Recombinant human collagen type xxiii, methods for its production and uses
By splicing and expressing optimized recombinant human type XXIII collagen, the problem of insufficient bioactivity in existing technologies has been solved, achieving efficient production and significantly enhanced cell adhesion and coagulation-promoting effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIAN GIANT BIOGENE TECH CO LTD
- Filing Date
- 2024-12-31
- Publication Date
- 2026-05-08
AI Technical Summary
Existing technologies lack recombinant human type XXIII collagen with good biological activity, especially recombinant collagen constructed by truncating and splicing, which is insufficient in promoting cell adhesion and coagulation activity.
By discovering and splicing six truncated proteins of natural human type XXIII collagen with good cell adhesion and coagulation activities, recombinant human type XXIII collagen was constructed and efficiently expressed and purified in the Pichia pastoris expression system. The purified protein was obtained by salting out and chromatographic chromatography.
It achieved a significant improvement in the cell adhesion and coagulation activities of recombinant human type XXIII collagen, and had bioactivity comparable to or better than commercially available recombinant collagen, while being produced efficiently in a commercial expression system.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of synthetic biology technology and relates to recombinant human type XXIII collagen, its production method and application. Background Technology
[0002] Collagen is the most abundant protein found in mammals. The 28 types of collagen that have been discovered so far can be divided into four categories: fibrous collagen, reticular collagen, fibrillary-associated collagen (FACITs) with discontinuous triple helix, and membrane-associated collagen (MACITs) with discontinuous triple helix.
[0003] Type XXIII collagen is a novel transmembrane collagen discovered in recent years. It is a type of type II transmembrane protein and structurally highly similar to type XIII and XXV collagen, all containing three triple-helix collagen domains flanked by four non-collagen domains. The transmembrane domain is located in the NC1 domain. The human type XXIII collagen transcript is 3067 bp in size, encoding a 540-amino acid sequence. When anchored to the cell surface, the protein size is 75 kDa, and it can be cleaved by furin protease to produce a 60 kDa soluble protein.
[0004] As a newly discovered collagen, research on the function of type XXIII collagen is scarce, with existing studies mostly focusing on its role as a marker for various metastatic tumors. There is virtually no research on constructing functional human type XXIII recombinant collagen using methods such as truncation or splicing. Summary of the Invention
[0005] In view of the technical problems existing in the prior art, the object of the present invention is to provide a truncated protein of natural human type XXIII collagen with good biological activity, and a recombinant human type XXIII collagen with better biological activity constructed by splicing the above-mentioned truncated protein.
[0006] The inventors discovered six truncated proteins of natural human type XXIII collagen with good cell adhesion and coagulation activity (comparable to commercially available recombinant human collagen), and spliced them together in a specific way to construct recombinant human type XXIII collagen with better cell adhesion and coagulation activity (superior to commercially available recombinant human collagen).
[0007] That is, the technical solution of the present invention includes:
[0008] 1. A recombinant human type XXIII collagen, the amino acid sequence of which is shown in any one of SEQ ID No: 1 to 7.
[0009] 2. The nucleic acid of the recombinant human type XXIII collagen described in code item 1.
[0010] 3. An expression vector comprising the nucleic acid described in item 2.
[0011] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, with Escherichia coli and Pichia pastoris being preferred, and Pichia pastoris being more preferred.
[0012] 5. A method for producing recombinant human type XXIII collagen as described in item 1, comprising the steps of culturing the host cells described in item 4 to express the recombinant human type XXIII collagen as described in item 1, and collecting the culture containing the recombinant human type XXIII collagen. The expression may be one or a combination of constitutive expression and induced expression. The inducer for induced expression may be an inducer such as IPTG, β-galactoside, methanol, or ethanol.
[0013] 6. The production method according to item 5 further includes the step of separating and purifying the culture to obtain purified recombinant human type XXIII collagen.
[0014] 7. The production method according to item 6, wherein the separation and purification employs one or more of the following methods in combination: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably a combination of chromatographic chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.
[0015] 8. Use of the recombinant human type XXIII collagen as described in item 1 in the preparation of tissue engineering materials.
[0016] 9. The use according to item 8, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bone, artificial skin, oral absorbable biofilms, bone implants, and bone repair scaffolds.
[0017] The beneficial effects of this invention include:
[0018] 1) The amino acid sequence of the recombinant human type XXIII collagen described in this invention is 100% homologous to the natural amino acid sequence and has no immunogenicity.
[0019] 2) The recombinant human type XXIII collagen described in this invention can be expressed at high yields in a commercially available Pichia pastoris expression system. This expression system features post-translational modification, facilitates efficient secretory expression, and produces very few endogenous secretory proteins, which is convenient for subsequent purification.
[0020] 3) The recombinant human type XXIII collagen described in this invention has been verified to have cell adhesion properties that are comparable to or better than those of commercially available recombinant human collagen, thus achieving the intended application. Attached Figure Description
[0021] Figure 1 The image shows the expression of recombinant human type XXIII collagen 1-6 as detected by SDS-PAGE.
