A deredundant recombinant humanized type III collagen, its preparation method and application

Abstract

This disclosure belongs to the field of genetic engineering technology, and provides a deredundant recombinant humanized type III collagen, its preparation method, and its applications. The protein is the core functional fragment of a humanized type III collagen with non-moisturizing redundant regions removed, and its amino acid sequence is shown in SEQ ID NO:1. The disclosure also includes the gene encoding the above protein; a recombinant expression vector containing the above gene; recombinant genetically engineered bacteria transformed into the above recombinant expression vector; a method for preparing the above protein; and the application of the above protein in the preparation of moisturizing skincare products, skin repair materials, and medical dressings. This disclosure obtains deredundant type III collagen by removing redundant regions that do not contribute to moisturizing, significantly reducing the protein molecular weight, reducing the expression burden, and greatly improving expression and purification efficiency. The raw materials are inexpensive and readily available, resulting in low cost and good biocompatibility; the process is simple, the reaction conditions are mild, and energy consumption is low.

Description

Technical Field

[0001] This disclosure relates to the field of genetic engineering technology, and in particular to a deredundant recombinant humanized type III collagen, its preparation method, and its application. Background Technology

[0002] Human type III collagen is an important structural protein in the dermis, mucous membranes, and soft tissues of the skin. It possesses excellent biocompatibility, moisturizing properties, and repair activity, and is widely used in skincare products, wound repair, and aesthetic medicine. Currently, type III collagen is mainly prepared through two methods: extraction from animal tissues and full-length expression from recombinant human sources. Animal-derived type III collagen carries risks of viral residue and immunogenicity, and its amino acid sequence differs from that of human-derived collagen, resulting in limited moisturizing and biological activity. Although human recombinant full-length type III collagen has higher safety and better sequence consistency, related technologies mostly use full-length sequence expression, which has obvious technical defects: (1) The full-length sequence contains a large number of redundant segments that do not contribute to moisture absorption and hydration, resulting in a larger protein molecular weight, which significantly increases the expression difficulty and purification cost of expression systems such as Pichia pastoris; (2) The water binding sites are only concentrated in specific loose regions of the Gly-XY repeat sequence. The redundant sequence does not improve the moisturizing performance, but reduces the protein expression level and water solubility; (3) Current products have not optimized the sequence and deredundant design for moisturizing function. The moisture absorption rate and hydration rate are difficult to meet the requirements of high-end skin care products for highly effective moisturizing ingredients, which limits their application effect and market competitiveness.

[0003] Therefore, developing a redundancy-free, low-molecular-weight, high-expression, and highly moisturizing recombinant humanized type III collagen (col3Δ) and its efficient preparation process has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] This disclosure provides a deredundancy-recombinant humanized type III collagen (col3Δ), its preparation method, and its application, in order to at least solve the above-mentioned technical problems existing in the prior art.

[0005] According to a first aspect of this disclosure, a deredundant recombinant humanized type III collagen is provided, wherein the collagen is a core functional fragment of humanized type III collagen with non-moisturizing redundant segments removed, and its amino acid sequence is as shown in SEQ ID NO:1: HHHHHHHHGGGGGGGGENLYFQGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER.

[0006] Specifically, this disclosure retains the core functional segments truly responsible for moisture absorption and hydration, while eliminating redundant fragments that do not contribute to hydration, thus achieving a small molecular weight and high hydration efficiency. The aforementioned collagen is based on the natural sequence of human type III collagen, retaining the core module with high hydrophilicity and high water-binding capacity in the Gly-XY repeat region, while deleting non-functional flexible regions and extension fragments that do not contribute to hydration. A His6-tev tag is added to the N-terminus, balancing soluble expression, affinity purification, and tag removability, ensuring that the final product contains the natural functional region without redundant residues affecting activity.

[0007] This disclosure defines and obtains for the first time a truncated humanized type III collagen that retains only its moisturizing core. It has a well-defined structure and unique sequence, belonging to a novel structural protein. It has a smaller molecular weight, better transdermal permeability and skin feel, higher expression levels, simpler purification, and significantly reduced industrialization costs.

