Recombinant III-type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. On the basis of human-derived III-type collagen COL3A1, an amino acid sequence suitable for efficient expression of pichia pastoris is obtained after optimization design and screening, a recombinant expression vector is provided on the basis, and recombinant engineering bacteria are constructed. The recombinant III-type micromolecular collagen is successfully expressed by adopting the constructed recombinant engineering bacteria, and the invention further provides a preparation method of the recombinant III-type micromolecular collagen. According to the present invention, the efficient expression of the recombinant III-type micromolecule collagen in the yeast is achieved, and the recombinant III-type micromolecule collagen has characteristics of high stability, and no significant degradation under different pH conditions, repeated freezing and thawing treatment, and different temperature and time duration treatment; the main band accounts for 90% or above in the fermentation stage, the purification difficulty is low, the sponge purity is 95% or above, and the cell adhesion activity is excellent while the stability is high.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to recombinant type III collagen, its preparation method, and its application. Background Technology

[0002] Collagen is an important component of the extracellular matrix (ECM) in animal organisms. It is a helical fibrous protein composed of three polypeptide chains. In the skin, it forms a dense elastic network, locking in moisture, supporting the skin, making cells plump and hydrated, resulting in elastic, moisturized, delicate, and smooth skin. In connective tissues such as cartilage, bone, tendons, and ligaments, the triple helix structure of collagen provides mechanical properties, constructing a fibrous scaffold that provides cells with high tensile strength and stability. Due to its excellent biocompatibility, bioactivity, and biodegradability, collagen is now widely used in many fields such as chemical engineering, pharmaceuticals, food, and cosmetics.

[0003] Research and application of recombinant collagen has a history of over 30 years. Existing literature and patents mainly focus on the expression of single-chain human collagen in different hosts and the expression of truncated single-chain collagen genes. A small number of studies focus on the co-expression of collagen with related post-expression modifying enzymes to obtain triple-helix collagen. Among various recombinant collagen expression methods, Pichia pastoris can perform certain post-translational modifications on the translated protein (especially glycosylation), strongly supporting the realization of protein biological functions. Using Pichia pastoris to establish an expression system offers advantages for large-scale industrial production, including high-density fermentation production, extremely low culture costs, short cycle time, and high expression levels. The target protein can be secreted extracellularly, and Pichia pastoris itself secretes very little protein, simplifying the purification process. The cell wall components do not contain endotoxins or peptidoglycans. With a clear genetic background, the Pichia pastoris expression system is an ideal method for the large-scale industrial expression and production of recombinant human collagen.

[0004] Recombinant collagen is currently used more extensively in the cosmetics industry. With the continuous advancement of the market, an increasing number of medical devices also utilize recombinant collagen as a primary raw material. Due to the safety requirements of medical devices, the purity and stability of recombinant collagen are crucial. However, most current recombinant collagen expression focuses on active sites or more efficient expression. Some studies also aim to increase the stability of recombinant collagen by mutating certain amino acids, but this cannot achieve 100% homology of the raw material, posing a greater challenge to its use as a raw material for medical devices.

[0005] Existing patents for expressing recombinant collagen in yeast mainly focus on the successful expression of collagen sequences, aiming to express recombinant collagen. Some also involve the expression of artificially designed collagen-like sequences, but the selected type III collagen fragments have a large number of unstable sites, which leads to risks such as deamidation, oxidation, and hydrolysis during the purification and production of the expressed protein. This manifests as technical problems such as difficult purification, extremely low yield of full-length protein from a single band, high requirements for storage environment, and short storage time.

[0006] Therefore, existing research on recombinant collagen expression focuses more on cellular active sites and efficient expression, neglecting the stability design of the protein itself. This leads to the protein being prone to degradation during expression and production, and it will also degrade in application scenarios due to poor stability. This makes it difficult to apply recombinant collagen to medical devices, especially Class III medical devices. Summary of the Invention

[0007] The purpose of this invention is to overcome some technical problems existing in the prior art. By directional selection of the amino acid sequence of existing type III collagen, potential enzyme cleavage sites and unstable sequence fragments are avoided in sequence design, so that it can be efficiently expressed in Pichia pastoris and the collagen obtained after purification has high stability. Furthermore, taking into account factors such as related biological activity sites and hydrophilicity, it has related biological activities while maintaining high stability.

[0008] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0009] The recombinant type III small molecule collagen provided by the present invention includes a repeating tandem fragment A, wherein fragment A includes multiple highly stable sequence fragments that are easy to express efficiently in Pichia pastoris and are not easily cleaved by enzymes. The sequence fragments are selected from one or more of human type III collagen COL3A1, namely 501-518AA, 966-983AA, 792-809AA, and 966-983AA.

