A triple-helix recombinant human type III collagen, its preparation method and application

Abstract

This invention discloses a triple-helix recombinant human type III collagen, its preparation method, and its applications, belonging to the field of biotechnology. The triple-helix recombinant human type III collagen is obtained by linking a leader peptide, a linker sequence, recombinant human type III collagen, and a proline hydroxylase. The leader peptide and proline hydroxylase are linked to the N-terminus or C-terminus of the recombinant human type III collagen via the linker sequence, respectively. The triple-helix recombinant human type III collagen prepared by this invention is expected to have significantly higher biological activity than small molecule collagen peptides with random coil structures or incomplete small molecule fragments, better mimicking the function of endogenous collagen in the human body. As a raw material for pharmaceuticals or cosmetics, it shows application potential in promoting fibroblast proliferation and stimulating endogenous collagen synthesis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a triple-helix recombinant human type III collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is one of the most abundant structural proteins in animals and a major component of the extracellular matrix, providing elastic support for tissues such as skin, bones, and tendons. To date, 28 different types of collagen have been discovered, among which human type III collagen is abundant in the dermis, playing a crucial role in maintaining skin suppleness and elasticity, especially in infant skin and the early stages of wound healing.

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

[0004] To overcome the shortcomings of animal-derived collagen, recombinant human collagen technology has emerged. This technology uses genetic engineering to transfer the gene encoding human collagen into microorganisms for expression. For example, patent publication number CN116284340A transforms recombinant type III collagen into Pichia pastoris for expression, thereby obtaining collagen with high purity, low immunogenicity, and good safety. However, recombinant expression technology faces two major challenges:

[0005] First, there is the issue of 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. The stability of this structure depends on the abundant glycine-proline-hydroxyproline repeating units in the collagen sequence, especially the hydroxylation of proline and lysine. Conventional recombinant expression systems lack modifying enzymes such as prolyl hydroxylase, which are specific to mammalian cells. This results in low proline hydroxylation in the expressed collagen peptide chains, making it difficult to spontaneously form a stable triple helix structure, thus making its biological activity far lower than that of natural collagen. In existing technologies, such as patent publication number CN115109795A, although there are attempts to solve this problem by co-expressing hydroxylases, how to achieve efficient and uniform hydroxylation and ensure the correct folding and long-term stability of the triple helix structure remains a technical challenge.

[0006] Secondly, there are issues with protein stability and transdermal absorption efficiency. Recombinant collagen polypeptide chains are easily degraded by proteases in host cells or by enzymes in the external environment during production and storage, leading to product fragmentation and affecting its structural and functional integrity. More importantly, as an active ingredient in cosmetics, collagen needs to effectively penetrate the stratum corneum to exert its effects of promoting fibroblast proliferation and improving skin condition. However, large molecular weight intact collagen or large fragments have extremely low transdermal absorption efficiency, limiting its skincare effects. Currently, many products are forced to hydrolyze collagen into small peptides to promote absorption, but this disrupts the triple helix structure, causing it to lose its specific biological activities based on its spatial structure.

[0007] Therefore, there is an urgent need in this field for a novel preparation technology for recombinant human type III collagen that can overcome the above-mentioned defects and improve the stability of the triple helix structure and transdermal absorption efficiency of type III collagen. Summary of the Invention

[0008] Therefore, the present invention provides a triple-helix recombinant human type III collagen, its preparation method, and its application to solve the related technical problems existing in the prior art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] According to a first aspect of the present invention, a triple-helix recombinant human type III collagen is obtained by linking a leader peptide, a linker sequence, recombinant human type III collagen, and a proline hydroxylase, wherein the sequence of the leader peptide is as shown in SEQ ID NO.1, the sequence of the recombinant human type III collagen is as shown in SEQ ID NO.2, and the amino acid sequence of the proline hydroxylase is SEQ ID NO.3;

[0011] The leader peptide and proline hydroxylase are respectively linked to the N-terminus or C-terminus of the recombinant human type III collagen via a linker sequence, and at different ends.

[0012] Furthermore, the Linker sequence is (GGGGS)n, where n is 1-5.

