Triple-helix recombinant human III-type collagen, preparation method and application

Abstract

The invention discloses a triple-helix recombinant human type III collagen, a preparation method and an application, and belongs to the technical field of biology, the triple-helix recombinant human type III collagen is obtained by connecting a leading peptide, a Linker sequence, a recombinant human type III collagen and proline hydroxylase, the leader peptide and the proline hydroxylase are respectively connected to the N end or the C end of the recombinant human source III type collagen through a Linker sequence. The expected biological activity of the prepared triple-helix recombinant human-derived III-type collagen is far higher than that of small-molecule collagen peptide with a random coil structure or a small-molecule fragment with an incomplete structure, the functions of endogenous collagen of a human body are better simulated, and the triple-helix recombinant human-derived III-type collagen can be used as a medicine or cosmetic raw material. The application potential is shown in the aspects of promoting fibroblast proliferation, stimulating self collagen synthesis and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a triple helix recombinant human type III collagen, a preparation method and application. BACKGROUND

[0002] Collagen is one of the most abundant structural proteins in animals, and is the main component of the extracellular matrix, providing elastic support for tissues such as skin, bone, and tendon. So far, 28 different types of collagen have been found, among which human type III collagen is abundant in the dermis of the skin, especially in the skin of infants and in the early stages of wound healing, and plays a crucial role in maintaining skin flexibility and elasticity.

[0003] Currently, commercially available collagen is mainly extracted from animal tissues, such as cattle, pigs, and fish. However, animal-derived collagen has many inherent defects, including: 1) potential viral and pathogenic contamination risks, posing safety hazards; 2) may trigger immune rejection reactions; 3) relatively low biocompatibility due to species differences; 4) complex extraction process, with production and quality greatly affected by raw material sources.

[0004] To overcome the shortcomings of animal-derived collagen, recombinant human collagen technology has emerged. This technology uses genetic engineering to transfer the coding gene of human collagen into microorganisms for expression, such as the patent publication CN116284340A, which converts recombinant type III collagen into Pichia pastoris for expression, thereby obtaining high-purity, low-immunogenicity, and safe collagen. However, recombinant expression technology faces two major challenges: First, the formation and stability of the natural triple helix structure. The characteristic triple helix structure of natural collagen is the basis of its biological function, and the stability of this structure depends on the large number of glycine-proline-hydroxyproline repeat units in the collagen sequence, especially the hydroxylation of proline and lysine. Conventional recombinant expression systems lack the modification enzymes specific to mammalian cells, such as prolyl hydroxylase, resulting in low hydroxylation of proline in the expressed collagen peptide chain, making it difficult to form a stable triple helix structure, and its biological activity is much lower than that of natural collagen. In existing technologies, such as patent publication CN115109795A, attempts have been made to solve this problem by co-expressing hydroxylase, but how to achieve efficient and uniform hydroxylation and ensure the correct folding and long-term stability of the triple helix structure remains a technical difficulty.

[0005] Second, the problem of protein stability and transdermal absorption efficiency. Recombinant expressed collagen polypeptide chains are prone to be degraded by proteases in host cells or enzymes in the external environment during production and storage, leading to product fragmentation and affecting its structural and functional integrity. More importantly, as a cosmetic active ingredient, collagen needs to be able to effectively penetrate the stratum corneum of the skin to exert its effect of promoting fibroblast proliferation and improving skin condition. However, the transdermal absorption efficiency of intact collagen or large fragments with a large molecular weight is extremely low, limiting its skin care effect. Currently, many products are forced to hydrolyze collagen into small molecular peptide segments to promote absorption, but this destroys the triple helix structure, causing it to lose its specific biological activity based on the spatial structure.

[0006] Therefore, there is an urgent need in the art for a new preparation technology of recombinant human type III collagen that can overcome the above-mentioned defects and improve the stability of the triple helix structure of type III collagen and the transdermal absorption efficiency. SUMMARY

[0007] To this end, the present application provides a triple helix recombinant human type III collagen, a preparation method and an application to solve the related technical problems existing in the prior art.

