Recombinant human collagen type iii and its preparation method and use

Abstract

The application relates to the technical field of genetic engineering, and discloses a recombinant human collagen type III and a preparation method and application thereof, the amino acid sequence of the recombinant human collagen type III comprises the amino acid sequence shown in SEQ ID NO. 1, the amino acid sequence of the recombinant human collagen type III provided by the application has the characteristics of high hydrophilicity and uniform charge distribution, and the characteristics greatly improve the transdermal absorption efficiency of the product, so that the product can penetrate into the bottom layer of the skin more deeply and has better efficacy. Meanwhile, the 100% humanized amino acid sequence design of the product ensures high affinity of the product with the human skin and maximally reduces the risk of allergic reaction.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant human type III collagen, its preparation method, and its uses. Background Technology

[0002] Collagen is the most abundant structural protein in animals, widely found in connective tissues such as skin, bones, tendons, and blood vessels, playing a vital role in support, repair, and protection. Type III collagen is a naturally occurring subtype of collagen in the human body, its core function being to provide tissue softness and elasticity. Compared to other collagens, type III collagen is more suitable for sensitive skin repair, wound healing after cosmetic procedures, and mucosal repair. Recombinant human type III collagen is a bioactive substance synthesized through genetic engineering technology, with a structure highly consistent with that of human type III collagen. It has enormous application potential in biomedical materials (such as artificial skin and tissue engineering scaffolds), high-end cosmetics (such as moisturizing and anti-aging skincare products), functional foods, and pharmaceuticals (such as drug carriers and hemostatic materials).

[0003] There are two main approaches to obtaining collagen: traditional methods derived from animal tissues and biosynthetic methods using recombinant expression of heterologous proteins. Animal tissue-based methods pose biosafety risks and cannot be used on a large scale. Heterologously expressed collagen includes full-length, truncated, and truncated peptides of natural collagen. Several companies have already obtained collagen by recombinantly expressing different collagen fragments, but many of these methods suffer from low activity and either fail to express or inhibit the expression of some beneficial target proteins. Summary of the Invention

[0004] The purpose of this invention is to overcome the technical problems of low activity of recombinant collagen and its ineffectiveness or inhibitory effect on the expression of some beneficial target proteins in the prior art. This invention provides a recombinant human type III collagen, its preparation method, and its uses. This recombinant human type III collagen has high hydrophilicity and uniform charge distribution, which greatly improves the transdermal absorption efficiency of the product, allowing it to penetrate deeper into the skin layer and exert better efficacy. Furthermore, this recombinant human type III collagen can significantly inhibit the expression of MMP-1 in fibroblasts, significantly promote the expression of HAS2 in keratinocytes, and extremely significantly promote the expression of HAS3 in keratinocytes.

[0005] To achieve the above objectives, the first aspect of the present invention provides a recombinant human type III collagen having an amino acid sequence as shown in SEQ ID NO.1.

[0006] A second aspect of the present invention provides a nucleic acid that encodes the recombinant human type III collagen.

[0007] A third aspect of the present invention provides a recombinant vector containing the aforementioned nucleic acid.

[0008] A fourth aspect of the present invention provides a recombinant cell containing the aforementioned nucleic acid, or a method for preparing the recombinant cell comprising: transforming a host cell using the aforementioned recombinant vector.

[0009] The fifth aspect of the present invention provides a method for preparing recombinant human type III collagen, comprising inducing expression in the recombinant cells.

[0010] The sixth aspect of the present invention provides a collagen product containing the recombinant human type III collagen and / or the recombinant human type III collagen prepared by the method described above.

[0011] The seventh aspect of the present invention provides the use of the recombinant human type III collagen and / or the recombinant human type III collagen prepared by the method in the preparation of collagen products.

[0012] Through the above technical solution, the recombinant human type III collagen provided by this invention possesses an amino acid sequence with high hydrophilicity and uniform charge distribution. This characteristic significantly improves the transdermal absorption efficiency of the product, allowing it to penetrate deeper into the skin's basal layer and exert better efficacy. Simultaneously, its 100% humanized amino acid sequence design ensures high affinity between the product and human skin, minimizing the risk of allergic reactions. The recombinant human type III collagen provided by this invention has broad application prospects in pharmaceuticals, skincare products, daily chemicals, biomaterials, and industrial materials.

