Recombinant type i collagen and use thereof

Abstract

Provided are a high-stability recombinant type I collagen and a use thereof, relating to the technical field of collagens. By means of the directional selection of existing amino acid sequences, potential enzyme cutting sites and unstable sequence fragments are avoided during sequence design, enabling efficient expression in Pichia pastoris; and in addition, after being purified, the recombinant type I collagen still has high stability. The recombinant type I collagen takes into account factors such as related biological active sites and hydrophilicity, has related biological activity while maintaining high stability, and has good practicability.

Description

Recombinant Type I Collagen and Its Applications Technical Field

[0001] This invention belongs to the field of collagen technology, specifically relating to highly stable recombinant type I collagen and its applications. 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] Currently, collagen is mainly obtained through animal tissue extraction or recombinant expression technology. While the technology for extracting collagen from animal tissues is relatively mature and has a long history of application, traditional collagen production typically uses acids and alkalis to extract it from animal tissues, which is difficult, results in uneven collagen properties, significant batch-to-batch variations, and poses a risk of viral infection. Research and application of recombinant collagen has a history of over thirty years. Existing literature and patents mainly focus on the expression of single-chain human collagen in different hosts, the expression of truncated single-chain collagen genes, and a small number of studies focus on the co-expression of collagen with related post-expression modifying enzymes to obtain triple-helix collagen.

[0004] Current research on recombinant expression of type I collagen focuses primarily on its effective secretory expression and biological activity, neglecting its stability. This results in most commercially available type I recombinant collagens being unstable and prone to degradation. This increases the immunogenicity of the raw material, making subsequent purification more difficult and reducing yield. Even when high-purity lyophilized sponges are obtained through purification processes, unstable recombinant collagen undergoes significant degradation upon remelting, increasing the risk to the subsequent use of the finished product and greatly limiting its applications. Therefore, there is a need to develop highly stable recombinant type I collagen. Summary of the Invention

[0005] To address some shortcomings in existing technologies, this invention provides a highly stable recombinant type I collagen and its applications. This invention aims to achieve efficient expression in Pichia pastoris by selectively choosing existing amino acid sequences and avoiding potential restriction enzyme sites and unstable sequence fragments during sequence design. Furthermore, the purified recombinant type I collagen exhibits high stability. This recombinant type I collagen considers relevant biological activity sites and hydrophilicity, possessing both high stability and relevant biological activity, thus demonstrating excellent practicality.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0007] This invention first provides a highly stable recombinant type I collagen, which includes a repeating tandem fragment A. Fragment A includes multiple highly stable sequence fragments that are easily and efficiently expressed by Pichia pastoris and are not easily cleaved by enzymes. The sequence fragments are one or more of the following in human type I collagen CO1A1: 302-322AA, 413-445AA, 485-532AA, 554-577AA, 593-613AA, 668-679AA, 740-763AA, 776-799AA, 899-922AA, 962-976AA, 1010-1039AA, and 1070-1102AA.

[0008] Preferably, fragment A comprises any of the following combinations of sequence fragments:

[0009] a) 554-577AA, 1070-1102AA;

[0010] b)668-679AA, 776-799AA, 1082-1102AA;

[0011] c)593-613AA, 776-799AA, 962-976AA;

[0012] d)593-613AA, 668-679AA, 776-799AA, 962-976AA, 1082-1102AA;

[0013] e)413-445AA, 899-922AA, 1082-1102AA;

[0014] f)302-322AA, 485-532AA, 740-763AA, 1010-1039AA;

[0015] g)302-322AA, 485-532AA, 776-799AA;

[0016] h)668-679AA, 740-763AA, 1010-1039AA, 1082-1102AA.

[0017] Preferably, the size of the highly stable recombinant type I collagen is 30-50 kDa.

[0018] Preferably, the N-terminus of the highly stable recombinant type I collagen is supplemented with LEKR.

[0019] Preferably, the highly stable recombinant type I collagen comprises amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, or amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0020] The present invention also provides nucleotides encoding the above-mentioned highly stable recombinant type I collagen.

[0021] The present invention also provides a recombinant expression vector comprising nucleotides encoding the aforementioned highly stable recombinant type I collagen.

[0022] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium contains nucleotides encoding the above-mentioned highly stable recombinant type I collagen or the above-mentioned recombinant expression vector.