[0008] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: According to a first aspect of the present application, a triple helix recombinant human type III collagen is obtained by connecting a leader peptide, a Linker sequence, a recombinant human type III collagen and a proline hydroxylase, wherein the sequence of the leader peptide is shown in SEQ ID NO. 1, the sequence of the recombinant human type III collagen is shown in SEQ ID NO. 2, and the amino acid sequence of the proline hydroxylase is SEQ ID NO. 3. The leader peptide and the proline hydroxylase are connected to the N-terminus or C-terminus of the recombinant human type III collagen through the Linker sequence, respectively, and are different ends.

[0009] Further, the Linker sequence is (GGGGS)n, wherein n is 1-5.

[0010] Further, the leader peptide is connected to the C-terminus of the recombinant human type III collagen through the Linker sequence, and the proline hydroxylase is connected to the N-terminus of the recombinant human type III collagen through the Linker sequence.

[0011] Further, the leader peptide is connected to the C-terminus of the recombinant human type III collagen through the Linker sequence, and the proline hydroxylase is connected to the N-terminus of the recombinant human type III collagen through the Linker sequence, to form a triple helix recombinant human type III collagen, and the protein sequence is shown in SEQ ID NO. 4.

[0012] According to a second aspect of the present application, a gene sequence encoding the triple helix recombinant human type III collagen is provided, and the gene sequence is shown as SEQ ID NO. 5.

[0013] According to a third aspect of the present application, a preparation method of the triple helix recombinant human type III collagen is provided, comprising the following steps: chemically synthesizing a gene sequence of the triple helix recombinant human type III collagen; constructing a recombinant expression plasmid of the triple helix recombinant human type III collagen; constructing a recombinant Pichia pastoris of the triple helix recombinant human type III collagen; inducing culture of the recombinant Pichia pastoris; harvesting the culture solution, centrifuging to obtain supernatant, and isolating and purifying to obtain the triple helix recombinant human type III collagen.

[0014] Further, the recombinant expression plasmid is selected from pPic9k.

[0015] Further, the recombinant Pichia pastoris is selected from GS115 Pichia pastoris.

[0016] According to a fourth aspect of the present application, a recombinant Pichia pastoris expressing the triple helix recombinant human type III collagen is provided, and the recombinant Pichia pastoris is transformed with the above-mentioned gene sequence.

[0017] According to a fifth aspect of the present application, the triple helix recombinant human type III collagen is used in the preparation of cosmetics.

[0018] The present application has the following advantages: The present application fuses and expresses the recombinant human type III collagen and the proline hydroxylase P4HA3 fragment, so that the proline hydroxylase can hydroxylate the proline residues in the collagen domain in situ while the target protein is expressed. The innovative hydroxylation modification method is more efficient and uniform than co-expression or in-vitro addition of hydroxylase, greatly improves the content of hydroxyproline, and provides support for the spontaneous correct folding and long-term stable activity of the triple helix structure. When designing the collagen sequence, we use protein structure prediction tools such as Alphafold to simulate and analyze the triple helix structure, and predictively remove the cleavage sites easily recognized by proteases. This AI-based rational design enhances the stability of the protein molecule from the source, effectively reduces degradation during fermentation production and subsequent storage, and ensures that the final product is a large functional protein with complete triple helix structure, rather than an inactive small fragment.

[0019] Meanwhile, on the basis of the previous research, a specific leader peptide is fused to the collagen, which is proved to have a similar function to cell penetrating peptide, and can effectively guide the whole fusion protein to pass through the skin keratin layer barrier, realize the good transdermal delivery efficiency while maintaining the integrity of the triple helix structure, and improve the interaction with the dermal fibroblasts.