[0013] Furthermore, tests revealed that the recombinant human type III collagen provided by this invention can significantly inhibit MMP-1 expression in fibroblasts, significantly promote HAS2 expression in keratinocytes, and extremely significantly promote HAS3 expression in keratinocytes; while existing recombinant collagens either do not affect the expression of the above target proteins or produce effects opposite to those of the recombinant human type III collagen provided by this invention. Attached Figure Description

[0014] Figure 1 The results are SDS-PAGE protein detection results of recombinant human type III collagen fermentation broth; Line 1 is the marker, and Line 2 is the fermentation broth induced by recombinant bacteria for 48 hours in Example 1.

[0015] Figure 2 These are the SDS-PAGE protein detection results of the purified recombinant human type III collagen sample; where Line 1 is the marker and Line 2 is the purified recombinant human type III collagen sample from Example 1.

[0016] Figure 3 This is a diagram showing the results of a fibroblast proliferation experiment;

[0017] Figure 4 This is a diagram showing the results of an experiment on extracellular matrix metabolism in fibroblasts;

[0018] Figure 5 This is a diagram showing the results of an experiment on the metabolism of matrix metalloproteinases in fibroblasts;

[0019] Figure 6 This is a diagram showing the results of a keratinocyte proliferation experiment;

[0020] Figure 7 This is a diagram showing the results of a keratinocyte migration experiment;

[0021] Figure 8 This is a diagram showing the results of an experiment on the metabolism of keratinocyte-related factors;

[0022] Figure 9 This is a diagram showing the results of an experiment on the metabolism of keratinocyte moisturizing-related factors. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below. It should be noted that the specific embodiments are only a detailed description of the present invention and should not be regarded as a limitation of the present invention.

[0024] The first aspect of the present invention provides a recombinant human type III collagen having the amino acid sequence shown in SEQ ID NO.1.

[0025] In this invention, the following amino acid sequence is designated as SEQ ID NO.1: GPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPRGDKGETG ERGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGE VGPAGSPSGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPRGDKGETGER.

[0026] The recombinant human type III collagen provided by this invention may have an amino acid sequence selected from any one of the following (a)-(d):

[0027] (a) The amino acid sequence shown in SEQ ID NO.1;

[0028] (b) The modified amino acid sequence of the amino acid sequence shown in SEQ ID NO.1;

[0029] (c) An amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b);

[0030] (d) An amino acid sequence to which a signal sequence is attached at the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b).

[0031] In some embodiments, the modification includes chemical modification and / or fluorescent modification.

[0032] Chemical modification refers to the addition of chemical groups to amino acid residues through chemical means. It is understandable that chemically modified proteins do not alter their amino acid sequence or peptide backbone structure, thus retaining the original protein's function and activity.

[0033] Chemical modification can be achieved through conventional methods in the field, such as utilizing the organism's own enzyme system or genetic engineering to complete the corresponding chemical modification within living cells, or by chemically modifying purified peptides or proteins through chemical or enzymatic reactions. The types of chemical modification can include at least one of phosphorylation, glycosylation, acetylation, methylation, ubiquitination, SUMOylation, lipidation, or sulfation.

[0034] Fluorescent modification refers to attaching fluorescent groups to amino acid residues. Unlike chemical modification, fluorescent groups do not increase the function and activity of the protein, nor do they affect the function and activity of the original protein. It can be used for in vitro studies, such as protein localization tracking, interaction detection, and fluorescence imaging.

[0035] Fluorescent modification can be achieved using conventional methods in the art, such as site-specific modification via specific enzymatic reactions. The site of fluorescence modification can be the amino group of lysine, the thiol group of cysteine, or the N-terminal amino group. The types of fluorescent modifications can include FITC, FAM, TRITC, Rhodamine B, Cy3, Cy5, GFP, or mCherry, as well as combinations of these fluorescent modifications.

[0036] Tags can be categorized by function into epitope tags, protein / domain tags, and novel smart tags. Epitope tags are typically composed of short peptides and can be used for immunoassay and affinity purification, such as the His tag (six consecutive histidine residues). Protein / domain tags usually have larger molecular weights and, in addition to purification, can enhance protein expression levels and solubility. Novel smart tags often combine synthetic biology and chemical biology techniques, and different novel smart tags typically have drastically different functions. It is understood that introducing a tag does not alter the amino acid sequence or peptide backbone structure, thus preserving the original protein's function and activity. Those skilled in the art can construct and apply tags using conventional methods in the field.

[0037] Signal sequences typically serve to target protein transport, guiding proteins to specific organelles or the extracellular environment to ensure their proper location and function. Those skilled in the art can construct and apply signal sequences using conventional methods in the field.