[0023] Preferably, the host bacteria of the recombinant engineered bacteria are eukaryotic bacteria or prokaryotic bacteria.

[0024] Preferably, the host bacteria is Pichia pastoris.

[0025] Preferably, the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 31952, CGMCC No. 31953, and CGMCC No. 31954.

[0026] The present invention also provides a composition comprising the above-described highly stable recombinant type I collagen.

[0027] The present invention also provides an article comprising the above-described highly stable recombinant type I collagen or composition.

[0028] This invention also provides the application of the above-mentioned highly stable recombinant type I collagen, composition or product in the preparation of medical devices, biomaterials, tissue engineering products and cosmetics.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) The recombinant type I collagen described in this invention has high stability, with the main band accounting for more than 90% during the fermentation stage. It is easy to purify and is not prone to degradation during the purification process, which greatly improves the yield of the full-length protein. The purity of the scale-up pilot sponge is above 95%.

[0031] (2) The sample obtained after purification of the recombinant type I collagen described in this invention performed better than the original protein 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.

[0032] (3) The recombinant protein involved in 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. Attached Figure Description

[0033] Figure 1 shows the SDS-PAGE of the supernatant from the recombinant type I collagen expression in shake flasks (induced for 48 h).

[0034] Figure 2 shows the SDS-PAGE of the supernatant from the fermentation of recombinant type I collagen in a 5L tank.

[0035] Figure 3 shows the sponge gel diagram of recombinant type I collagen 102-105.

[0036] Figure 4 shows gel images of recombinant type I collagen 102–105 at different pH values, where a represents 102, b represents 103, c represents 104, and d represents 105.

[0037] Figure 5 shows the repeated freeze-thaw gel diagram of recombinant type I collagen 102–104.

[0038] Figure 6 shows the results of circular dichroism spectroscopy detection of recombinant type I collagen from 102 to 105, where a represents 102, b represents 103, c represents 104, and d represents 105.

[0039] Figure 7 shows the cytotoxicity results of recombinant type I collagen 102–105, where a represents 102, b represents 103, c represents 104, and d represents 105.

[0040] Figure 8 is a bar chart showing the cell adhesion experiment results of recombinant type I collagen 102-105, where a represents 0.5 mg / mL and b represents 1.0 mg / mL.

[0041] Figure 9 is a statistical chart of the migration area ratio of recombinant type I collagen 102-105.

[0042] Figure 10 shows the magnified SDS-PAGE test results of three batches, with the gel running images of Marker, two samples from batch SF2406002, two samples from batch SF2406003, and two samples from batch SF2406004, respectively.

[0043] Figure 11 shows the mass spectrometry identification results of recombinant type I collagen 103 sponge.

[0044] Figure 12 shows the mass spectrometry identification results of recombinant type I collagen 105 sponge.

[0045] Figure 13 shows the mass spectrometry identification results of three batches of recombinant type I collagen 102 in pilot-scale sponge. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0048] Example 1: Design of highly stable recombinant type I collagen

[0049] Using human type I collagen CO1A1 as the parent sequence (https: / / www.uniprot.org / uniprotkb / P02452), sequence fragments with high stability, easy to be efficiently expressed by Pichia pastoris, and not easily digested by enzymes were selected. Any two or more of these fragments were selectively tandem and repeated a certain number of times, so that the size of the repeated sequence conformed to the optimal size for Pichia pastoris to express exogenous proteins. LEKR was added to the front end to ensure the correctness of the N-terminus.

[0050] Among them, the sequence fragments that are highly stable, easy to express efficiently in Pichia pastoris, and not easily digested by enzymes are the parent sequence 302-322AA, 413-445AA, 485-532AA, 554-577AA, 593-613AA, 668-679AA, 740-763AA, 776-799AA, 899-922AA, 962-976AA, 1010-1039AA, and 1070-1102AA.