[0020] The proline hydroxylase and the collagen are designed as a single fusion protein, and only one engineering strain needs to be constructed and fermentation culture is carried out, so that the active collagen with a triple helix structure and stable hydroxylation can be obtained. This simplifies the production process, avoids the cumbersome steps of co-expressed strain construction and in vitro enzymatic hydroxylation, reduces the production cost, and is conducive to realizing the uniformity and stability of the product quality.

[0021] In summary, the triple helix recombinant human type III collagen prepared by the application has a much higher biological activity than the small molecule collagen peptide or small molecule fragment with incomplete structure, and can better simulate the function of the endogenous collagen in the human body, and has great application potential in promoting fibroblast proliferation, stimulating self-collagen synthesis and repairing skin. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and those skilled in the art can obtain other implementation drawings according to the provided drawings without creating any creative labor.

[0023] The structures, proportions, sizes, etc. shown in the specification are only used to cooperate with the content disclosed in the specification, to be understood and read by those skilled in the art, and do not define the limiting conditions for the implementation of the application, so they do not have technical significance. Any modification of the structure, change of the proportion relationship or adjustment of the size, without affecting the effect and purpose that can be achieved by the application, should still fall within the scope of the technical content disclosed by the application.

[0024] Figure 1 Circular dichroism spectrum of the triple helix recombinant human type III collagen corresponding to the experimental example in the application; Figure 2 Fluorescence retention distribution diagram of the whole skin organ chip model of the triple helix recombinant human type III collagen corresponding to the experimental example in the application. DETAILED DESCRIPTION

[0025] The following embodiments of the present application are described by way of specific examples, and other advantages and effects of the present application will be readily understood by those skilled in the art from the disclosure. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0026] According to a first aspect of the present application, a triple helix recombinant human type III collagen is connected by a leader peptide, a Linker sequence, a recombinant human type III collagen and a proline hydroxylase, wherein the sequence of the leader peptide is shown in SEQ ID NO. 1, the sequence of the recombinant human type III collagen is shown in SEQ ID NO. 2, and the amino acid sequence of the proline hydroxylase is SEQ ID NO. 3. The leader peptide and the proline hydroxylase are respectively connected to the N-terminus or C-terminus of the recombinant human type III collagen through the Linker sequence, and the different ends.

[0027] Further, the Linker sequence is (GGGGS)n, wherein n is 1-5.

[0028] Further, the leader peptide is connected to the C-terminus of the recombinant human type III collagen through the Linker sequence, and the proline hydroxylase is connected to the N-terminus of the recombinant human type III collagen through the Linker sequence.

[0029] Further, the leader peptide is connected to the C-terminus of the recombinant human type III collagen through the Linker sequence, and the proline hydroxylase is connected to the N-terminus of the recombinant human type III collagen through the Linker sequence, to form a triple helix recombinant human type III collagen, and the protein sequence is shown in SEQ ID NO. 4.

[0030] According to a second aspect of the present application, a gene sequence encoding the above triple helix recombinant human type III collagen is provided, and the gene sequence is shown in SEQ ID NO. 5.

[0031] According to a third aspect of the present application, a preparation method of a triple helix recombinant human type III collagen is provided, comprising the following steps: Chemically synthesizing a gene sequence of the triple helix recombinant human type III collagen; Constructing a recombinant expression plasmid of the triple helix recombinant human type III collagen; Constructing a recombinant Pichia pastoris of the triple helix recombinant human type III collagen; Inducing the culture of the recombinant Pichia pastoris; Harvesting the culture solution, centrifuging to obtain supernatant, and isolating and purifying to obtain the triple-helical recombinant human type III collagen.

[0032] Further, the recombinant expression plasmid is selected from pPic9k.

[0033] Further, the recombinant Pichia pastoris is selected from GS115 Pichia pastoris.

[0034] According to a fourth aspect of the present application, a recombinant Pichia pastoris expressing the triple-helical recombinant human type III collagen is provided, wherein the recombinant Pichia pastoris is transformed with the gene sequence described above.