[0010] Furthermore, according to embodiments of the present invention, fragment A comprises any of the following combinations of sequence fragments:

[0011] a) 501-518AA, 966-983AA;

[0012] b) 792-809AA, 966-983AA.

[0013] Furthermore, according to an embodiment of the present invention, the number of times segment A is repeated in series is 10 or 14; when segment A is combination a), the number of times it is repeated in series is 10; when segment A is combination b), the number of times it is repeated in series is 14.

[0014] Furthermore, according to an embodiment of the present invention, LEKR is added to the N-terminus of the recombinant type III small molecule collagen.

[0015] Preferably, the recombinant type III small molecule collagen comprises the amino acid sequence described in SEQ ID No:1 or SEQ ID No:2.

[0016] The present invention also provides a polynucleotide encoding the above-mentioned recombinant type III small molecule collagen.

[0017] Furthermore, according to embodiments of the present invention, the polynucleotide sequence of the recombinant type III small molecule collagen is as shown in SEQ ID No:3 or SEQ ID No:4, or a degenerate sequence thereof.

[0018] The present invention also provides a recombinant expression vector comprising a polynucleotide encoding the above-mentioned collagen.

[0019] This invention also provides recombinant engineered bacteria or cells constructed from the above-mentioned recombinant expression vector, wherein the host cell of the recombinant engineered bacteria or cells is preferably *Pichia pastoris*. The bacterial strain preservation numbers of the recombinant engineered bacteria or cells are: CGMCC No. 39031 and CGMCC No. 39032; preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; preservation date: October 31, 2025; classification name: *Pichia pastoris* Komagataella phaffii.

[0020] The present invention also provides a method for preparing the recombinant type III small molecule collagen, comprising the following steps:

[0021] The recombinant engineered bacteria were inoculated into seed culture medium YPG to obtain seed liquid; the seed liquid was then inoculated into fermentation medium and the pH was adjusted; glycerol was added as feed during fermentation, and methanol and glycerol were used as a mixed carbon source for induction culture to obtain recombinant type III small molecule collagen.

[0022] According to embodiments of the present invention, the obtained collagen was treated under different pH conditions, repeated freeze-thaw cycles, and different temperatures and durations. Cell adhesion activity, cytotoxicity, and cell migration rate were tested on the collagen. The experiments verified that the recombinant protein had high stability, with no significant degradation under any of the stability test conditions. During the fermentation stage, the main band accounted for more than 90%, purification was easy, and the sponge purity was above 95%. Furthermore, it exhibited good cell adhesion activity, no potential cytotoxicity, and good biological activity.

[0023] The present invention also provides a composition comprising the recombinant type III small molecule collagen, or the recombinant expression vector, or the recombinant engineered bacteria or cells.

[0024] The present invention also provides an article comprising the recombinant type III small molecule collagen, or the recombinant expression vector, or the recombinant engineered bacteria or cells, or the recombinant type III small molecule collagen prepared by the method, or the composition thereof; preferably, the article is selected from pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0025] The present invention also provides the use of the recombinant type III small molecule collagen, or the polynucleotide, or the recombinant expression vector, or the recombinant engineered bacteria, or the recombinant type III small molecule collagen prepared by the method, or the composition, or the product thereof in the preparation of pharmaceuticals, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The S12 and S15 recombinant proteins provided by this invention have high stability, with the main band accounting for more than 90% during the fermentation stage, and are easy to purify. The purity of the sponges is more than 95%.

[0028] (2) The S12 and S15 recombinant proteins provided by the present invention, after purification, showed better performance than the original proteins in the stability test. No significant degradation occurred under any conditions of the stability test, which reduced the difficulty of storage and extended the storage time.

[0029] (3) The recombinant protein provided by this invention has similar physicochemical properties and biological functions to the original sequence. When used as a biological material, it has higher stability, high batch-to-batch consistency, and is easy to control in terms of quality, which is beneficial to product stability. Moreover, the method is simple and easy to operate, suitable for large-scale industrial production, and has broad market application prospects. Attached Figure Description

[0030] Figure 1 SDS-PAGE images of shake flask expression supernatant (induced for 48 h) of recombinant type III small molecule collagen S12 and S15.

[0031] Figure 2 SDS-PAGE image of the supernatant from the fermentation of recombinant engineered bacterial strains expressing recombinant type III small molecule collagen S12 and S15 in a 5L tank.

[0032] Figure 3 The SDS-PAGE results of recombinant type III small molecule collagen S12 and S15 lyophilized sponge solutions were obtained under different pH conditions and placed at 25°C for different numbers of days. The top figure shows the results for S12, and the bottom figure shows the results for S15.

[0033] Figure 4The results of SDS-PAGE validation of recombinant type III small molecule collagen S12 and S15 after repeated freeze-thaw cycles.

