

[0060] The results are as follows Figure 5 , 6As shown, at concentrations of 0.4–250 μg / ml, recombinant human type III collagen SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5, after 48 h of interaction with HFF-1 cells, significantly promoted the proliferation of human skin fibroblasts (HFF-1 cells). The ability of SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 to promote HFF-1 cell proliferation was approximately 1.3 times that of commercially available human type III collagen (abcam, ab7535) at the same concentration, indicating that SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 possess good biological activity.

[0061] Example 5. Detection of the cell adhesion-promoting ability of recombinant humanized type III collagen The ability of collagen to promote NIH / 3T3 cell adhesion was detected using a fluorescent labeling method. The specific steps included: Collagen coating: Dilute SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5 samples and commercially available human type III collagen (abcam, ab7535) to 0.5 mg / ml using DPBS. Add 100 μL of different concentrations of SYJY-PRO3-3, SYJY-PRO3-4, SYJY-PRO3-5, D-PBS control, and commercially available human type III collagen to each well of a 96-well plate, coating three wells with each concentration. Incubate at 37°C for 2 h. Discard excess coating solution from the wells, add 100 μL / well of 1% BSA-PBS solution, and incubate at 37°C in a 5% CO2 incubator for 1 h. Wash three times with PBS afterwards.

[0062] Cell seeding and adhesion: Prepare complete culture medium containing 1% Hoechst 33342 fluorescent dye. Use the complete culture medium with fluorescent dye to prepare a solution containing 5 × 10⁶ cells per ml. 4 A cell suspension of 100 μl was seeded into a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 1 hour.

[0063] Microscope photography: Take out the 96-well cell culture plate, adjust the microscope's luminescence module to the UV module, and take pictures of the cells in the wells.

[0064] Wash away unattached cells: Add 200 μl of DPBS buffer to each well, cover with the cover plate membrane, invert, and centrifuge at 350g for 5 min. Discard the liquid in the wells, wash once with DPBS, and add 100 μl of DPBS buffer to each well.

[0065] Fluorescence detection: Images were taken using a fluorescence microscope.

[0066] Results Analysis: An automated cell counting program was used to count the number of fluorescently labeled cell nuclei to determine the cell count before and after centrifugation. The adhesion percentage was calculated. A higher adhesion percentage indicates better sample adhesion.

[0067] V=N 后 ÷N 前 ×100%, where V is the percentage of cell adhesion; N 前 N represents the number of cells before centrifugation. 后 This represents the number of cells after centrifugation.

[0068] Experimental results are as follows Figure 7 , 8 As shown, after 1 hour of cell adhesion, the number of cells in each well was similar and showed no difference. After centrifugation and washing, at a concentration of 0.5 mg / ml, the number of cells in the wells coated with recombinant human type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 was significantly higher than that in the DPBS control group; the number of cells in the wells was comparable to that in the wells coated with commercially available collagen, with no significant difference. This indicates that recombinant human type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 can significantly promote cell adhesion, and their adhesion properties are comparable to those of commercially available collagen.

[0069] Example 6. Detection of the cell migration-promoting ability of recombinant humanized type III collagen The ability of collagen to promote cell migration was tested using a scratch assay, which included the following steps: Cell culture: HFF-1 cells were cultured in DMEM medium containing 15% fetal bovine serum to induce logarithmic growth. Cells were cultured in a 37°C, 5% CO2 incubator.

[0070] Materials and reagents: 6-well plate, pipette, sterile 10μL pipette tip, PBS, serum-free culture medium, and protein solution to be tested.

[0071] Cell seeding: Seed cells at an appropriate density in 6-well plates, allowing the cells to reach near confluence (approximately 90% - 100%) after overnight culture.

[0072] Scratching: Use a sterile 10μL pipette tip to draw a vertical straight line on the cell monolayer, trying to ensure that the scratch width is uniform.

[0073] Washing: Gently rinse the cells three times with PBS to remove cell debris caused by the scratches.

[0074] Protein addition: Add 2 ml of serum-free culture medium containing different concentrations of the protein to be tested to each well. At the same time, set up a blank control group (only DMEM medium added) and commercial collagen (human type III collagen, abcam, ab7535).