[0008] According to a second aspect of the present disclosure, there is provided a gene encoding the above-mentioned redundant recombinant humanized type III collagen, the nucleotide sequence of which is as shown in SEQ ID NO:2: CAYCAYCAYCAYCAYCAYCAYCAYGGNGGNGGNGGNGGNGGNGGNGGNGARAAYYTNTAYTTYCARGGNGARMGNGGNGGNCCNGGNGGNCCNGGNCCNCARGGNCCNCCNGGNAARAAYGGNGARACNGGNCCNCARGGNCCNCCNGGNCCNACNGGNCCNGGNGGNGAYAARGGNGAYACNGGNCCNCCNGGNCCNCARGGNYTNCARGGNYTNCCNGGNACNGGNGGNCCNCCNGGNGARAAYGGNAARCCNGGNGARCCNGGNCCNAARGGNGAYGCNGGNGCNCCNGGNGCNCCNGGNGGNAARGGNGAYGCNGGNGCNCCNGGNGARMGNGGNCCNCCNGGNYTNGCNGGNGCNCCNGGNYTNMGNGGNGGNGCNGGNCCNCCNGGNCCNGARGGNGGNAARGGNGCNGCNGGNCCNCCNGGNCCNCCNGGNGCNGCNGGNACNCCNGGNYTNCARGGNATGCCNGGNGARMGNGGNGGNYTNGGNWSNCCNGGNCCNAARGGNGAYAARGGNGARCCNGGNGGNCCNGGNGCNGAYGGNGTNCCNGGNAARGAYGGNCCNMGNGGNCCNACNGGNCCNATHGGNCCNCCNGGNCCNGCNGGNCARCCNGGNGAYAARGGNGARGGNGGNGCNCCNGGNYTNCCNGGNATHGCNGGNCCNMGNGGNWSNCCNGGNGARMGNGGNGARACNGGNCCNCCNGGNCCNGCNGGNTTYCCNGGNGCNCCNGGNCARAAYGGNGARCCNGGNGGNAARGGNGARMGNGGNGCNCCNGGNGARAARGGNGARGGNGGNCCNCCNGGNGTNGCNGGNCCNCCNGGNGGNWSNGGNCCNGCNGGNCCNCCNGGNCCNCARGGNGTNAARGGNGARMGN;Where R is selected from A or G, Y from C or T, S from G or C, W from A or T, K from G or T, M from A or C, B from C, G or T, D from A, G or T, H from A, C or T, V from A, C or G, and N from A, T, C or G.

[0009] Specifically, the above gene sequences employ a degenerate base design, based on codon degeneracy, covering all nucleotide sequences that can encode redundant recombinant humanized type III collagen, facilitating codon optimization and efficient expression.

[0010] According to a third aspect of this disclosure, a recombinant expression vector is provided, comprising the aforementioned gene.

[0011] According to a fourth aspect of this disclosure, a recombinant genetically engineered bacterium is provided, wherein the recombinant genetically engineered bacterium is transferred into the aforementioned recombinant expression vector.

[0012] In one embodiment, the recombinant genetically engineered bacteria is Pichia pastoris (Pichia pastoris). Pichia pastoris ).

[0013] According to the fifth aspect of this disclosure, a method for preparing the above-mentioned deredundant recombinant humanized type III collagen is provided, comprising the following steps: S1: Construct a recombinant expression vector containing the gene encoding the deredundant recombinant humanized type III collagen; S2: Transform the recombinant expression vector into host cells to obtain positive recombinant bacteria; S3: Induce expression in the positive recombinant bacteria to obtain the expression product; S4: Purify the expression product to obtain the deredundant recombinant humanized type III collagen.

[0014] In one embodiment, the conversion method in step S2 is selected from at least one of electroconversion, lithium acetate conversion, protoplast conversion, and chemicompetent conversion.

[0015] In a preferred embodiment, the conversion method in step S2 is selected from electroconversion.

[0016] In one embodiment, the host cell in step S2 is Pichia pastoris.

[0017] In one embodiment, step S2 uses resistant plates to screen for positive clones, thereby obtaining positive recombinant bacteria.

[0018] In one embodiment, step S3 uses methanol-induced expression at a temperature of 30-35°C for 48-72 hours, maintaining dissolved oxygen ≤30% and methanol concentration ≤1% during the induction process.

