Humanized III-type collagen with stable triple-helix structure as well as preparation method and application of humanized III-type collagen

Abstract

The invention belongs to the field of bioengineering, and discloses a soluble humanized III-type collagen with a stable triple helix structure, and a preparation method and application thereof. According to the invention, a bioinformatics method is adopted to preferably splice functional fragments from a humanized III-type collagen sequence, and a coding gene is integrated into a pichia pastoris genome in a multi-copy form to obtain a high-expression humanized III-type collagen strain; high-expression target protein is obtained through biological fermentation, the target protein with the purity of 95% or above can be obtained through one-step chromatography, the production efficiency is obviously improved, the process is simple and easy to operate, and the method is suitable for industrial amplification. The prepared humanized III-type collagen has a triple helix structure, is stable in structure, has the activities of promoting cell migration, promoting cell proliferation, resisting inflammation and the like, and has great value in the fields of medical beauty and medicine.

Description

Technical Field

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

[0002] Collagen is the most abundant and essential protein in the human body, found in various tissues such as skin, bones, tendons, cartilage, and blood vessels, playing a vital role in cellular and tissue life activities. It accounts for approximately 30% of the total protein in the body. The collagen family includes at least 28 different types of collagen, with type III collagen accounting for about 5% to 15% of the total collagen in the human body. It is an important structural protein, mainly distributed in the collagen microlayer between the epidermis and dermis of the skin.

[0003] Type III collagen, a triple-helix structure composed of three α1 chains, is a key component in maintaining skin elasticity and repair capabilities. Together with type I collagen, it forms the skin's basement membrane, providing strength and elastic support. During the regeneration of skin and other tissues, it helps promote wound healing and cell migration. Simultaneously, type III collagen plays a crucial role in the development and maintenance of blood vessels. As a major component of the blood vessel wall, it, along with elastic fibers, forms the "elastic scaffold" of blood vessels, enabling them to expand and contract with blood pressure fluctuations and preventing damage caused by pressure changes.

[0004] Current technologies for preparing type III collagen primarily rely on animal cartilage extraction or genetic engineering. Currently, 90% of collagen is extracted from animal tissues, including bovine, swine, fish, mouse, or human (embryo) sources. However, animal collagen suffers from various problems and risks, such as heterologous side effects due to species differences, animal virus risks, and low abundance making extraction difficult, significantly limiting the development of the collagen industry. In contrast, type III collagen prepared using genetic engineering techniques offers advantages such as a designable DNA sequence, 100% homology with human amino acid sequences, avoidance of viral risks, high yield, high purity, and ease of industrial production. Given the numerous functions of type III collagen, it shows immense application potential for improving skin aesthetics, promoting wound healing, and in biomedical materials.

[0005] Recombinant collagen expression systems are mainly divided into prokaryotic and eukaryotic expression systems, represented by *Escherichia coli* and *Pichia pastoris*, respectively. *E. coli* expression systems are mature and have short culture cycles, but they cannot perform complex protein modifications (such as glycosylation), resulting in inactive proteins with high endotoxin content. *Pichia pastoris*, on the other hand, possesses many advantages of eukaryotic expression systems, such as protein processing, folding, and post-translational modifications. It often uses secretory expression, which facilitates subsequent purification and large-scale production.

[0006] There is considerable research on type III collagen in the market, currently focusing primarily on humanized collagen. This involves using genetic engineering techniques to repeatedly replicate small sequences, or splicing small sequences and then repeating them multiple times. However, this method results in humanized collagen with a limited variety of amino acid fragments, high fragment similarity, and significant differences from human type III collagen sequences. Most humanized collagen lacks a triple helix structure, exhibits poor thermal stability, and shows considerable differences in biological activity or function compared to human type III collagen. Taking Pichia pastoris as an example, conventional large-scale fermentation expression yields are limited, making it difficult to achieve high expression levels at the laboratory level. Conventional separation methods mainly use disc centrifuges, which suffer from expensive equipment and increased workload for subsequent purification due to the increased final volume of the feed solution.

[0007] CN115521372A discloses a recombinant humanized type III collagen with a triple helix structure. Its sequence is an n-repetition of three basic amino acid units (Gly-XY), containing characteristic collagen peptides or functional peptides. Its sequence can only match the human collagen sequence via the Gly-XY tripeptide. The sequence of CN115521372A is not derived from characteristic or functional fragments of the original human collagen sequence.

[0008] CN112194720A discloses a recombinant human type III collagen with a triple helix structure, which is formed by co-expression of the proline hydroxylase gene of Chlorella paramecium virus 1, and by initiating the formation of a triple helix structure from a single amino acid chain through proline hydroxylation. Its sequence differs significantly from that of human collagen.