[0013] Furthermore, the leader peptide is linked to the C-terminus of the recombinant human type III collagen via a linker sequence, and the proline hydroxylase is linked to the N-terminus of the recombinant human type III collagen via a linker sequence.

[0014] Furthermore, the leader peptide is linked to the C-terminus of the recombinant human type III collagen via a linker sequence, and the proline hydroxylase is linked to the N-terminus of the recombinant human type III collagen via a linker sequence, forming a triple-helix recombinant human type III collagen, the protein sequence of which is shown in SEQ ID NO.4.

[0015] According to a second aspect of the invention, a gene sequence encoding a triple-helix recombinant human type III collagen as described above is provided, the gene sequence being shown in SEQ ID NO.5.

[0016] According to a third aspect of the present invention, a method for preparing triple-helical recombinant human type III collagen is provided, comprising the following steps:

[0017] Gene sequence of chemically synthesized triple-helix recombinant human type III collagen;

[0018] Construct a recombinant expression plasmid for triple-helix recombinant human type III collagen;

[0019] Recombinant Pichia pastoris strain used to construct triple-helix recombinant human type III collagen;

[0020] Induced culture of the recombinant Pichia pastoris;

[0021] The culture medium was harvested, the supernatant was collected by centrifugation, and the triple-helix recombinant human type III collagen was obtained by separation and purification.

[0022] Furthermore, the recombinant expression plasmid is selected from pPic9k.

[0023] Furthermore, the recombinant Pichia pastoris strain is selected from GS115 Pichia pastoris.

[0024] According to a fourth aspect of the present invention, a recombinant Pichia pastoris expressing the triple-helix recombinant human type III collagen as described above, said recombinant Pichia pastoris being transformed with the above-described gene sequence.

[0025] According to a fifth aspect of the invention, the use of the triple-helix recombinant human type III collagen as described above in the preparation of cosmetics is provided.

[0026] The present invention has the following advantages:

[0027] This invention expresses recombinant human type III collagen by fusing it with a proline hydroxylase P4HA3 fragment. Simultaneously with target protein expression, the proline hydroxylase performs in-situ hydroxylation of proline residues in the collagen domain. This innovative hydroxylation modification method is more efficient and uniform than co-expression or in vitro addition of the hydroxylase, significantly increasing the hydroxyproline content and supporting the spontaneous correct folding and long-term stable activity of the triple helix structure. In designing the collagen sequence, we used protein structure prediction tools such as Alphafold to simulate and analyze the triple helix structure and predictively removed cleavage sites easily recognized by proteases. This AI-based rational design enhances the stability of the protein molecule from the source, effectively reducing degradation during fermentation and subsequent storage, ensuring that the final product is a large functional protein with a complete triple helix structure, rather than an inactive small fragment.

[0028] Meanwhile, based on previous research, a specific leader peptide was fused to collagen. This leader peptide was shown to have a function similar to cell-penetrating peptides, which can effectively guide the entire fusion protein through the skin's stratum corneum barrier. This achieves good transdermal delivery efficiency while maintaining the complete triple helix structure and improving the interaction with dermal fibroblasts.

[0029] By designing proline hydroxylase and collagen as a single fusion protein, production requires only the construction of one engineered strain and a single fermentation culture to obtain stable hydroxylated, triple-helix active collagen. This simplifies the production process, avoids cumbersome steps such as co-expression strain construction and in vitro enzymatic hydroxylation, reduces production costs, and facilitates the uniformity and stability of product quality.

[0030] In summary, the triple-helix recombinant human type III collagen innovatively prepared in this invention is expected to have significantly higher biological activity than small molecule collagen peptides with random coil structures or structurally incomplete small molecule fragments when used as a raw material for pharmaceuticals or cosmetics. It can better mimic the function of endogenous human collagen, demonstrating great application potential in promoting fibroblast proliferation, stimulating endogenous collagen synthesis, and repairing skin. Attached Figure Description

[0031] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0032] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0033] Figure 1 The circular dichroism spectroscopy of the triple-helix recombinant human type III collagen corresponding to the experimental example in this invention;

[0034] Figure 2 This is a fluorescence retention distribution map of a whole-skin organ-on-a-chip model of triple-helix recombinant human type III collagen corresponding to the experimental example in this invention. Detailed Implementation

[0035] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] According to a first aspect of the present invention, a triple-helix recombinant human type III collagen is obtained by linking a leader peptide, a linker sequence, recombinant human type III collagen, and a proline hydroxylase, wherein the sequence of the leader peptide is as shown in SEQ ID NO.1, the sequence of the recombinant human type III collagen is as shown in SEQ ID NO.2, and the amino acid sequence of the proline hydroxylase is SEQ ID NO.3.