[0075] Observation and photography: At 0 hours and 24 hours after the scratch, cell migration in the scratched area was recorded by photographing under the same field of view using an inverted microscope. Multiple fields of view were selected during photography to ensure the accuracy of the results.

[0076] Results Analysis: The width of the scratch region at different time points was measured using ImageJ image analysis software, and the cell migration rate was calculated. Cell migration rate = (0-hour scratch width – 24-hour scratch width) ÷ 0-hour scratch width × 100%. The effect of protein on cell migration was analyzed by comparing the cell migration rates of the experimental group and the control group.

[0077] Experimental results are as follows Figure 9 As shown in the cell scratch assay, cells supplemented with recombinant human type III collagen SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5 exhibited significant cell migration and full cell extension 24 hours after scratching. Compared to the blank control group, recombinant human type III collagen molecules demonstrated a good ability to promote cell migration, comparable to commercially available collagen.

[0078] Example 7. Detection of Cytotoxicity of Recombinant Humanized Type III Collagen The cytotoxicity of recombinant humanized type III collagen SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5 was evaluated using the Cell Counting Kit-8 (CCK-8) assay. 100 μL of HFF-1 cell suspension was cultured at 5 × 10⁻⁶ cells per well. 5Cells were placed at a density of 100 μL in 96-well cell culture plates and incubated at 37 °C and 5% CO2 for 24 hours. The culture medium was discarded, and the cells were washed three times with PBS. Then, 100 μL of recombinant humanized collagen at different concentrations (0.01, 0.05, 0.1, and 0.5 mg / mL) was added. The experiment included a cell control group, a positive control group, and a blank group. The cell control group contained cells and 100 μL of DMEM medium; the positive control group contained cells and 100 μL of DMEM medium containing 5% DMSO; and the blank group contained only 100 μL of DMEM medium without cells. After incubating the cell culture plates at 37 °C and 5% CO2 for 24 hours, the liquid in the wells was aspirated, and 100 μL of DMEM medium containing 10% CCK-8 was added to each well. The plates were then incubated in a cell culture incubator for another 2 hours. The absorbance at 450 nm was measured using a multi-mode microplate reader. Each experiment was performed in triplicate. The absorbance value of the recombinant humanized collagen group was At, the absorbance value of the cell control group was Ac, and the absorbance value of the blank group was A0. Cell viability (%) = (At - A0) ÷ (Ac - A0) × 100%.

[0079] Experimental results are as follows Figure 10 As shown, significant cell death was observed in the positive control group wells, with a survival rate of 0%. The effects of different concentrations of recombinant humanized type III collagen (SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5) and blank solvent on cell viability were compared. Referring to ISO 10993-5-2009 Part 5: Standards for in vitro cytotoxicity assays, it can be seen that cell viability remained between 90% and 110%, with minimal fluctuations, meeting the requirement that "cytotoxicity reaction grade should be ≤ 2, and relative cell proliferation / viability ≥ 70%". This indicates that recombinant humanized type III collagen did not exhibit cytotoxicity, fully demonstrating its good biocompatibility.

[0080] Example 8. Stability of recombinant humanized type III collagen The recombinant humanized type III collagen (SYJY-PRO3-3 / SYJY-PRO3-4 / SYJY-PRO3-5) prepared in Example 2 was prepared into a 0.1% solution with deionized water and placed in a constant temperature stable chamber at 50°C. Samples were taken out at 0, 1, 3 and 5 days respectively, and the stability of the samples was investigated by SDS-PAGE detection method.

[0081] Experimental results are as follows Figure 11As shown, SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 were placed at 50℃ for 5 days. The SDS-PAGE results showed that the purity of each protein molecule was high, no degradation fragments were observed, and there were no significant differences between the different observation days, indicating good stability.