[0009] CN119331080A describes a recombinant human type III collagen protein assembled into collagen fibers using a triple helix structure. It is mainly composed of 8-20 tandem repeats of the C-terminal propeptide. It is expressed using a high-cost mammalian cell expression system and its sequence differs significantly from that of human collagen. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides a stable method for preparing and applying triple-helical humanized type III collagen. The preparation method and application have been studied in pilot-scale trials. This invention directly extracts a longer characteristic or functional fragment from the original human collagen sequence, specifically the N-terminal 12 amino acids and the C-terminal 24 consecutive amino acids. This amino acid sequence has a specific function in forming a triple-helical structure. This invention uses a low-cost Pichia pastoris expression system, and the expressed sequence ultimately forms a triple-helical structure through self-assembly. Therefore, the triple-helical humanized type III collagen sequence of this invention is closer to the human collagen sequence.

[0011] In a first aspect of the invention, a polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen is provided, wherein the polynucleotide sequence is as shown in SEQ ID NO.1.

[0012] In a second aspect of the invention, a triple-helix recombinant humanized type III collagen is provided, wherein its amino acid sequence is shown in SEQ ID NO.2.

[0013] In one embodiment of the second aspect of the present invention, the triple-helix recombinant humanized type III collagen is obtained by encoding and expressing the polynucleotide sequence or its degenerate sequence described in the first aspect.

[0014] In a third aspect of the invention, a vector is provided that carries a polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen as described in the first aspect. The vector is preferably a plasmid carrying the polynucleotide sequence, and more preferably pPIC9K.

[0015] In a fourth aspect of the invention, a microorganism is provided comprising the polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen as described in the first aspect, or the vector as described in the third aspect. The microorganism is preferably Pichia pastoris GS115.

[0016] In a fifth aspect of the present invention, a method for preparing recombinant humanized type III collagen with a stable triple helix structure is provided, characterized by comprising the following steps: culturing a strain containing the polynucleotide sequence shown in SEQ ID NO.1 in a culture medium; and separating and purifying the fermentation broth obtained from the culture to obtain recombinant humanized type III collagen with a triple helix structure.

[0017] In one embodiment of the fifth aspect of the present invention, the strain is a Pichia pastoris strain.

[0018] In one embodiment of the fifth aspect of the present invention, the culture includes: inoculating a strain containing the polynucleotide sequence shown in SEQ ID NO.1 into BMGY medium, shaking and culturing at 30°C until the OD reaches about 10 at 600nm, transferring to BSM medium for fermentation, culturing for 16-20 h, and ending fermentation after methanol-induced fermentation for 42 h.

[0019] In one embodiment of the fifth aspect of the present invention, the separation and purification includes: centrifuging the fermentation broth by continuous flow sedimentation, collecting the supernatant, clarifying and filtering it with a hollow fiber membrane and then changing the liquid, performing ion exchange chromatography and then desalting and concentrating it.

[0020] In one embodiment of the fifth aspect of the invention, 0.5-2.0 g / L Ly and 0.6-2.4 g / L Pro are added to the BSM culture medium.

[0021] In one embodiment of the fifth aspect of the present invention, the separation factor of the continuous flow sedimentation centrifugation is 1000 to 2000; the continuous feed rate is 2 L / min to 5 L / min.

[0022] In one embodiment of the fifth aspect of the present invention, the ion exchange chromatography employs one-step cation exchange chromatography.

[0023] In a sixth aspect of the invention, a recombinant humanized type III collagen with a triple helix structure is provided, wherein it remains stable at room temperature and shows no structural change after being subjected to a high temperature of 95°C for 1 hour.

[0024] In a seventh aspect of the invention, the use of the recombinant humanized type III collagen with the triple helix structure described herein in the preparation of compositions for use in the fields of cosmetic skincare, tissue engineering materials, and pharmaceuticals is provided.

[0025] Beneficial effects: This invention uses long peptide segments from natural collagen to splice them together, thereby maximizing the preservation of the biological activity and function of collagen.

[0026] This invention provides a humanized type III collagen with a stable triple helix structure, which has significant stability and biological functions compared to traditional humanized type III collagen, and is more widely used in tissue engineering and medicine.

[0027] This invention optimizes the traditional BSM culture medium by adding Gly and L-Pro, which significantly increases their expression levels. The expression level was detected by BCA and was greater than 10 mg / ml.

[0028] This invention employs a continuous flow sedimentation centrifugation method, which not only reduces equipment costs but also reduces process time costs.

[0029] This invention employs one-step strong cation exchange chromatography and one-step elution, which shortens the chromatography time and reduces the complexity of the chromatography operation while ensuring high purity and lowering the cost of chromatography packing materials. Attached Figure Description

[0030] Figure 1 This is a simplified plasmid map of recombinant humanized type III collagen.

