CN119331080A

CN119331080A - Recombinant human type III collagen with triple helical structure assembled into collagen fibers and its application

Info

Publication number: CN119331080A
Application number: CN202411482731.4A
Authority: CN
Inventors: 梁重阳
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2024-10-23
Filing date: 2024-10-23
Publication date: 2025-01-21

Abstract

The present invention provides a recombinant human type III collagen with a triple helix structure and assembled into collagen fibers and its application, belonging to the field of bioengineering technology. The present invention screens out collagen fragments that can form a triple helix structure through a bioinformatics algorithm, and connects these fragments in series, combines the key C-terminal domain and a specific amino acid sequence, and effectively promotes the formation of a stable triple helix higher structure and the assembly of collagen fibers. The recombinant human type III collagen is efficiently expressed through a eukaryotic expression system and shows excellent stability, low immunogenicity and good biocompatibility. The experimental results show that the prepared recombinant human type III collagen not only has a natural triple helix 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

Recombinant human-derived III-type collagen with triple-helix structure and assembled into collagen fibers and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant human-derived III-type collagen which has a triple-helical structure and is assembled into collagen fibers and application thereof.

Background

Collagen is an important structural protein, which is widely present in human tissues and organs. It has excellent biocompatibility and biodegradability, and has been widely used in biomedical fields. However, the conventional animal-derived collagen has problems of low purity, high immunogenicity, unstable quality, and the like in the extraction process. In addition, the sequence and structure of animal collagen are different from those of human collagen, which may cause rejection reaction, and limit the clinical application of animal collagen.

Typical structures of collagen are G-X-Y sequences (where G is glycine, X and Y are usually proline and hydroxyproline), which are capable of forming stable triple helix structures. In natural collagen, the triple helix structure is critical to the stability, functionality and biocompatibility of collagen. However, due to the long length of natural collagen and the complex post-translational modifications, there is a degree of difficulty in achieving recombinant expression of full-length collagen.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant human type III collagen assembled into collagen fibers with triple helix structure and application thereof, wherein a mammalian cell expression system is used to construct and screen a stable cell line, so that the recombinant human type III collagen can be correctly folded in cells, and is ensured to have the triple helix structure of natural collagen, and complete collagen fibers can be formed. The recombinant collagen has high stability and low immunogenicity, is suitable for the biomedical field, and has the advantages of low cost and strong expandability.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a recombinant human-source III-type collagen assembled into a collagen fiber, which is prepared by connecting any one of collagen fragments capable of forming a triple-helical structure with a specific amino acid sequence after being connected in series for 8-20 times and then connecting a C-terminal domain.

Preferably, the sequence of the three-helix collagen fragment is shown as SEQ ID No. 7-SEQ ID No. 20.

Preferably, the C-terminal domain comprises a human type III collagen C-terminal propeptide sequence, an XV collagen C-terminal propeptide sequence, an XIX collagen NC2 domain sequence or a stabilizing peptide sequence (GPP) 4, wherein the human type III collagen C-terminal propeptide sequence is shown as SEQ ID NO. 3, the XV collagen C-terminal propeptide sequence is shown as SEQ ID NO. 4, the 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.

Preferably, the specific amino acid sequence is shown as SEQ ID NO. 2.

Preferably, the amino acid sequence of the recombinant humanized III type collagen is shown as SEQ ID No. 26-SEQ ID No. 28.

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

The invention provides a recombinant expression vector, which comprises a nucleotide sequence of recombinant human-derived type III collagen.

The invention provides a host cell comprising the recombinant expression vector, wherein the host cell is selected from CHO cells, pichia pastoris or Saccharomyces cerevisiae eukaryotic expression systems.

The invention also provides application of the recombinant human source III type collagen in preparing cosmetics and medical tissue engineering products.

The invention also provides application of the recombinant human source III type collagen in preparing medical materials for promoting skin repair and improving biocompatibility.

Compared with the prior art, the invention has the following beneficial effects that the fragments capable of forming the triple helix structure are obtained by utilizing the characteristics of the G-X-Y sequence and adopting algorithm screening, and are connected in series, and the formation of the stable triple helix high-level structure and the assembly of the collagen fiber are effectively promoted by combining a key C-terminal structural domain and a specific amino acid sequence. By concatenating these optimized fragments and expressing them in mammalian cells, they show excellent stability, low immunogenicity and good biocompatibility, and finally a recombinant human type III collagen with high stability and low immunogenicity is obtained, and can be further assembled to form complete collagen fibers. The invention adopts a mammalian cell expression system to construct and screen a stable cell strain, so that the recombinant humanized III type collagen can be correctly folded in cells, ensure that the recombinant humanized III type collagen has a triple helix structure of natural collagen, and can form complete collagen fibers. The recombinant collagen has high stability and low immunogenicity, is suitable for the biomedical field, and has the advantages of low cost and strong expandability.