[0051] The sequence fragments that are highly stable, easily expressed by Pichia pastoris, and not easily digested by enzymes are shown below:

[0052] 302-322AA: GPRGLPGERGRPGAPGPAGAR (SEQ ID NO. 17);

[0053] 413-445AA:GARGPSGPQGPGGPPPGPKGNSGEPGAPGSKGDT (SEQ ID NO. 18);

[0054] 485-532AA: ERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEA (SEQ ID NO. 19);

[0055] 554-577AA:PPGPAGQDGRPGPPGPPGARGQA (SEQ ID NO. 20);

[0056] 593-613AA: KAGERGVPGPPGAVGPAGKD (SEQ ID NO. 21);

[0057] 668-679AA:GAPGPSGARGER(SEQ ID NO.22);

[0058] 740-763AA: GPKGDRGDAGPKGADGSPGKDGVR (SEQ ID NO. 23);

[0059] 776-799AA: GAPGDKGESGPSGPAGPTGARGAP (SEQ ID NO. 24);

[0060] 899-922AA:GPAGKEGGKGPRGETGPAGRPGEV(SEQ ID NO.25);

[0061] 962-976AA: GLPGQRGERGFPGLP (SEQ ID NO. 26);

[0062] 1010-1039AA: GESGREGAPGAEGSPGRDGSPGAKGDRGET (SEQ ID NO. 27);

[0063] 1070-1102AA: GPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQ (SEQ ID NO.28). 554-577AA and 1070-1102AA were selected and tandemly linked, then repeated 6 times with LEKR added to the leading end to obtain recombinant type I collagen 101. The amino acid sequence of 101 is shown in SEQ ID NO.1.

[0064] SEQ ID NO.1:

[0065]

[0066] Select 668-679AA, 776-799AA, and 1082-1102AA and tandem them, then repeat 6 times and add LEKR to the front to obtain recombinant type I collagen 102. The amino acid sequence of the recombinant type I collagen 102 is shown in SEQ ID NO.2.

[0067] SEQ ID NO.2:

[0068]

[0069] 593-613AA, 776-799AA, and 962-976AA were selected and tandemly combined, then repeated 6 times, with LEKR added to the front end to obtain recombinant type I collagen 103. The amino acid sequence of the recombinant type I collagen 103 is shown in SEQ ID NO.3.

[0070] SEQ ID NO.3:

[0071]

[0072] The amino acid sequences 593-613AA, 668-679AA, 776-799AA, 962-976AA, and 1082-1102AA were selected, tandemly, and then repeated 5 times. LEKR was added to the front segment to obtain recombinant type I collagen 104. The amino acid sequence of recombinant type I collagen 104 is shown in SEQ ID NO.4.

[0073] SEQ ID NO.4:

[0074]

[0075] The amino acid sequences of recombinant type I collagen 105 were obtained by tandemly selecting 413-445AA, 899-922AA, and 1082-1102AA, repeating the process 6 times, and adding LEKR to the front end. The amino acid sequence of recombinant type I collagen 105 is shown in SEQ ID NO.5.

[0076] SEQ ID NO.5:

[0077]

[0078] The amino acids 302-322AA, 485-532AA, 740-763AA, and 1010-1039AA were selected, tandemly, and then repeated 4 times. LEKR was added to the front end to obtain recombinant type I collagen 106. The amino acid sequence of recombinant type I collagen 106 is shown in SEQ ID NO.6.

[0079] SEQ ID NO.6:

[0080]

[0081] 302-322AA, 485-532AA, and 776-799AA were selected and tandemly combined, then repeated 5 times, with LEKR added to the front end to obtain recombinant type I collagen 107. The amino acid sequence of the recombinant type I collagen 107 is shown in SEQ ID NO.7.

[0082] SEQ ID NO.7:

[0083]

[0084] The amino acid sequences of 668-679AA, 740-763AA, 1010-1039AA, and 1082-1102AA were selected, tandemly, and then repeated 5 times. LEKR was added to the front end to obtain recombinant type I collagen 108. The amino acid sequence of the recombinant type I collagen 108 is shown in SEQ ID NO.8.

[0085] SEQ ID NO.8:

[0086]

[0087] Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to optimize the codons based on the preferences of Pichia pastoris, and then synthesized nucleotides encoding 101, 102, 103, 104, 105, 106, 107, and 108, the sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.16.

[0088] SEQ ID NO.9:

[0089]

[0090] SEQ ID NO. 10:

[0091]

[0092] SEQ ID NO. 11:

[0093]

[0094] SEQ ID NO. 12:

[0095]

[0096] SEQ ID NO. 13:

[0097]

[0098] SEQ ID NO. 14:

[0099]

[0100] SEQ ID NO. 15:

[0101]

[0102] SEQ ID NO. 16:

[0103]

[0104] Example 2: Construction of recombinant expression vector and recombinant engineered bacteria

[0105] (1) Construction of recombinant expression vector:

[0106] The nucleotides synthesized in Example 1 were cloned into the EcoRI and NotRI spaces of the pPIC9K empty vector (purchased from Thermo Fisher Scientific), so that the target fragment was accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, and recombinant expression vectors expressing 101, 102, 103, 104, 105, 106, 107, and 108 were obtained.

