CN-119798414-B - Recombinant human XXIII type collagen, and production method and application thereof

Abstract

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

Inventors

• FAN DAIDI
• WANG PENG
• YUWEN WEIGANG
• YANG LIUQING
• ZHANG YUANPU
• XU RU
• LIU LIN
• JIA LIPING
• YAN YUBO

Assignees

• 西安巨子生物基因技术股份有限公司

Dates

Publication Date

20260508

Application Date

20241231

Claims (8)

1. 1. A recombinant human XXIII type collagen has an amino acid sequence shown in SEQ ID No. 7.
2. 2. A nucleic acid encoding the recombinant human XXIII type collagen of claim 1.
3. 3. An expression vector comprising the nucleic acid of claim 2.
4. 4. A host cell into which the expression vector of claim 3 has been introduced.
5. 5. A method of producing recombinant human XXIII type collagen according to claim 1, comprising the steps of culturing the host cell of claim 4 so that it expresses the recombinant human XXIII type collagen of claim 1, and collecting a culture comprising the recombinant human XXIII type collagen.
6. 6. The production method according to claim 5, further comprising the step of subjecting the culture to separation and purification, thereby obtaining purified recombinant human XXIII type collagen.
7. 7. Use of recombinant human XXIII type collagen according to claim 1 in the preparation of tissue engineering material.
8. 8. The use according to claim 7, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bone, artificial skin, orally absorbable biological membranes, bone implants, bone repair scaffolds.

Description

Recombinant human XXIII type collagen, and production method and application thereof Technical Field The invention belongs to the technical field of synthetic biology, and relates to recombinant human XXIII type collagen, a production method and application thereof. Background Collagen is the most abundant protein found in mammals, and the 28 types of collagen found at present can be divided into four major classes, fibrillar collagen, reticular collagen, fibril related collagens with discontinuous triple helices (FACITs), and membrane related collagens with discontinuous triple helices (MACITs). Type XXIII collagen is a novel transmembrane collagen found in recent years, is a type II transmembrane protein, has high similarity to type XIII and type XXV collagens in structure, comprises three triple helix collagen domains, is provided with four non-collagenous domains on two sides, and is located in the NC1 domain. The size of the human XXIII type collagen transcript is 3067bp, the human XXIII type collagen transcript encodes 540 amino acid sequences, the anchoring and cell-indicated protein size is 75KD, and the human XXIII type collagen transcript can be cleaved by furin to generate a 60KD soluble protein. As a newly discovered collagen, few studies on XXIII type collagen function have been conducted, and the current studies have focused on it as a marker of various metastatic tumors. There is essentially no study to construct functional human XXIII-type recombinant collagen by means of truncation, splicing, etc. Disclosure of Invention In view of the technical problems existing in the prior art, the invention aims to provide a truncated protein of natural human XXIII type collagen with good biological activity and recombinant human XXIII type collagen with better biological activity constructed by splicing the truncated proteins. The inventors found 6 truncated proteins of natural human XXIII type collagen with good cell adhesion promoting and procoagulant activities (comparable to commercially available recombinant human collagen) and spliced them in a specific manner to construct recombinant human XXIII type collagen with better cell adhesion promoting and procoagulant activities (superior to commercially available recombinant human collagen). Namely, the technical scheme of the invention comprises the following steps: 1. a recombinant human XXIII type collagen has an amino acid sequence shown in any one of SEQ ID No. 1-7. 2. Nucleic acid encoding recombinant human XXIII type collagen of item 1. 3. An expression vector comprising the nucleic acid of item 2. 4 A host cell into which the expression vector of item 3 has been introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, bacillus subtilis, bacillus licheniformis, etc., eukaryotic hosts such as Pichia pastoris, saccharomyces cerevisiae, animal cells, plant cells, etc., preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris. 5. A method of producing the recombinant human XXIII type collagen of item 1, comprising the steps of culturing the host cell of item 4, expressing the recombinant human XXIII type collagen of item 1, and collecting a culture comprising the recombinant human XXIII type collagen. The expression may be one or a combination of constitutive expression and inducible expression. Wherein the inducer for inducing expression can be IPTG, beta-galactoside, methanol, ethanol, etc. 6. The production method according to item 5, further comprising the step of subjecting the culture to separation and purification to thereby obtain the purified recombinant human XXIII type collagen. 7. The production method according to item 6, wherein the separation and purification are performed by one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation. 8 Use of recombinant human XXIII-type collagen according to item 1 in the preparation of a tissue engineering material. 9. The use according to item 8, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bones, artificial skin, orally absorbable biofilms, bone implants, bone repair scaffolds. The beneficial effects of the invention include: 1) The amino acid sequence of the recombinant human XXIII type collagen is 100% homologous with the natural amino acid sequence, and has no immunogenicity. 2) The recombinant human XXIII type collagen can be expressed in high yield in a commercial pichia pastoris expression system. The expression system has post-translational modification, is easy to secrete and express with high efficiency, and is convenient for subsequent purification due to few endogenous secreted proteins. 3) The recombinant human XXIII type collagen provid