[0035] According to a fifth aspect of the present application, the triple-helical recombinant human type III collagen described above is used in the preparation of a cosmetic product.

[0036] In the present application, the culture media used in the following examples, including MD culture medium, YPD culture medium, BMGY culture medium, and BMMY culture medium, are all commonly used culture media for culturing Pichia pastoris in the art, the formulations of which are known and can be obtained through conventional commercial channels, and thus the specific formulations are not described herein.

[0037] In the present application, the molecular cloning operations involved in the following examples are all conventional operations in the technical field, or are operated according to the product's operation manual, and thus the specific operations are not described herein.

[0038] Example 1 In the present example, the triple-helical recombinant human type III collagen is obtained by connecting a leader peptide, a Linker sequence, a recombinant human type III collagen, and a proline hydroxylase, wherein the sequence of the leader peptide is shown in SEQ ID NO. 1, the sequence of the recombinant human type III collagen is shown in SEQ ID NO. 2, and the amino acid sequence of the proline hydroxylase is SEQ ID NO. 3. Based on the previous research (patent publication No. CN116284340A) and experience, the leader peptide is connected to the C-terminal of the recombinant human type III collagen through the Linker sequence, and the proline hydroxylase is connected to the N-terminal of the recombinant human type III collagen through the Linker sequence, to form a fusion expression protein, forming the experimental example.

[0039] Meanwhile, in the above fusion process, the Linker sequence between the leader peptide and the C-terminal of the recombinant human type III collagen is (GGGGS)n, wherein n is 1; and the Linker sequence between the proline hydroxylase and the N-terminal of the recombinant human type III collagen is (GGGGS)n, wherein n is 5, so as to ensure that the proline hydroxylase can maximize the hydroxylation of the proline residues and improve the stability of the triple-helical structure.

[0040] Based on the above sequence information, in this application, the expression vector is pPic9k or pPicza (purchased from Shanghai Generay Biotech Co., Ltd.), and the expression strain is GS115 Pichia pastoris (purchased from Shanghai Generay Biotech Co., Ltd.).

[0041] According to the above description, the leader peptide sequence (described in SEQ ID NO. 1), the linker sequence (n≤5), the recombinant human type III collagen (as shown in SEQ ID NO. 2) and the proline hydroxylase P4HA3 (as shown in SEQ ID NO. 3) are connected by SnapGene software (downloaded from https: / / www.snapgene.com / ); at the same time, based on AlphaFold structure optimization (https: / / alphafold.ebi.ac.uk / ) and protein sequence optimization (https: / / www.jcat.de / ) to reduce protease cleavage sites, the optimized protein sequence is as shown in SEQ ID NO. 4, and the gene sequence is as shown in SEQ ID NO. 5.

[0042] In order to verify the effect of the leader peptide and the proline hydroxylase P4HA3 on improving the permeability and folding stability of the recombinant human type III collagen, on the basis of the above experimental examples, deletion example 1 (without fusing the leader peptide sequence), deletion example 2 (without fusing the proline hydroxylase P4HA3 sequence) and deletion example 3 (without fusing the leader peptide sequence and the proline hydroxylase P4HA3 sequence) are also set, and other treatment methods are the same as the experimental examples.

[0043] The fusion gene fragment of the triple helix recombinant human type III collagen (deletion example 1, deletion example 2 and deletion example 3 are also obtained by chemical synthesis) is obtained by gene synthesis, and then the fusion gene fragment is inserted into the yeast vector pPic9k to obtain a recombinant plasmid; then the linear recombinant plasmid obtained by SgII linearization is purified by a DNA recovery kit, and the linear plasmid is transformed into the GS115 Pichia pastoris (purchased from Shanghai Generay Biotech Co., Ltd.) competent cells by electroporation. The positive clones are screened by MD culture plates, and the high copy strain is screened by different concentrations of G418.

