Recombinant human type III collagen with a triple helical structure assembled into collagen fibers and its application

Abstract

The present invention provides a recombinant human type III collagen protein that has a triple helical structure and is assembled into collagen fibers, and its application, which belongs to the field of bioengineering technology. The present invention screens out collagen fragments that can form a triple helical structure through a bioinformatics algorithm, and connects these fragments in series, combines the key C-terminal domain and the specific amino acid sequence, and effectively promotes the formation of a stable triple helical higher-order structure and the assembly of collagen fibers. The recombinant human type III collagen protein is efficiently expressed through a eukaryotic expression system and shows excellent stability, low immunogenicity and good biocompatibility. Experimental results show that the prepared recombinant human type III collagen protein not only has a natural triple helical structure, but can also be further assembled into macromolecular collagen fibers, which is suitable for multiple fields such as medical beauty, bioengineering and tissue repair, and has broad application prospects and market value.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to a recombinant human type III collagen protein having a triple helical structure and assembled into collagen fibers, and applications thereof. Background Art

[0002] Collagen is an important structural protein, widely present in human tissues and organs. Its excellent biocompatibility and biodegradability have led to its widespread application in biomedical applications. However, traditional animal-derived collagen suffers from issues such as low purity, high immunogenicity, and inconsistent quality during extraction. Furthermore, the sequence and structure of animal collagen differ from human collagen, potentially leading to xenotransplantation rejection and limiting its clinical application.

[0003] The typical structure of collagen is a GXY sequence (where G is glycine, and X and Y are usually proline and hydroxyproline), which forms a stable triple helical structure. In natural collagen, the triple helical structure is crucial for collagen stability, functionality, and biocompatibility. However, due to the long length of natural collagen and the complex post-translational modifications, it is difficult to achieve recombinant expression of full-length collagen. Summary of the Invention

[0004] In light of this, the present invention aims to provide a recombinant human type III collagen with a triple-helical structure that assembles into collagen fibers, and its use. This invention utilizes a mammalian cell expression system to construct and screen stable cell lines, enabling the recombinant human type III collagen to correctly fold in cells, ensuring it retains the triple-helical structure of native collagen and forms intact collagen fibers. This recombinant collagen exhibits high stability and low immunogenicity, making it suitable for biomedical applications. Furthermore, its production method offers the advantages of low cost and strong scalability.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a recombinant human type III collagen with a triple helical structure that is assembled into collagen fibers. The recombinant human type III collagen is prepared by connecting any one of the collagen fragments capable of forming a triple helical structure in series 8 to 20 times, connecting it with a specific amino acid sequence, and then connecting it to a C-terminal domain.

[0007] Preferably, the sequences of the triple helical collagen fragments are shown as SEQ ID No. 7 to SEQ ID No. 20.

[0008] Preferably, the C-terminal domain includes a human type III collagen C-terminal propeptide sequence, a type XV collagen C-terminal propeptide sequence, a type XIX collagen NC2 domain sequence or a stabilizing peptide sequence (GPP) 4; the human type III collagen C-terminal propeptide sequence is shown as SEQ ID NO: 3; the type XV collagen C-terminal propeptide sequence is shown as SEQ ID NO: 4; the type XIX collagen NC2 domain sequence is shown as SEQ ID NO: 5; and the stabilizing peptide (GPP) 4 sequence is shown as SEQ ID NO: 6.

[0009] Preferably, the specific amino acid sequence is shown in SEQ ID NO: 2.

[0010] Preferably, the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID No. 26 to SEQ ID No. 28.

[0011] Preferably, the optimized nucleotide sequence of the amino acid sequence SEQ ID No. 27 of the recombinant human type III collagen is shown as SEQ ID No. 32.

[0012] The present invention provides a recombinant expression vector comprising a nucleotide sequence of recombinant human type III collagen.

[0013] The present invention provides a host cell comprising the recombinant expression vector, wherein the host cell is selected from a CHO cell, a Pichia pastoris or a Saccharomyces cerevisiae eukaryotic expression system.