[0107] (2) Construction of recombinant engineered bacteria:

[0108] 10 μg of the recombinant expression vectors expressing 101, 102, 103, 104, 105, 106, 107, and 108 were linearized by digestion with SalI (purchased from TaKaRa in Dalian, with specific procedures following the kit instructions) at 37°C overnight. The linearized plasmids were then recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), with the volume controlled at approximately 10 μL.

[0109] 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.

[0110] 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 / mL G418, inverted, and incubated at 30°C for 3–4 days until single colonies appeared, yielding Pichia pastoris engineered strains expressing 101, 102, 103, 104, 105, 106, 107, and 108.

[0111] Example 3: Induced Expression and Identification of Recombinant Type I Collagen

[0112] Pichia pastoris engineered strains expressing 101, 102, 103, 104, 105, 106, 107, and 108, respectively, and simultaneously, engineered strains from patent CN201911135958.0 (full-length human type I α1 chain sequence), were placed in 100 mL Erlenmeyer flasks containing 10 mL of BMGY medium and cultured at 28-30℃ and 220 rpm until the OD600 reached 10-15 (18-24 h). The cells were centrifuged at 1500-3000 g for 5 min at room temperature, collected, and resuspended in 10 mL of BMM2 medium to adjust the OD600. 600The culture medium was kept at approximately 200 mg / L and placed on a shaker at 28-30°C and 220 rpm for further growth and induction expression for 2 days. 100% methanol was added to the culture medium every 24 hours until the final concentration reached 1.0%. After induction with methanol for 48 hours, a 1 mL sample was collected and placed in a 1.5 mL EP tube. The tube was centrifuged at 12000 g for 5 min at 4°C, and the supernatant was collected. Samples to be tested were stored at -80°C for later use.

[0113] The expression supernatant was collected and 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added. The mixture was then heated in a 100℃ metal bath for 10 min, followed by SDS-PAGE analysis. The results are shown in Figure 1. Figure 1 shows that the gel image conforms to the expected molecular weight, indicating successful acquisition of recombinant type I collagen. Furthermore, compared to the engineered strain in patent CN201911135958.0, all seven sequences from 101 to 108 (except for 101) were expressed efficiently. Considering purity as a priority, sequences 102, 103, 104, and 105 showed superior performance.

[0114] Based on small-scale experiments, three recombinant type I collagen proteins, 102, 103, and 105, were selected. Samples of the three engineered bacteria expressing 102, 103, and 105 were sent to the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC No. 31952, CGMCC No. 31953, and CGMCC No. 31954, respectively. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Accession date: September 12, 2024; Classification name: *Pichia pastoris* *Komagataella phaffii*.

[0115] Example 4: Fermentation purification of recombinant type I collagen

[0116] (1) Fermentation:

[0117] The Pichia pastoris engineered strains expressing 102, 103, 104, and 105, selected in Example 3, were subjected to a high-temperature fermentation experiment in a 5L jar, and lyophilized sponge samples with four collagen sequences were obtained after purification for further verification.

[0118] The culture media used include:

[0119] YPG seed culture medium: yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L;

[0120] Fermentation medium: NH4H2PO4 190.4 g / L, KH2PO4 10.06 g / L, CaSO4·2H2O 1.18 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, glycerol 40 g / L;

[0121] Feeding medium: 50% w / v glycerol, with 12 mL PTM1 micronutrients per liter;

[0122] Induction medium: 100% methanol, with 12 mL of trace elements added per liter;

[0123] PTM1: Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0124] After sterilizing the fermentation medium at high temperature, PTM1 was added after the temperature dropped to room temperature. The pH was adjusted with ammonia water. The selected strains 102, 103, 104, and 105 were induced at pH 4 and 5, respectively, to confirm the appropriate induction pH.