Drawings

FIG. 1 is a sequence design of recombinant human type III collagen;

FIG. 2 is a schematic representation of the sequence bioinformatics calculation for recombinant human type III collagen;

FIG. 3 is a diagram of the genetic construction of recombinant human type III collagen;

FIG. 4 is a SDS-PAGE map (R/NR) of the supernatant of CHO 48h cells transfected with host cells;

FIG. 5 is a drawing of Pool cell expression supernatant R/NR-SDS-PAGE;

FIG. 6 shows the result of denaturing and non-reducing electrophoresis of recombinant human type III collagen, wherein A in FIG. 6 shows the result of denaturing and non-reducing electrophoresis of recombinant human type III collagen RHCol and B in FIG. 6 shows the result of denaturing and non-reducing electrophoresis of different recombinant human type III collagen;

FIG. 7 is a liquid chromatography-mass spectrometry (LC-MS) total ion flow diagram of recombinant human type III collagen RHCol;

FIG. 8 is a mass spectrum of a key peptide fragment of recombinant human type III collagen RHCol;

FIG. 9 is a graph showing the effect of recombinant human type III collagen RHCol2 on cell behavior.

Detailed Description

In the invention, the method for obtaining the collagen fragment with the triple-helix structure comprises the steps of screening and designing the collagen fragment capable of forming the triple-helix structure through a bioinformatics algorithm. The screening and designing are to cut off the whole sequence (SEQ ID NO: 1) of the human type III collagen by adopting G-X-Y tripeptide repeat sequence characteristics and using bioinformatics algorithms such as Tm calculation and molecular dynamics simulation, design a plurality of peptide fragments of 12-40 amino acids, preferably 15-34 amino acids, more preferably 24-30 amino acids, calculate the melting temperature (Tm value) of each peptide fragment by using the Tm algorithm, combine amino acid composition analysis and immunogenicity prediction, screen out peptide fragments with high solubility and low immunogenicity, wherein the Tm value of the peptide fragments is 0-80, preferably 30-75, more preferably 50-70, the peptide fragments with high solubility are peptide fragments containing more than 45% of hydrophilic amino acids, the peptide fragments with low immunogenicity are peptide fragments without T cell epitope or peptide fragments with binding affinity with MHC molecules higher than 1000nM, and finally obtain the peptide fragments with high solubility and stability of the recombinant type III collagen by molecular dynamics simulation verification.

In the invention, the sequence of the three-helix collagen fragment is shown as SEQ ID No. 7-SEQ ID No.20, and specifically comprises the following steps:

SEQ ID No.7:GESGRPGRPGERGLPGPPGIKGPAGIPGFP;

SEQ ID No.8:GERGAPGFRGPAGPNGIPGEKGPAGERGAP;

SEQ ID No.9:GAPGPMGPRGAPGERGRPGLP;

SEQ ID No.10:GAPGPMGPRGAPGER;

SEQ ID No.11:GPMGPRGAPGERGRPGLPGAA;

SEQ ID No.12:GAPGPMGPRGAPGERGRP;

SEQ ID No.13:GRPGERGLPGPPGIKGPAGIP;

SEQ ID No.14:GERGLPGPPGIKGPA;

SEQ ID No.15:GESGRPGRPGERGLPGPPGIK;

SEQ ID No.16:GPRGAPGERGRPGLP;

SEQ ID No.17:GPMGPRGAPGERGRP;

SEQ ID No.18:GKDGESGRPGRPGERGLPGPP;

SEQ ID No.19:GENGAPGPMGPRGAPGERGRPGLP;

SEQ ID No.20:GESGRPGRPGERGLPGPPGIKGPAGIP;

The number of the series connection is preferably 10 to 18, more preferably 12 to 16.