[0014] The present invention also provides the use of the recombinant human type III collagen in the preparation of cosmetics and medical tissue engineering products.

[0015] The present invention also provides the use of the recombinant human type III collagen in the preparation of medical materials that promote skin repair and improve biocompatibility.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention utilizes the characteristics of the GXY sequence, adopts an algorithm to screen and obtain fragments that can form a triple helical structure, and connects these fragments in series, combines the key C-terminal domain and the specific amino acid sequence, and effectively promotes the formation of a stable triple helical higher-order structure and the assembly of collagen fibers. By connecting these optimized fragments in series and expressing them in mammalian cells, excellent stability, low immunogenicity and good biocompatibility are shown, and finally a recombinant human type III collagen with high stability and low immunogenicity is obtained, which can be further assembled to form complete collagen fibers. The present invention uses a mammalian cell expression system to construct and screen stable cell lines, so that the recombinant human type III collagen can be correctly folded in the cell, ensuring that it has the triple helical structure of natural collagen and can form complete collagen fibers. This recombinant collagen has high stability and low immunogenicity, is suitable for biomedical applications, and its production method has the advantages of low cost and strong scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the sequence design of recombinant human type III collagen;

[0018] Figure 2 This is a schematic diagram of sequence bioinformatics calculation of recombinant human type III collagen;

[0019] Figure 3 This is the gene map for recombinant human type III collagen;

[0020] Figure 4 The SDS-PAGE image of the supernatant of CHO cells transfected for 48 h (R / NR);

[0021] Figure 5 This is the R / NR-SDS-PAGE image of Pool cell expression supernatant;

[0022] Figure 6 The results of denatured non-reducing electrophoresis of recombinant human type III collagen are shown in Figure 2. Figure 6 A in the figure is the result of denaturing non-reducing electrophoresis of purified recombinant human type III collagen RHCol2. Figure 6 B in the figure is the denaturing non-reducing electrophoresis result of different recombinant human type III collagens;

[0023] Figure 7 This is the total ion chromatogram of recombinant human type III collagen RHCol2 by liquid chromatography-mass spectrometry (LC-MS);

[0024] Figure 8 This is the mass spectrum of the key peptide segment of recombinant human type III collagen RHCol2;

[0025] Figure 9 The effect of recombinant human type III collagen RHCol2 on cell behavior. DETAILED DESCRIPTION

[0026] The present invention provides a recombinant human type III collagen with a triple helical structure that is assembled into collagen fibers. The recombinant human type III collagen is prepared by connecting any one of the collagen fragments capable of forming a triple helical structure in series 8 to 20 times, connecting it with a specific amino acid sequence, and then connecting it to a C-terminal domain.

[0027] In the present invention, the method for obtaining the triple helical collagen fragment is: screening and designing the collagen fragment that can form a triple helical structure by using a bioinformatics algorithm. The screening and design is to use the GXY tripeptide repeat sequence characteristics, and use bioinformatics algorithms such as Tm calculation and molecular dynamics simulation to analyze the overall sequence of human type III collagen (SEQ ID NO:1) is truncated to design multiple peptides of 12 to 40 amino acids, preferably peptides of 15 to 34 amino acids, and more preferably peptides of 24 to 30 amino acids; the melting temperature (Tm value) of each peptide is calculated using the Tm algorithm, and peptides with high solubility and low immunogenicity are screened out in combination with amino acid composition analysis and immunogenicity prediction; the Tm value of the peptide is 0 to 80, preferably 30 to 75, and more preferably 50 to 70, the peptide with high solubility is a peptide containing more than 45% hydrophilic amino acids, and the peptide with low immunogenicity is a peptide that does not contain T cell epitope peptides or has a binding affinity to MHC molecules greater than 1000 nM; the feasibility of the screened peptides is verified by molecular dynamics simulation, and finally multiple collagen fragments with triple helical structures are obtained, ensuring that the recombinant human type III collagen has high stability and low immunogenicity and can form a stable triple helical structure.