[0044] Example 2 The high copy strain obtained in Example 1 is inoculated into YPD medium and cultured at 28°C with a shaking speed of 220 rpm overnight. The next day, 10 mL of the recovered seed liquid is transferred to 1 L of BMGY medium at a volume ratio of 1:100, and cultured at 28°C with a shaking speed of 220 rpm, until the OD 600When the OD600 = 2, the bacteria were collected by centrifugation. The bacteria were resuspended in BMMY medium and continued to be cultured at 28°C, 220 rpm, and 1 wt% methanol was added every 24 h. The expression of the protein was detected by sampling. After 72 h of fermentation, the supernatant was collected by centrifugation, and the supernatant was dialyzed against PB buffer until the conductivity was lower than 2 ms / cm.

[0045] The protein was purified using CM cation exchange resin, and trace amounts of cell residues were further removed using Q anion exchange resin. After desalination by dialysis, the protein was freeze-dried into a collagen sponge. The freeze-dried sponge can be quickly and completely dissolved in water. Experiments show that the expression methods in Example 1 above can correctly express the triple-helical recombinant human type III collagen.

[0046] In the description, the accompanying drawings show Figure 1 The circular dichroism analysis of the triple-helical recombinant human type III collagen corresponding to the experimental examples is shown. It can be seen that the circular dichroism spectrum of the collagen has positive and negative peak patterns, specifically, a negative peak near 199 nm and a positive peak near 222 nm, and has a triple-helical structure.

[0047] Example 3 The purified triple-helical recombinant human type III collagen obtained in Example 2 (including experimental examples, deletion example 1, deletion example 2, and deletion example 3) and commercial recombinant collagen (purchased from Shanghai Generay) were prepared into a 10 μg / mL solution, and a blank control was used physiological saline. 100 μL of the solution was added to each well of a 96-well plate coated with no polylysine, and incubated at 37°C for 30 min, and then washed twice with PBS. Then 1% BSA was added, and after blocking at 37°C for 30 min, rat fibroblasts (cultured in serum-free medium) were added; after 1 h, the culture medium in the wells was gently aspirated, and the unabsorbed cells were gently washed with PBS. The number of living cells adsorbed at the bottom of the plate was detected by the CCK8 method (purchased from Shanghai Generay) to verify the activity of the recombinant collagen in promoting adhesion, and the results are shown in Table 1: Table 1: Activity of recombinant collagen in promoting cell adhesion

[0048] The experimental results show that the experimental examples and deletion example 1 of the triple-helical recombinant human type III collagen fused with the leader peptide both have the ability to promote cell adhesion, which is the same as the commercial recombinant collagen, indicating that the protein has its specific biological activity. In deletion examples 2 and 3, the recombinant human type III collagen cannot be folded due to the deletion of proline hydroxylase P4HA3, which affects its activity.

[0049] Example 4 The thickness of the pig skin was 0.5 mm, and the subcutaneous fat was removed and fixed on the Franz diffusion cell (effective diffusion area 1.0 cm 2 , receiving chamber volume 5 mL) with PBS buffer at pH 7.4 at 37°C constant temperature stirring. The experimental and missing examples 1 of triple helix recombinant human type III collagen were labeled with FITC (labeling rate > 90%), and prepared as 1 mg / mL PBS solution, 200 μL of which was evenly applied to the surface of the pig skin. The receiving liquid was collected at 2h, 6h, 12h and 24h, and an equal volume of fresh PBS solution was added. The fluorescence intensity in the receiving liquid was measured by fluorescence spectrophotometer (excitation wavelength 490 nm, emission wavelength 520 nm), and the cumulative penetration amount was calculated.

[0050] This example aims to evaluate the transdermal absorption efficiency of the triple helix recombinant human type III collagen prepared in Example 2 by Franz diffusion cell method, and the cumulative amount of transdermal absorption (μg / cm 2 ) is used to represent the experimental results as shown in Table 2: Table 2: Cumulative transdermal absorption amount of collagen

[0051] As shown in Table 2, the cumulative transdermal amount of triple helix recombinant human type III collagen with fusion leader peptide within 24h is significantly higher than that of missing example 1, indicating that the leader peptide effectively promotes the skin penetration ability of the protein, which has potential application in cosmetics.