In the invention, the specific amino acid sequence is shown as SEQ ID NO. 2, and is specifically as follows:

CGGVGAAAIAGIGGEKAGGFAPYYGD;

The C-terminal domain comprises a humanized type III collagen C-terminal propeptide sequence, an XV type collagen C-terminal propeptide sequence, an XIX type collagen NC2 domain sequence or a stabilizing peptide sequence (GPP) 4, wherein the humanized type III collagen C-terminal propeptide sequence is shown as SEQ ID NO:3, and the specific steps are as follows:

EPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

the C-terminal propeptide sequence of the XV-type collagen is shown as SEQ ID NO. 4, and is specifically as follows:

NLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA;

The sequence of the NC2 structural domain of the type XIX collagen is shown as SEQ ID NO. 5, and is specifically as follows:

ADAVSFEEIKKYINQEVLRIFEERMAVFLSQLKLPAAMLAAQAY;

The sequence of the stabilizing peptide (GPP) 4 is shown as SEQ ID NO. 6, and is specifically as follows:

GPPGPPGPPGPP。

In the invention, the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID No. 26-SEQ ID No.28, wherein SEQ ID No.26 is formed by connecting SEQ ID No.7 sequence 12 times in series and then with SEQ ID No.2 and then with SEQ ID No.3, SEQ ID No.27 is formed by connecting SEQ ID No.8 sequence 12 times in series and then with SEQ ID No.2 and then with SEQ ID No.3, the nucleotide sequence of the optimized SEQ ID No.27 is shown as SEQ ID No.32, SEQ ID No.28 is formed by connecting SEQ ID No.9 sequence 16 times in series and then with SEQ ID No.2 and then with SEQ ID No.3, and the specific sequence is as follows:

SEQ ID No.26:

GESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPGESGRPGRPGERGLPGPPGIKGPAGIPGFPCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

SEQ ID No.27:

GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

SEQ ID No.28:

GAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPGAPGPMGPRGAPGERGRPGLPCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL;

SEQ ID No.32:

GGGGAGCGAGGAGCACCAGGCTTCAGAGGACCCGCCGGACCAAATGGGATTCCAGGAGAAAAAGGACCCGCCGGAGAGCGAGGAGCACCAGGGGAACGGGGTGCCCCTGGGTTTCGGGGGCCCGCTGGACCTAACGGGATTCCAGGGGAAAAAGGACCTGCTGGTGAACGCGGAGCTCCAGGGGAGCGGGGCGCCCCAGGCTTCAGAGGACCAGCTGGACCAAATGGAATCCCAGGAGAAAAGGGCCC CGCAGGTGAACGGGGGGCACCCGGCGAGCGTGGCGCTCCTGGTTTTCGGGGCCCAGCTGGACCAAATGGGATACCTGGTGAAAAGGGGCCAGCCGGTGAACGAGGGGCACCTGGGGAGAGAGGTGCTCCAGGATTCAGAGGCCCAGCTGGTCCAAACGGTATTCCTGGCGAAAAAGGTCCAGCCGGAGAGAGAGGGGCACCTGGAGAACGGGGAGCCCCTGGCTTTCGAGGGCCTGCTGGACCAAATGGTATTCCAGGAGAAAAGGGGCCAGCTGGGGAAAGGGGAGCCCCAGGAGAACGTGGAGCCCCTGGGTTCAGAGGGCCCGCTGGGCCAAACGGGATTCCCGGGGAGAAAGGACCAGCAGGTGAACGGGGTGCACCAGGAGAAAGAGGAGCTCCTGGTTTCCGCGGCCCAGCCGGTCCCAACGGCATACCTGGGGAAAAAGGTCCAGCTGGGGAAAGGGGTGCTCCCGGAGAGCGAGGAGCTCCCGGGTTTCGGGGTCCTGCAGGTCCCAACGGGATCCCCGGTGAAAAAGGTCCCGCTGGTGAAAGGGGCGCTCCCGGAGAGCGGGGGGCTCCTGGCTTCAGGGGTCCAGCTGGCCCCAACGGTATTCCAGGCGAGAAGGGCCCAGCCGGCGAAAGAGGTGCTCCAGGGGAACGTGGCGCCCCAGGGTTTCGTGGTCCCGCTGGGCCTAACGGAATCCCAGGAGAGAAGGGACCAGCTGGGGAAAGAGGCGCACCTGGTGAACGTGGAGCCCCAGGGTTCCGGGGTCCCGCTGGCCCAAACGGTATTCCCGGCGAAAAGGGCCCAGCAGGCGAGCGGGGTGCACCATGTGGTGGCGTGGGGGCTGCAGCTATAGCCGGAATAGGCGGAGAAAAGGCTGGCGGTTTCGCCCCATACTATGGAGACGAACCCATGGATTTCAAGATTAACACCGATGAAATAATGACCAGCCTGAAATCAGTGAATGGTCAAATCGAGAGCCTGATATCACCAGATGGATCCCGGAAAAATCCAGCTCGAAATTGCCGGGACCTGAAATTCTGCCACCCTGAACTTAAGAGCGGGGAGTACTGGGTCGATCCTAACCAGGGCTGTAAACTGGACGCTATCAAGGTGTTTTGCAATATGGAAACAGGCGAGACTTGTATATCTGCTAACCCCCTTAATGTGCCTCGCAAGCATTGGTGGACCGATTCCTCAGCTGAGAAGAAGCATGTTTGGTTCGGGGAAAGCATGGATGGGGGATTTCAGTTTTCCTATGGTAACCCTGAGTTGCCAGAGGATGTTCTTGACGTGCACCTTGCCTTTCTGAGATTGCTTTCCAGCCGTGCAAGCCAGAACATTACCTATCACTGCAAAAATTCTATCGCCTACATGGATCAGGCCTCTGGCAATGTTAAGAAGGCTCTGAAGCTGATGGGGTCCAATGAAGGAGAGTTTAAAGCTGAAGGGAACAGTAAGTTTACTTACACCGTGCTTGAGGACGGGTGTACTAAGCATACAGGCGAATGGTCAAAGACCGTGTTTGAGTACCGAACACGGAAGGCTGTGAGGCTCCCTATAGTGGACATCGCTCCCTATGATATTGGGGGACCCGACCAGGAGTTTGGCGTGGATGTGGGACCAGTGTGCTTTCTG.