[0038] A second aspect of the present invention provides a nucleic acid that encodes the recombinant human type III collagen.

[0039] It is well known in the art that of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon usually does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences.

[0040] This invention improves the expression efficiency of recombinant human type III collagen by adjusting the gene sequence. Specifically, it modifies synonymous codons in the gene to better match the codon preferences of Pichia pastoris, thereby eliminating rare codons and improving translation efficiency. The inventors have found that using the above-described preferred embodiments can achieve higher expression levels of the recombinant human type III collagen provided by this invention.

[0041] Preferably, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO.2.

[0042]

[0043] In this invention, the nucleic acid may contain only the sequence shown in SEQ ID NO.2, or it may be a DNA molecule with the sequence shown in SEQ ID NO.2 as the coding region and additional components added. These other components may include any components required in the art for artificially synthesizing gene sequences and expressing them via expression vectors, such as promoters, enhancers, Kozak sequences, etc.; or, for example, nucleotide sequences encoding tag or signal sequences coupled to the N-terminus or C-terminus of an amino acid sequence, which may be coupled to or inserted into the sequence shown in SEQ ID NO.2 as components.

[0044] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. Typically, the obtained nucleotide sequence is cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods. The nucleotide sequence shown in SEQ ID NO.2 of this invention was synthesized by Jiangsu Saisofe Biotechnology Co., Ltd. Alternatively, the relevant nucleotide sequence can also be synthesized using known artificial chemical synthesis methods.

[0045] A third aspect of the present invention provides a recombinant vector containing the aforementioned nucleic acid.

[0046] In this invention, the "vector" used in the recombinant vector can be any vector known in the art, such as various commercially available plasmids, granules, bacteriophages, and retroviruses.

[0047] The "vector" used in the recombinant vector of the present invention is preferably the pPIC9K plasmid.

[0048] A fourth aspect of the present invention provides a recombinant cell containing the aforementioned nucleic acid, or a method for preparing the recombinant cell comprising: transforming a host cell using the aforementioned recombinant vector.

[0049] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0050] According to the present invention, preferably, the recombinant cell is a yeast, and more preferably, Pichia pastoris strain GS115.

[0051] In this invention, the recombinant vector can be transformed, transduced, or transfected into host cells using conventional methods in the art, such as chemical transformation by calcium chloride method or high-voltage electroporation transformation.

[0052] The fifth aspect of the present invention provides a method for preparing recombinant human type III collagen, comprising inducing expression in the recombinant cells.

[0053] In this invention, "inducing expression in the recombinant cells" can be any conventional method in the art. For example, a suitable induction method can be selected based on the type of recombinant vector and recombinant cells. According to this invention, it is preferred to use the Pichia pastoris strain GS115 (hereinafter referred to as the recombinant strain) transformed with the recombinant vector pPIC9K-2E for induction culture. Conventional methods in the art can be adopted. For example, BMMY medium (Buffered Methanol-complex Medium) is a commonly used liquid culture medium in the art for expressing the target protein in methanol-induced Pichia pastoris recombinant strains. The Pichia pastoris recombinant strain can be first amplified in large quantities in BMGY medium (Buffered Glycerol-complex Medium) compatible with BMMY medium to obtain high-density cells, and then transferred to BMMY medium. The time and amount of methanol added are reasonably set to induce the expression of the target protein. The resulting fermentation broth is centrifuged, and the supernatant is collected to obtain a liquid containing recombinant human type III collagen.

[0054] In this invention, the fermentation broth obtained by inducing expression of the above-mentioned recombinant strain can be used directly as a collagen product or its raw material, or the recombinant human type III collagen obtained by separating and purifying the fermentation broth can be used as a collagen product or its raw material. The separation and purification can be carried out using conventional protein separation methods in the art.

[0055] The sixth aspect of the present invention provides a collagen product containing the recombinant human type III collagen and / or the recombinant human type III collagen prepared by the method described above.

[0056] In this invention, the recombinant human type III collagen can be made into corresponding collagen products. Specifically, the collagen products can exist in solid, semi-solid or liquid form. The collagen products can contain excipients or additives for preparing collagen products, etc. Those skilled in the art can choose according to their needs, which will not be elaborated here.

[0057] In some embodiments, the collagen product is selected from at least one of pharmaceuticals, skincare products, daily chemical products, biomaterials, and industrial materials.