[0031] Figure 2 This is the electrophoresis diagram of the fermentation optimization in Example 2. Figure 2Line 1 represents the protein molecular weight standard 26610, line 2 represents the protein before optimization, line 3 represents the protein after optimization, and line 4 represents the protein molecular weight standard 26610.

[0032] Figure 3 This is an electrophoresis image of humanized type III collagen obtained by SP chromatography in Example 3. Figure 3 Line 1 is the protein molecular weight standard 26616, line 2 is the sample before chromatography, line 3 is column pass-through 1, line 4 is column pass-through 2, and line 5 is SP chromatography elution.

[0033] Figure 4 This is the result of HPLC purification and identification of humanized type III collagen SP in Example 3.

[0034] Figure 5 These are the SDS-PAGE detection results after treatment at different temperatures in Example 4. Figure 5 Line 1 represents a sample boiled in water at 95℃ for 1 hour; line 2 represents a sample bathed in water at 80℃ for 1 hour; line 3 represents a sample bathed in water at 60℃ for 1 hour; line 4 represents a sample bathed at room temperature for 1 hour; and line 5 represents the protein molecular weight standard 26616.

[0035] Figure 6 The results are HPLC analysis results of samples from room temperature and boiling water at 95°C for 1 hour in Example 4. Figure 6 The upper middle section shows the HPLC results of the sample after being placed at room temperature, while the lower section shows the HPLC results of the sample after being boiled in water at 95°C for 1 hour.

[0036] Figure 7 This is the identification result of the N-terminus of humanized type III collagen in Example 5.

[0037] Figure 8 This is the identification result of the C-terminus of humanized type III collagen in Example 5.

[0038] Figure 9 The results are the characterization and identification of the triple helix structure of humanized type III collagen in Example 5.

[0039] Figure 10 This is the result of the cytotoxicity experiment of the humanized collagen in Example 6.

[0040] Figure 11 This is the result of the humanized type III collagen cell proliferation experiment in Example 6.

[0041] Figure 12 This is the result of the humanized type III collagen cell adhesion experiment in Example 6. Figure 12In the middle, the top four images, from left to right, show the SC group, the 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml recombinant humanized type III collagen sample groups, respectively; the bottom four images, from left to right, show the SC group, the 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml bovine type I collagen sample groups, respectively.

[0042] Figure 13 These are the results of the cell migration-promoting experiment using humanized type III collagen in Example 6. The left two columns of eight images show the SC group, and the 0.5 mg / ml, 0.1 mg / ml, and 0.05 mg / ml recombinant humanized type III collagen sample groups at different time points; the right two columns of eight images show the SC group, and the 0.5 mg / ml, 0.1 mg / ml, and 0.05 mg / ml bovine type I collagen sample groups at different time points.

[0043] Figure 14 This is the statistical result of cell migration promotion by humanized type III collagen in Example 6 (the sample group is humanized type III collagen lyophilized sponge, and the control group is bovine type I collagen lyophilized powder). .

[0044] Figure 15 This is the result of the regulation of firming and anti-wrinkle genes by humanized type III collagen in Example 6.

[0045] Figure 16 This is the result of the regulation of inflammatory factor genes by humanized type III collagen in Example 6.

[0046] Figure 17 This refers to the inhibitory effect of humanized type III collagen on HeLa cells in Example 6.

[0047] Biological sequence: Detailed Implementation

[0048] To obtain stable triple-helix humanized type III collagen, this invention uses the full-length sequence of human type III collagen [sequence number P02461 in Genebank] as a reference. Amino acids 174-185 from the N-terminus of human type III collagen are extracted, along with the 219 amino acid sequences in between [specifically positions 201-218, 354-365, 429-443, 459-476, 540-560, 648-680, 696-719, 735-752, 768-785, 843-857, 882-908, totaling 219 amino acids], and repeats this process twice. Then, 24 amino acids from positions 1173-1196 at the C-terminus are extracted and tandemly linked to obtain the amino acid sequence shown in SEQ ID NO.2 above.

[0049] Based on the designed amino acid sequence, obtain the corresponding polynucleotide sequence information.

[0050] In one embodiment of the present invention, a natural polynucleotide sequence is obtained based on the amino acid sequence of humanized type III collagen, an Xho I restriction endonuclease site is added to its 5' end, an EcoR I restriction endonuclease site is added to its 3' end, and then the polynucleotide sequence is optimized by Pichia pastoris codons and named Col3a1.

[0051] The Xho I site at 5708 bp of the pPIC9K vector was mutated from CTCGAG to CACGAG. The entire Col3a1 gene was synthesized, digested, ligated, and transformed into the pPIC9K vector to construct the pPIC9K-Col3a1 plasmid. The pPIC9K-Col3a1 plasmid was linearized with the restriction endonuclease Sal I, transformed into Pichia pastoris GS115 competent cells, and screened for histidine deficiency and 4 mg / mL genimycin. Positive clones, GS115-pPIC9K-Col3a1, were obtained. Finally, shake-flask selection was performed.