[0034] Figure 5 The results of SDS-PAGE verification of recombinant type III small molecule collagen S12 and S15 samples placed at different temperatures for different times are shown; the top figure shows the results of S12, and the bottom figure shows the results of S15.

[0035] Figure 6 The results of cytotoxicity experiments for recombinant type III small molecule collagen S12 and S15 are shown; the top figure shows the results for S12, and the bottom figure shows the results for S15.

[0036] Figure 7 The bar chart shows the cell adhesion results of recombinant type III small molecule collagen S12 and S15. The top bar chart shows the results at a concentration of 0.5 mg / mL, and the bottom bar chart shows the results at a concentration of 1.0 mg / mL.

[0037] Figure 8 The statistical results show the cell migration area ratio of recombinant type III small molecule collagen S12 and S15.

[0038] Figure 9 The recombinant type III small molecule collagen S12 obtained by expression was analyzed by Nano-HPLC-MS / MS mass spectrometry to compare the peptide sequence with the theoretical sequence.

[0039] Figure 10 The recombinant type III small molecule collagen S15 obtained by expression was analyzed by Nano-HPLC-MS / MS mass spectrometry to compare the peptide sequence with the theoretical sequence. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0041] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments, unless otherwise described, can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0042] The Pichia pastoris expression system is used in this embodiment of the invention, but it can be extended to other yeasts and other expression systems in the same way; it involves Pichia pastoris engineered strains that can express S12 and S15 (including but not limited to the expression vectors mentioned in this invention); and the same effect can be achieved by using different numbers of repetitions of the sequence of the present invention; the same effect can also be achieved by using different plasmid vectors and different engineered strains for the sequence involved in the present invention; the same effect can also be achieved by correspondingly lengthening or shortening the sequence involved in the present invention; simple adjustments to the relevant content are considered simple substitutions of the present invention. In addition, the terms "approximately" and "around" used in this embodiment do not indicate an unclear meaning, but are expressions based on the numerical fluctuations in the actual production process, and the errors of the values ​​corresponding to "approximately" and "around" are all within ±1%.

[0043] Example 1:

[0044] (1) Design of amino acid sequences

[0045] Using human type III collagen COL3A1 as the parent sequence (https: / / www.uniprot.org / uniprotkb / P02461), sequences with high stability, ease of efficient expression in Pichia pastoris, and resistance to enzyme digestion were selected. The specific design was as follows: Two fragments from the parent sequence, 501-518AA and 966-983AA, were tandemly linked and repeated 10 times to ensure their size matched the optimal size for Pichia pastoris expression of exogenous proteins, thus designing recombinant type III small molecule collagen S12. Two fragments from the parent sequence, 792-809AA and 966-983AA, were tandemly linked and repeated 14 times to ensure their size matched the optimal size for Pichia pastoris expression of exogenous proteins, thus designing recombinant type III small molecule collagen S15. LEKR was added before each sequence to ensure the correctness of the N-terminus. The recombinant type III small molecule collagen S12 and S15 sequences are as follows:

[0046] Amino acid sequence of recombinant type III small molecule collagen S12:

[0047] SEQ ID No:1:

[0048] LEKRGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKPGEKGPAGERGAPGPAGPRGPRGSPGPQGVKGESGKP

[0049] Amino acid sequence of recombinant type III small molecule collagen S15:

[0050] SEQ ID No:2:

[0051] LEKRGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKPGPRGSPGERGETGPPGPAGPRGSPGPQGVKGESGKP

[0052] (2) DNA sequence synthesis and construction of recombinant expression vector

[0053] The synthesis of the DNA sequence and the construction of the recombinant expression vector were both entrusted to Nanjing Genscript Biotech Co., Ltd.

[0054] First, codon optimization was performed based on the preferences of Pichia pastoris. Then, DNA fragments expressing recombinant type III small collagen S12 and S15 were synthesized. The synthesized gene fragments were cloned into the EcoRI and NotRI restriction sites of the pPIC9K empty vector (purchased from Thermo Fisher Scientific), so that the target fragments were accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, and recombinant plasmids expressing recombinant type III small collagen S12 and S15 were obtained.

[0055] The DNA sequences of recombinant type III small molecule collagen S12 and S15 are as follows:

[0056] DNA sequence of recombinant type III small molecule collagen S12:

[0057] SEQ ID No:3:

[0058]

[0059] DNA sequence of recombinant type III small molecule collagen S15:

[0060] SEQ ID No:4:

[0061]

[0062] (3) Construction of recombinant engineered strains

[0063] 10 μg of the recombinant expression plasmid obtained in step (2) was digested with SalⅠ (purchased from Dalian TaKaRa Company, the specific operation is performed according to the kit instructions) at 37℃ overnight to linearize it. Then, the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the volume was controlled at about 10 μL.