[0019] In one embodiment, the purification steps in step S4 sequentially include: Ni-NTA affinity chromatography, enzyme digestion, ion exchange chromatography, molecular sieve desalting, and lyophilization.

[0020] In one embodiment, the expression level of the deredundant recombinant humanized type III collagen is ≥10 g / L.

[0021] According to the sixth aspect of this disclosure, the above-mentioned deredundant recombinant humanized type III collagen is provided for use in the preparation of any one of moisturizing skin care products, skin repair materials, and medical dressings.

[0022] According to one possible implementation of this disclosure, at least the following beneficial effects are achieved: This disclosure obtains deredundant recombinant humanized type III collagen by removing redundant segments that do not contribute to moisturizing, significantly reducing the protein molecular weight, decreasing the expression burden, and greatly improving the expression and purification efficiency of Pichia pastoris. High-density fermentation with Pichia pastoris can achieve expression levels of over 10 g / L, with stable yields and controllable processes, making it suitable for large-scale industrial preparation.

[0023] The collagen disclosed herein has short fibers and a tight, dense structure, making it easier to disperse in skincare products and dressings. It also has good film-forming properties, a gentle feel on the skin, and significantly improved moisture absorption and hydration rates, making it suitable for high-end moisturizing and skin repair applications.

[0024] This disclosure uses recombinant human sequences, which are free of animal-derived viruses and sensitization risks, exhibit good biocompatibility, and can be safely used in cosmetics, medical dressings, and other fields. The fermentation and purification conditions are mild, requiring no extreme temperatures or pressures; the purification steps are highly efficient and controllable; and the overall process is green, low-cost, and easily scalable.

[0025] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0026] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which: In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0027] Figure 1 The diagram shows the results of the initial screening of strain expression level by sampling 24 h after induction of Pichia pastoris with high expression of col3Δ in Example 1 of this disclosure; Figure 2A cryo-scanning electron micrograph of the purified detagged col3Δ collagen from Example 1 of this disclosure is shown. Detailed Implementation

[0028] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0029] Example 1 This embodiment prepared a deredundant recombinant humanized type III collagen (col3Δ), as detailed below: (1)PCR amplification. Based on the gene encoding the redundant-free recombinant humanized type III collagen (hereinafter referred to as col3Δ, whose nucleotide sequence is as shown in SEQ ID NO:2: CAYCAYCAYCAYCAYCAYCAYCAYGGNGGNGGNGGNGGNGGNGGNGGNGARAAYYTNTAYTTYCARGGNGARMGNGGNGGNCCNGGNGGNCCNGGNCCNCARGGNCCNCCNGGNAARAAYGGNGARACNGGNCCNCARGGNCCNCCNGGNCCNACNGGNCCNGGNGGNGAYAARGGNGAYACNGGNCCNCCNGGNCCNCARGGNYTNCARGGNYTNCCNGGNACNGGNGGNCCNCCNGGNGARAAYGGNAARCCNGGNGARCCNGGNCCNAARGGNGAYGCNGGNGCNCCNGGNGCNCCNGGNGGNAARGGNGAYGCNGGNGCNCCNGGNGARMGNGGNCCNCCNGGNYTNGCNGGNGCNCCNGGNYTNMGNGGNGGNGCNGGNCCNCCNGGNCCNGARGGNGGNAARGGNGCNGCNGGNCCNCCNGGNCCNCCNGGNGCNGCNGGNACNCCNGGNYTNCARGGNATGCCNGGNGARMGNGGNGGNYTNGGNWSNCCNGGNCCNAARGGNGAYAARGGNGARCCNGGNGGNCCNGGNGCNGAYGGNGTNCCNGGNAARGAYGGNCCNMGNGGNCCNACNGGNCCNATHGGNCCNCCNGGNCCNGCNGGNCARCCNGGNGAYAARGGNGARGGNGGNGCNCCNGGNYTNCCNGGNATHGCNGGNCCNMGNGGNWSNCCNGGNGARMGNGGNGARACNGGNCCNCCNGGNCCNGCNGGNTTYCCNGGNGCNCCNGGNCARAAYGGNGARCCNGGNGGNAARGGNGARMGNGGNGCNCCNGGNGARAARGGNGARGGNGGNCCNCCNGGNGTNGCNGGNCCNCCNGGNGGNWSNGGNCCNGCNGGNCCNCCNGGNCCNCARGGNGTNAARGGNGARMGN;Where R is selected from A or G, Y from C or T, S from G or C, W from A or T, K from G or T, M from A or C, B from C or G or T, D from A or G or T, H from A or C or T, V from A or C or G, and N from A or T or C or G, forward and reverse primers are designed with BamHI and EcoRI restriction sites added to the 5' end of each primer. A synthetic gene template (1 ng μL) is used. -1 PCR was performed in a 50 μL reaction system; program: 95℃, 3 min → (95℃, 30 s; 60℃, 30 s; 72℃, 30 s) × 30 cycles → 72℃, 5 min. The PCR product was detected as a single band by 1% agarose gel electrophoresis and then recovered using a PCR product purification kit.