The preparation method of the recombinant human-derived type III collagen comprises the steps of inserting any one of the polynucleotides from SEQ ID No.26 to SEQ ID No.28 into a recombinant expression vector, constructing a stable recombinant expression system, transferring the constructed stable recombinant expression vector into host cells, screening stable cell lines, culturing the host cells, enabling the host cells to efficiently express the recombinant human-derived type III collagen, and separating and purifying to obtain the recombinant human-derived type III collagen with high purity.

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

Example 1

The G-X-Y sequence in the recombinant human type III collagen fragment (SEQ ID NO: 1) was intercepted and analyzed using bioinformatics algorithms, and the specific procedure for the analysis is shown in FIG. 2. First, a plurality of peptide fragments were cut out according to different lengths of the sequences, see Table 1, and their thermal denaturation midpoint temperature values (Tm) were calculated. The algorithm evaluates peptide length, amino acid composition, pairwise interactions, and peptide end functions to obtain a predicted stability value for each peptide.

TABLE 1 interception of peptide fragment Length

Length of cut-out peptide	12	15	18	21	24	27	30
								Number of truncated peptide fragments	339	338	337	336	335	334	333

In order to verify the effectiveness of combining an AI algorithm with machine learning, calculation and experimental verification are carried out on peptide fragments with different lengths, and the predicted Tm value has higher correlation with an actual experimental result when the length of the peptide fragment is between 20 and 40 amino acids. Based on this, peptide stretches of 24 to 30 amino acids in length were selected for further optimization and sequence design.

In addition, hydroxyproline in these peptide fragments was subjected to mutation substitution, and the thermal denaturation midpoint temperature value after mutation was recalculated using a bioinformatics algorithm. The results show that hydroxyproline mutation significantly improves the stability of the peptide fragment. Therefore, the optimized peptide fragments are introduced into the finally designed recombinant human type III collagen sequence, and the efficient expression of the optimized peptide fragments in mammalian cells and the formation of stable triple helix structures are verified through experiments.