[0064] Linearized plasmids were electroporated into competent Pichia pastoris GS115 cells (purchased from Thermo Fisher Scientific). The electroporated bacterial culture was spread on MD plates, with 100-200 μL per plate. The plates were incubated at room temperature for 10 min and then inverted at 30°C for 2-5 days until single colonies (positive transformants) appeared.

[0065] Add 2 mL of sterile double-distilled water to the surface of an MD plate, then gently scrape off the His+ transformants from the plate surface using a sterile triangular spreader and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water. 5 One cell was spread on a YPD plate containing 0.5 mg / mLG418, inverted, and incubated at 30°C for 3-4 days until a single colony appeared, thus obtaining engineered bacteria expressing recombinant type III small molecule collagen S12 and S15.

[0066] Subsequently, the engineered bacterial samples expressing recombinant type III small molecule collagen S12 and S15 were sent to the China General Microbiological Culture Collection Center (CGMCC), with the following accession numbers: CGMCC No: 39031 and CGMCC No: 39032, respectively; accession address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession date: October 31, 2025; classification and name: *Pichia pastoris* Komagataella phaffii.

[0067] (4) Induced expression and identification of recombinant collagen

[0068] Pichia pastoris engineered strains expressing recombinant type III small molecule collagen S12 and S15 were taken separately, and the engineered strain disclosed in the patent (i.e., the Pichia pastoris genetically engineered strain disclosed in all of the applicant's patents CN201310033299.6, accession number: CGMCC No:7189) were placed in 100mL Erlenmeyer flasks containing 10mL BMGY medium and cultured at 28-30℃ and 220rpm until the OD600 reached 10-15 (culture time 18-24h). The cells were centrifuged at 1500-3000g for 5min at room temperature, and the cells were collected and resuspended in 10mL BMM2 medium to adjust the OD600.600 The culture medium was kept at approximately 2000 mg / L and placed on a shaker at 28-30°C and 220 rpm for 2 days to continue growth and induce expression. Every 24 hours, 100% methanol was added to the culture medium until the final concentration was 1.0%. After 48 hours of methanol induction, bacterial culture samples were collected in 1 mL volume and placed in a 1.5 mL EP tube. The tubes were centrifuged at 12000 g for 5 min at 4°C to collect the expression supernatant. The samples to be tested were stored at -80°C for later use.

[0069] The collected expression supernatant was added to 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), and heated in a 100℃ metal bath for 10 min before SDS-PAGE analysis. The results are as follows: Figure 1 As shown, the results indicate that both the Pichia pastoris genetically engineered strain in the published patent CN201310033299.6 and the Pichia pastoris engineered strain expressing recombinant type III small molecule collagen S12 and S15 provided by this invention can efficiently express recombinant type III small molecule collagen S12 and S15 (sequences shown in SEQ ID No:1 or SEQ ID No:2). Furthermore, the recombinant type III small molecule collagen S12 and S15 expressed by the Pichia pastoris engineered strain expressing recombinant type III small molecule collagen S12 and S15 provided by this invention have superior purity.

[0070] (5) Fermentation purification pilot test

[0071] High-temperature fermentation experiments were conducted on strains S12 and S15 expressing recombinant type III small molecule collagen in 5L tanks, and lyophilized sponge samples of the two collagen sequences were obtained after purification for further validation.

[0072] The culture media used are as follows:

[0073] Seed culture medium YPG: contains 10 g / L yeast extract, 20 g / L peptone, and 10 g / L glycerol.

[0074] Fermentation medium: containing 190.4 g / L NH4H2PO4, 10.06 g / L KH2PO4, 1.18 g / L CaSO4•2H2O, 18.2 g / L K2SO4, 14.9 g / L MgSO4•7H2O, and 40 g / L glycerol.

[0075] Feeding medium: 50% w / v glycerol, with 12 mL PTM1 (trace elements) per liter of glycerol.

[0076] Induction medium: 100% methanol, with 12 mL of PTM1 (trace elements) added per liter of methanol.

[0077] PTM1 (trace elements): Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0078] After the fermentation medium was sterilized at high temperature, PTM1 was added after the temperature dropped to room temperature. Ammonia was added during the fermentation process to adjust the pH. Induction was carried out at pH 4 and 5 respectively to confirm the appropriate induction pH.