[0037] The leader peptide and proline hydroxylase are linked to the N-terminus or C-terminus of recombinant human type III collagen via a linker sequence, and at different ends.

[0038] Furthermore, the Linker sequence is (GGGGS)n, where n is 1-5.

[0039] Furthermore, the leader peptide is linked to the C-terminus of the recombinant human type III collagen via a linker sequence, and the proline hydroxylase is linked to the N-terminus of the recombinant human type III collagen via a linker sequence.

[0040] Furthermore, the leader peptide is linked to the C-terminus of the recombinant human type III collagen via a linker sequence, and the proline hydroxylase is linked to the N-terminus of the recombinant human type III collagen via a linker sequence, forming a triple-helix recombinant human type III collagen, the protein sequence of which is shown in SEQ ID NO.4.

[0041] According to a second aspect of the invention, a gene sequence encoding the above-described triple-helix recombinant human type III collagen is provided, the gene sequence being shown in SEQ ID NO. 5.

[0042] According to a third aspect of the present invention, a method for preparing triple-helical recombinant human type III collagen is provided, comprising the following steps:

[0043] Gene sequence of chemically synthesized triple-helix recombinant human type III collagen;

[0044] Construct a recombinant expression plasmid for triple-helix recombinant human type III collagen;

[0045] Recombinant Pichia pastoris strain used to construct triple-helix recombinant human type III collagen;

[0046] Induced culture of recombinant Pichia pastoris;

[0047] The culture medium was harvested, the supernatant was collected by centrifugation, and the triple-helix recombinant human type III collagen was obtained by separation and purification.

[0048] Furthermore, the recombinant expression plasmid was selected from pPic9k.

[0049] Furthermore, the recombinant Pichia pastoris was selected from GS115 Pichia pastoris.

[0050] According to a fourth aspect of the present invention, a recombinant Pichia pastoris strain expressing the above-described triple-helix recombinant human type III collagen, wherein the recombinant Pichia pastoris strain is transformed with the above-described gene sequence.

[0051] According to a fifth aspect of the invention, the use of the above-described triple-helix recombinant human type III collagen in the preparation of cosmetics is provided.

[0052] In this application, the culture media used in the following examples, including MD medium, YPD medium, BMGY medium and BMMY medium, are all commonly used culture media for culturing Pichia pastoris in the art. Their formulations are known and they can all be obtained through conventional commercial channels. The specific formulations will not be described in detail here.

[0053] In this application, the molecular cloning operations involved in the following embodiments, unless otherwise specified, are all conventional operations in this technical field, or operations performed according to the product's instruction manual, and the specific operations will not be described in detail here.

[0054] Example 1

[0055] In this embodiment, the triple-helix recombinant human type III collagen is obtained by linking a leader peptide, a linker sequence, recombinant human type III collagen, and proline hydroxylase. 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 prior research (patent publication number CN116284340A) and experience, this application links the leader peptide to the C-terminus of the recombinant human type III collagen via a linker sequence, and the proline hydroxylase to the N-terminus of the recombinant human type III collagen via a linker sequence, forming a fusion expression protein, thus creating an experimental example.

[0056] Meanwhile, during the fusion process, the linker sequence between the leader peptide and the C-terminus of recombinant human type III collagen is (GGGGS)n, where n is 1; the linker sequence between the proline hydroxylase and the N-terminus of recombinant human type III collagen is (GGGGS)n, where n is 5, thereby ensuring that the proline hydroxylase can maximally hydroxylate proline residues and improve the stability of the triplet structure.

[0057] Based on the above sequence information, in this application, the expression vector is pPic9k or pPicza (purchased from Shanghai Sangon Biotech), and the expression strain is GS115 Pichia pastoris (purchased from Shanghai Sangon Biotech).