[0028] In the present invention, the triple helical collagen fragment sequences are shown in SEQ ID No. 7 to SEQ ID No. 20, specifically:

[0029] SEQ ID No.7: GESGRPGRPGERGLPGPPGIKGPAGIPGFP;

[0030] SEQ ID No.8: GERGAPGFRGPAGPNGIPGEKGPAGERGAP;

[0031] SEQ ID No.9:GAPGPMGPRGAPGERGRPGLP;

[0032] SEQ ID No.10: GAPGPMGPRGAPGER;

[0033] SEQ ID No.11:GPMGPRGAPGERGRPGLPGAA;

[0034] SEQ ID No.12:GAPGPMGPRGAPGERGRP;

[0035] SEQ ID No.13: GRPGERGLPGPPGIKGPAGIP;

[0036] SEQ ID No.14: GERGLPGPPGIKGPA;

[0037] SEQ ID No.15:GESGRPGRPGERGLPGPPGIK;

[0038] SEQ ID No.16: GPRGAPGERGRPGLP;

[0039] SEQ ID No.17:GPMGPRGAPGERGRP;

[0040] SEQ ID No.18: GKDGESGRPGRPGERGLPGPP;

[0041] SEQ ID No.19: GENGAPGPMGPRGAPGERGRPGLP;

[0042] SEQ ID No.20: GESGRPGRPGERGLPGPPGIKGPAGIP;

[0043] The number of series connections is preferably 10 to 18 times, more preferably 12 to 16 times.

[0044] In the present invention, the specific amino acid sequence is shown in SEQ ID NO: 2, which is as follows:

[0045] CGGVGAAAIAGIGGEKAGGFAPYYGD;

[0046] The C-terminal domain includes a human type III collagen C-terminal propeptide sequence, a type XV collagen C-terminal propeptide sequence, a type XIX collagen NC2 domain sequence or a stabilizing peptide sequence (GPP) 4; the human type III collagen C-terminal propeptide sequence is shown in SEQ ID NO: 3, specifically as follows:

[0047] EPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPE LPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

[0048] The C-terminal propeptide sequence of type XV collagen is shown in SEQ ID NO: 4, and is specifically as follows:

[0049] NLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA;

[0050] The type XIX collagen NC2 domain sequence is shown in SEQ ID NO: 5, and is specifically as follows:

[0051] ADAVSFEEIKKYINQEVLRIFEERMAVFLSQLKLPAAMLAAQAY;

[0052] The stabilizing peptide (GPP) 4 sequence is shown in SEQ ID NO: 6, and is specifically as follows:

[0053] GPPGPPGPPGPP.

[0054] In the present invention, the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID No. 26 to SEQ ID No. 28, wherein SEQ ID No. 26 is formed by concatenating SEQ ID No. 7 12 times, connecting it with SEQ ID NO: 2, and then connecting it with SEQ ID NO: 3; SEQ ID No. 27 is formed by concatenating SEQ ID No. 8 12 times, connecting it with SEQ ID NO: 2, and then connecting it with SEQ ID NO: 3. The optimized nucleotide sequence of SEQ ID No. 27 is shown as SEQ ID No. 32; SEQ ID No. 28 is formed by concatenating SEQ ID No. 9 16 times, connecting it with SEQ ID NO: 2, and then connecting it with SEQ ID NO: 3. The specific sequences are as follows:

[0055] SEQ ID No. 26:

[0056] ;

[0057] SEQ ID No. 27:

[0058] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

[0059] SEQ ID No.28:

[0060] GAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

[0061] SEQ ID No.32:

[0062]

[0063] The present invention provides a recombinant expression vector comprising a nucleotide sequence of recombinant human type III collagen.

[0064] The present invention provides a host cell comprising the recombinant expression vector, wherein the host cell is selected from a CHO cell, a Pichia pastoris or a Saccharomyces cerevisiae eukaryotic expression system.