[0079] Except for pH, the batch culture and induction expression conditions for the engineered strains were consistent: a fed-batch culture method was used, and the culture temperature was 30℃. Each engineered strain was inoculated into two 1L shake flasks containing 200ml of seed culture medium YPG, and cultured at 220rpm and 30℃ for 18-20h until OD600 = 2~10. A 5L fermenter (Baoxing Biotechnology) was used, filled with 2L of fermentation medium. 2% glycerol was sterilized separately. Before inoculation, the fermentation speed was adjusted to 300rpm, the aeration rate to 4L / min, and the temperature to 30℃. The pH was adjusted to 4.5 using a alkali solution prepared with concentrated ammonia. Then, 0.9mL of PTM1 was added first, followed by 200ml of the prepared seed culture (flame ring inoculation). The dissolved oxygen electrode was then calibrated, and fermentation began. When the dissolved oxygen level dropped to 30% for the first time, the dissolved oxygen cascade speed function was used to maintain 30%; wait for the glycerol to be depleted, the dissolved oxygen to rebound, and the dissolved oxygen level to be greater than 70% (OD600 = 2~10). 600 (Value approximately 20), cancel dissolved oxygen cascade speed, increase stirring speed to 650 rpm, use 30% glycerol in a linked feeding process, 80 ml of feed per batch. Stop glycerol feeding, and after dissolved oxygen rebounds to above 70%, set pH values ​​of 4 and 5 for each strain, keeping other conditions consistent, temperature 29℃, and induce culture using a methanol-glycerol mixed carbon source (methanol:50% glycerol volume ratio = 7:3). Manually add 5 mL of the methanol-glycerol mixed carbon source. After dissolved oxygen rebounds to above 70%, set the feeding rate (addition of feed medium) to 8 mL / h, increase to 10 mL / h after one hour, and then increase again to 20 mL / h after another hour. When the dissolved oxygen value drops below 30%, stop feeding and wait for dissolved oxygen to rebound. After dissolved oxygen rises back to 30%, resume linked feeding. Induction for 40-60 hours, when UV measurement shows no significant increase or decrease in protein concentration, the culture can be removed from the tank. UV protein quantification formula: C (mg / mL) = 0.144 * (A215 - A225), A215 < 1.5, where A215 and A225 are the absorbance values ​​of the protein at wavelengths of 215 nm and 225 nm, respectively, C is the concentration of the protein solution, and 0.144 is an empirical correction coefficient.

[0080] Collect the fermentation supernatant for SDS-PAGE electrophoresis analysis, such as... Figure 2As shown, under high-density fermentation conditions, the Pichia pastoris engineered strains expressing recombinant type III small molecule collagen S12 and S15 constructed in this invention can efficiently express the target proteins, and the expression levels are consistent under two different pH induction conditions.

[0081] Based on previous experiments, a small-scale purification trial was conducted:

[0082] The fermentation supernatant of the strains expressing recombinant type III small molecule collagen S12 and S15 was collected and purified to obtain lyophilized sponges of the two proteins. Based on the properties of the two proteins, ion exchange chromatography was used for purification. The raw materials and excipients used are shown in Table 1, and the purification equipment used is shown in Table 2.

[0083] Prepare the chromatography buffers: Buffer A: 20mM KH2PO4, pH 4.0; Buffer B: 20mM KH2PO4, 1M NaCl, pH 4.0.

[0084] Process flow: fermentation supernatant ultrafiltration desalting → chromatography → eluent ultrafiltration desalting → freeze drying into sponge; fermentation supernatant pretreatment: ultrafiltration desalting of fermentation broth to conductivity 7 mS / cm, and pH adjustment to 4.0.

[0085] The chromatography process is as follows:

[0086] Column equilibration: Manual mode, flow rate set to 35 ml / min, inlet A1 (Buffer A), column position valve positive flush, continuously monitor the Cond conductivity curve and pH curve on the spectrum interface until the conductivity curve drops to its lowest point and flattens out (about 3 column volumes). After the pH curve flattens out, it means that the column has been equilibrated. Click pause.

[0087] Sample loading: Manual mode, flow rate set to 30ml / min, inlet A2 (sample pH 4.0, 1000ml), click pause when the injection volume reaches the required volume.

[0088] Rebalancing: Manual mode, flow rate set to 35 ml / min, inlet A1 (Buffer A), click continue, run the program to continuously monitor the chromatogram curves, and continue balancing after the UV curve, conductivity curve and pH curve have stabilized, then click pause.

[0089] Elution: Manual mode, flow rate set to 35 ml / min, inlet B1 (Buffer B), set 25% B and 100% B for elution and collection respectively, click Continue. When the A215 spectrum curve rises, click Outlet1, click Continue to start eluting and collecting the target component until UV215 drops to its lowest point, at which point collection ends. Observe the eluent volume and detect the concentration.

[0090] The collected eluent was desalted by ultrafiltration until the conductivity was below 1 ms, then lyophilized and the sponge was collected.

[0091] Through the above purification experiments, two corresponding recombinant type III small molecule collagen S12 and S15 lyophilized sponges were successfully obtained.