[0030] (2) Double enzyme digestion and purification. The recovered col3Δ fragment and pAOX1-His plasmid were digested with BamHI and EcoRI at 37℃ for 1 h, respectively. The enzyme digestion products were separated into target bands by 1% agarose gel electrophoresis, and the gel was purified and the DNA concentration was determined.

[0031] (3) Ligation and preliminary cloning. The vector and the insert were ligated using the T4 DNA ligase method; 5 μL of the ligation mixture was transformed into DH5α competent cells, heat-shocked at 42℃ for 45 s, and then recovered at 37℃ for 45 min.

[0032] (4) Resistance screening. 100 μL of conversion solution was coated with a solution containing zeocin (concentration of 100 μg / mL). -1 LB plates were incubated at 37°C for 12-16 hours, and single colonies were picked.

[0033] (5) Colony PCR, plasmid extraction and sequencing. Colony PCR was performed using primers at both ends of the insertion region. The positive clone band should be consistent with the theoretical length. The positive clone was inoculated into 10 mL of LB-Zeocin and cultured at 37℃ and 200 rpm for 12-15 h. The plasmid was extracted according to the Miniprep kit and sent to Sanger sequencing to verify the correctness and directionality of the sequence.

[0034] (6) Preparation of linearized vector. The recombinant plasmid with correct sequencing was digested with SacI at 37℃ for 30 min, and electrophoresis confirmed that only one linearized main band was obtained; the linearized DNA (≥500 ng μL) was recovered and purified. -1 ), electro-transfer Pichia pastoris GS115-Δku70 expression. 100 μL of transformation buffer was spread onto a medium containing 100 μg / mL Zeocin. -1 LB flat plate.

[0035] (7) High-density fermentation and purification. The above LB plates were incubated at 37℃ for 12-16 h, and 8 single colonies were picked and verified as positive clones. The 8 positive clones were inoculated into 2L Erlenmeyer flasks containing 500 mL of BMGY and cultured at 30℃ with shaking at 200 rpm until OD. 600 =1~1.5. Transfer the culture medium to a 1L centrifuge cup, centrifuge at 3000×g for 5min to harvest the cells; wash 2~3 times with 50mL sterile water to remove glycerol. Finally, resuspend in BMMY to adjust OD. 600 Adjust the concentration to 0.3 (approximately 1-2 L). Transfer the resuspended solution to a 5 L Erlenmeyer flask and begin methanol-induced expression at 30°C and 200 rpm. The initial methanol concentration is 0.5% (v / v), with subsequent additions of methanol every 24 hours to maintain a methanol concentration <1% (v / v). Maintain dissolved oxygen at 20%-30% during induction. Initially, samples were taken 24 hours after induction for SDS-PAGE electrophoresis to compare expression levels across multiple candidate strains and screen for high-yielding strains (see [link to relevant documentation]). Figure 1 (The second and eighth strains were high-yielding dominant strains). Based on the protein expression monitoring results at multiple time points throughout the 0-72h cycle, the optimal total induction expression time for this system was determined to be 72h. Fermentation was terminated after 72h of induction, and subsequent product purification was carried out.

