A recombinant humanized type iii collagen and its preparation method and use

Abstract

This invention belongs to the field of biotechnology, specifically relating to a recombinant type III humanized collagen, its preparation method, and its uses. Based on the amino acid sequence of type III human collagen, this invention utilizes bioinformatics and other methods to design the amino acid sequence, enabling the recombinant collagen to achieve higher expression efficiency and a triple helix structure while maintaining high biological activity. A novel collagen sequence containing 180 amino acids was designed, resulting in a small molecular weight and better skin penetration. Compared with commercially available collagen products, the recombinant humanized collagen prepared using the above technical solution exhibits better stability, cell proliferation and migration promotion, and barrier repair capabilities, thus possessing significant practical application value.

Description

Technical Field

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

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25%–30% of total protein. Currently, approximately 29 different types of collagen have been identified, which can be classified into type I, type II, type III, etc., according to the order of their discovery. Type III collagen is the main collagen in human skin, fascia, and tendons, distributed at the epidermal-dermal junction, providing elasticity and stress resistance to the skin. Furthermore, type III collagen is abundant in blood vessels, maintaining vascular strength and tension with its excellent mechanical properties and promoting angiogenesis. It also has significant effects on promoting wound repair and activating clotting factors to promote coagulation mechanisms. However, with age, type III collagen is continuously and rapidly lost and cannot be regenerated by the body. In recent years, with the deepening research on its functional activity, type III collagen has been used as a key raw material in fields such as medical aesthetics, medical devices, skincare, and food applications.

[0004] Collagen can be categorized into animal-derived collagen and recombinant collagen based on its origin. Collagen obtained from animal connective tissue exhibits quality variations due to animal-derived viruses and individual animal differences, which limits its application and development for new products. Recombinant collagen, a non-natural collagen synthesized through genetic engineering, possesses higher bioactivity and biocompatibility, lower immunogenicity, lower risk of missed pathogen detection, better water solubility, and the potential for further processing and optimization. Therefore, its applications are broader, attracting significant market attention. Currently, most commercially available recombinant collagen is derived from microbial expression systems; the primary expression hosts are *E. coli* and *Pichia pastoris*. However, recombinant collagen from *E. coli* generally exhibits low expression levels, and various fusion tags in the protein sequence may affect the protein's activity. Furthermore, most expressed recombinant collagen cannot form a triple helix structure. Summary of the Invention

[0005] In response to the aforementioned existing technologies, the inventors, through long-term technical and practical exploration, have provided a recombinant type III humanized collagen, its preparation method, and its uses. Experiments have demonstrated that the recombinant type III collagen produced by this invention has high yield and good safety; it possesses excellent cell proliferation, migration, and barrier repair bioactivity; and its purification cost is low, making it suitable for large-scale industrial production. Based on the above research results, this invention has been completed.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a recombinant type III humanized collagen, said recombinant type III humanized collagen being any one of the following A1)-A3):

[0008] A1) A protein composed of the amino acid sequence shown in SEQ ID NO.1;

[0009] A2) A protein related to the recombinant type III humanized collagen obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1).

[0010] A3) is a protein that shares more than 85% identity with A1) or A2) and is associated with the recombinant type III humanized collagen.

[0011] The recombinant type III humanized collagen has a triple helix structure.

[0012] In a second aspect, the present invention provides a polynucleotide capable of encoding the aforementioned recombinant type I and II humanized collagen.

[0013] Specifically, the polynucleotide is either B1) or B2) as follows:

[0014] B1) The nucleotide sequence is the DNA molecule described in SEQ ID NO.2;

[0015] B2) A DNA molecule that has more than 85% identity with and has the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or substituting and / or deleting and / or adding one or more nucleotides.

[0016] A third aspect of the present invention provides a carrier comprising the aforementioned polynucleotide.

[0017] In a fourth aspect, the present invention provides a host cell containing the above-mentioned polynucleotides and the above-mentioned vector; or, the host cell is capable of expressing the above-mentioned recombinant type I and II humanized collagen.

[0018] A fifth aspect of the present invention provides a method for preparing recombinant type I and II human collagen, comprising the steps of:

[0019] The recombinant type III human collagen was expressed using the aforementioned host cells, and then separated and purified to obtain the final product.

[0020] A sixth aspect of the present invention provides the use of the above-described recombinant type I / II humanized collagen, the recombinant type I / II humanized collagen encoded by the aforementioned polynucleotide, or the recombinant type I / II humanized collagen produced by the aforementioned host cells in the preparation of food, cosmetic, or pharmaceutical products.