[0082] Example 9. Application of recombinant humanized type III collagen in cosmetics 1. Preparation of aqueous solutions for promoting wound healing The recombinant humanized type III collagen (SYJY-PRO3-3, SYJY-PRO3-4, SYJY-PRO3-5) obtained in Example 2 were mixed with 1% sodium hyaluronate, 0.1% panthenol, 3% glycerol and deionized water at a concentration of 0.5% (w / w), and the pH was adjusted to 6.5 to prepare transparent aqueous solutions.

[0083] Commercially available human type III collagen (abcam, ab7535) was prepared into a transparent aqueous solution using the same method and used as a control group.

[0084] 2. Animal testing Thirty-six female BALB / c mice were procured. After shaving the backs of the mice with an electric shaver, depilatory cream was evenly applied to remove hair from the backs of the necks. For the model animals (30 mice), a mixture of acetone and ether in equal volumes (2:1) was used to moisten cotton pads and lightly applied to the exposed skin on the backs of the necks for 15 seconds, followed immediately by a cotton pad soaked in distilled water for 30 seconds. This was repeated twice daily for five consecutive days to establish a dry skin model. The control group (6 mice) only received a cotton pad soaked in distilled water for 30 seconds, twice daily for five consecutive days.

[0085] On day 6 of model construction, the moisture content of the modeled area was measured in the animals. Based on the skin moisture content, the animals were randomly divided into 5 groups of 6 animals each: a model group and 4 experimental groups. After grouping, the number of times the animals scratched was measured and recorded. The model group received a matrix formula without recombinant humanized type III collagen. The 4 experimental groups received matrices containing commercially available collagen, SYJY-PRO3-3, SYJY-PRO3-4, and YJY-PRO3-5, respectively. The control group (no modeling) received a matrix formula without recombinant humanized type III collagen. All animals were treated once daily for 28 consecutive days.

[0086] After the final application, the skin moisture content of the modeling area in each group of animals was measured (using a Corneometer® CM825 to determine the stratum corneum moisture content) and the number of times the animals scratched. After the tests, the animals were euthanized, and approximately 1 cm of skin tissue from the modeling area was taken. 2After weighing, the tissue was quickly placed in liquid nitrogen. According to the mass-volume ratio of 0.1 mg: 1 ml, an appropriate amount of physiological saline was added based on the tissue weight. Then, the tissue was homogenized, and the content of type I collagen in the skin tissue was detected.

[0087] Number of scratches: such as Figure 12-14 The results showed that skin moisture decreased significantly after modeling, and the number of scratches in mice increased significantly. After applying a transparent aqueous solution for 28 days, the number of scratches in all experimental groups decreased significantly, while the number of scratches in the model group decreased slightly, and the numbers in each experimental group were similar to those in the control group. There were significant differences in the number of scratches between the experimental groups and the model group. P＜ 0.05 No significant differences were observed between the groups containing SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 and the group containing commercially available collagen.

[0088] Skin moisture content: After 28 days of treatment with the control group's clear aqueous solution, the skin moisture content of the experimental group mice was significantly higher than that of the control group. The differences in skin moisture content between the experimental groups and the model group were statistically significant. P＜0.05 No significant difference in skin hydration was observed between the experimental group containing SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 and the group containing commercially available collagen. This indicates that the use of SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 can significantly increase skin hydration, with effects comparable to commercially available collagen, and has the effect of improving skin condition and reducing wrinkles.

[0089] Type I collagen content in skin tissue: The type I collagen content in the animal tissues of each experimental group was significantly higher than that in the control group and the model group. Compared with the model group, the type I collagen content in the skin tissues of each experimental group was significantly increased, and the differences were statistically significant. P＜0.05 The type I collagen content in the skin tissue of each experimental group was approximately 30% higher than that of the control group. The type I collagen content in the skin tissue of each experimental group was also 20% higher than that of commercially available collagen.

[0090] The experimental results show that SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 can significantly repair the skin barrier, increase skin moisture content and type I collagen content, and significantly improve skin wrinkles, elasticity, and barrier function.

[0091] 3. Efficacy Testing Test subjects: 40 healthy female volunteers aged 25-50 (10 in the control group and 10 in the experimental group × 3 groups) with fine lines and loose skin on their faces were randomly assigned to groups. The control group used an aqueous solution containing 0.5% commercially available collagen, while the experimental groups used aqueous solutions containing 0.5% recombinant humanized type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5, respectively. They were applied to their faces after cleansing in the morning and evening for 28 days.

