Recombinant human III-type collagen with triple-helix structure and assembled into collagenous fiber and application of recombinant human III-type collagen

Abstract

The invention provides recombinant human III-type collagen which is of a triple-helix structure and assembled into collagenous fibers and application of the recombinant human III-type collagen, and belongs to the technical field of bioengineering. Collagen fragments capable of forming a triple helix structure are screened out through a bioinformatics algorithm, the fragments are connected in series, a key C-terminal structural domain and a specific amino acid sequence are combined, and formation of a stable triple helix advanced structure and assembly of collagenous fibers are effectively promoted. The recombinant human III type collagen 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 III-type collagen not only has a natural triple helix structure, but also can be further assembled into macromolecular collagen fibers, is suitable for multiple fields such as medical beauty, bioengineering and tissue repair, and has wide application prospects and market values.

Description

Technical Field

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

[0002] Collagen is an important structural protein that is widely present in human tissues and organs. It has excellent biocompatibility and biodegradability and has been widely used in the biomedical field. However, traditional animal-derived collagen has problems such as low purity, high immunogenicity, and unstable quality during the extraction process. In addition, the sequence and structure of animal collagen are different from those of human collagen, which may lead to xenogeneic rejection, 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 can form a stable triple helix structure. In natural collagen, the triple helix structure is essential for the stability, functionality and biocompatibility of collagen. 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 view of this, the purpose of the present invention is to provide a recombinant human type III collagen with a triple helical structure and assembled into collagen fibers and its application. The present invention uses a mammalian cell expression system to construct and screen a stable cell line, so that the recombinant human type III collagen can be correctly folded in the cell, ensuring that it has a triple helical structure of natural collagen and can form complete collagen fibers. This recombinant collagen has high stability and low immunogenicity, is suitable for the biomedical field, and its production method has 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 helix structure and 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 helix structure in series 8 to 20 times, connecting with a specific amino acid sequence, and then connecting with a C-terminal domain.

[0007] Preferably, the sequence of the collagen fragment with triple helix structure is 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 as shown in SEQ ID NO: 3; the type XV collagen C-terminal propeptide sequence is as shown in SEQ ID NO: 4; the type XIX collagen NC2 domain sequence is as shown in SEQ ID NO: 5; the stabilizing peptide (GPP) 4 sequence is as shown in 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 in 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, which contains the 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 for promoting skin repair and improving 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 helix 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 helix higher 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, and it 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 helix structure of natural collagen and can form complete collagen fibers. This recombinant collagen has high stability and low immunogenicity, is suitable for the biomedical field, and its production method has the advantages of low cost and strong scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The sequence design diagram 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 construction map of recombinant human type III collagen;

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

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

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

[0023] Figure 7 It is the total ion current 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 peptides of recombinant human type III collagen RHCol2;

[0025] Fig. 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 helix structure and 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 helix structure in series 8 to 20 times, connecting with a specific amino acid sequence, and then connecting with 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 bioinformatics algorithm. The screening and design is to use the GXY tripeptide repeat sequence characteristics, 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 with MHC molecules higher than 1000nM; the feasibility of the screened peptides is verified by molecular dynamics simulation, and finally a plurality of 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, and 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, and is 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 sequence of the stabilizing peptide (GPP) 4 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 in SEQ ID No.26 to SEQ ID No.28, wherein SEQ ID No.26 is formed by connecting SEQ ID NO:2 and SEQ ID NO:3 after 12 concatenations of SEQ ID No.7; SEQ ID No.27 is formed by connecting SEQ ID NO:2 and SEQ ID NO:3 after 12 concatenations of SEQ ID No.8; the optimized nucleotide sequence of SEQ ID No.27 is shown in SEQ ID No.32; SEQ ID No.28 is formed by connecting SEQ ID NO:2 and SEQ ID NO:3 after 16 concatenations of SEQ ID No.9; the specific sequence is 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, which contains the 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 for promoting skin repair and improving 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 SEQID No.28 into a recombinant expression vector to construct a stable recombinant expression system, transferring the constructed stable recombinant expression vector into a host cell, screening a stable cell line, culturing the host cell, allowing the host cell 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope 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 As shown. First, multiple peptides were extracted according to the different lengths of the sequence, as shown in Table 1, and their thermal denaturation midpoint temperatures (Tm) were calculated. The algorithm evaluates the peptide length, amino acid composition, pairwise interactions, and the function of the peptide termini to obtain a stability prediction value for each peptide.

[0071] Table 1 Length of peptide segments extracted

[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] In order to verify the effectiveness of AI algorithm combined with machine learning, peptides of different lengths were calculated and experimentally verified, and it was found that when the peptide length was between 20 and 40 amino acids, the predicted Tm value had a high correlation with the actual experimental results. Based on this, peptides with a length of 24 to 30 amino acids were selected for further optimization and sequence design.