[0052] Example 5 In order to ensure the rigor and repeatability of the experiment, the transdermal effect was verified by Yeyetang Detection Technology (Shanghai) Co., Ltd. The specific process is as follows: I. Test purpose Quantitative evaluation of the penetration efficiency of triple helix recombinant human type III collagen in full skin organ chip model, and provide data support for cosmetic formulation design.

[0053] II. Test principle The full skin organ chip model is based on fluorescence labeling technology: the sample is labeled with FITC and applied to the epidermis side, the concentration of fluorescent substances penetrating through the skin barrier reflects the penetration ability, laser confocal microscope captures the fluorescence signal distribution, and ImageJ software semi-quantitatively analyzes the penetration depth and intensity.

[0054] III. Test materials and instruments Reagents: DPBS, triple helix recombinant human type III collagen, fluorescein isothiocyanate (FITC), 4% paraformaldehyde, sucrose (10%, 20%, 30% solution), OCT embedding agent.

[0055] Model: Full-thickness skin organ chip model.

[0056] Instrument: NIKON AX laser confocal microscope.

[0057] Four, experimental steps 4.1 Main operation steps ① Sample preparation and fluorescence labeling: triple helix recombinant human type III collagen was labeled with FITC for subsequent experiments to quantify and visualize the material.

[0058] ② Experimental treatment: 200 μL of sample was evenly coated on the surface of the model, 500 μL of medium solution was added to the lower chamber (to receive permeate and provide nutrition), and the model was incubated at 37°C, 5% CO2 for 2 hours. Then, the residual material was removed by gently washing with pre-warmed DPBS for 3 times.

[0059] ③ Fixation: the model tissue was fixed with 4% paraformaldehyde (PFA) at 4°C for 2 hours.

[0060] ④ Dehydration, embedding and sectioning: gradient dehydration was performed in 10%, 20%, and 30% sucrose solutions, respectively, until the model sank in the 30% sucrose solution, indicating that the dehydration was complete. The dehydrated skin model was embedded with OCT embedding agent and frozen sectioned with a thickness of 10 μm.

[0061] ⑤ Observation: the distribution of FITC fluorescence signal in the section was observed using a laser confocal microscope to evaluate the transdermal penetration and skin retention effect of the fluorescent marker.

[0062] 4.2 Data processing The fluorescence signal was collected and semi-quantitatively analyzed using the software provided with the laser confocal microscope and ImageJ image processing software. The data was processed and plotted using Excel and GraphPad Prism 9.0 software, and the data was represented as mean ± SEM.

[0063] Five, experimental results The fluorescence penetration distribution map of the full-thickness skin organ chip model is shown in Figure 2 As can be observed, the fluorescence signal of the triple helix recombinant human type III collagen on the skin surface is strong and evenly distributed, indicating that the retention amount on the skin surface is more. As the skin level deepens, the fluorescence signal gradually weakens, indicating that the triple helix recombinant human type III collagen mainly penetrates to the epidermis layer, and part reaches the dermis layer. This distribution characteristic shows that the triple helix recombinant human type III collagen has certain transdermal ability.

[0064] The average value of the total fluorescence intensity of the triple helix recombinant human type III collagen in the full skin organ chip model was 53669±1080. This higher fluorescence intensity value reflects the overall retention amount in the skin, indicating that the triple helix recombinant human type III collagen molecules can form a relatively thick retention layer on the skin surface. From the data of each parallel experimental group, the fluorescence intensity value is relatively stable with a small fluctuation range, showing good repeatability and reliability of the experimental results.