[0022] Figure 2 The image shows the expression of recombinant human type XXIII collagen 7 detected by SDS-PAGE. Detailed Implementation
[0023] Example 1: Preparation of recombinant human type XXIII collagen in Pichia pastoris expression system
[0024] I. Experimental Methods
[0025] 1) Preparation of recombinant human type XXIII collagen expression vector
[0026] According to the amino acid sequences shown in SEQ ID No:1-SEQ ID No:7, codon optimization was performed on the yeast expression system to obtain the target gene sequence. The obtained target gene sequence was then synthesized by Qingke Biotechnology Co., Ltd. The synthesized gene was ligated into the pPicZαA plasmid to obtain the recombinant human type XXIII collagen expression vector pPicZαA-XXIII1-7. It should be noted that SEQ ID No:1-6 are truncated proteins of natural human type XXIII collagen (referred to as recombinant human type XXIII collagen 1-6), while SEQ ID No:7 is the recombinant human type XXIII collagen (referred to as recombinant human type XXIII collagen 7) assembled from the above truncated proteins in a specific sequence.
[0027] 2) Preparation of recombinant human type XXIII collagen-expressing yeast strain
[0028] pPicZαA-XXIII 1-7 were linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Transformants were screened using bleomycin resistance as a selection marker to obtain the corresponding recombinant human XXIII type collagen yeast expression strains.
[0029] 3) Induced expression of recombinant human type XXIII collagen
[0030] (1) Preparation of primary culture: Pick a single colony from the plate and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate at 30℃ and 200 rpm for 48 h for activation;
[0031] (2) Secondary seed preparation: 1% inoculum was transferred to 200ml YPD liquid medium and cultured at 30℃ and 200rpm for 24h as the seed for the upper tank;
[0032] (3) Prepare 3L of BSM medium in a 5L fermenter, sterilize at 121℃ for 20min, and after cooling to 30℃, adjust the pH to 5.0 with ammonia water. Add the secondary seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture.
[0033] (4) Cultivate to OD 600 When the OD value is 50, start adding 50% glycerin until it reaches 50%. 600 Stop adding methanol when the dissolved oxygen reaches 120, and wait for the dissolved oxygen to rebound to 100% before starting to add methanol for induction.
[0034] (5) During the induction process, the DO should be controlled to be no less than 30% and the pH should be around 5.0. After induction for 40 hours, the fermentation should be stopped. The culture medium should be centrifuged at 8000 rpm for 10 minutes to collect the supernatant. The protein purity and concentration should be detected by SDS-PAGE.
[0035] 4) Purification of recombinant human XXIII collagen
[0036] The collected supernatant was decolorized by passing it through a hollow fiber column, purified by ion exchange and molecular sieve, and then freeze-dried under vacuum to obtain purified recombinant human XXIII collagen 1-7.
[0037] II. Results
[0038] SDS-PAGE analysis of the supernatant after fermentation showed successful expression of recombinant human XXIII collagen proteins 1-7, with high yield and good purity. Protein electrophoresis results are as follows... Figure 1 and 2 As shown.
[0039] Example 2: Detection of cell adhesion-promoting activity of recombinant human type XXIII collagen
[0040] I. Experimental Methods
[0041] Cell adhesion promotion experiments were conducted on HSF (human skin fibroblast) cells using various recombinant human XXIII collagens, commercially available recombinant human collagens, and bovine serum albumin (purchased from Beijing Solarbio) as described in Example 1. Human HSF cells were seeded in sterile 96-well plates with 200 μL of culture medium per well. The blank control group received only PBS. After incubation at 37°C for 2 hours, each group was washed three times with PBS to remove unadhered cells. 100 μL of culture medium and 50 μL of MTT solution were added to each well, and the mixture was shaken well and incubated for 4 hours. The mixture was then aspirated, and 150 μL of DMSO was added to dissolve the purple formazan crystals. The mixture was shaken for 10 minutes and then placed in a microplate reader. The absorbance of each well was measured at a wavelength of 570 nm.
[0042] Cell adhesion promotion rate = (OD of experimental group - OD of blank group) × 100% / blank group.
[0043] II. Results
[0044] Table 1 shows the data on HSF cell adhesion promotion by recombinant human XXIII collagen, commercially available recombinant human collagen, and bovine serum albumin.
[0045] Table 1. Effects of different protein concentrations on cell adhesion promotion of skin fibroblasts (cell adhesion rate values)
[0046]
[0047] As shown in Table 1, recombinant human type XXIII collagen 1-6 are basically equivalent to commercially available recombinant human collagen in promoting cell adhesion. However, the spliced protein, namely recombinant type XXIII collagen 7, has a stronger effect on promoting cell adhesion than the commercially available protein. In other words, recombinant human type XXIII collagen 7 has a better ability to promote cell adhesion.