[0092] Table 1: Raw and auxiliary materials for purification pilot test

[0093]

[0094] Table 2: Equipment for Small-Scale Purification

[0095]

[0096] (6) Stability verification of freeze-dried sponge

[0097] The recombinant type III small molecule collagen sponges S12 and S15 obtained in the above experiments were prepared into 1 mg / mL solutions with ddH2O. The stability of the two recombinant type III collagens was tested under repeated freeze-thaw cycles, different temperatures (4℃, 25℃, 40℃, 60℃), and different pH conditions (4, 7, 9 corresponding to acidic, neutral, and alkaline conditions) to determine their stability.

[0098] Solutions of recombinant type III small molecule collagen S12 and S15 were adjusted to pH values ​​of 4.0, 7.0, and 9.0, respectively, and incubated at 25°C. Samples were then subjected to SDS-PAGE analysis at different time points. The results are as follows: Figure 3 As shown.

[0099] Take 1 mL each of solutions of recombinant type III small molecule collagen S12 and S15, and repeatedly freeze and thaw three times, each time at -80℃ for 10 min, followed by heating at 99℃ for 10 min. Samples are then taken and verified by SDS-PAGE. The results are as follows: Figure 4 As shown.

[0100] Solutions of recombinant type III small molecule collagen S12 and S15 were sampled and placed at 4℃, 25℃, 40℃, and 60℃ for 7 days. Samples from different time points were then subjected to SDS-PAGE verification. The results are as follows: Figure 5 As shown.

[0101] Preliminary stability verification was performed using purified small-scale sponge samples, combined with... Figures 3 to 5 The experimental results showed that the stability of recombinant type III small molecule collagen S12 and S15 sponges met the stability requirements of collagen, and no significant degradation occurred under different pH, temperature and repeated freeze-thaw conditions.

[0102] (7) Cell experiments

[0103] The experiments were conducted according to the standards of GB / T16886.5-2017 In Vitro Cytotoxicity Tests, GB / T16886.12-2017 Sample Preparation and Reference Materials, and YY / T 1849-2022 Recombinant Collagen. Three cell experiments were performed on two lyophilized sponge samples of recombinant type III small molecule collagen S12 and S15, including cytotoxicity, cell adhesion, and cell migration.

[0104] Cytotoxicity assay—MTT method:

[0105] The extract used in this embodiment is a stock solution with a concentration of 1% prepared with sterile water, which is then diluted with sterile water to the required concentration according to experimental needs. 1% is the concentration of the recombinant type III small molecule collagen S12 and S15 lyophilized sponge samples.

[0106] The entire procedure was performed in a clean bench to ensure aseptic operation. L-929 cells (cell line from the Chinese Academy of Sciences Cell Bank, SCSP-5039) were cultured in MEM medium (manufacturer: Gibco, catalog number: 6125391; containing 10% FBS and 1% penicillin and streptomycin) at 37°C and 5% CO2. Cells that had reached the logarithmic growth phase were digested with 0.25% trypsin (containing EDTA). After digestion, the cell suspension was centrifuged (1000 rpm, 5 minutes), the supernatant was discarded, and the cells were resuspended in MEM medium. Cells were counted to obtain 1×10⁶ cells. 5 Cell suspension per mL. For culture, 100 μL of cell suspension was seeded into each well of a 96-well plate and cultured in a cell culture incubator (37°C, 5% CO2, >90% humidity). Cell morphology was observed under a microscope.

[0107] After 24 hours of incubation, when the cells had adhered to the plate and grown to approximately 70% confluence, the original culture medium in the 96-well plate was discarded. 100 μL of extraction buffer (final concentrations of 100%, 75%, 50%, and 25%) was added to each well of the 96-well plate to create sample groups. Blank control samples (MEM medium), negative control samples (MEM medium with 100 mg / mL Negative RM-C added), and positive control samples (MEM medium with 10% DMSO added) were also included. The 96-well plates were incubated in a cell culture incubator (37°C, 5% CO2, >90% humidity) for 24 hours, with six replicates per group.

[0108] After 24 hours of culture, the 96-well plates were removed, and cell morphology was observed under a microscope. The liquid was then removed, and 50 μL of MTT (final concentration 1 mg / mL) was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator. After 2 hours, the supernatant was removed, and 100 μL of isopropanol was added to each well to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader, and the cytotoxicity was calculated. The results are as follows: Figure 6 As shown, the results indicate that under the experimental conditions, the quantitative evaluation results suggest that the relative survival rates of the sample-recombinant type III small molecule collagen S12, sample-recombinant type III small molecule collagen S15, and the 100% concentration group of the extract (concentration: 1%, where 100% of the extract concentration is the initial concentration of the dissolved sample is 1%) are greater than 70%, indicating that the test substances have no potential cytotoxicity to L-929 cells.