[0058] The following are examples of collagen products. Drugs can be oral or topical medications. Oral medications can utilize the high bioavailability of collagen through intestinal absorption to promote skin, joint, and bone health. Topical medications can be medical dressings, tissue engineering scaffolds, hemostatic or anti-adhesion materials, and injectable formulations. Skincare products can be creams, serums, masks, or beauty dressings, or injectable beauty products. Daily chemical products can be shampoos or hair care products. Biomaterials can be cell culture coating materials, biological reagent delivery carriers, etc. Industrial materials can be biodegradable packaging, industrial coatings, etc.

[0059] The seventh aspect of the present invention provides the use of at least one of the recombinant human type III collagen and the recombinant human type III collagen prepared by the method in the preparation of collagen products.

[0060] In some embodiments, the application includes the preparation of collagen products having the efficacy of inhibiting MMP-1 expression in fibroblasts, promoting HAS2 expression in keratinocytes, and promoting HAS3 expression in keratinocytes.

[0061] Tests have shown that existing recombinant collagens have the negative effects of promoting MMP-1 expression in fibroblasts, not affecting HAS2 expression in keratinocytes, and inhibiting HAS3 expression in keratinocytes. The recombinant human type III collagen provided by this invention can significantly inhibit MMP-1 expression in fibroblasts, significantly promote HAS2 expression in keratinocytes, and extremely significantly promote HAS3 expression in keratinocytes.

[0062] Specifically, the recombinant human type III collagen provided by the present invention simultaneously promotes fibroblast proliferation, promotes COL4 expression in fibroblasts, promotes COL7 expression in fibroblasts, promotes FN expression in fibroblasts, inhibits MMP-1 expression in fibroblasts, promotes keratinocyte proliferation, promotes keratinocyte migration, promotes K1 expression in keratinocytes, promotes K10 expression in keratinocytes, promotes TGM-1 expression in keratinocytes, promotes HAS2 expression in keratinocytes, and promotes HAS3 expression in keratinocytes.

[0063] This invention experimentally verified that the recombinant human type III collagen provided by this invention promotes fibroblast proliferation, keratinocyte proliferation, and keratinocyte migration. The expression levels of multiple genes in fibroblasts and keratinocytes were tested, revealing that the expression levels of multiple genes were regulated. The following provides a detailed description of fibroblasts and keratinocytes and their various genes:

[0064] Fibroblasts are the core cells of the dermis, responsible for synthesizing extracellular matrix components, such as collagen, elastic fibers, and glycosaminoglycans. Increased fibroblast proliferation means more cells participate in the synthesis of extracellular matrix components, macroscopically manifested as increased dermal thickness and accelerated wound healing.

[0065] Type IV collagen (COL4) is a major component of the basement membrane's dense layer, forming a reticular structure connecting the epidermis and dermis. COL4 expression in fibroblasts promotes, macroscopically, enhanced skin structural support and improved barrier function.

[0066] Type VII collagen (COL7) forms anchoring fibers that fix the basement membrane to the dermal collagen fiber network. COL7 expression in fibroblasts promotes, macroscopically, enhanced skin resistance to friction damage and delayed photoaging.

[0067] Fibronectin (FN) mediates cell adhesion, migration, and extracellular matrix assembly, and is a key regulator of wound healing. FN expression in fibroblasts promotes accelerated wound repair and enhances skin elasticity and resilience by promoting the orderly arrangement of collagen fibers.

[0068] Matrix metalloproteinase-1 (MMP-1) specifically degrades type I and type III collagen, and is one of the direct causes of photoaging. Inhibition of MMP-1 expression in fibroblasts macroscopically results in reduced collagen loss, improved wrinkle depth and skin firmness, reduced inflammatory responses induced by collagen degradation products, and relief of skin redness and sensitivity.

[0069] Keratinocytes make up more than 80% of epidermal cells, and their proliferation is fundamental to epidermal renewal. For example, in wound healing, keratinocytes migrate from the wound edge to cover the exposed dermis, a crucial step in re-epithelialization. Therefore, promoting keratinocyte proliferation helps repair thinning of the stratum corneum caused by barrier damage, enhances the body's ability to resist external stimuli, and accelerates the migration and shedding of melanin granules into the epidermis, improving pigmentation. Promoting keratinocyte migration can also shorten wound healing time and reduce the risk of infection.