[0065] The present invention also provides the use of the recombinant human type III collagen in the preparation of cosmetics and medical tissue engineering products.

[0066] The present invention also provides the use of the recombinant human type III collagen in the preparation of medical materials that promote skin repair and improve biocompatibility.

[0067] In the present invention, the preparation method of the recombinant human type III collagen is as follows: inserting any one of the polynucleotides from SEQ ID No. 26 to SEQ ID No. 28 into a recombinant expression vector to construct a stable recombinant expression system, transferring the constructed stable recombinant expression vector into host cells, screening for stable cell lines, culturing the host cells, allowing the host cells to efficiently express the recombinant human type III collagen, and obtaining high-purity recombinant human type III collagen through separation and purification.

[0068] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0069] Example 1

[0070] The GXY sequence in the recombinant human type III collagen fragment (SEQ ID NO: 1) was extracted and analyzed using a bioinformatics algorithm. The specific analysis procedure is as follows: Figure 2 First, multiple peptides of varying sequence lengths were extracted (see Table 1), and their thermal denaturation midpoint temperatures (Tm) were calculated. The algorithm evaluated peptide length, amino acid composition, pairwise interactions, and the functionality of the peptide termini to produce a stability prediction for each peptide.

[0071] Table 1 Length of truncated peptides

[0072] Cut-off peptide length 12 15 18 21 24 27 30 Number of peptide segments that can be intercepted 339 338 337 336 335 334 333

[0073] To validate the effectiveness of the AI ​​algorithm combined with machine learning, computational and experimental verification was performed on peptides of varying lengths. The team found that the predicted Tm values ​​for peptides between 20 and 40 amino acids showed a high correlation with the actual experimental results. Based on this, peptides between 24 and 30 amino acids in length were selected for further optimization and sequence design.

[0074] Furthermore, the proline residues in these peptides were mutated with hydroxyproline residues, and the thermal denaturation midpoint temperatures of these residues were recalculated using a bioinformatics algorithm. The results showed that the hydroxyproline mutations significantly improved the stability of the peptides. Consequently, these optimized peptides were incorporated into the final designed recombinant human type III collagen sequence, and their efficient expression and stable triple helical structure in mammalian cells were experimentally verified.

[0075] Example 2

[0076] When the present invention uses three sequences comprising SEQ ID NOs: 7 to 9 and two sequences comprising SEQ ID NOs: 19 to 20 in mammalian cell expression, they are connected to the linker sequence human type III collagen C-terminal propeptide sequence (SEQ ID NO: 3), that is, the linker sequence is placed at the C-terminus of the tandem collagen sequence to obtain a recombinant triple-helical collagen sequence (SEQ ID NOs: 21 to 25). These terminal domains stabilize the associated α chains and promote the formation of triple helices by guiding chain selection and disulfide bond formation. Experimental results show that the recombinant protein designed without the C-terminal propeptide has poor solubility and cannot produce a triple helical structure, which further verifies the necessity of the terminal domain in improving protein solubility and functionality. In addition, the C-terminal propeptide sequence (SEQ ID NO: 3) is replaced with the C-terminal propeptide sequence of type XV collagen (SEQ ID NO: 4), the NC2 domain sequence of type XIX collagen (SEQ ID NO: 5) or the stabilizing peptide sequence (GPP) 4 (SEQ ID NO: 6), that is, three sequences containing SEQ ID NO: 7 to 9 are used in mammalian cell expression, which are respectively connected to the C-terminal domains SEQ ID NO: 4 to SEQ ID NO: 6. The specific connection sequences are shown in SEQ ID NO: 33 to 41. The resulting sequence also improves the ability to form a triple helical structure.