[0109] Cell adhesion assay—centrifugation method:

[0110] Add 100 μL of sample (recombinant type III small molecule collagen-S12, recombinant type III small molecule collagen-S15) to each well of a 96-well plate, with two concentration gradients of 0.5 mg / mL and 1 mg / mL for each sample. Prepare four wells for each sample coating and incubate at 37°C in a 5% CO2 incubator 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. After removing the liquid from the wells, wash three times with D-PBS, discard the washing solution, seal with sealing film, and store at 4°C for later use.

[0111] NIH / 3T3 cells (from the Cell Bank of the Chinese Academy of Sciences, SCSP-515) were cultured in a 37°C, 5% CO2 incubator. Cell density and status were observed daily under an inverted microscope. When the cells reached 80%–90% confluence in the culture flask, they were passaged or seeded. Cells were diluted to 5 × 10⁻⁶ cells / mL using MEM medium premixed with Hoechst-33342 fluorescent staining agent (10%). 4 Cells / mL. Add 100 μL of cells to each well, cover with aluminum foil, and incubate at 37°C and 5% CO2 for 1 h. Measure three replicates; the fourth well is used to adjust microscope parameters and its measurement is not used.

[0112] At least 2×2 digital tiled images (fluorescence) of each well in three wells were captured using an inverted microscope at 4x magnification. Each well was filled with D-PBS to form an "inverted meniscus," air bubbles were removed, and the wells were sealed with a sealing film. The plates were centrifuged at 300 g relative centrifugation (RCF) at 22°C (inverted) for 5 min. After centrifugation, the sealing film was discarded, and the supernatant was removed from the wells. The plates were washed once with D-PBS, and then 100 μL of D-PBS was added. A total of 12 fluorescent tiled digital images were captured for each of the three wells (at least a 2×2 matrix is ​​recommended, with 10% overlap). The number of cells per sample was calculated to be approximately 2400 to 3600 (800–1200 cells / well × 3 wells). The sample from the published patent (i.e., the recombinant human collagen prepared in Example 1 of patent application number CN201310033299.6) was used as a control (i.e., the sample control group in Table 3).

[0113] The amino acid sequence of the sample in Example 1 of application number CN201310033299.6 is shown as SEQ ID No: 5 (the amino acid sequence of the sample in patent CN201310033299.6 is SEQ ID No: 1).

[0114] SEQ ID No:5:

[0115] AGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGS PGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAEFTAGNTGAPG SPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKG DRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPADHHHHHHTGLARF

[0116] Group setting instructions:

[0117] The sample groups were: Recombinant Type III Small Molecule Collagen S12-0.5 mg / mL, Recombinant Type III Small Molecule Collagen S15-0.5 mg / mL, Recombinant Type III Small Molecule Collagen S12-1 mg / mL, and Recombinant Type III Small Molecule Collagen S15-1 mg / mL.

[0118] The blank control group consisted of serum-free DMEM (solution medium).

[0119] The negative control group consisted of PBS.

[0120] The sample control group used recombinant human collagen prepared in Example 1 of application number 201310033299.6 as a control, with corresponding sample control of 0.5 mg / mL and sample control of 1 mg / mL.

[0121] Test results are shown Figure 7 As shown in Table 3, the results indicate that recombinant type III small molecule collagen S12 and S15 both have a positive effect on cell adhesion and the effect is better than that of the sample control.

[0122] Table 3: Summary of Cell Adhesion Experiment Results

[0123]

[0124] Note 1: * indicates a significant difference in migration rate compared to the blank control group (no serum) (P<0.05), T-test method.

[0125] Cell migration assay—scratch assay:

[0126] First, use a marker pen to draw evenly spaced horizontal lines on the back of the 6-hole board, approximately every 0.5cm to 1cm, passing through each hole. Pass three lines through each hole, and insert approximately 5×10 mm holes. 5 One NIH / 3T3 cell (from the Chinese Academy of Sciences Cell Bank, SCSP-515).

[0127] On the second day of cell culture, using a pipette tip aligned with a ruler, make a cut as perpendicular as possible to the horizontal line on the back of the cell. The pipette tip must be vertical, not tilted. Wash the cells three times with PBS to remove any cut cells. Add serum-free medium containing the test sample to create the experimental group, setting up a gradient concentration of 0.05% and 0.1%. Incubate at 37°C in a 5% CO2 incubator. Take samples and photographs at 0h and 24h.

[0128] The scratch area of ​​each image was calculated using ImageJ image processing software. The cell migration rate of each group was calculated by dividing the total area of ​​migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area. A graph was plotted with the sample as the x-axis and the migration rate ratio as the y-axis (in %), and the photos of the experimental and control groups at the initial 0 and the end of the experiment were compared. One-way ANOVA was used to analyze the differences in data between the experimental groups, followed by the chi-square test.