Example 2

When three sequences containing SEQ ID NO 7-9 and two sequences containing SEQ ID NO 19-20 are used in mammalian cell expression, the recombinant triple helix collagen sequence (SEQ ID NO 21-25) is connected with a linker sequence human type III collagen C-terminal propeptide sequence (SEQ ID NO 3), namely the linker sequence is placed at the C end of a tandem collagen sequence, and the end domains stabilize related alpha chains and promote formation of triple helix through guidance chain selection and disulfide bond formation. Experimental results show that the recombinant protein designed without adding the C-terminal propeptide has poor solubility and cannot generate a triple helix structure, which further verifies the necessity of the end domain in improving the protein solubility and the functionality. In addition, the C-terminal propeptide sequence (SEQ ID NO: 3) is replaced by an XV-type collagen C-terminal propeptide sequence (SEQ ID NO: 4), an XIX-type collagen NC2 domain sequence (SEQ ID NO: 5) or a stabilizing peptide sequence (GPP) 4 (SEQ ID NO: 6), namely three sequences comprising SEQ ID NO: 7-9 are used in mammalian cell expression and are respectively connected with the C-terminal domain SEQ ID NO: 4-SEQ ID NO:6, the specific connection sequence is shown as SEQ ID NO: 33-41, and the obtained sequences also improve the formation capacity of a triple helix structure.

Example 3

In the invention, a key amino acid sequence (SEQ ID NO: 2) is added into the collagen sequences SEQ ID NO: 21-25 in series in example 2 to obtain a new sequence (SEQ ID NO: 26-30), namely recombinant human collagen RHCol1, RHCol2, RHCol3, RHCol4 and RHCol5, and the five recombinant collagens are purified respectively and verified by denaturing non-reducing electrophoresis. Through experiments, it was found that a part of the protein to which the key sequence (SEQ ID NO: 2) was not added could form a triple helix structure, but could not be assembled into a collagen fiber. After the sequence is added, the recombinant human-derived III-type collagen not only successfully forms a stable triple helix structure, but also can be further assembled into a macromolecular collagen fiber with functionality, and the sequences of SEQ ID NO. 29 and SEQ ID NO. 30 cannot form the triple helix structure.

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 is formed by concatenating the SEQ ID NO.8 sequence 12 times in series with the 8 repeated SEQ ID NO. 2 sequence. The experimental results show that the protein cannot migrate normally in the denatured non-reduced SDS-PAGE analysis, and is completely accumulated in the loading well, and cannot form a band. This phenomenon suggests that in the case of 8 repeats of SEQ ID NO. 2, the protein may form aggregates of high molecular weight, which may not enter the gel. This aggregation phenomenon suggests that SEQ ID NO 2 plays a very critical role in collagen assembly and that its proper repetition is necessary to maintain protein function and structural stability.

Example 4

The experimental procedure is specifically described by taking RHCol2 as an example, the amino acid sequence obtained by codon optimization of SEQ ID NO:27 for ensuring high-efficiency expression of recombinant protein in mammalian cells is shown as SEQ ID NO:32, ecoRI and HindIII cleavage sites are added according to the gene sequence, sequence synthesis is carried out by Nanjin St Biotechnology Co., ltd., and cloned into a universal vector pUC57, and the plasmid name is pUC57-Col. The fragment of interest, approximately 1900bp in size, was recovered for assembly of the expression plasmid by double digestion with EcoRI and HindIII. The expression vector was used as an own vector, the expression vector was digested with EcoRI and HindIII, and a 5920bp vector frame fragment was recovered for construction of the expression vector.

The 5920bp fragment of the vector is connected with a plurality of 1900bp fragments of the recombinant collagen, the recombinant vector is subjected to transformation, cloning and colony PCR identification, and the recombinant vector map is shown in figure 3. And selecting positive clones which are identified to be correct by PCR, inoculating the positive clones into an LB culture medium, and culturing thalli overnight to obtain large plasmids. The correct positive clones were identified by restriction enzyme testing and sequenced by Beijing qingke biosciences, inc. The correct positive clone is identified by sequencing and enzyme digestion, and the large plasmid is used for constructing stable cell strains.

Example 5

Pool transfection the plasmid obtained in example 4 was transfected into the host cell CHO by electrotransfection, co-transfecting 2 batches. Recombinant plasmids harbor the GS resistance gene and can be screened by MSX. After electrotransformation, the cells were transferred to a 6-well plate, placed in a 37 ℃ and 5% CO ₂ incubator for static culture, and after 48 hours, the supernatant was concentrated 8 times and subjected to R/NR-SDS-PAGE detection, and the SDS-PAGE diagram of the 48-hour transfected cell supernatant is shown in FIG. 4. The results showed that the supernatant contained the correctly expressed protein of interest.