[0070] Keratin 1 (K1) and keratin 10 (K10) are the main structural proteins of the granular layer and stratum corneum, forming an intermediate filament network. Expression of K1 and K10 in keratinocytes promotes macroscopic manifestations of enhanced mechanical barrier function and improved stratum corneum integrity. For example, the K1 / K10 cross-linking structure enhances the skin's resistance to friction and stretching, thus preventing blisters and cracking. It also helps reduce transepidermal water loss, thereby improving dry skin and flaking. Transglutaminase-1 (TGM-1) catalyzes the cross-linking of keratinocyte capsule proteins, forming the final barrier structure of keratinocytes. Expression of TGM-1 in keratinocytes promotes macroscopic manifestations of enhanced keratinocyte capsule stability and improved moisturizing capacity. For example, the cross-linking of keratinocyte capsule proteins resists enzymatic degradation and physical damage, maintaining skin barrier function. Furthermore, the capsule structure of keratinocyte capsule proteins promotes the retention of natural moisturizing factors, improving skin hydration.

[0071] Hyaluronic acid synthase 2 (HAS2) primarily synthesizes high molecular weight hyaluronic acid (HMW-HA), while hyaluronic acid synthase 3 (HAS3) generates low molecular weight hyaluronic acid (LMW-HA). The expression of HAS2 and HAS3 in keratinocytes promotes macroscopic effects including: increased production of HMW-HA, which forms a gel network in the extracellular matrix, providing mechanical support and binding water, thus improving skin elasticity and hydration; and increased production of LMW-HA, which participates in inflammation regulation and cell migration, accelerating wound healing and promoting epidermal regeneration.

[0072] It is worth noting that, as verified, the recombinant human type III collagen provided by this invention possesses all the above-mentioned effects. The expression of the various proteins involved is simultaneously regulated and can also exert a synergistic effect. For example, inhibiting MMP-1 expression and enhancing COL7 expression can synergistically reduce UV-induced collagen destruction, while enhancing HAS3 expression produces more LMW-HA, which neutralizes free radicals and reduces inflammatory damage. Therefore, the recombinant human type III collagen provided by this invention can be prepared as a reagent with anti-photoaging effects, such as skincare products and dressings. Furthermore, by promoting FN expression in fibroblasts, thereby mediating cell adhesion, migration, and extracellular matrix assembly, and synergistically promoting keratinocyte migration, it shortens wound healing time and reduces the risk of infection. The recombinant human type III collagen provided by this invention can be prepared as a reagent to aid wound healing, such as facial fillers and tissue engineering materials. The above are merely illustrative examples, and practical applications are not limited to these examples.

[0073] To further understand the present invention, specific embodiments are described below. The scope of protection of the present invention is not limited by the following embodiments.

[0074] In the following examples, the competing product is recombinant type III collagen (TTA01-02PC) from Jiangsu Chuangjian Medical Technology Co., Ltd.; the pPIC9K plasmid was provided and cloned by Jiangsu Saisofe Biotechnology Co., Ltd.; Sal I enzyme was purchased from Thermo Fisher Scientific, catalog number FD0644; GS115 host bacteria were purchased from Saisofe Biotechnology Co., Ltd.; fibroblasts were purchased from Guangdong Boxi Biotechnology, catalog number PC2031; and keratinocytes were purchased from the Chinese Academy of Sciences, catalog number SCSP-5091.

[0075] The components or sources of the culture medium used in the examples are as follows:

[0076] YPD medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L.

[0077] MD medium: glucose 20 g / L, YNB 13.4 g / L, biotin 4 × 10⁻⁶ 4 g / L.

[0078] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, Na₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 13.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ 4 g / L, glycerol 10 g / L.

[0079] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, Na₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ 4 g / L, methanol 10 mL / L.

[0080] The DMEM medium was purchased from Beijing Solarbio Science & Technology Co., Ltd., product model number 11995. n% FBS DMEM refers to DMEM medium containing n% FBS (fetal bovine serum).

[0081] In the accompanying drawings corresponding to the following examples, the "*" mark indicates that the difference in the experimental results between the experimental group below the "*" mark and its corresponding control group (Control) is significantly statistically significant (0.01 < p ≤ 0.05); the "**" mark indicates that the difference in the experimental results between the experimental group below the "**" mark and its corresponding control group (Control) is extremely significantly statistically significant (0.001 < p ≤ 0.01); the "***" mark indicates that the difference in the experimental results between the experimental group below the "***" mark and its corresponding control group (Control) is extremely significantly statistically significant (p ≤ 0.001); the method of significance analysis is the t-test.