[0092] Test method: Skin moisture content: The moisture content of the stratum corneum was determined using a Corneometer® CM825.

[0093] Skin elasticity: R2 value (elasticity recovery rate) was measured using Cutometer® dual suction mode.

[0094] Wrinkle volume: 3D imaging system (Primos®) analyzes wrinkle areas.

[0095] Irritation test: Irritation test shall be conducted by patch test (ISO 10993-10).

[0096] The results are shown in the table below:

[0097] Statistical methods: Independent samples t-test was used, and the significance threshold was set at p<0.05.

[0098] Test results Skin moisture content: all experimental groups showed a significant increase compared to the control group (p<0.01); Skin elasticity: the experimental groups showed a significant increase (p<0.05). Wrinkle depth: the experimental groups showed a significant reduction in wrinkle volume (p<0.01), while the control group showed no significant change. Irritation test: the patch test (ISO 10993-10) showed no erythema or edema at a concentration of 0.5%, with a sensitization rate of 0%.

[0099] This embodiment demonstrates that an aqueous solution containing 0.5% recombinant humanized type III collagen SYJY-PRO3-3, SYJY-PRO3-4, and SYJY-PRO3-5 can significantly improve skin wrinkles, elasticity, and barrier function, and its safety meets cosmetic regulations, making it suitable for skincare product development.

Claims

1. A highly active recombinant humanized type III collagen, characterized in that, The recombinant humanized type III collagen includes a first basic repeating unit, the amino acid sequence of which is shown in SEQ ID NO:

1. The recombinant humanized type III collagen is formed by repeating the first basic repeating unit 1-10 times.

2. A nucleic acid molecule encoding the recombinant humanized type III collagen of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule includes a second basic repeat unit, the nucleotide sequence of which is shown in SEQ ID NO:

2. The nucleic acid molecule is formed by repeating the second basic repeat unit 1 to 10 times. When the number of repetitions exceeds 2, the second basic repeat units are directly linked together.

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

5. The carrier according to claim 4, characterized in that, The vector comprises an expression control element operatively linked to the nucleic acid molecule, a purification tag nucleotide, and / or a leader sequence nucleotide, wherein the purification tag is selected from one or both of His tags and Sumo tags.

6. A cell, characterized in that, The cell carries the nucleic acid molecule as described in claim 2 or 3, or contains the vector as described in claim 4 or 5, to express the recombinant humanized type III collagen as described in claim 1.

7. The engineered *E. coli* strain used to produce the recombinant humanized type III collagen according to claim 1, characterized in that, The carrier comprises the one described in claim 4 or 5, wherein the Escherichia coli is strain BL21.

8. A method for preparing recombinant humanized type III collagen according to claim 1, characterized in that, Includes the following steps: 1) Pre-culture engineered Escherichia coli in LB medium at 37°C until OD. 600 ≥0.6; 2) Cool to 16-30℃ and add 0.5-2 mM IPTG to induce expression for 10-20 hours; 3) After lysing the bacterial cells, the target protein was captured by Ni-NTA affinity chromatography; 4) After purification, the target collagen with a purity of ≥95% is obtained, which is the recombinant humanized type III collagen.

9. The preparation method according to claim 8, characterized in that, The purification process includes, but is not limited to, one or a combination of several of the following: salting out, chromatographic chromatography, acid-base precipitation, and membrane separation.

10. An aqueous solution for promoting wound healing, characterized in that, The mixture comprises the recombinant humanized type III collagen and sodium hyaluronate as described in claim 1, wherein the concentration of the recombinant humanized type III collagen is 0.1-2% w / v, the concentration of the sodium hyaluronate is 0.5-2% w / v, and the recombinant humanized type III collagen and the sodium hyaluronate form a clear and transparent aqueous solution under pH 6.5-7.5 conditions.

11. Use of the recombinant humanized type III collagen as described in claim 1 in the manufacture of skin care products, tissue engineering materials, or medical devices.

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