[0036] (8) Protein extraction and detection. After induction, the protein was centrifuged at 5000 rpm for 15 min and the supernatant was collected. The clarified liquid was filtered through a 0.45 μm filter and then subjected to Ni-NTA nickel column affinity chromatography (20 mM → 250 mM Imidazole gradient) to obtain the target protein. The untagged col3Δ collagen was obtained by TEV enzyme digestion. The protein was analyzed by SDS-PAGE (12%), and the target band was located at ~37 kDa. Coomassie staining showed a purity of >90%.

[0037] The obtained high-purity unlabeled col3Δ collagen solution was then ultrafiltered to a concentration that was adjusted to prepare a protein-water solution. This solution was injected into a mold and then freeze-dried to obtain a solid-state freeze-dried collagen sample. After gold sputtering for conductivity, the sample's microstructure was observed using a cryo-scanning electron microscope. The results are as follows: Figure 2 As shown. Figure 2 The results showed that the obtained collagen fibers were approximately 100 μm long, with a tight and dense overall structure, exhibiting a continuous, interwoven three-dimensional network structure.

[0038] (9) Protein purification and yield determination. The Ni-NTA eluent was further purified by Q-Sepharose Fast Flow ion exchange (pH 8.0 → NaCl 0~0.5M gradient) and Superdex 200 16 / 600 molecular sieve chromatography. After collecting the target peak, it was concentrated to 5 mg / mL by 10 kDa ultrafiltration. -1 Based on the molecular weight (37kDa) and the theoretical ε... 280 =0.85mL mg -1 cm -1 On NanoDrop TM 2000 protein concentration; in this example, the batch yield of the 5L fermenter was 53g of lyophilized powder, which is equivalent to ~10g / L.

[0039] Comparative Example 1 This comparative example provides commercially available recombinant human type III collagen (full length), specifically from the brand AdvancedBioMatrix (USA) (product number 5019).

[0040] Comparative Example 2 This comparative example provides fish-derived type III collagen, specifically from the brand Vital Proteins (catalog number MC07U).

[0041] Comparative Example 3 This comparative example provides bovine type III collagen, specifically from the brand Chondrex (product number 1302).

[0042] Comparative Example 4 This comparative example provides porcine type III collagen, specifically from the brand Chondrex (product number 1303).

[0043] Test case The hygroscopic and moisturizing properties of type III collagen in Example 1 and Comparative Examples 1-4 were tested.

[0044] Take approximately 20 mg (W0) of freeze-dried collagen, equilibrate it at 25°C and 75% humidity for 24 hours, and weigh it (W0). s Then, equilibrate in a 20% humidity environment for 24 hours and weigh (W). d Calculate the moisture absorption rate and moisture retention rate using the following formulas: Moisture absorption rate = (W s -W0) / W0×100%; Moisturizing rate = (W d -W0) / (W s -W0)×100%.

[0045] The test results are shown in Table 1.

[0046] Table 1

[0047] Note: Tukey's test following ANOVA was used. Different letters indicate significant differences (P < 0.05). Table 1 shows that the deredundant recombinant humanized type III collagen (col3Δ) of Example 1 significantly outperformed various commercially available control collagens in both moisture absorption and moisturizing rates, demonstrating excellent potential for skin moisturizing applications. This indicates that by removing the non-moisturizing redundant segments of humanized type III collagen, the core moisturizing active sequence is more concentrated, avoiding the dilution of protein water solubility and activity by redundant sequences. This achieves a double improvement in moisture absorption (+52.95%) and moisturizing rate (+59.31%), solving the technical pain point of low moisturizing efficiency in traditional recombinant human and animal collagens. The excellent moisture absorption and moisturizing properties indicate that the collagen of this disclosure can rapidly absorb moisture from the environment and the skin surface while maintaining long-lasting moisture retention. It is particularly suitable for high-end moisturizing skincare products, skin repair dressings, and medical wound healing materials, significantly improving the moisturizing efficacy and user experience of the products.

[0048] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

[0049] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0050] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A deredundant recombinant humanized type III collagen, characterized in that, The collagen is the core functional fragment of humanized type III collagen with non-moisturizing redundant segments removed, and its amino acid sequence is shown in SEQ ID NO:1: HHHHHHHHGGGGGGGGENLYFQGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER.