[0052] The selected GS115-pPIC9K-Col3a1 engineered strain was inoculated into 400 ml of BMGY medium and cultured at 30 ℃ with shaking until the OD600 reached approximately 10. It was then transferred to a 5 L fermenter containing 3 L of BSM medium, where the temperature was maintained at 29 ℃. The pH was adjusted with ammonia to maintain around 5.0, and dissolved oxygen was controlled at 20%–30%. Fermentation was carried out for approximately 86 hours to obtain the fermentation broth. The target protein, humanized type III collagen, was found in the supernatant.

[0053] The fermentation broth obtained from the above process was subjected to continuous flow sedimentation centrifugation, and the supernatant was collected. After clarification and filtration using a hollow fiber membrane, the liquid was changed, and ion exchange chromatography was performed. After the liquid was changed, humanized type III collagen was obtained.

[0054] Preferably, the BSM culture medium for expression contains 1.2 g / L of Gly and 1.2 g / L of Pro.

[0055] Preferably, the continuous flow sedimentation centrifugal separation factor is 1330; the continuous feed rate is 2.5 L / min.

[0056] Preferably, the chromatography step is a one-step SP strong cation exchange chromatography using SP Sepharose Fast Flow, and only one-step elution of the target protein is performed. The specific conditions are as follows: first, the chromatography column is equilibrated with loading buffer, then the target protein is loaded and bound, then reequilibrated with loading buffer, and finally the target protein is eluted directly with elution buffer, without any other impurity elution steps; wherein the loading buffer is pH 4.0 20mM acetate-sodium acetate + 0.03M NaCl; the elution buffer is 20mM pH 6.0 PB + 0.05M NaCl.

[0057] In addition, the present invention provides a recombinant humanized type III collagen with a stable triple helix structure, which has good heat resistance and shows no significant change when boiled in water at 95°C for 1 hour.

[0058] Finally, this invention provides the application of recombinant humanized type III collagen with a stable triple helix structure in the preparation of compositions for use in the fields of beauty and skincare, tissue engineering materials, and medicine.

[0059] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. The present invention is not limited to these embodiments.

[0060] Example 1

[0061] The preparation steps of humanized type III collagen engineered bacteria are as follows: 1. Obtain the humanized type III collagen polynucleotide sequence Twelve amino acids (positions 174-185) from the N-terminus of human type III collagen were extracted, and a 225-amino acid sequence (positions 210-908) was added and tandemly repeated twice. This was then followed by tandem repetition of the N-terminal sequence (positions 1173-1196), resulting in a total of 474 amino acids (SEQ ID NO. 2). The molecular weight was 39997.2 Da, and the polynucleotide sequence was 1422 bp. Pichia pastoris codon optimization yielded the humanized type III collagen polynucleotide sequence (SEQ ID NO. 1). An Xho I restriction site (CTCGAG) was added to the 5' end, and an EcoR I restriction site was added to the 3' end. This sequence was named Col3a1. The entire Col3a1 polynucleotide sequence was synthesized.

[0062] 2. Acquisition of pPIC9K-Col3a1 plasmid

[0063] The pPIC9K and Col3a1 genes were double-digested with Xho I and EcoRI, and then ligated into *E. coli* DH5α competent cells. *E. coli* strains containing the pPIC9K-Col3a1 plasmid were obtained through ampicillin resistance selection. The construction and sequencing of pPIC9K-Col3a1 were performed using standard techniques in this field. The pPIC9K-Col3a1 plasmid map can be found in [link to image]. Figure 1 .

[0064] 3. Transformation screening and shake-flask experiment

[0065] The pPIC9K-Col3a1 plasmid was linearized with the restriction endonuclease Sal I using conventional techniques in the art. It was then transformed into Pichia pastoris GS115 competent cells and screened for histidine deficiency and 4 mg / mL genimycin to obtain positive clones, specifically the GS115-pPIC9K-Col3a1 strain. The GS115-pPIC9K-Col3a1 engineered strain was inoculated into BMGY medium for shake-flask screening of high-expression strains.

[0066] Example 2

[0067] The fermentation expression process of humanized type III collagen is as follows: 1. Fermentation expression of humanized type III collagen: The selected GS115-pPIC9K-Col3a1 engineered strain was inoculated into 400 mL of BMGY medium and cultured at 30 ℃ with shaking until OD. 600 The culture medium was approximately 10 mg / L, then transferred to a 5 L fermenter containing 2 L of BSM medium. The temperature was maintained at 30 °C, and the pH was adjusted with ammonia to maintain around 5.0. Dissolved oxygen was controlled at 20%–30%. After 20 hours of culture in the small tank, the culture was transferred to a 150 L fermenter for scale-up fermentation. Methanol-induced fermentation lasted approximately 66 hours. After fermentation, the fermentation broth was centrifuged to separate the solid and liquid components, retaining the supernatant, which contained the target protein. BCA protein content analysis showed an expression level exceeding 3.5 g / L.