[0125] The batch culture and induction conditions for Pichia pastoris engineered strains expressing 102, 103, 104, and 105 were consistent except for pH: 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, 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 concentrated ammonia solution. Then, 0.9mL of LPTM1 was inoculated first, followed by 200mL of the prepared seed culture (flame ring inoculation). The dissolved oxygen electrode was then calibrated, and fermentation began after calibration. Once the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade rotation function to maintain it at 30%; wait for the glycerol to be depleted, and for the dissolved oxygen to rebound and exceed 70% (OD). 600(Value approximately 20), cancel dissolved oxygen cascade speed, increase stirring speed to 650 rpm, use 30% glycerol for continuous feeding, 80 mL of feed. Stop glycerol feeding, after dissolved oxygen rebounds to above 70%, set pH 4 and pH 5 for each strain, keeping other conditions consistent, temperature 29℃, and induce culture with a mixed carbon source of methanol and glycerol (methanol: 50% glycerol = 7:3). Manually add 5 mL, after dissolved oxygen rebounds to above 70%, set the feeding rate to 8 mL / h, increase to 10 mL / h after one hour, and increase again to 20 mL / h after one hour. When the dissolved oxygen value is below 30%, stop feeding, wait for dissolved oxygen to rebound, and resume continuous feeding after dissolved oxygen rises back to 30%. Induce for 40-60 hours, and when the increase in protein concentration measured by UV is not obvious or decreases, the culture can be removed from the tank. UV protein quantification formula: C (mg / mL) = 0.144*(A215-A225), A215 < 1.5.

[0126] The fermentation supernatant was collected and analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 2. As can be seen from Figure 2, under high-density fermentation conditions, all strains were able to express the target protein efficiently, and the expression was even better at pH 5.

[0127] (2) Purification:

[0128] Collect the fermentation supernatant of each strain in step (1) for purification. Based on the properties of the four proteins 102, 103, 104, and 105, ion exchange was used for purification. The raw materials and excipients used are shown in Table 1. The purification equipment used was a Bio-Lab100 chromatography instrument (Hanbang), a BONA-GM-18 ultrafiltration unit (Bona), and a Pilot2-4MC lyophilizer (Boyikang). Prepare the chromatography buffers: Buffer A: 20 mM KH2PO4, pH 4.0; Buffer B: 20 ​​mM KH2PO4, 1 M NaCl, pH 4.0.

[0129] Table 1: Purification Raw Materials and Auxiliaries

[0130]

[0131] The purification process is as follows: fermentation supernatant ultrafiltration desalting → chromatography → eluent ultrafiltration desalting → freeze drying into sponge;

[0132] Fermentation supernatant pretreatment: The fermentation broth was desalted by ultrafiltration to a conductivity of 7 mS / cm and the pH was adjusted to 4.0;

[0133] Chromatography process:

[0134] 1) 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 the 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.

[0135] 2) Sample loading: Manual mode, flow rate set to 30ml / min, inlet A2 (sample pH 4.0, 1000ml), click pause when the volume of the injected liquid reaches the required volume.

[0136] 3) 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. Click pause.

[0137] 4) 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.

[0138] Eluent treatment:

[0139] The collected eluent was ultrafiltered and desalted until the conductivity was below 1 ms, then lyophilized. Sponges at 102–105 ppm were collected, and appropriate amounts of sponges were subjected to SDS-PAGE for verification. The results are shown in Figure 3. Figure 3 shows that 104 ppm has a lower purity, while 102, 103, and 105 ppm can all yield lyophilized sponges with higher purity.

[0140] Example 5: Stability test of recombinant type I collagen

[0141] (1) Stability test of recombinant type I collagen:

[0142] The sponges 102-105 obtained in Example 4 were prepared into 1 mg / mL solutions with ddH2O. Then, their stability was tested under repeated freeze-thaw cycles, different temperatures (4℃, 25℃, 40℃, 60℃), and different pH conditions (4, 7, 9) to determine the sequence with the best stability among the four recombinant type I collagens.

[0143] The pH of solutions containing sponges 102–105 was adjusted to 3.0, 7.0, and 9.0, respectively, and incubated at 25°C. Samples were taken after different number of days for SDS-PAGE verification, and the results are shown in Figure 4. Figure 4 shows that among the four lyophilized sponges, sponge 102 exhibited the best stability at different pH levels.

[0144] One mL of each solution containing sponges 102 to 105 was taken and subjected to three freeze-thaw cycles, each time freezing at -80°C for 10 min and then heating at 99°C for 10 min. Samples were then taken and verified by SDS-PAGE. The results are shown in Figure 5. As can be seen from Figure 5, among the four freeze-dried sponges, sponge 102 exhibited the best stability at different temperatures.