[0021] A seventh aspect of the present invention provides a cosmetic product comprising at least the above-described recombinant type I and II humanized collagen.

[0022] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects:

[0023] The above technical solution is based on the amino acid sequence of type III human collagen. Using bioinformatics and other methods, the amino acid sequence is designed to achieve higher expression efficiency and a triple helix structure in recombinant collagen while maintaining high biological activity. A novel collagen sequence containing 180 amino acids is designed, resulting in a small molecular weight and better skin penetration. The nucleotide sequence of this recombinant collagen is optimized using E. coli codon preference, thereby constructing a high-expression recombinant strain. The recombinant collagen produced by fermentation of this strain contains no exogenous amino acids such as tags, making it a recombinant humanized collagen with good safety. This invention also provides a low-cost and efficient protein purification process suitable for large-scale production. Compared with commercially available collagen products, the recombinant humanized collagen prepared using the above technical solution has better stability, cell proliferation and migration promotion, and barrier repair capabilities, thus possessing good practical application value. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is an SDS-PAGE image of recombinant E. coil BL21 / pET28a-F15 after induction and expression; lane M represents the standard protein with a molecular weight of 180 kDa; lane 1 represents the cell lysis supernatant of recombinant E. coil BL21 / pET28a-F15.

[0026] Figure 2SDS-PAGE protein electrophoresis image of the protein obtained after high-density culture of recombinant strain E.coil BL21 / pET28a-F15 in a 20L fermenter; where lane M represents the standard protein with a molecular weight of 180kDa; lane 1 represents the purified F15 collagen.

[0027] Figure 3 The bar chart shows the DPPH free radical scavenging rate of different collagen proteins; where the comparative example represents the experimental group of commercially available recombinant collagen; F15 represents the experimental group of recombinant humanized collagen prepared using Example 5 of the present invention.

[0028] Figure 4 The bar chart shows the relative expression levels of FLG mRNA in HaCaT cells under the action of different collagen proteins; where control group 1 represents the blank control group; control group 2 represents the commercially available recombinant collagen control group; Col represents the recombinant humanized collagen F15 group prepared in Example 5 of this invention; in the figure, compared with control group 1, P<0.001(***) and P<0.0001(****) are considered to be significantly different.

[0029] Figure 5 This is a circular dichroism chromatogram of recombinant collagen F15. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] In a typical embodiment of the present invention, a recombinant type III humanized collagen is provided, wherein the recombinant type III humanized collagen is any one of the following A1)-A3):

[0033] A1) A protein composed of the amino acid sequence shown in SEQ ID NO.1;

[0034] A2) A protein related to the recombinant type III humanized collagen obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1).

[0035] A3) is a protein that shares more than 85% identity with A1) or A2) and is associated with the recombinant type III humanized collagen.

[0036] The proteins shown in A1)-A3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0037] In this invention, the recombinant type III humanized collagen has a triple helix structure.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In this invention, the term "identity" refers to the percentage of identical (i.e., the same) amino acids between two or more polypeptides, which can be calculated by dividing the number of positions containing the same amino acid residues by the total number of amino acid residues in the polypeptide and multiplying by 100.

[0042] In another aspect, the present invention provides a polynucleotide capable of encoding the aforementioned recombinant type I and II humanized collagen.

[0043] In this invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or the like. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function.

[0044] Specifically, the polynucleotide is either B1) or B2) as follows:

[0045] B1) The nucleotide sequence is the DNA molecule described in SEQ ID NO.2;

[0046] B2) A DNA molecule that has more than 85% identity with and has the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or substituting and / or deleting and / or adding one or more nucleotides.

[0047] In another aspect, the present invention provides a carrier comprising the aforementioned polynucleotide.

[0048] 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 pET28a), which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through 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-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome after introduction and replicate along with the host genome. Furthermore, some vectors can direct the expression of a target gene. Such vectors are referred to herein as "expression vectors." Recombinant expression vectors may contain a form of nucleic acid suitable for expression in host cells, which means 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.

[0049] In another aspect, the present invention provides a host cell containing the aforementioned polynucleotide and the aforementioned vector; or, the host cell is capable of expressing the aforementioned recombinant type I and II humanized collagen.

[0050] In this invention, the term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be any cell useful in the production of recombinant humanized collagen according to this application. To produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. The host cell can be a prokaryotic or eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli.