[0068] 2. Optimization of the composition of BSM medium: The optimized BSM medium composition is based on the basic inorganic salt content (CaSO4·2H2O is 0.93 g / L). S The optimal concentrations of BCA protein were 18.2 g / L, MgSO4·2H2O was 14.9 g / L, KOH was 4.13 g / L, 1.2 g / L ly, 1.2 g / L Pro, carbon source (glycerol 40 g / L), pH adjuster (phosphate 26.8 ml / L), and trace elements were added at 8 ml / L according to the PTM1 formula ratio (added after sterilization of the culture medium). The pH was adjusted to 5.0, and fermentation was carried out in the optimized culture medium with the addition of induction medium (50% (w / v) sorbitol (sterilized): methanol = 1:6:, PTM1 4 ml / L) for induction expression. The expression level increased to 10 g / L according to the BCA protein content detection. The optimized fermentation expression results are shown in the figure. Figure 2 .

[0069] The results showed that the expression level was significantly increased after adding 1.2 g / L Lly and 1.2 g / L Pro to the original BSM medium.

[0070] Example 3

[0071] 1. Crude separation of humanized type III collagen

[0072] After fermentation, 110L of fermentation broth was collected, and a sedimentation centrifuge was run. After the centrifuge frequency was stabilized at 50HZ (separation factor of 1333), the position of the centrifuge skimming tube was adjusted, and the fermentation broth was pumped into the centrifuge at a flow rate of 2.5L / min. 75L of centrifugal supernatant was collected from the skimming tube. 75L of supernatant was filtered through a hollow fiber membrane, and then washed with 30L of purified water. The filtrates were combined and collected to 100L. The filtrate was concentrated 10 times using a 10kd ultrafiltration membrane, and then washed 5 times with pH 4.2 10mM acetate-sodium acetate buffer. 15L of ultrafiltration retentate was collected.

[0073] 2. One-step SP chromatography purification

[0074] 10L of SPFF chromatography packing material was packed into a 200mm... A 300 mm chromatography column was rinsed with purified water for 10 column volumes (10 CV) to remove residual ethanol. The SP column was then equilibrated to 5 CV with loading buffer (pH 4.0, 20 mM acetate-sodium acetate + 0.03 M NaCl). 15 L of the collected ultrafiltration retentate was loaded at a flow rate of 400 mL / min. After loading, the SP column was equilibrated again with loading buffer to wash away unbound impurities. The target analyte was then eluted with elution buffer (20 mM pH 6.0 PB + 0.05 M NaCl). 10 L of the eluent was collected as the target protein and analyzed. The electrophoresis results are shown below. Figure 3 The HPLC analysis results of the target analyte are as follows: Figure 4 .

[0075] 3. Preparation of sterile humanized type III collagen stock solution

[0076] The collected SP chromatography eluent was ultrafiltered using a 10kDa ultrafiltration membrane, concentrated to 2L, and then purified water was added for 8-fold desalting. The final concentrate was about 3L, which was then filtered through a 0.2µm sterile filter membrane to obtain a sterile solution of humanized type III collagen.

[0077] In Comparative Example 1, the SPFF chromatography packing material was replaced with MMC multimode chromatography packing material. The loading buffer was pH 4.4 20mM acetate-sodium acetate, elution buffer 1 was pH 4.4 20mM acetate-sodium acetate + 0.05M NaCl, and elution buffer 2 was pH 4.4 20mM acetate-sodium acetate + 0.2M NaCl. The chromatography column was first equilibrated with the loading buffer, and after equilibration, the sample was loaded. After loading, the column was reequilibrated with the loading buffer, and then eluted with elution buffer 1, which contained some of the target protein and a large amount of other proteins. Finally, elution buffer 2 was used to elute the target protein.

[0078] In Comparative Example 2, SPFF chromatography packing material was used, and impurities were eluted using pH 4.0 20mM citric acid-sodium citrate as loading buffer and pH 4.0 20mM citric acid-sodium citrate + 0.05M NaCl as elution buffer 1. Then, the target analyte was eluted using pH 4.0 20mM citric acid-sodium citrate + 0.15M NaCl as elution buffer 2.

[0079] In Comparative Example 3, PhenylFF was used, and column equilibration was performed using pH 7.0 20mM MPB + 1.2M (NH4)2SO4 as the loading buffer. The crude separated sample was supplemented with (NH4)2SO4 to prepare a protein sample containing 1.2M (NH4)2SO4, and the sample pH was adjusted to 7.0-7.5 with NaOH. After column equilibration, the sample was loaded, and impurities were eluted with pH 7.0 20mM MPB + 0.8M (NH4)2SO4. Finally, the target protein was eluted with pH 7.0 20mM PB + 0.45M (NH4)2SO4.