[0145] Solutions containing sponges 102 to 105 were taken and placed at 4℃, 25℃, 40℃, and 60℃ for 7 days. Samples were taken at different time points during this period for SDS-PAGE verification. The results showed that among the four lyophilized sponges, sponge 102 exhibited the best stability at different temperatures.

[0146] In summary, the stability of sponge 102 meets the stability requirements, and it does not undergo significant degradation under different conditions.

[0147] (2) Structural verification of recombinant type I collagen:

[0148] The purified sponges 102–105 were dissolved in appropriate amounts with ddH2O to a concentration of 1 mg / mL, and then equilibrated at 4°C for 48 hours. After equilibration, the solutions were detected using a circular dichroism spectroscopy (CDS). Before detection, the CDS was calibrated with air and ddH2O. The solutions containing sponges 102–105 were then detected sequentially, and the results are shown in Figure 6. Figure 6 shows that sponge 102 exhibits a certain higher-order structure. In addition to the primary and secondary structures, the CDS shows a characteristic absorption peak at 221, indicating a higher-order tertiary structure. The other three sponges do not show a characteristic absorption peak at 221, indicating primarily primary and secondary structures.

[0149] Example 6: Cellular Experiments with Recombinant Type I Collagen

[0150] This embodiment refers to GB / T16886.5-2017 In Vitro Cytotoxicity Tests, GB / T16886.12-2017 Sample Preparation and Reference Materials, YY / T 1849-2022 Recombinant Collagen, etc., to conduct three cell experiments on four small-scale lyophilized sponge samples of 102-105. The specific steps are as follows.

[0151] (1) Cytotoxicity test:

[0152] 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 (containing 10% FBS and 1% penicillin-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 Cells / mL cell suspension. 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.

[0153] After 24 hours of culture, 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%), blank control, negative control, and positive control samples were added to the corresponding wells of the 96-well plate. The 96-well plate was then incubated in a cell culture incubator (37°C, 5% CO2, >90% humidity) for 24 hours, with six replicates per group.

[0154] After culturing for 24 hours, the 96-well plate was removed, and cell morphology was observed under a microscope. The liquid was then removed, and 50 μL MTT (final concentration 1 mg / mL) was added to each well. The plate was then 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 shown in Figure 7.

[0155] Figure 7 shows that, under the experimental conditions, the quantitative evaluation results indicate that the relative survival rate of the 100% concentration groups (concentration: 1%) of sample-102, sample-103, and sample-105 extracts was greater than 70%, indicating that sample-102, sample-103, and sample-105 had no potential cytotoxicity to L-929 cells. The relative survival rate of the 100% concentration group (concentration: 1%) of sample-104 extract was less than 70%, indicating that sample-104 has potential cytotoxicity to L-929 cells. Furthermore, at a certain concentration, 102 has a proliferative effect on cells.

[0156] (2) Cell adhesion test:

[0157] Add 100 μL of sample (sample / blank control) to each well of a 96-well plate, preparing two concentration gradients of 0.5 mg / mL and 1 mg / mL for each sample. Coat four wells with each sample and incubate at 37°C in a 5% CO2 incubator for 2 hours. 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 hour. 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.

[0158] NIH / 3T3 cells (cell line 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 complete culture 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.

[0159] At least 4×4 digital tiled images (fluorescence) of each well in three wells were captured using an inverted microscope. Each well was filled with D-PBS to form an "inverted meniscus," air bubbles were removed, and the well was sealed with a sealing film. The plates were centrifuged at 22°C (inverted) for 5 min at a relative centrifugation force (RCF) of 300 g. 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. For each of the three wells, a total of 25 fluorescent tiled digital images were captured (at least a 4×4 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 results are shown in Figure 8 and Table 2. Combining Figure 8 and Table 2, it can be seen that 10²–10⁵ cells all have a positive effect on cell adhesion, with 10² showing the best performance.

[0160] Table 2. Results of Cell Adhesion Experiment

[0161]

[0162] Note: * indicates a significant difference in migration rate compared to the serum-free (blank) group (P<0.05), using the chi-square test.

[0163] (3) Cell migration assay:

[0164] 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.

[0165] 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 the cut cells. Add serum-free medium containing the test sample as 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.