[0051] Another aspect of the present invention provides a method for preparing recombinant type I and II human collagen, comprising the steps of:

[0052] The recombinant type III human collagen was expressed using the aforementioned host cells, and then separated and purified to obtain the final product.

[0053] The separation and purification steps include removing impurities from the protein, centrifugation, hollow fiber membrane clarification, ultrafiltration membrane concentration, and ion exchange chromatography. Using these steps, the obtained protein has a purity greater than 95% and a yield greater than 60%, making it highly suitable for industrial production applications.

[0054] In another aspect, the present invention provides the use of the above-mentioned recombinant type I / II humanized collagen, the recombinant type I / II humanized collagen encoded by the aforementioned polynucleotide, or the recombinant type I / II humanized collagen produced by the aforementioned host cells in the preparation of food, cosmetic or pharmaceutical products.

[0055] In another aspect, the present invention provides a cosmetic product comprising at least the aforementioned recombinant type I and II humanized collagen. Compared with commercially available collagen products, the recombinant humanized collagen of the present invention exhibits better stability, cell proliferation and migration promotion, and barrier repair capabilities, and therefore can be used as a raw material in cosmetics, especially skincare products.

[0056] Of course, the cosmetics may also contain any other raw materials permitted in the cosmetic field, including but not limited to dispersants, skin feel modifiers, emulsifiers, emollients, humectants, fragrances, colorants, film-forming agents, thickeners, and preservatives. Those skilled in the art can choose to add these ingredients according to the actual situation, and no specific limitations are made here.

[0057] 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.

[0058] Example 1: Design of recombinant humanized collagen sequence

[0059] The amino acid sequence of human type III collagen α1 chain protein was obtained from the NCBI database (GenBank: KAI2526099.1). Through in-depth mining and analysis of the sequence information, various techniques such as bioinformatics were used to simulate and design the charge characteristics, hydrophilicity and hydrophobicity of amino acids and the three-dimensional spatial structure of the protein. The aim was to ensure that the recombinant collagen maintains high biological activity while achieving higher expression efficiency and optimizing its triple helix structure.

[0060] The recombinant humanized collagen in this embodiment contains 180 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.1. This sequence does not contain any exogenous amino acids such as tags, and contains multiple bioactive sites of human type III collagen. It has high biocompatibility and low risk of use, thereby effectively ensuring the realization of the biological function of recombinant collagen.

[0061] Example 2: Construction of recombinant strain E. coil BL21 / pET28a-F15

[0062] Based on the codon bias of *E. coli*, the coding gene for recombinant humanized collagen shown in SEQ ID NO.1 of Example 1 was codon optimized. The optimized coding gene nucleotide sequence is shown in SEQ ID NO.2 and named F15. The nucleotide sequence of F15 was synthesized by BGI Genomics and cloned into the *E. coli* expression vector pET28a. After transformation into *E. coli* TOP10, the recombinant expression plasmid pET28a-F15 was obtained without frameshifting. DNA sequencing alignment confirmed the successful construction of the recombinant expression plasmid pET28a-F15.

[0063] The validated recombinant expression vector pET28a-F15 was transformed into E. coli BL21(DE3) according to the E. coli protocol. Transformants were screened using kanamycin antibiotics, and further validated by PCR. The recombinant strain E. coli BL21 / pET28a-F15 expressing recombinant humanized collagen was successfully constructed.

[0064] Example 3: Induced expression of recombinant E. coil BL21 / pET28a-F15

[0065] Single colonies were picked from recombinant E. coil BL21 / pET28a-F15 and placed in LB liquid medium (1% NaCl, 0.5% yeast extract, 1% peptone) containing 50 μg / mL kanamycin. The culture was incubated overnight at 37°C and 200 rpm. Then, a 2% (v / v) inoculum was transferred to TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K₂HPO₄, 0.22% KH₂PO₄, 0.4% glycerol) and incubated at 37°C and 200 rpm until OD₀. 600The concentration was 0.7. At this point, IPTG (isopropyl-β-D-thiogalactoside) was added to the culture medium to a final concentration of 0.5 mM to induce protein expression. The culture was incubated at 25°C for 16 h. Afterward, the bacterial cells were collected by centrifugation, resuspended in Tris buffer (20 mM Tris, pH 7.5), and then sonicated. The supernatant and precipitate were analyzed by SDS-PAGE. Based on theoretical prediction, the molecular weight of this recombinant humanized collagen should be 15.6 kDa. The SDS-PAGE results are as follows: Figure 1 As shown, a distinct protein expression band is present around 15.6 kDa. These results demonstrate that the recombinant humanized collagen in this embodiment has been successfully induced to express.