[0082] Example 1: Design, Preparation and Purification of Recombinant Human Type III Collagen

[0083] I. Design of Recombinant Human Type III Collagen Sequence

[0084] The amino acid sequence of the natural human type III collagen α1 chain protein (GenBank: AGL34959.1) was obtained through the NCBI database, and the biological information of this sequence was deeply mined and analyzed. The specific process was as follows: First, different fragments of type III collagen were intercepted and recombined, and the recombinant configurations with high interaction network density with type III collagen, type IV collagen, fibronectin and elastin were selected through protein interaction analysis, obtaining 30 candidate protein sequences. Further optimization design was carried out on the natural human type III collagen α1 chain, and a recombinant type III humanized collagen with higher expression efficiency and structural stability while maintaining high biological activity was obtained from the 30 candidate sequences, named 2E. The amino acid sequence of 2E is shown in SEQ ID NO.1. The analysis results of surface charge distribution and hydrophilicity showed that 2E had excellent water solubility and structural stability.

[0085] II. Construction of Recombinant Human Type III Collagen Expression Strains

[0086] According to the codon preference of Pichia pastoris, the amino acid sequence shown in SEQ ID NO.1 was codon-optimized, and the nucleotide sequence of the optimized coding gene was shown in SEQ ID NO.2. The DNA sequence was commissioned to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis and cloned into the Pichia pastoris expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-2E. After linearizing the recombinant plasmid pPIC9K-2E with Sal I enzyme, it was transformed into the GS115 host bacteria. After resuscitation, the bacterial solution was spread on the nutrient-deficient MD plate and cultured statically at 30 °C for 3 d.

[0087] III. Induced Expression of Recombinant Bacteria

[0088] Single colonies were randomly selected and inoculated into YPD medium, and cultured overnight at 30°C with shaking at 220 rpm. The colonies were then transferred to BMGY medium at a 10% inoculum and cultured overnight at 30°C with shaking at 220 rpm. Finally, the colonies were transferred to BMMY medium at a 10% inoculum for induction. 1% methanol was added every 24 hours for induction. The culture was terminated after 72 hours. The fermentation broth was collected, and the supernatant was collected by centrifugation for electrophoresis. The results are as follows: Figure 1 As shown, the target protein is expressed in the fermentation broth.

[0089] IV. Recombinant bacterial induction expression

[0090] The fermentation broth was centrifuged, and the supernatant was collected as the feed solution. The feed solution was clarified by encapsulating it in a 300 kDa membrane, then concentrated in a 10 kDa membrane. Buffer was added for head washing until the conductivity reached 130 mg / cm, and the pH was adjusted to 7.0. This was the loading solution. The loading solution was purified by hydrophobic chromatography using Phenyl Purose 6FF to obtain the sample. Results are as follows... Figure 2 As shown, the purity of the sample reached over 90%. After the purified sample was replaced with PBS buffer and the protein concentration was determined by the BCA method, it was stored for later use. This sample was used for testing in subsequent examples.

[0091] Example 2: Fibroblast proliferation experiment using recombinant human type III collagen

[0092] On Day 0, fibroblasts in logarithmic growth phase were resuspended in 10% FBS DMEM at a ratio of 5E3 / well and added to 96-well plates. On Day 1, fibroblasts with the supernatant removed were used as the test cells in this example. Sample group (2E), competitor group, positive control group (10% FBS), and blank control group (Control) were set up: Sample group (2E) was the test cells with sample diluted with 0.1% FBS DMEM (this sample was prepared in Example 1, divided into three groups, diluted to 100ppm, 10ppm and 1ppm respectively), competitor group was the test cells with competitor product diluted with 0.1% FBS DMEM (divided into three groups, diluted to 100ppm, 10ppm and 1ppm respectively), positive control group (10% FBS) was the test cells with 10% FBS DMEM, and blank control group (Control) was the test cells with 0.1% FBS DMEM. Each group had 3 parallel controls, and the cells were incubated at 37°C for 48 hours. After incubation, the supernatant was removed, cells were washed once with PBS, and CCK8 solution prepared with DMEM basal medium was added. Incubation was carried out at 37°C for 1 hour. OD values ​​were obtained using a microplate reader, cell viability was calculated, and plotted using Grapdhpad. Results are shown below. Figure 3As shown, 2E at concentrations of 100 ppm, 10 ppm, and 1 ppm all exhibited good effects in promoting fibroblast proliferation, with the promoting effect being superior to that of competing products at concentrations of 10 ppm and 1 ppm.