[0129] The results are as follows Figure 8 As shown, the sample in the published patent (i.e., the recombinant human collagen prepared in Example 1 of the patent application number CN201310033299.6) was used as a control. The results showed that S12 and S15 both had a positive effect on cell adhesion and the effect was better than the control.

[0130] The above cell experiments showed that the recombinant type III small molecule collagen S12 and S15 sequences both have excellent biological activity, and the effect is significantly better than that of the control.

[0131] (8) Mass spectrometry identification

[0132] The supernatant obtained from the fermentation of recombinant type III small molecule collagen S12 and S15 through the above steps was subjected to gel electrophoresis. After dye destaining, the main band was excised and sent to Suzhou Putai Biomedical Co., Ltd. for mass spectrometry detection and verification. The detection results of recombinant type III small molecule collagen S12 are as follows: Figure 9 As shown, the detection results of recombinant type III small molecule collagen S15 are as follows: Figure 10 As shown.

[0133] from Figure 9 , Figure 10 As can be seen, the mass spectrometry results of recombinant type III small molecule collagen S12 and S15 are highly consistent with the theoretical sequences SEQ ID No:1 and SEQ ID No:2, indicating that the results are in line with expectations.

[0134] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Recombinant type III small molecule collagen, characterized in that, The recombinant type III small molecule collagen contains a tandemly repeated fragment A, which is highly stable, easily expressed by Pichia pastoris, and not easily cleaved by enzymes; fragment A includes one or more of human type III collagen COL3A1, namely 501-518AA, 966-983AA, 792-809AA, and 966-983AA.

2. The recombinant type III small molecule collagen according to claim 1, characterized in that, The fragment A includes either the fragment formed by linking 501-518AA and 966-983AA in the human type III collagen COL3A1 sequence, or the fragment formed by linking 792-809AA and 966-983AA.

3. The recombinant type III small molecule collagen according to claim 2, characterized in that, The number of times segment A is repeated in series is 10 or 14.

4. The recombinant type III small molecule collagen according to claim 1, characterized in that, The recombinant type III small molecule collagen has LEKR added to its N-terminus.

5. The recombinant type III small molecule collagen according to any one of claims 1 to 4, characterized in that, The recombinant type III small molecule collagen includes the amino acid sequence shown in SEQ ID No:1 or SEQ ID No:

2.

6. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant type III small molecule collagen as described in any one of claims 1-5.

7. The polynucleotide according to claim 6, characterized in that, The polynucleotide sequence is such as the nucleotide sequence shown in SEQ ID No:3 or SEQ ID No:4, or its degenerate sequence.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide as described in claim 6 or 7.

9. Recombinant engineered bacteria or cells, characterized in that, The recombinant engineered bacteria or cells contain the polynucleotides of claim 6 or 7, or the recombinant expression vector of claim 8, and preferably the host cell of the recombinant engineered bacteria or cells is Pichia pastoris.

10. The recombinant engineered bacteria or cells according to claim 9, wherein the strain preservation numbers of the recombinant engineered bacteria or cells are: CGMCC No. 39031 and CGMCC No. 39032.

11. A method for preparing recombinant type III small molecule collagen according to any one of claims 1-5, comprising the following steps: The recombinant engineered bacteria of claim 9 or 10 are inoculated into seed culture medium YPG to obtain seed liquid; the seed liquid is inoculated into fermentation culture medium and the pH value is adjusted; glycerol is added as feed during fermentation and induced culture is carried out with a mixed carbon source of methanol and glycerol to obtain recombinant type III small molecule collagen.

12. A composition, characterized in that, The composition comprises the recombinant type III small molecule collagen as described in any one of claims 1-5, or the recombinant expression vector as described in claim 8, or the recombinant engineered bacteria or cells as described in claim 9 or 10.

13. The product, characterized in that, The product comprises the recombinant type III small molecule collagen as described in any one of claims 1-5, or the recombinant expression vector as described in claim 8, or the recombinant engineered bacteria or cells as described in claim 9 or 10, or the recombinant type III small molecule collagen prepared by the method described in claim 11, or the composition as described in claim 12; preferably, the product is selected from pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, or health products.

14. Use of the recombinant type III small molecule collagen as described in any one of claims 1-4 or 5, or the polynucleotide as described in claim 6 or 7, or the recombinant expression vector as described in claim 8, or the recombinant engineered bacteria as described in claim 9 or 10, or the recombinant type III small molecule collagen prepared by the method described in claim 11, or the composition as described in claim 12, or the article as described in claim 13 in the preparation of pharmaceuticals, pharmaceutical compositions, medical devices, biomaterials, tissue-engineered products, cosmetics, and health products.

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