Recombinant human XXIII type collagen as well as production method and application thereof

Abstract

The invention discloses recombinant human XXIII type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human XXIII type collagen is shown as any one of SEQ ID No: 1-7, the SEQ ID No: 1-6 are truncated proteins of natural human XXIII type collagen, and the SEQ ID No: 7 is the recombinant human XXIII type collagen formed by splicing the truncated proteins according to a specific sequence. The cell adhesion promotion of the recombinant human XXIII type collagen is superior to or equivalent to that of commercially available recombinant human collagen, the amino acid sequence of the recombinant human XXIII type collagen is 100% homologous with that of natural human XXIII type collagen, and the recombinant human XXIII type collagen has no immunogenicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology and relates to recombinant human type XXIII collagen, a production method and application thereof. Background Art

[0002] Collagen is the most abundant protein found in mammals. The 28 types of collagen discovered so far can be divided into four major categories: fibrillar collagen, reticular collagen, fibril-associated collagens with interrupted triple helix (FACITs) and membrane-associated collagens with interrupted triple helix (MACITs).

[0003] Type XXIII collagen is a new type of transmembrane collagen discovered in recent years. It is a type II transmembrane protein. Its structure is highly similar to that of type XIII and type XXV collagen. They all contain three triple helical collagen domains, flanked by four non-collagen domains, and the transmembrane domain is located in the NC1 domain. The transcript size of human type XXIII collagen is 3067 bp, encoding a sequence of 540 amino acids. The protein size anchored to the cell surface is 75 KD, which can be cleaved by furin to produce a 60 KD soluble protein.

[0004] As a newly discovered collagen, there are few studies on the function of type XXIII collagen, and existing studies mostly focus on its role as a marker for various metastatic tumors. There is basically no research on constructing functional human type XXIII recombinant collagen by truncation, splicing, etc. 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 truncated protein.

[0006] The inventors discovered six truncated proteins of natural human type XXIII collagen with good cell adhesion and procoagulant activities (comparable to commercially available recombinant human collagen), and spliced ​​them in a specific way to construct recombinant human type XXIII collagen with better cell adhesion and procoagulant activities (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 Nos: 1 to 7.

[0009] 2. A nucleic acid encoding the recombinant human type XXIII collagen described in 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 may 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, preferably Escherichia coli and Pichia pastoris, and more preferably Pichia pastoris.

[0012] 5. A method for producing the recombinant human type XXIII collagen described in item 1, comprising the steps of culturing the host cell described in item 4 to express the recombinant human type XXIII collagen described in item 1, and collecting the culture containing the recombinant human type XXIII collagen. The expression can be constitutive expression, induced expression, or a combination of the two. The inducer for inducing expression can be an inducer such as IPTG, β-galactoside, methanol, ethanol, etc.

[0013] 6. The production method according to item 5, further comprising 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 adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.

[0015] 8. Use of the recombinant human type XXIII collagen according to 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 bones, artificial skin, oral absorbable biomembranes, bone implants, and bone repair scaffolds.

[0017] The beneficial effects of the present invention include:

[0018] 1) The amino acid sequence of the recombinant human type XXIII collagen described in the present invention is 100% homologous to the natural amino acid sequence and has no immunogenicity.

[0019] 2) The recombinant human type XXIII collagen of the present invention can be expressed in high yield in a commercial Pichia pastoris expression system. The expression system has post-translational modification and is easy to secrete and express efficiently. The minimal endogenous secretory protein facilitates subsequent purification.

[0020] 3) The recombinant human type XXIII collagen described in the present invention has been verified to have cell adhesion comparable to or better than commercially available recombinant human collagen, and can achieve the application purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows the expression of recombinant human type XXIII collagen 1-6 detected by SDS-PAGE.

[0022] Figure 2 The figure shows the expression of recombinant human type XXIII collagen 7 detected by SDS-PAGE. DETAILED DESCRIPTION

[0023] Example 1: Preparation of recombinant human type XXIII collagen in Pichia pastoris expression system

[0024] 1. Experimental Methods

[0025] 1) Preparation of recombinant human type XXIII collagen expression vector

[0026] According to the amino acid sequence shown in SEQ ID No: 1-SEQ ID No: 7, the codon of the yeast expression system was optimized to obtain the target gene sequence, and the target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to 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 to 6 are truncated proteins of natural human type XXIII collagen (referred to as recombinant human type XXIII collagen 1 to 6), and SEQ ID No: 7 is a recombinant human type XXIII collagen (referred to as recombinant human type XXIII collagen 7) formed by splicing the above truncated proteins in a specific order.

[0027] 2) Preparation of yeast expression strain for recombinant human type XXIII collagen

[0028] pPicZαA-XXIII 1-7 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain the corresponding recombinant human type XXIII collagen yeast expression strain.

[0029] 3) Inducible expression of recombinant human type XXIII collagen

[0030] (1) Primary seed preparation: 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 culture at 30°C, 200 rpm for 48 h for activation;

[0031] (2) Preparation of secondary seeds: transfer 1% of the inoculum to 200 ml of YPD liquid medium and culture at 30°C and 200 rpm for 24 h to serve as seeds for tanks;

[0032] (3) 3 L of BSM medium was prepared in a 5 L fermenter, sterilized at 121° C. for 20 min, cooled to 30° C., adjusted to pH 5.0 with aqueous ammonia, and the secondary seeds prepared in (2) were added to the fermenter by flame inoculation for fermentation;

[0033] (4) Cultivate until OD 600 When OD is 50, add 50% glycerol until 600 When the concentration reaches 120, stop adding, wait until the dissolved oxygen rebounds to 100%, and start adding methanol for induction;

[0034] (5) During the induction process, DO was controlled to be no less than 30% and pH was about 5.0. The fermentation was terminated after 40 h of induction. The culture medium was centrifuged at 8000 rpm for 10 min to collect the supernatant, and the protein purity and concentration were detected by SDS-PAGE.