[0074] In addition, the proline in these peptides was mutated with hydroxyproline, and the thermal denaturation midpoint temperature value after mutation was recalculated using a bioinformatics algorithm. The results showed that the hydroxyproline mutation significantly improved the stability of the peptide. Therefore, these optimized peptides were introduced into the final designed recombinant human type III collagen sequence, and their efficient expression in mammalian cells and the formation of a stable triple helix structure were verified by experiments.

[0075] Example 2

[0076] When the present invention uses three sequences including SEQ ID NO:7-9 and two sequences of SEQ ID NO:19-20 in mammalian cell expression, they are connected with 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 NO:21-25), and these terminal domains stabilize the related α chains and promote the formation of triple helices by guiding chain selection and disulfide bond formation. The experimental results show that the recombinant protein without the C-terminal propeptide design 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, and the specific connection sequences are shown in SEQID NO: 33 to 41. The resulting sequence also improves the ability to form a triple helix structure.

[0077] Example 3

[0078] The present invention adds a key amino acid sequence (SEQ ID NO: 2) to the collagen sequences SEQ ID NO: 21 to 25 in series in Example 2 to obtain new sequences (SEQ ID NO: 26 to 30), namely recombinant human collagen RHCol1, RHCol2, RHCol3, RHCol4, and RHCol5, and purifies the five recombinant collagens respectively, and verifies them by denaturing non-reducing electrophoresis. Through experiments, it was found that although some proteins without the addition of the key sequence (SEQ ID NO: 2) can form a triple helix structure, they cannot be assembled into collagen fibers. After adding this sequence, the recombinant human type III collagen not only successfully forms a stable triple helix structure, but can also be further assembled into functional macromolecular collagen fibers, while the sequences of SEQ ID NO: 29 and SEQ ID NO: 30 cannot form a triple helix 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 specifically described. To ensure the efficient expression of recombinant proteins in mammalian cells, SEQ ID NO:27 is codon-optimized to obtain an amino acid sequence as shown in SEQ ID NO:32. EcoRI and HindIII restriction sites are added according to the gene sequence, and Nanjing GenScript Biotechnology Co., Ltd. is commissioned to synthesize the sequence and clone it into the universal vector pUC57. The plasmid name is pUC57-Col. After double digestion with EcoRI and HindIII, the target fragment of about 1900bp is recovered for assembly of expression plasmids. The expression vector uses its own vector, which is digested with EcoRI and HindIII, and the 5920bp vector framework fragment is recovered for the 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. The positive clones identified by PCR were selected and inoculated into LB medium. After overnight culture, the plasmids were extracted. The positive clones identified by restriction digestion were entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The positive clones identified by sequencing and restriction digestion were extracted and used for stable cell line construction.

[0083] Example 5

[0084] Pool transfection: The plasmid obtained in Example 4 was transfected into the host CHO cells by electroporation, and two batches were transfected in total. 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 supernatant of cells transfected 48 hours later is shown in Figure 4 The results showed that the supernatant contained the correctly expressed target protein.

[0085] Cell pool screening: plate 96-well plates, plate 10 96-well plates in 2 batches and place them in a 5% CO2 incubator for static culture. Culture the 96-well cells for about two weeks, take the cell culture supernatant for ELISA quantitative screening, transfer the high-expressing clones to 24-well plates, and use the same method to screen and amplify 6-well plates and shake flasks. The shake flask expression test results showed high-expressing 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. The R / NR-SDS-PAGE of the Pool cell expression supernatant is shown in Figure 2. Figure 5 .

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

[0087] Summary of cell pool screening: Based on the above experimental results and comprehensive evaluation of cell growth and protein expression results, 6 candidate pool cells (3F5, 6C9, 1F8, 6C5, 1B7, 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 3L 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 Observe the cell status and record the cell density every day until the total volume in the fermenter expands 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 keep the sugar concentration at 3.0 g / L. 6 When the fermentation temperature reached 1.34 °C, the fermenter 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 expected density, stop the culture. Transfer the fermentation broth to a centrifuge cup, balance it, and centrifuge it using a low-temperature high-speed centrifuge. Centrifugation conditions: 3500 rpm / min, 30 minutes, 4°C.

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

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

[0095] The purity and impurities of the centrifuged samples were tested by SDS-PAGE electrophoresis. The concentration of the electrophoresis separation gel was 8% or 10%, and the concentration of the concentrated 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 (pH 7.5), subjected to column chromatography using an MMC composite filler, and eluted step by step using a 1 M NaCl phosphate buffer (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) Sample freeze drying: 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, 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, slowly restore the pressure of the freeze-drying chamber, take out the freeze-dried bottle, and immediately seal it with a sealing cap to prevent the sample from absorbing moisture.

[0101] (5) Storage: The freeze-dried samples should be placed in a dry, sealed container and can be stored at a low temperature of -20°C or -80°C to ensure the long-term stability and activity of the samples.