Cell pool screening, namely, paving 96-well plates, paving 10 96-well plates in 2 batches, placing the plates in a 5% CO ₂ incubator for static culture, culturing 96-well cells for about two weeks, taking Cell culture supernatant for ELISA quantitative screening, transferring high-expression clones into 24-well plates, and screening and amplifying 6-well plates and shake flasks by adopting the same method. The detection results of shake flask expression show high expression cloning, 6 candidate Pool cells (3F 5, 6C9, 1F8, 6C5, 1B7 and 7F 5) are screened out totally according to cell growth and protein expression by 96-well plates, 24-well plates, 6-well plates and shake flask gradient screening, the detection results of R-SDS-PAGE and NR-SDS-PAGE of the supernatants of the candidate Pool cells confirm that the candidate Pool cells correctly express target proteins, and the R/NR-SDS-PAGE of the supernatants of the Pool cells is shown in figure 5.

The 6 strains of pool cells screened were mixed and used for 3L reactor protein preparation. Meanwhile, when candidate pool cells were expanded to a sufficient amount in shake flasks, cell seeds were frozen at a cell density of 1×10 ⁷ cells/mL.

Cell pool Cell selection summary based on the above experimental results, 6 candidate pool cells (3F 5, 6C9, 1F8, 6C5, 1B7, 7F 5) were selected for subsequent development based on comprehensive evaluation of Cell growth and protein expression results.

Example 6

And (3) expanding the recombinant CHO cells, namely selecting clones with highest batch expression quantity for shake flask culture. 500mL shake flasks were prepared and 50mL of medium (ActiCHO P Powder CD) was added and inoculated at a cell density of 1.0X10 ⁶ cells/mL. When the cell density reached about 4.0X10 ⁶ cells/mL, the cells were all transferred to a 3L fermenter and the medium was supplemented to a cell density of 2.0X10 ⁶ cells/mL.

3L fermenter run-on experiments in a 3L fermenter, the cell density was maintained at 2.0X10 ⁶ cells/mL all the time by the addition of medium. And (3) observing the cell state every day, recording the cell density until the total volume in the fermentation tank is expanded to about 1L, and starting to supplement the fed-batch culture medium (CD EFFICIENT FEED CAG) when the cell density is 4.0-6.0X10 ⁶ cells/mL, so that the sugar concentration is kept at 3.0g/L. When the cell density reached 8X 10 ⁶ cells/mL, the fermenter temperature was lowered to 34℃for protein expression. The expression level of the protein in the fermentation supernatant was determined by SDS-PAGE electrophoresis.

Collecting and treating fermentation liquor:

(1) When the cells cultured in the fermenter reached the desired density, the culture was stopped. Transferring the fermentation broth into a centrifugal cup, balancing, and centrifuging by using a low-temperature high-speed centrifuge. Centrifugation conditions were 3500rpm/min,30 min, 4 ℃.

(2) After centrifugation, the supernatant was collected, re-trimmed, and centrifuged again in a low temperature, high speed centrifuge using an angular rotor. The centrifugation conditions were 5000rpm/min,4℃and 30 minutes.

(3) After two centrifugation, the cell pellet was discarded, and the supernatant of the fermentation broth was collected and stored in a-20 ℃ refrigerator.

The purity and impurities of the centrifuged samples were detected by SDS-PAGE, the concentration of the electrophoresis separation gel was selected to be 8% or 10%, and the concentration of the concentration gel was selected to be 5%.

And (3) separating and purifying:

(1) Passing the supernatant of the fermentation broth through a 0.22 μm filter membrane, diluting with phosphate buffer (pH 7.5) for 2 times, performing column chromatography with MMC composite filler, eluting with 1M NaCl phosphate buffer (pH 7.5) step by step, and collecting the components;

(2) The collected components were chromatographed through a molecular sieve with 4FF packing, using 0.9% nacl solution as the mobile phase;

(3) Sample concentration was performed using a concentration tube and sample concentration was quantitatively analyzed using BCA protein quantification.

(4) And (3) freeze-drying the sample, namely filling the concentrated and quantified collagen solution into a freeze-drying bottle, and pre-freezing the freeze-drying bottle filled with the sample in a freezer at the temperature of-40 ℃ or lower for 2-4 hours so as to ensure that the sample is completely frozen. And placing the pre-frozen sample in a freeze dryer, setting the temperature of a cold trap to be between 50 ℃ below zero and 80 ℃ below zero, and performing primary drying for 24-48 hours under the vacuum degree of 10-20 Pa. After the primary drying is finished, gradually increasing the temperature of the freeze-drying chamber to 20-25 ℃, and keeping the vacuum degree unchanged for 12-24 hours so as to remove residual moisture. After the completion, the pressure of the freeze-drying chamber is slowly restored, the freeze-drying bottle is taken out, and the freeze-drying bottle is immediately sealed by a sealing cover to prevent the sample from absorbing moisture.