[0066] Example 4: High-density culture of recombinant E. coil BL21 / pET28a-F15 in a 5L tank

[0067] The recombinant strain E. coil BL21 / pET28a-F15 was cultured at high density in a 5L tank. The bacterial culture in the glycerol tube was inoculated into 60mL of seed culture medium (1% peptone, 1% sodium chloride, 0.5% yeast extract) containing 50μg / mL kanamycin at a 1‰ inoculation rate, and cultured at 37℃ and 180rpm for about 15h. The cultured seed culture was transferred to a 5L fermenter containing 3L of fermentation medium (3% yeast extract, 1.5% glycerol, 3.25% ammonium sulfate, 0.2% sodium chloride, 1.0% disodium hydrogen phosphate dodecahydrate, 0.31% potassium dihydrogen phosphate, 0.17% magnesium sulfate heptahydrate, 0.02% defoamer, 0.43% trace elements, pH adjusted to 7.0 with sodium hydroxide; 500mL trace element formula: 1.625g boric acid, 0.275g manganese sulfate monohydrate, 0.3325g copper chloride dihydrate, 0.1375g zinc sulfate monohydrate, 0.211g cobalt chloride, 0.1735g sodium molybdate).

[0068] Initial fermentation parameters were set as follows: stirring speed 150 rpm, fermentation temperature 37℃, aeration rate 4 L / min, dissolved oxygen adjusted by stirring and controlled at 20%, and pH maintained at 7.0 by automatic addition of ammonia during fermentation. Once dissolved oxygen rose to 35%, feeding was initiated (feeding medium formula: 3% ammonium sulfate, 0.22% magnesium sulfate heptahydrate, 30% glycerol, 0.5% trace elements), with an initial feeding rate of 0.8 mL / min. Dissolved oxygen was maintained at 20% by increasing aeration. 600For the 80-year-old strain, 0.5 mM IPTG was added, the temperature was adjusted to 25℃, and the dissolved oxygen was controlled at around 30%. The induction time was 10-20 hours. Samples were taken periodically after the induction period, and the fermentation broth was analyzed by SDS-PAGE. Using BioAnaly software, the grayscale values ​​of the standard protein and recombinant humanized collagen were determined. It was calculated that when the recombinant strain E. coil BL21 / pET28a-F15 was used for high-density fermentation in a 5L tank for 15 hours, the yield of recombinant humanized collagen was 8.6 g / L, achieving high expression efficiency of recombinant humanized collagen.

[0069] Example 5: Purification and preparation of recombinant humanized collagen

[0070] Recombinant bacteria E. coil BL21 / pET28a-F15 were subjected to high-density fermentation in a 5L tank. After 15 hours of induction culture, the fermentation broth was centrifuged at 8000 rpm to obtain bacterial sludge. The bacterial sludge was then purified using the following steps to obtain pure recombinant humanized collagen:

[0071] (1) Removal of impurities and proteins: The bacterial solution obtained after resuspending homogenized bacteria in the bacterial mud is adjusted to pH 6.5 with low concentration HCl. During the acid addition process, the bacterial solution is continuously stirred to denature and precipitate recombinant humanized collagen.

[0072] (2) Centrifugation: Remove acid-precipitated proteins, and adjust the pH of the supernatant to 7.0 with low concentration NaOH;

[0073] (3) Hollow fiber membrane clarification: The centrifuged supernatant obtained in step (2) is filtered through a 250 kDa hollow fiber membrane for sterilization and removal of macromolecular impurities;

[0074] (4) Ultrafiltration membrane concentration: The feed solution is concentrated using a 5 kDa ultrafiltration membrane pack, while removing small molecule impurities;

[0075] (5) Ion exchange chromatography: Anion exchange chromatography is used. Recombinant humanized collagen is positively charged. During chromatographic loading, the flow-through is collected directly, while negatively charged impurities such as other proteins are adsorbed onto the column, thus removing impurities. The resulting flow-through is the purified stock solution. The protein purified by the above steps has a purity greater than 95% and a yield greater than 60%.