[0093] Example 3: Extracellular matrix metabolism experiment of recombinant human type III collagen in fibroblasts

[0094] Fibroblasts were resuspended in 10% FBS DMEM medium and added to 6-well plates at a ratio of 3E5 / well. After cell adhesion, these cells served as the test cells in this example. Sample groups (2E), competitor groups, positive control groups (TGF-β), and blank control groups (Control) were set up: Sample group (2E) consisted of the sample diluted with 10% FBS DMEM medium (prepared in Example 1); competitor group consisted of the competitor product diluted with 10% FBS DMEM medium; positive control group (TGF-β) consisted of the sample with 5 ng / mTGF-β added; and blank control group (Control) consisted of no treatment. Each group had three parallel controls. After thorough mixing, the cells were incubated at 37°C. After 24 hours, RNA was extracted and reverse transcribed to obtain cDNA, followed by qPCR experiments. The relative expression levels of COL4 (type IV collagen), COL7 (type VII collagen), and FN (fibronectin) were calculated based on the experimental results. The results are as follows: Figure 4 As shown, 2E has a significant effect on promoting COL4 (type IV collagen), COL7 (type VII collagen) and FN (fibronectin), and its promoting effect on COL7 and FN is better than that of competing products.

[0095] Example 4: Matrix metalloproteinase metabolism experiment of recombinant human type III collagen

[0096] Fibroblasts were resuspended in 10% FBS DMEM medium and added to 6-well plates at a ratio of 3E5 / well. After cell adhesion, these were used as the test cells in this example. Sample group (2E), competitor group, positive control group (TGF-β), and blank control group (Control) were set up: Sample group (2E) consisted of the sample diluted with 10% FBS DMEM medium (prepared in Example 1); competitor group consisted of the competitor product diluted with 10% FBS DMEM medium; positive control group (TGF-β) consisted of the sample with 5 ng / mTGF-β added; and blank control group (Control) consisted of no treatment. Each group had three parallel controls. After thorough mixing, the cells were incubated at 37°C. After 24 hours, RNA was extracted and reverse transcribed to obtain cDNA, followed by qPCR experiments. The relative expression level of MMP-1 was calculated based on the experimental results. The results are as follows: Figure 5As shown, 2E has a significant inhibitory effect on MMP-1 (matrix metalloproteinase-1), and its effect is better than that of competing products.

[0097] Example 5: Keratinocyte proliferation experiment using recombinant human type III collagen

[0098] Keratinocytes were resuspended in 10% FBS DMEM medium and added to 6-well plates at a ratio of 3E5 / well. After cell adhesion, these cells were used as the test cells in this embodiment. Sample group (2E), competitor group, positive control group (10% FBS), and blank control group (Control) were set up. The sample group (2E) was the test cells with the addition of sample diluted with 0.1% FBS DMEM (this sample was prepared in Example 1 and divided into three groups, diluted to 100ppm, 10ppm and 1ppm respectively). The competitor group was the test cells with the addition of competitor product diluted with 0.1% FBS DMEM (divided into three groups, diluted to 100ppm, 10ppm and 1ppm respectively). The positive control group (10% FBS) was the test cells with the addition of 10% FBS DMEM. The blank control group (Control) was the test cells with the addition of 0.1% FBS DMEM. Each group had three parallel controls. The cells were incubated at 37°C for 48 hours. After incubation, the supernatant was removed, cells were washed once with PBS, and CCK8 solution prepared with DMEM basal medium was added. Incubation was carried out at 37°C for 1 hour. OD values ​​were obtained using a microplate reader, cell viability was calculated, and plotted using Grapdhpad. Results are shown below. Figure 6 As shown, 2E has a good effect on promoting the proliferation of keratinocytes.

[0099] Example 6: Keratinocyte migration assay using recombinant human type III collagen

[0100] Day 0: First, streak the bottom of the empty 24-well plate by drawing two parallel straight lines through each well, parallel to the edge of the plate. Then, resuspend the keratinocytes in 10% FBS DMEM complete medium and seed them into the 24-well plate at a ratio of 1E5 / well. After the cells adhere overnight, streak the wells with a 200µl pipette tip perpendicular to the center of the two parallel straight lines to create scratches that indicate cell damage. After all cells were streaked, the supernatant was removed, and the cells were washed twice with PBS to serve as the test cells for this example. Sample group (2E), competitor group, positive control group (10% FBS), and blank control group (Control) were set up: Sample group (2E) consisted of adding the sample diluted with 0.1% FBS DMEM (prepared in Example 1) to the test cells; competitor group consisted of adding the competitor product diluted with 0.1% FBS DMEM to the test cells; positive control group (10% FBS) consisted of adding 10% FBS DMEM to the test cells; and blank control group (Control) consisted of adding 0.1% FBS DMEM to the test cells. Three parallel controls were set up for each group. The cells were thoroughly mixed and photographed at 0h. Afterwards, the cells were incubated at 37℃ for 24h and photographed again. ImageJ was used to process the healed areas and calculate cell migration rate. The cell migration rate test results are as follows: Figure 7 As shown, 2E has a better effect on promoting keratinocyte migration, and the effect is better than that of competing products.