[0077] Example 3

[0078] The present invention adds a key amino acid sequence (SEQ ID NO: 2) to the collagen sequence SEQ ID NO: 21 to 25 in series in Example 2 to obtain a new sequence (SEQ ID NO: 26 to 30), namely recombinant human collagen RHCol1, RHCol2, RHCol3, RHCol4, and RHCol5, and the five recombinant collagens are purified respectively, and the denaturing non-reducing electrophoresis is verified. Through experiments, it was found that although some proteins to which the key sequence (SEQ ID NO: 2) was not added were able to form a triple helical structure, they could not be assembled into collagen fibers. After adding this sequence, the recombinant human type III collagen not only successfully formed a stable triple helical structure, but was also able to be further assembled into functional macromolecular collagen fibers, while the sequences of SEQ ID NO: 29 and SEQ ID NO: 30 could not form a triple helical structure.

[0079] In order to further verify the function of SEQ ID NO:2 in collagen assembly, SEQ ID NO:31 was recombinantly expressed, wherein SEQ ID NO:31 was formed by connecting SEQ ID No.8 sequence 12 times in series with SEQ ID NO:2 sequence repeated 8 times. The experimental results showed that it failed to migrate normally in the denaturing non-reducing SDS-PAGE analysis, and the protein was completely accumulated in the loading well and could not form a band. This phenomenon indicates that when SEQ ID NO:2 was repeated 8 times, the protein may have formed high-molecular-weight aggregates, which prevented it from entering the gel. This aggregation phenomenon shows that SEQ ID NO:2 plays a very critical role in collagen assembly, and that its appropriate repetition is necessary to maintain protein function and structural stability.

[0080] Example 4

[0081] Taking RHCol2 as an example, the experimental process is described in detail. To ensure efficient expression of the recombinant protein in mammalian cells, SEQ ID NO: 27 was codon-optimized to obtain an amino acid sequence as shown in SEQ ID NO: 32. EcoRI and HindIII restriction sites were added according to the gene sequence. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the sequence and cloned into the universal vector pUC57. The plasmid name is pUC57-Col. After double digestion with EcoRI and HindIII, a target fragment of approximately 1900bp was recovered for assembly of the expression plasmid. The expression vector used its own vector, which was digested with EcoRI and HindIII, and a 5920bp vector framework fragment was recovered for construction of the expression vector.

[0082] The vector 5920bp fragment was connected with multiple recombinant collagen 1900bp fragments, transformed, cloned and identified by colony PCR. The recombinant vector map is shown in Figure 3 As shown. Positive clones identified by PCR were selected and inoculated into LB medium. After overnight culture, the cells were cultured for plasmid extraction. Positive clones identified by enzyme digestion were commissioned for sequencing by Beijing Qingke Biotechnology Co., Ltd. Positive clones identified by sequencing and enzyme digestion were purified for plasmid extraction and used to construct stable cell lines.

[0083] Example 5

[0084] Pool transfection: The plasmid obtained in Example 4 was transfected into the host CHO cells by electroporation, with a total of 2 batches transfected. The recombinant plasmid carries the GS resistance gene and can be screened by MSX. After electroporation, the cells were transferred to a 6-well plate and placed in a 37°C, 5% CO2 incubator for static culture. After 48 hours, the supernatant was concentrated 8 times and then subjected to R / NR-SDS-PAGE detection. The SDS-PAGE of the cell supernatant after 48 hours of transfection is shown in the figure. Figure 4 The results showed that the supernatant contained the correctly expressed target protein.

[0085] Cell pool screening: 96-well plates were plated, 10 96-well plates were plated in 2 batches and placed in a 5% CO2 incubator for static culture. The 96-well cells were cultured for about two weeks, and the cell culture supernatant was taken for ELISA quantitative screening. The high-expression clones were transferred to 24-well plates, and the same method was used for screening and amplification of 6-well plates and shake flasks. The shake flask expression test results showed high-expression clones; after gradient screening in 96-well plates, 24-well plates, 6-well plates and shake flasks, a total of 6 candidate pool cells (3F5, 6C9, 1F8, 6C5, 1B7, 7F5) were screened based on cell growth and protein expression. The R-SDS-PAGE and NR-SDS-PAGE test results of the candidate pool cell supernatant confirmed that they correctly expressed the target protein. Pool cell expression supernatant R / NR-SDS-PAGE is shown in Figure 2. Figure 5 .