[0058] As described above, the leader peptide sequence (described in SEQ ID NO.1), linker sequence (n≤5), recombinant human type III collagen (shown in SEQ ID NO.2), and proline hydroxylase P4HA3 (shown in SEQ ID NO.3) were linked together using SnapGene software (downloaded from https: / / www.snapgene.com / ). Simultaneously, based on AlphaFold structure optimization (https: / / alphafold.ebi.ac.uk / ) and protein sequence optimization with reduced protease cleavage sites (https: / / www.jcat.de / ), the optimized protein sequence is shown in SEQ ID NO.4, and the gene sequence is shown in SEQ ID NO.5.

[0059] To verify the effects of the leader peptide and proline hydroxylase P4HA3 on improving the permeability and folding stability of recombinant human type III collagen, in addition to the above experimental examples, deletion examples 1 (unfused leader peptide sequence), deletion example 2 (unfused proline hydroxylase P4HA3 sequence), and deletion example 3 (unfused leader peptide sequence and proline hydroxylase P4HA3 sequence) were also set up, with other treatments being the same as in the experimental examples.

[0060] A triple-helix recombinant human type III collagen fusion gene fragment was obtained through gene synthesis (deletion examples 1, 2, and 3 were also obtained through chemical synthesis). This fusion gene fragment was then inserted into the yeast vector pPic9k to obtain a recombinant plasmid. The recombinant plasmid obtained was linearized using SgII, and the linear plasmid was purified using a DNA recovery kit. The linear plasmid was then transformed into GS115 Pichia pastoris (purchased from Shanghai Sangon Biotech) competent cells via electroporation. Positive clones were screened using MD culture plates, and high-copy strains were further screened using different concentrations of G418.

[0061] Example 2

[0062] The high-copy strain obtained in Example 1 was inoculated into YPD medium and cultured overnight at 28°C and 220 rpm with shaking. The next day, 10 mL of the revived seed culture was transferred to 1 L of BMGY medium at a 1:100 volume ratio and cultured at 28°C and 220 rpm with shaking until OD... 600 =2 hours, centrifuge to collect bacterial cells. Resuspend the bacterial cells in BMMY medium and continue to culture at 28°C and 220 rpm with shaking. Add 1 wt% methanol every 24 hours and take samples to detect protein expression levels. After 72 hours of fermentation, centrifuge the bacterial culture to collect the supernatant, and dialyze the supernatant with PB buffer until the conductivity is below 2 mS / cm.

[0063] The protein was purified using CM cation exchange resin, and trace cellular residues were further removed using Q anion exchange resin. After dialysis to remove salts, the protein was freeze-dried into collagen sponges. The freeze-dried sponges could be rapidly and completely dissolved in water. Experiments showed that the expression methods in Example 1 above could correctly express triple-helix recombinant human type III collagen.

[0064] The instruction manual includes Figure 1 The circular dichroism chromatogram (CD) analysis of the triple-helix recombinant human type III collagen corresponding to the experimental example is shown. It can be seen that there are positive and negative peaks of collagen on the CD spectrum. Specifically, there is a negative peak near 199 nm and a positive peak near 222 nm, which shows that it has a triple-helix structure.

[0065] Example 3

[0066] The purified triple-helix recombinant human type III collagen obtained in Example 2 (including experimental example, deletion example 1, deletion example 2, and deletion example 3) and commercially available recombinant collagen (purchased from Shanghai Sangon Biotech) were prepared into a 10 μg / mL solution. Physiological saline was used as the blank control. 100 μL of the solution was added to each well of a 96-well plate without poly-L-lysine coating, and incubated at 37°C for 30 min. The plates were then washed twice with PBS. 1% BSA was added, and the plates were blocked at 37°C for 30 min. Rat fibroblasts (cultured in serum-free medium) were then added. After 1 h, the culture medium was gently aspirated from the wells, and unadsorbed cells were gently washed with PBS. The number of viable cells adsorbed at the bottom of the wells was detected using the CCK8 assay (purchased from Shanghai Sangon Biotech) to verify the adhesion-promoting activity of the recombinant collagen. The results are shown in Table 1.