[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 with 20% B eluent, E2 is the elution fraction with 30% B eluent, E3 is the elution fraction with 50% B eluent, E4 is the elution fraction with 60% B eluent, E5 is the elution fraction with 70% B eluent, and E6 is the elution fraction with 100% B eluent. Figure 6 As shown in 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 denaturing 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 (acetonitrile was 80%). Use AcclaimPepMap TM RSLC column (50μm*150mm, Thermo Scientific) was balanced with 92% A solution, and the injection volume was 1μL. After separation by column, the enzymatic hydrolysate was separated by nano-liquid chromatography and then analyzed by mass spectrometry using Thermo QE HF mass spectrometer (Thermo Fisher). The analysis time was 120 minutes. Detection method: positive ion mode. The mass-to-charge ratio of peptides and peptide fragments was collected according to the following method: 20 fragment spectra (MS2scan) were collected after each full scan. The scanning range was 400-1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE28eV.

[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 It can be seen that the mass spectrometry analysis results show that the purity of RHCol2 collagen is high, the peptides are evenly distributed and the peaks are clear, indicating that the protein produces the expected peptide fragments after enzymatic hydrolysis. These fragments are highly consistent with the theoretical values, further proving that the amino acid sequence of the recombinant collagen is correct and the structure is complete. These results show that RHCol2 has high structural fidelity and consistency, and can meet the needs of subsequent functional testing and applications.

[0107] Circular dichroism (CD): Perform circular dichroism analysis on the recombinant human type III collagen sample. Take an appropriate amount of collagen sample and prepare it into a 1 mg / mL solution with an appropriate buffer (such as 20 mM phosphate buffer, pH 7.5); scan in the range of 190-260 nm with a step length of 1 nm, a bandwidth of 1 nm, and a temperature controlled at 25°C; measure and subtract the background, draw a CD spectrum, and analyze the secondary structure characteristics of collagen.

[0108] The structures of recombinant human type III collagen RHCol1, RHCol2, and RHCol3 were characterized by circular dichroism spectroscopy. The results showed a negative peak near 207-208nm and a positive peak near 221-222nm, indicating that the prepared recombinant collagen had a triple helix 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 Figure 2. Fig. 9 As shown. Fig. 9 It can be seen that the cell proliferation rate of the experimental group with recombinant human type III collagen RHCol2 was significantly higher than that of the blank control group, and the cell proliferation rate increased further with the increase of collagen concentration. This shows 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 experiment: 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: Irritation and Delayed Hypersensitivity Tests". Each rabbit was injected with 0.5 mL of recombinant human type III collagen RHCol2 solution on the right side of the back, and normal saline was injected on the left side as a control. The erythema and edema reactions at the injection site were observed and recorded at 24 hours, 48 ​​hours and 72 hours. The results showed that the overall average score of the recombinant human type III collagen solution was 0, which was the same as the control group, indicating that the solution did not cause any erythema or edema reaction. This shows that the recombinant human type III collagen RHCol2 solution did not show obvious skin irritation or delayed hypersensitivity in the rabbit model, proving that it has good biocompatibility.

[0111] Rat implantation experiment: The implantation 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 Test". 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 amount 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 was no significant difference between the recombinant human type III collagen RHCol2 solution group and the control group in terms of redness, swelling, nodules, infection and tissue necrosis, and no obvious inflammatory response or tissue damage was found in histological examination. This shows 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 principle of the present invention. These improvements and modifications should also be regarded as 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 recombinant human type III collagen is prepared by connecting any one of the collagen fragments capable of forming a triple helix structure in series 8 to 20 times, connecting with a specific amino acid sequence, and then connecting with a C-terminal domain.

2. The recombinant human type III collagen according to claim 1, characterized in that: The sequences of the collagen fragments with triple helical structure are shown in SEQ ID No.7 to SEQ ID No.

20.

3. The recombinant human type III collagen according to claim 1, characterized in that: 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; The XV type collagen C-terminal propeptide sequence is shown in SEQ ID NO: 4; The type XIX collagen NC2 domain sequence is shown in SEQ ID NO: 5; The sequence of the stabilizing peptide (GPP) 4 is shown in SEQ ID NO:

6.

4. The recombinant human type III collagen according to claim 1, characterized in that: The specific amino acid sequence is shown in SEQ ID NO:

2.

5. The recombinant human type III collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No.26 to SEQ ID No.

28.

6. The recombinant human type III collagen according to claim 5, characterized in that: The optimized nucleotide sequence of the amino acid sequence SEQ ID No.27 of the recombinant human type III collagen is shown in SEQ ID No.

32.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleotide sequence of the recombinant human type III collagen according to claim 5.

8. A host cell comprising the recombinant expression vector according to claim 7, characterized in that: The host cell is selected from CHO cells, Pichia pastoris or Saccharomyces cerevisiae eukaryotic expression systems.

9. Use of the recombinant human type III collagen according to any one of claims 1 to 6 in the preparation of cosmetics and medical tissue engineering products.

10. Use of the recombinant human type III collagen according to any one of claims 1 to 6 in the preparation of medical materials for promoting skin repair and improving biocompatibility.

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