[0086] The 6 pool cells screened were mixed and used for protein production in 3L reactor. At the same time, when the candidate pool cells were expanded to a sufficient amount in the shake flask, 1×10 7 Cells were seeded at a cell density of 10 cells / mL.

[0087] Cell pool screening summary: Based on the above experimental results and comprehensive evaluation of cell growth and protein expression results, six candidate pool cells (3F5, 6C9, 1F8, 6C5, 1B7, and 7F5) were selected for subsequent development.

[0088] Example 6

[0089] Expansion of recombinant CHO cells: Select the clone with the highest expression level in the batch for shake flask culture. Prepare a 500 mL shake flask, add 50 mL of culture medium (ActiCHO P Powder CD), and the cell density is 1.0×10 6 cells / mL. When the cell density reaches 4.0×10 6 When the cell density was about 2.0 × 10 cells / mL, all cells were transferred to a 3 L fermenter and medium was added to reach a cell density of 2.0 × 10 6 cells / mL.

[0090] 3L fermenter fed-batch experiment: In a 3L fermenter, the cell density was maintained at 2.0×10 6 cells / mL. Observe the cell status and record the cell density every day until the total volume of the fermenter is expanded to about 1L; when the cell density grows to 4.0-6.0×10 6 cells / mL, feed medium (CD Efficient Feed CAG) was added to maintain the sugar concentration at 3.0 g / L. 6 cells / mL, the fermentation tank temperature was lowered to 34°C for protein expression. The protein expression level in the fermentation supernatant was determined by SDS-PAGE electrophoresis.

[0091] Fermentation broth collection and processing:

[0092] (1) When the cells in the fermenter reach the desired density, stop the culture. Transfer the fermentation broth to a centrifuge cup, balance it, and centrifuge it in a low-temperature high-speed centrifuge. Centrifugation conditions: 3500 rpm / min, 30 minutes, 4°C.

[0093] (2) After centrifugation, collect the supernatant, rebalance the solution, and centrifuge again in a low-temperature high-speed centrifuge using an angle rotor. Centrifugation conditions: 5000 rpm / min, 4°C, for 30 minutes.

[0094] (3) After two centrifugations, discard the cell pellet, collect the fermentation supernatant, and store it in a -20°C refrigerator.

[0095] The purity and impurities of the centrifuged samples were detected by SDS-PAGE electrophoresis. The concentration of the electrophoresis separation gel was 8% or 10%, and the concentration of the stacking gel was 5%.

[0096] Separation and purification:

[0097] (1) The fermentation broth supernatant was filtered through a 0.22 μm filter membrane, diluted 2-fold and passed through a phosphate buffer solution (pH 7.5), and then subjected to column chromatography using an MMC composite filler. The elution was performed step by step using a 1 M NaCl phosphate buffer solution (pH 7.5), and the fractions were collected.

[0098] (2) The collected fractions were subjected to molecular sieve chromatography using 4FF filler and a 0.9% NaCl solution as the mobile phase;

[0099] (3) Concentrate the sample using a concentrator tube; use BCA protein quantification to quantify the sample concentration.

[0100] (4) Freeze-drying of samples: The concentrated and quantified collagen solution is placed in a freeze-drying bottle, and the freeze-drying bottle containing the sample is placed in a freezer at -40°C or lower for pre-freezing for 2 to 4 hours to ensure that the sample is completely frozen. The pre-frozen sample is placed in a freeze dryer, and the cold trap temperature is set to -50°C to -80°C, and the vacuum degree is 10 to 20 Pa, and preliminary drying is performed for 24 to 48 hours. After the preliminary drying is completed, the temperature of the freeze-drying chamber is gradually increased to 20 to 25°C, and the vacuum degree is maintained unchanged for 12 to 24 hours to remove residual moisture. After completion, the pressure of the freeze-drying chamber is slowly restored, the freeze-drying bottle is taken out, and it is immediately sealed with a sealing cap to prevent the sample from absorbing moisture.