(5) Storage, in which the lyophilized sample is placed in a dry sealed container, optionally in a low temperature environment at-20 ℃ or-80 ℃ to ensure long-term stability and activity of the sample.

Recombinant human type III collagen RHCol was purified by denaturing non-reducing electrophoresis, as shown in FIG. 6A, F was the flow-through fraction during purification, E1 was 20% B eluate fraction, E2 was 30% B eluate fraction, E3 was 50% B eluate fraction, E4 was 60% B eluate fraction, E5 was 70% B eluate fraction, and E6 was 100% B eluate fraction. As can be seen from FIG. 6A, RHCol, E6 show good assembly properties under denaturing non-reducing conditions mainly in the form of trimers, i.e. the eluted fraction obtained using 70%1M NaCl phosphate buffer as eluent and the eluted fraction obtained using 100%1M NaCl phosphate buffer as eluent.

SDS-PAGE under denaturing and non-reducing conditions, and subjecting the obtained recombinant human type III collagen sample to SDS-PAGE analysis.

As shown in FIG. 6, lanes 1,2 and 3 show the results of denaturing non-reducing electrophoresis of recombinant human type III collagen, lanes RHCol, RHCol, RHCol3 show supermolecular weights in the case of denaturing non-reducing electrophoresis, lanes 4,5 show RHCol, RHCol, no trimer and trimer assembly are found in the case of denaturing non-reducing electrophoresis, lane 6 shows RHCol4 denaturing reducing electrophoresis, and lanes 7, 8 and 9 show trimer assembly in the case of denaturing reducing electrophoresis, as shown in lanes RHCol, RHCol, RHCol.

The liquid chromatography-mass spectrometry (LC-MS) method comprises the steps of taking 150 mug of recombinant human source III type collagen, placing the recombinant human source III type collagen in a 37 ℃ water bath for enzymolysis overnight (18-20 hours), desalting, and taking supernatant for nano-scale liquid chromatography separation and high-resolution mass spectrometer detection. The liquid phase used was 0.1% aqueous formic acid solution A and 0.1% aqueous acetonitrile formic acid solution B (80% acetonitrile). The enzymolysis product is subjected to mass spectrometry by a Thermo QE HF mass spectrometer (Thermo Fisher) after being separated by nano-upgrading liquid chromatography by balancing with a liquid A of 92 percent and feeding the sample by 1 mu L of the liquid A by using a ACCLAIMPEPMAP ^TM RSLC chromatographic column (50 mu m: 150mm,Thermo Scientific), wherein the analysis duration is 120 minutes. The detection mode is positive ion mode. The mass-to-charge ratio of the polypeptide and fragments of the polypeptide was determined by taking 20 fragment patterns (MS 2 scan) after each full scan (full scan). Scanning range is 400-1800, primary resolution is 60000, secondary resolution is 15000, and collision energy is CE28eV.

The total ion flow and mass spectrum of the recombinant human III-type collagen prepared by the invention are shown in figures 7 and 8. As can be seen from fig. 7 and 8, the mass spectrometry analysis results show that RHCol2 collagen has high purity, uniformly distributed peptide fragments and clear peak, and the protein generates the expected peptide fragment after enzymolysis. The fragments are highly coincident with the theoretical values, further proving that the amino acid sequence of the recombinant collagen is correct and the structure is complete. These results indicate that RHCol has high structural fidelity and consistency, and can meet the requirements of subsequent functional tests and applications.

Circular dichroism spectrum (CD) is carried out by performing circular dichroism spectrum analysis on recombinant human source III type collagen sample, taking proper amount of collagen sample, preparing 1mg/mL solution with proper buffer solution (such as 20mM phosphate buffer solution, pH 7.5), scanning within 190-260 nm, 1nm step length, 1nm bandwidth, controlling temperature at 25deg.C, measuring background and subtracting, drawing CD spectrogram, and analyzing secondary structure characteristics of collagen.

The structure of recombinant human type III collagen RHCol, RHCol and RHCol is characterized by using a circular dichroism spectrum, and the result shows that a negative peak appears near 207-208 nm and a positive peak appears near 221-222 nm, which indicates that the prepared recombinant collagen has a triple helix structure, and RHCol and RHCol have no phenomenon.