[0048] Example 3: In vivo coagulation test of recombinant human type XXIII collagen
[0049] I. Preparation of Recombinant Human Type XXIII Collagen Sponge
[0050] Accurately weigh 0.005% of purified TG enzyme and disperse it in a certain volume of purified water. After it is fully dissolved, weigh 7% (2%) of recombinant human XXIII collagen prepared in Example 1 and place it in a mixer at room temperature (25°C) and stir until fully dissolved. After standing to remove bubbles, fill and disperse it into molds. Place it in a refrigerator at 2-8°C for 48 hours to crosslink. After the crosslinking is completed, place the collagen sponge in a freeze dryer and freeze dry it to obtain the collagen sponge.
[0051] II. In vivo coagulation test of recombinant human type XXIII collagen
[0052] Sixty Kunming mice (purchased from the Experimental Animal Center of Xi'an Jiaotong University School of Medicine) were used. A blank control group (distilled water), a commercially available collagen sponge group (Wuxi Bedy Biotechnology Co., Ltd.), and a recombinant human type XXIII collagen sponge group were set up. The collagen sponge thickness was 5 mm.
[0053] Ten Kunming mice were used in each treatment group, with half male and half female. The scalpel was sterilized with medical alcohol and a flame. An incision was made on the inner thigh of each mouse. After bleeding, the blood was absorbed with gauze. Simultaneously, a collagen sponge (cut to twice the size of the wound) was quickly applied to the wound. A 20g weight was used for vertical pressure, and the timing was recorded. The wound was observed every 20 seconds, and the timing was stopped when no further bleeding occurred; this was the time required for hemostasis. The blank control group was sprayed with the corresponding distilled water (sprayed for 30 seconds), and its clotting time and process were observed. The comparison of the clotting effects of the three groups is shown in Table 2 below.
[0054] Table 2 Comparison of hemostatic effects of recombinant human type XXIII collagen sponge
[0055]
[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the essence and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
[0057] Sequence information:
[0058] SEQ ID No:1:GRRGKPGRRGDPGPPGQSGRDGYPGPLGLDGKPGLPGPKGEKGAPGDFGPRGDQGQD
[0059] SEQ ID No:2:GAAGPPGPPGPPGARGPPGDTGKDGPRGAQGPAGPKGEPGQDGEMGPKGPPGPKGEPGVPGKK
[0060] SEQ ID No:3:QPGPPPGPKGEPGSMGPRGENGVDGAPGPKGEPGHRGTDGAAGPRG APGLKGEQGD
[0061] SEQ ID No:4: GPPGPQGPPGPPGIPGAKGELGLPGAPGIDGEKGPKGQKGDPGEPGPAGLKGEAGEMGLS
[0062] SEQ ID No:5:GPPGPPGPPGPMGLQGIQGPKGLDGAKGEKGASGERGSGLPGPVGP PG
[0063] SEQ ID No:6:GLPGTKGEKGRPGEPGLDGFPGPRGEKGDRSERGEKGERGVPGRKGVKGQKGEPGPPGLD
[0064] SEQ ID No:7:GRRGKPGRRGDPGPPGQSGRDGYPGPLGLDGKPGLPGPKGEKGAPG DFGPRGDQGQDGAAGPPGPPGPPGARGPPGDTGKDGPRGAQGPAGPKGEPGQDGEMGPKGPPGPKGEPGVPGKKQPGPPGPKGEPGSMGPRGENGVDGAPGPKGEPGHRGTDGAAGPRGAPGLKGEQGDGPPGPQGPPGPPGIPGAKGELGLPGAPGIDGEKGPKGQKGDPGEPGPAGLKGEAGEMGLSGPPGPPGPPGPMGLQGIQGPKGLDGAKGEKGASGERGPSGLPGPVGPPGGLPGTKGEKGRPGEPGLDGFPGPRGEKGDRSERGEKGERGVPGRKGVKGQKGEPGPPGLD
Claims
1. A recombinant human type XXIII collagen, the amino acid sequence of which is shown in SEQ ID No:
7.
2. Nucleic acid encoding the recombinant human type XXIII collagen as described in claim 1.
3. An expression vector comprising the nucleic acid of claim 2.
4. The host cell into which the expression vector of claim 3 is introduced.
5. A method for producing recombinant human type XXIII collagen according to claim 1, comprising the steps of culturing the host cells according to claim 4 to express the recombinant human type XXIII collagen according to claim 1, and collecting the culture containing the recombinant human type XXIII collagen.
6. The production method according to claim 5, further comprising the step of separating and purifying the culture to obtain purified recombinant human type XXIII collagen.
7. The use of the recombinant human type XXIII collagen according to claim 1 in the preparation of tissue engineering materials.
8. The use according to claim 7, wherein, The tissue engineering materials are selected from hemostatic sponges, subcutaneous fillers, artificial bone, artificial skin, oral absorbable biomembranes, bone implants, and bone repair scaffolds.