[0080] Chromatographic purification of recombinant humanized type III collagen was performed using Examples 3, 1, 2, and 3. As shown in Table 1, all three comparative examples exhibited low purity, and the fractional elution resulted in significant loss of the target protein, leading to low yields. Example 1 demonstrated the achievement of high purity and high yield of the target protein during the purification of humanized type III collagen.

[0081] Table 1. Comparative Analysis of Results from Different Chromatography Processes

[0082] Example 4

[0083] High-temperature resistance test of human type III collagen

[0084] Humanized type III collagen stock solution was diluted with 20mM PB at pH 7.0 to prepare 100ml samples of 1mg / ml each. These samples were then divided into four groups, with four experimental settings: room temperature, 60℃, 80℃, and boiling water at 95℃. The samples were treated at each of the four temperatures for 1 hour. SDS-PAGE analysis showed no significant differences, indicating good high-temperature stability. Figure 5 As shown, size exclusion chromatograms of water at room temperature and boiling water at 95℃ for 1 hour were compared simultaneously. Figure 6 There was no significant difference, indicating that the recombinant type III collagen has a stable structure and good thermal stability.

[0085] Example 5

[0086] Identification and structural characterization of recombinant humanized type III collagen

[0087] 1. Protein sequence identification

[0088] Take an appropriate amount of the test sample, heat at 95℃ for 5 min, cool to room temperature, add Asp-N enzyme digestion at 37℃ for 2 h, and add 10% FA to terminate the reaction. Take half of the digested product, add TCEP for reduction at room temperature for 1 h, separate the digested peptides using ultra-high performance liquid chromatography (UHPLC), and then perform high-resolution mass spectrometry (HMS) detection and analysis. Data retrieval and analysis were performed using UNIFI software, and the N-terminal and C-terminal sequences were finally calculated. The results show that the N-terminal sequence is GPAGPPGPPGPPGPPGEPGQAGPSGPPGPPGPAGSPGSNGAPGGAGEPGKNGAKGEPGAKGE; the C-terminal sequence is DGPPGPAGSEGSPGHPGQPGPPGPPGAPGPC. The identification results are shown in the figure. Figure 7 and Figure 8 This indicates that the N-terminal and C-terminal sequences of humanized type III collagen are consistent with the theoretical sequences.

[0089] 2. Structural Characterization

[0090] A collagen sample was prepared to a concentration of 0.2 mg / ml. A 0.5 mm cuvette was used, and a blank control was prepared using a buffer. Detection was performed using a circular dichroism chromatograph. The sample showed a negative peak at 190-200 nm and a positive absorption peak near 220 nm. The results are as follows. Figure 9 As shown, this indicates that the humanized type III collagen has a triple helix structure.

[0091] Example 6

[0092] biological function

[0093] All tests involved in this embodiment used software to perform T-tests on logarithmic values. A p-value < 0.05 was considered statistically significant compared to the control group. "P < 0.01" indicates a highly statistically significant difference compared to the control group. "express.

[0094] 1. Cytotoxicity detection

[0095] Cytotoxicity assay: Cytotoxicity was detected using the CCK-8 assay. Fibroblasts (HFB) in logarithmic growth phase were cultured at a concentration of 2.5 × 10⁻⁶ cells / year. 4 Cells were seeded at 100 μL / well in 96-well plates and cultured for 24 h. When the cell deposition rate reached 40%–60%, the following treatments were administered: ① Sample group: 200 μL of culture medium containing different concentrations (10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL) of the sample was added to each well; ② Blank group: 200 μL of culture medium was added; ③ Zeroing group: no cells were added, only 200 μL of culture medium was added; ④ Positive control group (PC, 4% DMSO). After 24 h of culture, the medium was discarded and replaced with 100 μL / well of culture medium containing CCK-8 reagent (10%). Culture was continued for 2 h, and OD was measured using a microplate reader. 450 The relative cell viability is calculated using the following formula (1): (1) The calculation results are shown below. Figure 10 The results showed that samples with a concentration of ≤10 mg / mL had no significant cytotoxicity on fibroblasts (HFB) after 24 h of treatment.

[0096] 2. Cell proliferation activity assay

[0097] Fibroblasts (HFB) were resuscitated and then cultured in DMEM medium containing 10% FBS. HFB cells in the logarithmic growth phase were cultured at a rate of 5 x 10⁻⁶ cells / year. 4 Cells were seeded at 100 μL / well in 96-well plates. Humanized type III collagen was diluted with purified water to three concentrations: 0.5 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. Similarly, a control (bovine type I collagen) was prepared at concentrations of 0.5 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. Cell viability was assessed using the MTT assay after 0 h, 24 h, 48 h, and 72 h. Cell proliferation results are shown in the figure below. Figure 11The results showed that humanized type III collagen at concentrations of 0.5 mg / mL, 0.1 mg / mL, and 0.05 mg / mL significantly promoted cell proliferation in HFB cells at 0 h, 24 h, 48 h, and 72 h.