Cell proliferation experiments the effect of recombinant human type III collagen RHCol2 on L929 cell proliferation was studied using Cell Counting Kit-8 method. L929 cells were inoculated into 96-well plates at 5000 cells/well, cultured overnight in DMEM complete medium, then replaced with medium containing 0.01mg/mL, 0.10mg/mL, 1.00mg/mLRHCol, and after culturing for 1, 3, 5, and 7 days, the proliferation of cells was evaluated by measuring absorbance of the solution at 450nm using CCK-8 kit, and a parallel group without collagen was additionally provided as a blank, and the results are shown in FIG. 9. As can be seen from fig. 9, the experimental group to which the recombinant human type III collagen RHCol2 was added had significantly higher cell proliferation rate than the control group, and the cell proliferation rate was further improved with the increase of the collagen concentration. This indicates that RHCol can effectively promote the proliferation of L929 cells, shows good bioactivity and biocompatibility, and shows potential application value in tissue repair and regeneration medicine.

Biocompatibility experiments the intradermal reactivity of recombinant human type III collagen RHCol2 solutions in rabbits was evaluated according to GB/T16886.10-2017 section 10 biological evaluation of medical devices, stimulus and delayed hypersensitivity test. Each rabbit was injected with 0.5mL of recombinant human type III collagen RHCol solution on the right side of the back and physiological saline on the left side as a control. The erythema and edema response at the injection site was observed and recorded at 24 hours, 48 hours and 72 hours, and 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 response. This demonstrates that the recombinant human type III collagen RHCol solution did not exhibit significant skin irritation or delayed type hypersensitivity in the rabbit model, demonstrating good biocompatibility.

Rat implantation experiments the implantation reactivity of recombinant human type III collagen solutions in rats was evaluated according to GB/T16886.6-2022 medical device biological evaluation section 6 implantation experiments. The recombinant human type III collagen RHCol solution was injected into the subcutaneous tissue of the back of the rat, and the control group was injected with an equal amount of physiological saline. Local response and behavioral changes in rats were observed and recorded post-operatively on days 1, 7, 14 and 21. The results show that the recombinant human type III collagen RHCol solution group and the control group have no obvious difference in red swelling, induration, infection, tissue necrosis and the like, and no obvious inflammatory reaction or tissue injury is found in the histological examination. This indicates that the recombinant human type III collagen RHCol solution has good biocompatibility in the rat model.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The recombinant human-derived type III collagen assembled into the collagen fiber is characterized in that the recombinant human-derived type III collagen is prepared by connecting any one of collagen fragments capable of forming a triple-helical structure with a specific amino acid sequence after being connected in series for 8-20 times and then connecting a C-terminal domain.

2. The recombinant human-derived type III collagen according to claim 1, wherein the sequence of the triple-helix collagen fragment is shown in SEQ ID No. 7-SEQ ID No. 20.

3. The recombinant human type III collagen according to claim 1, wherein the C-terminal domain comprises a human type III collagen C-terminal propeptide sequence, an XV type collagen C-terminal propeptide sequence, an XIX type collagen NC2 domain sequence, or a stabilizing peptide sequence (GPP) 4;

The C-terminal propeptide sequence of the humanized III-type collagen is shown as SEQ ID NO. 3;

The C-terminal propeptide sequence of the XV-type collagen is shown as SEQ ID NO. 4;

The sequence of the NC2 structural domain of the type XIX collagen is shown as SEQ ID NO. 5;

The sequence of the stabilizing peptide (GPP) 4 is shown as SEQ ID NO. 6.

4. The recombinant human type III collagen according to claim 1, wherein the specific amino acid sequence is shown in SEQ ID No. 2.

5. The recombinant human-derived type III collagen according to claim 1, wherein the amino acid sequence of the recombinant human-derived type III collagen is shown as SEQ ID No. 26-SEQ ID No. 28.

6. The recombinant human type III collagen according to claim 5, wherein the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID No. 32.

7. A recombinant expression vector comprising the nucleotide sequence of recombinant human type III collagen according to claim 5.

8. A host cell comprising the recombinant expression vector of claim 7, wherein the host cell is selected from CHO cells, pichia pastoris, or saccharomyces cerevisiae eukaryotic expression systems.

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

10. The use of the recombinant human-derived type III collagen according to any one of claims 1 to 6 for the preparation of a medical material for promoting skin repair and improving biocompatibility.