[0101] (5) Storage: Place the freeze-dried sample in a dry, sealed container and store it at a low temperature of -20°C or -80°C to ensure the long-term stability and activity of the sample.

[0102] Denaturing non-reducing electrophoresis was performed on the purified recombinant human type III collagen RHCol2. Figure 6 As shown in A, F is the flow-through fraction in the purification process, E1 is the elution fraction of 20% B eluent, E2 is the elution fraction of 30% B eluent, E3 is the elution fraction of 50% B eluent, E4 is the elution fraction of 60% B eluent, E5 is the elution fraction of 70% B eluent, and E6 is the elution fraction of 100% B eluent. Figure 6 As shown in Figure A, RHCol2 mainly exists in the form of a trimer under denaturing non-reducing conditions, and eluents E5 and E6 show good assembly properties, that is, the elution fractions obtained using 70% 1M NaCl phosphate buffer as the eluent and the elution fractions obtained using 100% 1M NaCl phosphate buffer as the eluent have good assembly properties.

[0103] Denaturing non-reducing SDS-PAGE: The obtained recombinant human type III collagen samples were subjected to SDS-PAGE analysis under denaturing non-reducing conditions.

[0104] The results of denatured non-reducing electrophoresis of recombinant human type III collagen are as follows Figure 6As shown in B, lanes 1, 2, and 3 are RHCol1, RHCol2, and RHCol3, respectively, which showed super molecular weight under denaturing non-reducing electrophoresis, lanes 4 and 5 are RHCol4 and RHCol5, in which no trimer or trimer assembly was found, lane 6 is RHCol4 denaturing reduction electrophoresis, and lanes 7, 8, and 9 are RHCol1, RHCol2, and RHCol3, which showed trimer assembly under denaturing reduction electrophoresis.

[0105] Liquid chromatography-mass spectrometry (LC-MS): 150 μg of recombinant human type III collagen was enzymatically hydrolyzed overnight (18 to 20 hours) in a 37°C water bath. After desalting, the supernatant was used for nanoliter liquid chromatography separation and high-resolution mass spectrometry detection. Liquid A used in the liquid phase was 0.1% formic acid aqueous solution, and liquid B was 0.1% formic acid acetonitrile aqueous solution (80% acetonitrile). Acclaim PepMap was used. TM A 1 μL injection volume was injected onto a 50 μm x 150 mm RSLC column (Thermo Scientific) equilibrated with 92% solution A. The digests were separated by nano-LC and analyzed by mass spectrometry on a Thermo QE HF mass spectrometer (Thermo Fisher) for 120 minutes. Detection was performed in positive ion mode. The mass-to-charge ratios of peptides and peptide fragments were acquired using the following method: 20 fragmentation spectra (MS2scans) were acquired after each full scan. The scan range was 400–1800, with a primary resolution of 60,000 and a secondary resolution of 15,000. The collision energy was CE 28 eV.

[0106] The total ion current and mass spectrum of the recombinant human type III collagen prepared by the present invention are as follows: Figure 7 、 8 As shown. Figure 7 、 Figure 8 Mass spectrometry analysis revealed high purity of the RHCol2 collagen, with uniform peptide distribution and distinct peaks, indicating that enzymatic hydrolysis produced the expected peptide fragments. These fragments closely matched the theoretical values, further confirming the correct amino acid sequence and structural integrity of the recombinant collagen. These results demonstrate that RHCol2 possesses high structural fidelity and consistency, meeting the requirements of subsequent functional testing and applications.

[0107] Circular dichroism (CD): Analyze the recombinant human type III collagen sample by circular dichroism. Take an appropriate amount of collagen sample and prepare a 1 mg / mL solution with an appropriate buffer (such as 20 mM phosphate buffer, pH 7.5). Scan within the range of 190-260 nm with a step size of 1 nm, a bandwidth of 1 nm, and a temperature controlled at 25°C. Measure and subtract the background, plot the CD spectrum, and analyze the secondary structure characteristics of the collagen.