[0067] Table 1: Activity of recombinant collagen in promoting cell adhesion

[0068]

[0069] The experimental results showed that both the experimental example (containing a leader peptide and a triple-helix recombinant human type III collagen) and the deletion example 1 exhibited the ability to promote cell adhesion, similar to commercially available recombinant collagen, indicating that the protein possesses its specific biological activity. In deletion examples 2 and 3, the absence of proline hydroxylase P4HA3 prevented the recombinant human type III collagen from triplating, thus affecting its activity.

[0070] Example 4

[0071] A 0.5mm thick sample of detached pigskin was taken, subcutaneous fat was scraped off, and the sample was fixed in a Franz diffusion cell (with an effective diffusion area of ​​1.0cm²). 2 The receiving chamber (5 mL) was filled with PBS buffer (pH 7.4) and kept at a constant temperature of 37°C with stirring. The experimental cases of triple-helical recombinant human type III collagen and deletion case 1 were fluorescently labeled with FITC (labeling rate > 90%), and a 1 mg / mL PBS solution was prepared. 200 μL of this solution was evenly spread onto the surface of pig skin. The receiving solution was collected at 2 h, 6 h, 12 h, and 24 h, and an equal volume of fresh PBS solution was added. The fluorescence intensity in the receiving solution was measured using a fluorescence spectrophotometer (excitation wavelength 490 nm, emission wavelength 520 nm), and the cumulative permeation was calculated.

[0072] This embodiment aims to evaluate the transdermal absorption efficiency of the triple-helix recombinant human type III collagen prepared in Example 2 using the Franz diffusion cell method, expressed as the cumulative transdermal absorption (μg / cm³). 2 The experimental results are shown in Table 2.

[0073] Table 2: Cumulative amount of collagen absorbed transdermally

[0074]

[0075] As shown in Table 2, the cumulative transdermal amount of the triple helix recombinant human type III collagen with the lead peptide fused within 24 hours was significantly higher than that of the absence case 1, indicating that the lead peptide effectively promotes the protein's ability to penetrate the skin and has the potential for application in cosmetics.

[0076] Example 5

[0077] To ensure the rigor and repeatability of the experiment, Yiweitang Testing Technology (Shanghai) Co., Ltd. was commissioned to verify the transdermal effect. Details are as follows:

[0078] I. Purpose of the Test

[0079] Quantitatively evaluate the penetration efficiency of triple-helix recombinant human type III collagen in a whole-skin organ-on-a-chip model to provide data support for cosmetic formulation design.

[0080] II. Testing Principles

[0081] The whole-skin organ-on-a-chip model is based on fluorescent labeling technology: after the sample is labeled with FITC, it is applied to the epidermis. The concentration of fluorescent substances through the skin barrier reflects the permeability. The distribution of fluorescence signals is captured by laser confocal microscopy, and the penetration depth and intensity are semi-quantitatively analyzed by ImageJ software.

[0082] III. Experimental Materials and Instruments

[0083] Reagents: DPBS, triple-helix recombinant human type III collagen, fluorescein isothiocyanate (FITC), 4% paraformaldehyde, sucrose (10%, 20%, 30% solutions), OCT embedding agent.

[0084] Model: Full-skin organ-on-a-chip model.

[0085] Instrument: NIKON AX laser confocal microscope.

[0086] IV. Test Procedure

[0087] 4.1 Main operating steps

[0088] ① Sample preparation and fluorescent labeling: Triple-helix recombinant human type III collagen was labeled with FITC to generate fluorescence for subsequent quantitative and visual analysis.

[0089] ② Experimental treatment: 200 μL of sample was evenly spread on the surface of the model. 500 μL of culture medium solution (to receive osmotic substances and provide nutrients) was added to the lower chamber. The mixture was incubated at 37°C and 5% CO2 for 2 hours. Then, it was gently rinsed 3 times with pre-warmed DPBS to remove residual substances.

[0090] ③ Fixation: The model tissue was fixed for 2 hours at 4°C using 4% paraformaldehyde (PFA).