[0035] 4) Purification of recombinant human collagen XXIII

[0036] The collected supernatant is decolorized by a hollow fiber column, purified by ion exchange and molecular sieve, vacuum frozen and desalted, and then freeze-dried to obtain purified recombinant human XXIII collagen 1-7.

[0037] 2. Results

[0038] The results of SDS-PAGE detection of the supernatant after fermentation showed that the corresponding recombinant human XXIII collagen 1 to 7 were successfully expressed with high yield and good purity. Figure 1 and 2 shown.

[0039] Example 2: Detection of the cell adhesion promoting activity of recombinant human type XXIII collagen

[0040] 1. Experimental Methods

[0041] The various recombinant human XXIII collagens, commercially available recombinant human collagens and bovine serum albumin (purchased from Beijing Solebao) described in Example 1 were used to act on HSF (human skin fibroblasts) cells to promote cell adhesion experiments. Human HSF cells were inoculated in a sterile 96-well plate, 200 μL of culture medium per well, and only PBS was added to the blank control group. After being placed at 37°C for 2 hours, each group was washed 3 times with PBS to wash away the non-adherent cells, 100 μL of culture medium and 50 μL of MTT solution were added to each well, shaken well, and placed in an incubator for incubation for 4 hours, then the mixed solution was aspirated, 150 μL of DMSO was added to dissolve the purple crystal formazan, shaken for 10 minutes, placed in an ELISA reader, and the detection wavelength was 570 nm. The absorbance value of each well was measured.

[0042] Cell adhesion promotion rate = (OD of experimental group - OD of blank group) × 100% / blank group.

[0043] 2. Results

[0044] The data of HSF cell adhesion promoted by recombinant human XXIII collagen, commercially available recombinant human collagen and bovine serum albumin are shown in Table 1:

[0045] Table 1 Cell adhesion promoting effect of different proteins at different concentrations on skin fibroblasts

[0046]

[0047] It can be seen from the results in Table 1 that recombinant human type XXIII collagen 1 to 6 are basically equivalent to commercially available recombinant human collagen in promoting cell adhesion, but the spliced ​​protein, i.e., recombinant type XXIII collagen 7, has a stronger cell adhesion effect than the commercially available protein, that is, recombinant human type XXIII collagen 7 has better cell adhesion ability.

[0048] Example 3: In vivo coagulation test of recombinant human type XXIII collagen

[0049] 1. Preparation of recombinant human type XXIII collagen sponge

[0050] Accurately weigh the refined TG enzyme (0.005%), disperse it in a certain volume of purified water, and after it is fully dissolved, weigh the recombinant human XXIII collagen 7 (2%) prepared in Example 1 and stir it in a blender at room temperature (25°C) until it is fully dissolved, let it stand to defoam, and then fill and disperse it into a mold; place it in a 2-8°C refrigerator and let it stand for 48 hours to cross-link; put the cross-linked collagen sponge into a freeze dryer and freeze-dry it to obtain a collagen sponge.

[0051] 2. In vivo coagulation test of recombinant human type XXIII collagen

[0052] 60 Kunming mice (purchased from the Experimental Animal Center of the School of Medicine of Xi'an Jiaotong University) were used to set up a blank control group (distilled water), a commercially available collagen sponge group (Wuxi Beidi Bioengineering Co., Ltd.), and a recombinant human type XXIII collagen sponge group. The thickness of the collagen sponge was 5 mm.

[0053] Each treatment group had 10 Kunming mice, half male and half female. The scalpel was disinfected with medical alcohol and flame, and an incision was made on the inner thigh of the mouse with the scalpel. After the blood flowed out, the blood was absorbed with gauze, and the above-mentioned collagen sponge was quickly applied to the wound. The size of the sponge was cut according to 2 times the size of the mouse wound, and a 20g weight was used to apply pressure vertically and timed. The wound was observed every 20 seconds, and the timing was stopped after no bleeding, and the time required for hemostasis was obtained. The blank control group was sprayed with distilled water (kept within 30 seconds), and its coagulation time and coagulation process were observed. The comparison of the coagulation effects of the three groups is shown in Table 2 below:

[0054] Table 2 Comparison of coagulation effects of recombinant human type XXIII collagen sponge

[0055]

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the essence and principles of the present invention shall be equivalent replacement modes and shall be included in 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. A recombinant human type XXIII collagen, the amino acid sequence of which is shown in any one of SEQ ID Nos: 1 to 6.

3. A nucleic acid encoding the recombinant human type XXIII collagen according to claim 1 or 2.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell into which the expression vector according to claim 4 has been introduced; the host cell is preferably Pichia pastoris.

6. A method for producing the recombinant human type XXIII collagen according to claim 1 or 2, comprising the steps of culturing the host cell 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. 7 . The production method according to claim 6 , further comprising the step of separating and purifying the culture to obtain purified recombinant human type XXIII collagen.

8. The production method according to claim 7, wherein: The separation and purification adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation; preferably, a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.

9. Use of the recombinant human type XXIII collagen according to claim 1 or 2 in the preparation of tissue engineering materials.

10. The use according to claim 9, wherein The tissue engineering material is selected from hemostatic sponge, subcutaneous filler, artificial bone, artificial skin, oral absorbable biomembrane, bone implant, and bone repair scaffold.

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