[0065] Example 6 The HSF cells (purchased from Shanghai Generay Biotech) were inoculated in a 96-well plate at 5×10 3 After 24h of culture in DMEM medium containing 10% FBS, the medium was replaced with serum-free medium, and the triple helix recombinant human type III collagen prepared in Example 2 was added (final concentrations were 10 μg / mL, 50 μg / mL and 100 μg / mL, respectively), and the blank medium was set as the negative control, and the commercial recombinant type III collagen (final concentration was 10 μg / mL) was set as the positive control. After 48h of continuous culture, 10 μL of CCK-8 reagent was added to each well, and the OD450nm value was measured after 2h of incubation to calculate the cell proliferation rate. At the same time, the cell supernatant was collected, and the collagen secretion amount was detected by using a human type I collagen ELISA kit (purchased from Shanghai Generay Biotech). This embodiment verifies the promotion of the triple helix recombinant human type III collagen on the proliferation of human skin fibroblasts (HSF) and the synthesis of collagen, and the results are shown in Table 3 below: Table 3: Collagen on the proliferation rate of human skin fibroblasts and collagen synthesis

[0066] As shown in Table 3, the cell proliferation rate and type I collagen secretion amount of the experimental examples at the concentrations of 50 μg / mL and 100 μg / mL were significantly higher than those of the negative control group, and were comparable to or better than those of the positive control group, indicating that the triple helix recombinant human type III collagen had good biological activity.

[0067] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

Claims

1. A triple-helical recombinant human collagen type III, characterized in that, The recombinant human type III collagen and prolyl hydroxylase are connected by a linker sequence, a leader peptide and a prolyl hydroxylase, wherein the sequence of the leader peptide is shown as SEQ ID NO. 1, the sequence of the recombinant human type III collagen is shown as SEQ ID NO. 2, and the amino acid sequence of the prolyl hydroxylase is shown as SEQ ID NO.

3. The leader peptide and the prolyl hydroxylase are connected to the N-terminal or C-terminal of the recombinant human type III collagen through a linker sequence, respectively.

2. The triple-helical recombinant human collagen type III of claim 1, wherein, The linker sequence is (GGGGS)n, wherein n is 1-5.

3. The triple-helical recombinant human collagen type III of claim 1, wherein, The leader peptide is connected to the C-terminal of the recombinant human type III collagen through a linker sequence, and the prolyl hydroxylase is connected to the N-terminal of the recombinant human type III collagen through a linker sequence.

4. The triple-helical recombinant human collagen type III of claim 3, wherein, The leader peptide is connected to the C-terminal of the recombinant human type III collagen through a linker sequence, and the prolyl hydroxylase is connected to the N-terminal of the recombinant human type III collagen through a linker sequence, to form a triple helix recombinant human type III collagen, and the protein sequence is shown as SEQ ID NO.

4.

5. A gene sequence encoding the triple helix recombinant human type III collagen according to any one of claims 1-4, and the gene sequence is shown as SEQ ID NO.

5.

6. A method for the preparation of a triple helical recombinant human collagen type III according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: chemically synthesizing a gene sequence of the triple helix recombinant human type III collagen; constructing a recombinant expression plasmid of the triple helix recombinant human type III collagen; constructing a recombinant Pichia pastoris of the triple helix recombinant human type III collagen; inducing culture of the recombinant Pichia pastoris; harvesting the culture solution, centrifuging to obtain supernatant, and isolating and purifying to obtain the triple helix recombinant human type III collagen.

7. The production method according to claim 6, wherein The recombinant expression plasmid is selected from pPic9k.

8. The production method according to claim 6, wherein The recombinant Pichia pastoris is selected from GS115 Pichia pastoris.

9. A recombinant Pichia pastoris bacterium expressing the triple-helical recombinant human collagen type III of any one of claims 1-4, characterized in that, The recombinant Pichia pastoris is transformed with the gene sequence according to claim 5.

10. Use of the triple helix recombinant human type III collagen according to any one of claims 1-4 in the preparation of a cosmetic product.

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