2. A gene encoding the deredundant recombinant humanized type III collagen as described in claim 1, characterized in that, The nucleotide sequence of the said gene is as shown in SEQ ID NO:2: CAYCAYCAYCAYCAYCAYCAYCAYGGNGGNGGNGGNGGNGGNGGNGGNGARAAYYTNTAYTTYCARGGNGARMGNGGNGGNCCNGGNGGNCCNGGNCCNCARGGNCCNCCNGGNAARAAYGGNGARACNGGNCCNCARGGNCCNCCNGGNCCNACNGGNCCNGGNGGNGAYAARGGNGAYACNGGNCCNCCNGGNCCNCARGGNYTNCARGGNYTNCCNGGNACNGGNGGNCCNCCNGGNGARAAYGGNAARCCNGGNGARCCNGGNCCNAARGGNGAYGCNGGNGCNCCNGGNGCNCCNGGNGGNAARGGNGAYGCNGGNGCNCCNGGNGARMGNGGNCCNCCNGGNYTNGCNGGNGCNCCNGGNYTNMGNGGNGGNGCNGGNCCNCCNGGNCCNGARGGNGGNAARGGNGCNGCNGGNCCNCCNGGNCCNCCNGGNGCNGCNGGNACNCCNGGNYTNCARGGNATGCCNGGNGARMGNGGNGGNYTNGGNWSNCCNGGNCCNAARGGNGAYAARGGNGARCCNGGNGGNCCNGGNGCNGAYGGNGTNCCNGGNAARGAYGGNCCNMGNGGNCCNACNGGNCCNATHGGNCCNCCNGGNCCNGCNGGNCARCCNGGNGAYAARGGNGARGGNGGNGCNCCNGGNYTNCCNGGNATHGCNGGNCCNMGNGGNWSNCCNGGNGARMGNGGNGARACNGGNCCNCCNGGNCCNGCNGGNTTYCCNGGNGCNCCNGGNCARAAYGGNGARCCNGGNGGNAARGGNGARMGNGGNGCNCCNGGNGARAARGGNGARGGNGGNCCNCCNGGNGTNGCNGGNCCNCCNGGNGGNWSNGGNCCNGCNGGNCCNCCNGGNCCNCARGGNGTNAARGGNGARMGN; wherein, R is selected from A or G, Y is selected from C or T, S is selected from G or C, W is selected from A or T, K is selected from G or T, M is selected from A or C, B is selected from C or G or T, D is selected from A or G or T, H is selected from A or C or T, V is selected from A or C or G, and N is selected from A or T or C or G.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes the gene described in claim 2.

4. A recombinant genetically engineered bacterium, characterized in that, The recombinant genetically engineered bacteria were transferred into the recombinant expression vector of claim 3.

5. The recombinant genetically engineered bacteria according to claim 4, characterized in that, The recombinant genetically engineered bacteria is Pichia pastoris.

6. The method for preparing the deredundant recombinant humanized type III collagen according to claim 1, characterized in that, Includes the following steps: S1: Construct a recombinant expression vector containing the gene encoding the deredundant recombinant humanized type III collagen; S2: Transform the recombinant expression vector into host cells to obtain positive recombinant bacteria; S3: Induce expression in the positive recombinant bacteria to obtain the expression product; S4: Purify the expression product to obtain the deredundant recombinant humanized type III collagen.

7. The preparation method according to claim 6, characterized in that, In step S2, the conversion method is selected from at least one of electroconversion, lithium acetate conversion, protoplast conversion, and chemicompetent conversion; The host cell is Pichia pastoris.

8. The preparation method according to claim 6, characterized in that, Step S3 involves methanol-induced expression at a temperature of 30-35°C for 48-72 hours, maintaining dissolved oxygen levels ≤30% and methanol concentration ≤1% during the induction process.

9. The preparation method according to claim 6, characterized in that, The purification steps described in step S4 include, in sequence: Ni-NTA affinity chromatography, enzyme digestion, ion exchange chromatography, molecular sieve desalting, and lyophilization.

10. The use of the deredundant recombinant humanized type III collagen as described in claim 1 in the preparation of any one of moisturizing skin care products, skin repair materials, and medical dressings.

Images