[0098] 3. Cell adhesion

[0099] Set up a blank control group: Add 100 μL of PBS to a 96-well plate and coat it at 37°C for 2 h, with three replicates per group. Sample group / control group: Add 100 μL of recombinant type III collagen sample solution and bovine type I collagen sample solution of different concentrations (0.1 mg / ml, 0.05 mg / ml, 0.01 mg / ml) to a 96-well plate and coat it at 37°C for 2 h. Remove excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate at 37°C in a 5% CO2 incubator for 1 h. Remove the liquid from the wells, wash three times with PBS, remove the washing solution, seal with sealing film, and store at 4°C for later use.

[0100] HFB cells in logarithmic growth phase were divided into groups of 2 x 10-1. 4 Seeds were inoculated at a density of cells / well into the coated and incubated 96-well plates prepared above. The plates were incubated at 37°C in a 5% CO2 incubator for 2 h. The supernatant was discarded, and the plates were washed with PBS, fixed with 4% paraformaldehyde for 10 min, washed to remove residual fixative, stained with Hoechst 33342 staining solution, and then photographed and counted. Results are as follows: Figure 12 The statistical results are shown in Table 2.

[0101] Table 2. Cell adhesion detection results

[0102] The results showed that, compared with the negative control group, after 24 hours of treatment on HFB cells, the percentage of cell adhesion in the sample groups with different concentrations (0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml) was increased by 80.02%, 63.35%, and 51.10%, respectively.

[0103] 4. Cell migration

[0104] HSF cells in logarithmic growth phase were divided into groups of 2.5 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight at 37°C with 5% CO2. Once the plating rate reached over 70%, a scratch assay was performed. Two vertical scratches were made in each well using a 10 μL pipette tip, with the vertical scratch serving as the baseline (the pipette tip perpendicular to the edge of the ruler). The spacing between the scratches was maintained at 2 cm. After making the vertical scratches, horizontal scratches were made near the center axis of the 6-well plate, perpendicular to the baseline. After scratching, cells were washed three times with PBS. Three concentrations (0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml) were prepared for the sample group and control group, with 2 ml of the drug administered to each well. The negative control group was treated with 2 ml of serum-free culture medium. Cells were incubated for another 24 hours at 37°C with 5% CO2. Cells were photographed after scratching. Cell migration results are shown below. Figure 13 and Figure 14 .

[0105] Depend on Figure 13 and Figure 14 It can be seen that, compared with the negative control group, humanized type III collagen at three experimental concentrations of 0.1 mg / ml, 0.05 mg / ml and 0.01 mg / ml can significantly promote HSF cell migration, increasing cell migration rate by 97%, 88% and 29% respectively compared with the negative control.

[0106] 5. Genetic testing

[0107] Using real-time quantitative PCR, HSF cells in the logarithmic growth phase were divided into groups of 6 × 10⁻⁶ cells. 5 Cells were seeded at a density of 10 cells / well into 6-well plates and incubated in an incubator (37℃, 5% CO2) for 24 hours. When the cell deposition rate in the 6-well plates reached 40%–50%, the cells were divided into two groups and treated with 1.25 mg / mL and 0.625 mg / mL, with 2 mL of drug per well. After drug administration, the 6-well plates were incubated in an incubator (37℃, 5% CO2) for 24 hours. After culture, the cells were washed three times with 2 mL / well PBS, and 1 mL of RNAisoPlus was added to each well. After cell lysis by pipetting, the cell lysate was collected for RNA extraction, reverse transcription, and quantitative real-time PCR to detect gene levels. 2-△△ The results were calculated using the CT method. See the results below. Figure 15 The results showed that after 24 hours of treatment on HSF cells, the levels of the firming and anti-wrinkle genes COLⅣ, COLⅦ, FN, and TIMP1 were significantly increased at a concentration of 1.25 mg / mL, by 330.7%, 375.7%, 243.2%, and 402.3%, respectively. At a concentration of 0.625 mg / mL, the levels of the same genes were also significantly increased by 510.8%, 299.8%, 462.1%, and 504.1%, respectively, indicating that the sample has firming and anti-wrinkle effects.