[0076] Example 6: Cell proliferation and migration promoting effects of recombinant humanized collagen

[0077] The effect of recombinant type III collagen F15 on promoting cell proliferation: The results of the CCK-8 assay showed that the cell proliferation rate of ESF cells at final concentrations of 0.5 mg / mL and 1.0 mg / mL was 117.3% and 121.8%, respectively, which was significantly higher than that of the negative control group (1× maintenance medium), indicating that it has a certain effect on promoting ESF cell proliferation.

[0078] The promoting effect of recombinant type III collagen F15 on cell migration: The results of the cell scratch assay showed that the wound healing percentage of the negative control group (NC) was 55.7% at 24 h. The wound healing percentage (Wound Closure%) of the 0.1 mg / mL and 0.2 mg / mL recombinant type III collagen sample groups at 24 h was significantly higher than that of the negative control group (NC), at approximately 68.8% and 80.5%, respectively. Therefore, it is believed that the 0.1 mg / mL and 0.2 mg / mL recombinant type III collagen sample groups have a significant promoting effect on HaCaT wound healing.

[0079] Example 7: Barrier Repair Experiment

[0080] 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 2.5 × 10⁻⁶ cells. 5 / well. The experiment included a blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and the recombinant humanized collagen F15 group prepared in Example 5 of this invention (experimental group), with 3 replicates for each group. The 6-well plate was incubated in a cell culture incubator (37℃, 5% CO2) for 24 h. 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 two 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 h, the cells in each well were washed twice with 2 mL of PBS buffer. Following the Total RNA Extraction Kit, 1 mL of RNAisoPlus 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 filaggrin (FLG). A 2... -△△CT The method is used for calculation.

[0081] Different collagen proteins promote the expression of FLG mRNA in HaCaT cells, such as Figure 4 As shown, compared with the blank control group (control group 1), both commercially available recombinant collagen (control group 2) and the recombinant humanized collagen F15 (experimental group) prepared in Example 5 of this invention can significantly increase the relative expression level of FLG mRNA. Among them, the recombinant humanized collagen prepared in this invention has a stronger ability to promote FLG mRNA expression than commercially available recombinant collagen. Therefore, the recombinant humanized collagen of this invention has obvious skin barrier repair function.

[0082] Example 8: Collagen Circular Dichroism Experiment

[0083] The obtained recombinant collagen F15 pure product was dissolved in ultrapure water to prepare a 0.1 mg / ml solution. Baseline correction was performed before spectral analysis to remove any system noise or other non-sample-related signals. CD detection was performed at a wavelength of 180–260 nm.

[0084] The circular dichroism chromatographic characteristics of triple-helix collagen are as follows: a negative absorption peak near 200 nm; and a weak positive absorption peak near 220 nm. The circular dichroism chromatogram of recombinant collagen F15 in this application is shown below. Figure 5 As shown, there is a negative absorption peak at 197 nm and a weak positive absorption peak at 218 nm, which is consistent with the absorption characteristics of the triple helix.

[0085] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A recombinant type III humanized collagen, characterized in that, The recombinant type III humanized collagen is a protein composed of the amino acid sequence shown in SEQ ID NO.1; the recombinant type III humanized collagen has a triple helix structure.

2. A polynucleotide, characterized in that, The polynucleotide can encode the recombinant type III humanized collagen of claim 1.

3. The polynucleotide as described in claim 2, characterized in that, The polynucleotide is either B1) or B2) below: B1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; B2) A DNA molecule that has more than 85% identity with and has the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or substituting and / or deleting and / or adding one or more nucleotides.

4. A carrier, characterized in that, The vector comprises the polynucleotide of claim 2 or 3.

5. A host cell, characterized in that, The host cell contains the polynucleotide of claim 2 or 3 and the vector of claim 4; or the host cell is capable of expressing the recombinant type III humanized collagen of claim 1.

6. A method for preparing recombinant type III human collagen, characterized in that, Including the following steps: The recombinant type III human collagen is expressed using the host cell described in claim 5, and then separated and purified to obtain the final product.

7. The use of the recombinant type III humanized collagen of claim 1, the recombinant type III humanized collagen encoded by the polynucleotide of claim 2 or 3, or the recombinant type III humanized collagen produced by the host cell of claim 5 in the preparation of food, cosmetic or pharmaceutical products.

8. A cosmetic product, characterized in that, The cosmetic product contains at least the recombinant type III humanized collagen as described in claim 1.

9. The cosmetic product as described in claim 8, characterized in that, The cosmetics in question are skincare products.

10. The cosmetic product as described in claim 8, characterized in that, The cosmetics also contain any other raw material ingredients permitted to be added in any cosmetic field.

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