[0101] Example 7: Keratin Metabolism Experiment of Recombinant Human Type III Collagen

[0102] Keratinocytes were resuspended in 10% FBS DMEM medium and added to 6-well plates at a ratio of 3E5 / well. After cell adhesion, these cells served as the test cells in this embodiment. A sample group (2E), a competitor group, and a blank control group (Control) were set up: the sample group (2E) consisted of test cells treated with a sample diluted in 10% FBS DMEM (prepared in Example 1); the competitor group consisted of test cells treated with a competitor product diluted in 10% FBS DMEM; and the blank control group (Control) received no treatment. Each group had three parallel controls. After thorough mixing, the cells were incubated at 37°C for 24 hours. RNA was extracted and reverse transcribed to obtain cDNA, followed by qPCR experiments. The relative expression levels of K1 (keratin 1), K10 (keratin 10), and TGM-1 (transglutaminase-1) were calculated based on the experimental results. The results are as follows: Figure 8 As shown, 2E has a good effect on promoting the expression of keratin-related genes K1 (keratin 1), K10 (keratin 10) and TGM-1 (transglutaminase-1).

[0103] Example 8: Hyaluronic Acid-Related Factor Experiment of Recombinant Human Type III Collagen

[0104] Keratinocytes were resuspended in 10% FBS DMEM medium and added to 6-well plates at a ratio of 3E5 / well. After cell adhesion, these cells served as the test cells in this embodiment. A sample group (2E), a competitor group, and a blank control group (Control) were set up: the sample group (2E) consisted of test cells treated with a sample diluted in 10% FBS DMEM medium (prepared in Example 1); the competitor group consisted of test cells treated with a competitor product diluted in 10% FBS DMEM medium; and the blank control group (Control) received no treatment. Each group had three parallel controls. After thorough mixing, the cells were incubated at 37°C for 24 hours. RNA was extracted and reverse transcribed to obtain cDNA, which was then used in a qPCR experiment. The relative expression levels of HAS2 (hyaluronic acid synthase 2) and HAS3 (hyaluronic acid synthase 3) were calculated based on the experimental results. The results are as follows: Figure 9 As shown, 2E has a very good effect on promoting HAS2 (hyaluronic acid synthase 2) and HAS3 (hyaluronic acid synthase 3), and its effect is better than that of competing products.

[0105] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A recombinant human type III collagen, characterized in that, The amino acid sequence of this recombinant human type III collagen is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that, The nucleic acid encodes the nucleotide sequence of the recombinant human type III collagen as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid described in claim 2 or 3.

5. A recombinant cell, characterized in that, The cells comprise the nucleic acid as described in claim 2 or 3, or the method for preparing the recombinant cells comprises: transforming host cells using the recombinant vector as described in claim 4.

6. A method for preparing recombinant human type III collagen, characterized in that, This includes the induced expression of the recombinant cells as described in claim 5.

7. A collagen product, characterized in that, The collagen product includes the recombinant human type III collagen as described in claim 1 and / or the recombinant human type III collagen prepared by the method described in claim 6.

8. The collagen product according to claim 7, characterized in that, The collagen product is at least one of the following: a pharmaceutical product or a daily chemical product.

9. The collagen product according to claim 8, characterized in that, The collagen product is a skincare product.

10. The use of at least one of the recombinant human type III collagen of claim 1 and the recombinant human type III collagen prepared by the method of claim 6 in the preparation of collagen products, wherein the collagen products are daily chemical products.

11. The application according to claim 10, wherein the collagen product is a skin care product.

12. The application according to claim 10 or 11, characterized in that, The application includes at least one of the following: preparation of collagen products that inhibit MMP-1 expression in fibroblasts, collagen products that promote HAS2 expression in keratinocytes, and collagen products that promote HAS3 expression in keratinocytes.

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