[0166] The scratch area of ​​each image was calculated using ImageJ image processing software. The cell migration rate for 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 horizontal axis and the migration rate ratio as the vertical axis (in %). The experimental and control group photos were compared between the initial value at 0 and the end of the experiment. One-way ANOVA was used to analyze the differences in data between the experimental groups, and the chi-square test was performed. The results are shown in Figure 9. The results show that samples 102–105 all have a positive effect on cell migration, with sample 102 showing the best performance.

[0167] In summary, recombinant type I collagen 102–105 all possess biological activity, with 102 showing superior performance.

[0168] Example 7: Scale-up Verification

[0169] The shake-flask verification in Example 3 and the small-scale experiment in Example 4 demonstrate that the lyophilized sponge containing the recombinant type I collagen 102 sequence possesses a certain higher-order structure, high stability, and superior performance in cell experiments. This example further validates the recombinant engineered strain expressing recombinant type I collagen 102 through a 500L fermentation scale-up process, with three consecutive batches to verify expression stability and to outsource the identification of the sponge.

[0170] The 500L fermentation process was based on a small-scale trial, with the optimal pH for fermentation induction preferably set at 5.0. Three consecutive batches (batch numbers SF2406002, SF2406003, and SF2406004) were fermented, purified, and freeze-dried, yielding 119g, 134g, and 118g of freeze-dried sponges, respectively. SDS-PAGE analysis of the final sponges was performed, and the results are shown in Figure 10. The final sponges underwent SDS-PAGE analysis and grayscale analysis, yielding purities of 97.6%, 96.9%, and 100%, respectively. The gel images are shown in Figure 10. As can be seen from the figure, the three batches of 500L process yielded 102 freeze-dried sponges with a purity exceeding 95%.

[0171] Example 8: Characterization of highly stable recombinant type I collagen

[0172] In this embodiment, the lyophilized sponges containing recombinant type I collagen 103 and 105 obtained in Example 4 were subjected to gel electrophoresis experiments. After staining and destaining, the main bands were cut off and sent to Suzhou Putai Biomedical Co., Ltd. for mass spectrometry detection and verification. The detection results of 103 are shown in Figure 11, and the detection results of 105 are shown in Figure 12.

[0173] As can be seen from Figures 11 and 12, the mass spectrometry detection results of 103 and 105 are highly consistent with the theoretical sequences SEQ ID NO.3 and SEQ ID NO.5, indicating that the results are in line with expectations.

[0174] In this embodiment, three batches of 102 recombinant type I collagen freeze-dried sponges obtained in Example 7 were sent to Beijing Baitaipaike Biotechnology Co., Ltd. for mass spectrometry analysis. The results are shown in Figure 13. As can be seen from Figure 13, the full-sequence mass spectrometry identification results of the three batches of 102 trial production samples are completely consistent with the theoretical sequence, indicating that the results meet expectations and that the engineered strain can stably express the target product.

[0175] In summary, this invention, through targeted selection of existing amino acid sequences, avoids potential restriction enzyme sites and unstable sequence fragments during sequence design, enabling efficient expression in Pichia pastoris. Furthermore, the recombinant type I collagen obtained after purification also exhibits high stability. The recombinant type I collagen takes into account relevant biological activity sites and hydrophilicity, possessing both high stability and relevant biological activity, thus demonstrating excellent practicality.

[0176] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0177] 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 I collagen, characterized in that, The recombinant type I collagen has an amino acid sequence as shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

5.

2. A polynucleotide encoding the recombinant type I collagen of claim 1.

3. A recombinant expression vector comprising a polynucleotide encoding the recombinant type I collagen of claim 2.

4. A recombinant engineered bacterium, said recombinant engineered bacterium comprising a polynucleotide encoding the recombinant type I collagen of claim 2 or the recombinant expression vector of claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The host bacteria of the recombinant engineered bacteria are prokaryotic bacteria.

6. The recombinant engineered bacteria according to claim 4, characterized in that, The recombinant engineered bacteria is Pichia pastoris.

7. The recombinant engineered bacteria according to claim 4, characterized in that, The recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession numbers CGMCC No. 31952, CGMCC No. 31953, and CGMCC No. 31954.

8. A composition comprising the recombinant type I collagen of claim 1.

9. An article comprising the recombinant type I collagen of claim 1 or the composition of claim 8.

10. The use of the recombinant type I collagen of claim 1, the composition of claim 8, or the article of claim 9 in the preparation of medical devices, biomaterials, tissue-engineered products, and cosmetics.

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