[0108] The structures of recombinant human type III collagen RHCol1, RHCol2, and RHCol3 were characterized using circular dichroism spectroscopy. The results showed a negative peak near 207-208 nm and a positive peak near 221-222 nm, indicating that the prepared recombinant collagen had a triple helical structure, while this phenomenon was not found in RHCol4 and RHCol5.

[0109] Cell proliferation experiment: The Cell Counting Kit-8 method was used to study the effect of recombinant human type III collagen RHCol2 on L929 cell proliferation. L929 cells were seeded in 96-well plates at 5000 cells / well and cultured overnight in DMEM complete medium. The culture medium was then replaced with 0.01 mg / mL, 0.10 mg / mL, and 1.00 mg / mL RHCol2. After 1, 3, 5, and 7 days of culture, the CCK-8 kit was used to measure the absorbance of the solution at 450 nm to evaluate cell proliferation. A parallel group without collagen was set up as a blank control. The results are shown in the figure below. Figure 9 As shown. Figure 9 The results show that the cell proliferation rate in the experimental group supplemented with recombinant human type III collagen RHCol2 was significantly higher than that in the blank control group, and the cell proliferation rate further increased with increasing collagen concentration. This indicates that RHCol2 can effectively promote the proliferation of L929 cells, showing good bioactivity and biocompatibility, indicating its potential application value in tissue repair and regenerative medicine.

[0110] Biocompatibility testing: The intradermal reactivity of recombinant human type III collagen (RHCol2) solution in rabbits was evaluated according to GB / T 16886.10-2017, "Biological Evaluation of Medical Devices - Part 10: Tests for Irritation and Delayed-Type Hypersensitivity." Each rabbit received an injection of 0.5 mL of RHCol2 solution into the right side of the back, while saline was injected into the left side as a control. Erythema and edema reactions at the injection site were observed and recorded at 24, 48, and 72 hours. The results showed an overall mean score of 0 for the RHCol2 solution, the same as the control group, indicating that the solution did not cause any erythema or edema. This indicates that the RHCol2 solution did not exhibit significant skin irritation or delayed-type hypersensitivity reactions in the rabbit model, demonstrating its good biocompatibility.

[0111] Rat implantation experiment: The implant reactivity of recombinant human type III collagen solution in rats was evaluated according to GB / T 16886.6-2022 "Biological Evaluation of Medical Devices Part 6: Implantation Tests." The recombinant human type III collagen RHCol2 solution was injected into the subcutaneous tissue of the rat's back, and the control group was injected with an equal volume of normal saline. The local reactions and behavioral changes of the rats were observed and recorded on the 1st, 7th, 14th, and 21st days after surgery. The results showed that there were no significant differences in redness, swelling, induration, infection, and tissue necrosis between the recombinant human type III collagen RHCol2 solution group and the control group. Histological examination did not reveal any obvious inflammatory reaction or tissue damage. This indicates that the recombinant human type III collagen RHCol2 solution has good biocompatibility in the rat model.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A recombinant human type III collagen with a triple helical structure assembled into collagen fibers, characterized in that: The amino acid sequences of the recombinant human type III collagen are shown in SEQ ID No. 26 to SEQ ID No.

28.

2. The recombinant human type III collagen according to claim 1, characterized in that The nucleic acid encoding the amino acid sequence of the recombinant human type III collagen SEQ ID No. 27 is optimized as shown in SEQ ID No.

32.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a nucleotide sequence encoding the recombinant human type III collagen according to claim 1.

4. A host cell comprising the recombinant expression vector according to claim 3, characterized in that: The host cell is selected from CHO cells, Pichia pastoris or Saccharomyces cerevisiae eukaryotic expression systems.

5. Use of the recombinant human type III collagen according to claim 1 in the preparation of cosmetics and medical tissue engineering products.

6. Use of the recombinant human type III collagen according to claim 1 in the preparation of medical materials that promote skin repair and improve biocompatibility.

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