[0091] ④ Dehydration, embedding and sectioning: The model was sequentially dehydrated in 10%, 20% and 30% sucrose solutions until it sank to the bottom of the 30% sucrose solution, indicating that dehydration was complete. The dehydrated skin model was then embedded in OCT embedding agent and frozen sectioned with a section thickness of 10 μm.

[0092] ⑤ Observation: The distribution of FITC fluorescence signal in the sections was observed using a laser confocal microscope to evaluate the transdermal penetration and intradermal retention of the fluorescent label.

[0093] 4.2 Data Processing

[0094] Fluorescence signals were acquired and semi-quantitatively analyzed using the built-in software of the laser confocal microscope and image processing software such as ImageJ. Data were processed and plotted using Excel and GraphPad Prism 9.0 software. Data are expressed as mean ± SEM.

[0095] V. Experimental Results

[0096] Fluorescence permeation distribution map of whole-skin organ-on-a-chip model as shown in the figure. Figure 2 As shown, the fluorescence signal of triple-helix recombinant human type III collagen is strong and uniformly distributed on the skin surface, indicating a high retention rate. The fluorescence signal gradually weakens with increasing skin depth, suggesting that the main penetration depth of triple-helix recombinant human type III collagen is concentrated in the epidermis, with some reaching the dermis. This distribution characteristic indicates that triple-helix recombinant human type III collagen has a certain transdermal capacity.

[0097] The average total fluorescence intensity of triple-helix recombinant human type III collagen in a whole-skin organ-on-a-chip model was 53669 ± 1080. This high fluorescence intensity reflects its overall retention in the skin, indicating that triple-helix recombinant human type III collagen molecules can form a relatively thick retention layer on the skin surface. Data from parallel experimental groups showed relatively stable fluorescence intensity values ​​with small fluctuations, demonstrating good reproducibility and reliability of the experimental results.

[0098] Example 6

[0099] HSF cells (purchased from Shanghai Sangon Biotech) were stored at 5 × 10⁶ cells per well. 3Cells were seeded in 96-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free medium, and the triple-helix recombinant human type III collagen (final concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL) prepared in Example 2 were added to each well. Blank medium served as a negative control, and commercially available recombinant type III collagen (final concentration of 10 μg / mL) served as a positive control. After 48 h of further culture, 10 μL of CCK-8 reagent was added to each well, and after incubation for 2 h, the OD450nm value was measured to calculate the cell proliferation rate. Simultaneously, the cell supernatant was collected, and collagen secretion was detected using a human type I collagen ELISA kit (purchased from Shanghai Sangon Biotech). This example verifies the promoting effect of triple-helix recombinant human type III collagen on the proliferation and collagen synthesis of human skin fibroblasts (HSF). The results are shown in Table 3 below.

[0100] Table 3: Effects of collagen on the proliferation rate of human skin fibroblasts and collagen synthesis

[0101]

[0102] As shown in Table 3, the cell proliferation rate and type I collagen secretion in the experimental cases at concentrations of 50 μg / mL and 100 μg / mL were significantly higher than those in the negative control group, and were comparable to or better than those in the positive control group, indicating that the triple-helix recombinant human type III collagen of the present invention has good biological activity.

[0103] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A triple-helical recombinant human collagen type III, characterized in that, 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 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, and the protein sequence is shown as SEQ ID NO.

4. The Linker sequence is (GGGGS)n, wherein n is 1-5, 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 proline hydroxylase is shown as SEQ ID NO.

3.

2. The triple-helical recombinant human collagen type III of claim 1, wherein, The gene sequence encoding the protein sequence SEQ ID NO. 4 is shown as SEQ ID NO.

5.

3. A method for the preparation of a triple helical recombinant human collagen type III according to any one of claims 1-2, 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 the triple helix recombinant human type III collagen.

4. The production method according to claim 3, wherein The recombinant expression plasmid is selected from pPic9k.

5. The production method according to claim 3, wherein The recombinant Pichia pastoris is selected from GS115 Pichia pastoris.

6. A recombinant Pichia pastoris bacterium expressing the triple-helical recombinant human collagen type III of claim 2, characterized in that, The recombinant Pichia pastoris is transformed with the gene sequence of claim 2.

7. Use of the triple helix recombinant human type III collagen of any one of claims 1-2 in the preparation of a cosmetic product.

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