[0108] 6. Regulation of inflammatory cytokine genes

[0109] Real-time quantitative PCR was used. RAW264.7 cells in logarithmic growth phase were divided into groups of 6.4 × 10⁻⁶ cells. 5 Seeds were planted at a density of cells / well into 6-well plates and incubated overnight at 37°C with 5% CO2. The experiment consisted of a blank control group (BC), a model group (NC), a positive control group (20 μM dexamethasone), and a sample group. Fresh culture medium was added to the blank control group. The model group (NC) was cultured in medium containing 0.2 μg / mL LPS, while the experimental group was cultured in medium containing 0.2 μg / mL LPS and humanized type III collagen samples (concentrations of 0.313 mg / mL and 0.156 mg / mL). After 24 h of culture, the cells were washed twice with 2 mL / well of PBS, and 1 mL of RNAiso Plus was added to each well. After cell lysis by pipetting, the cell lysates were collected for RNA extraction, reverse transcription, and quantitative real-time PCR to detect the expression levels of related inflammatory factor genes. -△△ The results are calculated using the CT method.

[0110] After 24 hours of treatment on RAW264.7 cells, compared with the model group (NC), the levels of IL-6, TNF-α, IL-1α, and COX2 genes were significantly decreased at concentrations of 0.313 mg / mL and 0.156 mg / mL, respectively, by 19.7%, 31.3%, 38.7%, 49.8% and 36.2%, 46.2%, 54.2%, 64.2%, respectively. This indicates that recombinant type III collagen can significantly reduce the expression of inflammatory factors. The results of the regulation of inflammatory factor genes by humanized type III collagen are shown below. Figure 16 .

[0111] 7. Inhibitory effect on cancer cells

[0112] Using the MTT assay, HeLa cells in the logarithmic growth phase were divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded at 100 μL / well in 96-well plates. The experiment consisted of a control group (SC) and a sample / control group. Drug administration and culture methods were the same as in the cytotoxicity assay. Three concentration gradients (1 mg / mL, 0.5 mg / mL, 0.125 mg / mL, 0.025 mg / mL) were set up for the sample / control group, with three replicates for each gradient. Cell viability was measured at 0 h, 24 h, 48 h, and 72 h. Proliferation curves were plotted based on cell viability. (See figure). Figure 17 The results are shown in the "Average" section of Table 3.

[0113] The results showed that recombinant humanized type III collagen lyophilized sponges at concentrations of (1 mg / mL, 0.5 mg / mL, 0.125 mg / mL, and 0.025 mg / mL) significantly inhibited HeLa cell growth after 48 and 72 hours of treatment. This product's recombinant humanized type III collagen can inhibit the growth of some cancer cells to a certain extent.

[0114] Table 3. Results of cell proliferation assay for recombinant humanized type III collagen lyophilized sponge.

[0115] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Researchers in related fields can obtain other embodiments based on these embodiments without creative intent, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen, characterized in that: The polynucleotide sequence is shown in SEQ ID NO.

1.

2. A triple-helix recombinant humanized type III collagen, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. The triple-helix recombinant humanized type III collagen as described in claim 2, characterized in that: It is obtained by encoding the polynucleotide sequence or its degenerate sequence as described in claim 1.

4. A vector carrying a polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen as described in claim 1.

5. A microorganism comprising the polynucleotide sequence encoding a triple-helix recombinant humanized type III collagen as described in claim 1, or the vector as described in claim 4.

6. A method for preparing recombinant humanized type III collagen with a stable triple helix structure, characterized in that, Includes the following steps: The strain containing the polynucleotide sequence shown in SEQ ID NO.1 was cultured in a culture medium; and The fermentation broth obtained from the culture was separated and purified to obtain recombinant humanized type III collagen with a triple helix structure.

7. The preparation method according to claim 6, wherein, The strain is a Pichia pastoris strain; and / or The culture includes: inoculating a strain containing the polynucleotide sequence shown in SEQ ID NO.1 into BMGY medium, incubating at 30°C with shaking until the OD reaches approximately 10 at 600 nm, transferring to BSM medium for fermentation, incubating for 16-20 h, and then stopping fermentation after methanol-induced fermentation for 42 h; and / or The separation and purification process includes: centrifuging the fermentation broth through continuous flow sedimentation, collecting the supernatant, clarifying and filtering it with a hollow fiber membrane, changing the liquid, performing ion exchange chromatography, and then desalting and concentrating it.

8. The preparation method according to claim 7, characterized in that, Add 0.5-2.0 g / L Lly and 0.6-2.4 g / L Pro to the BSM medium; and / or The separation factor of the continuous flow sedimentation centrifuge is 1000 to 2000; the continuous feed rate is 2 L / min to 5 L / min.

9. The preparation method according to claim 6, characterized in that, The ion exchange chromatography employed a one-step strong cation exchange chromatography.

10. A recombinant humanized type III collagen with a triple helix structure, characterized in that, It remains stable at room temperature and shows no structural change after being exposed to 95°C for 1 hour.

11. The use of the recombinant humanized type III collagen with the triple helix structure as described in claim 2, 3 or 10 in the preparation of compositions for use in the fields of cosmetic skincare, tissue engineering materials and pharmaceuticals.

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