CN-121975001-A - Recombinant type II collagen for promoting cartilage repair, preparation method and application thereof

Abstract

The application discloses recombinant type II collagen for promoting cartilage repair, a preparation method and application thereof. The amino acid sequence of the recombinant type II collagen is constructed by repeating the amino acid sequence shown in SEQ ID NO. 1 for a plurality of times, wherein the repetition number is more than or equal to 2 and less than or equal to 20. The recombinant type II collagen has good activity of promoting cartilage cell proliferation, and particularly, the recombinant type II collagen of the recombinant type II collagen is superior to full-length type II collagen, and can be used for promoting cartilage repair. The recombinant type II collagen has 100% sequence identity with human type II collagen, strictly adheres to Gly-X-Y repeated sequence mode, has no introduction of heterologous amino acid residues, and has no rejection and sensitization risks.

Inventors

• FAN DAIDI
• GUO WENTAO
• MENG HONG
• YUWEN WEIGANG
• ZHANG JIANGRUI
• XU RU
• DUAN ZHIGUANG
• YAN YUBO

Assignees

• 西安巨子生物基因技术股份有限公司

Dates

Publication Date

20260505

Application Date

20251229

Claims (8)

1. 1. A truncated protein of type II collagen has the activity of promoting chondrocyte proliferation, and the amino acid sequence of the truncated protein is shown as SEQ ID NO. 1.
2. 2. A recombinant type II collagen is constructed by repeating the amino acid sequence shown in SEQ ID NO.1 for a plurality of times, wherein the number of times of repetition is more than or equal to 2 and less than or equal to 20.
3. 3. The recombinant type II collagen according to claim 2, wherein the number of repetitions is 4, and the amino acid sequence of the recombinant type II collagen is shown in SEQ ID No. 2.
4. 4. A nucleic acid encoding the truncated protein of claim 1 or the recombinant type II collagen of claim 2.
5. 5. An expression vector comprising the nucleic acid of claim 4.
6. 6. A host cell comprising the expression vector of claim 5. The host cell is preferably pichia pastoris.
7. 7. A method of producing the truncated protein of claim 1 or the recombinant type II collagen of claim 2 comprising the step of culturing the host cell of claim 6 and allowing expression of the truncated protein or recombinant type II collagen.
8. 8. A product comprising the truncated protein of claim 1 or the recombinant type II collagen of claim 2. The product may be, for example, a pharmaceutical, cosmetic or medical device. In particular to artificial cartilage, artificial cornea, vitreous body, wound repairing material, supporting biological material, medical beauty and shaping material, cosmetics and the like.

Description

Recombinant type II collagen for promoting cartilage repair, preparation method and application thereof Technical Field The invention belongs to the technical field of synthetic biology. In particular to recombinant human type II collagen which is produced by using pichia pastoris and has the effect of promoting cartilage repair, and a preparation method and application thereof. Background Collagen is the highest protein content in mammals, accounting for about 30% of the total protein. Up to now, at least 40 more collagen chain-encoding genes have been found. These different collagen chains, combined in different ways, can form more than 20 different types of collagen molecules. Wherein, the II-type collagen consists of 3 identical alpha 1 chains, is mainly distributed in cartilage and vitreous, and accounts for more than 90% of the total amount of human cartilage matrix collagen. With rapid development of genetic engineering and synthetic biology in recent years, protein expression systems based on animals, plants, fungi and bacteria have been established in a dispute, and recombinant collagen is synthesized by utilizing a genetic engineering means, so that the immunogenicity problem of the collagen can be effectively solved, and the collagen can meet more clinical requirements and has a wide application prospect. Compared with other technologies, the microbial expression system has the advantages of high yield, short production period, simple culture, low cost, easy acquisition of high-density fermentation and the like, and hosts applied to collagen expression at present mainly comprise pichia pastoris, saccharomyces cerevisiae, hansenula polymorpha, escherichia coli and the like. However, type II collagen has a large molecular weight and is easily degraded, and no matter what recombinant expression system is adopted, the problem that the expression is impossible or the yield is low exists at present. The man skilled in the art can construct various short proteins derived from the type II collagen by technical means such as truncating, repeating and splicing the amino acid sequence of the type II collagen, so that the short proteins can be easily produced in a recombinant expression system. However, the biological activity of such short proteins is often inferior to that of full length type II collagen. Thus, it is a technical problem facing the skilled person how to obtain short proteins derived from type II collagen with a biological activity substantially comparable to that of full length type II collagen. Disclosure of Invention In view of the above-described problems of the prior art, an object of the present invention is to provide a truncated protein derived from type II collagen having excellent chondrocyte proliferation promoting activity, and a recombinant type II collagen constructed by repeated amino acid sequences of the truncated protein, which have chondrocyte proliferation promoting activity equivalent to or superior to that of full-length type II collagen. The inventor has conducted intensive studies to solve the above technical problems, and has completed the present invention by obtaining a truncated protein derived from type II collagen having a good chondrocyte proliferation promoting activity, which is comparable to full-length type II collagen, through activity screening, and further obtaining recombinant type II collagen having a chondrocyte proliferation promoting activity superior to full-length type II collagen through repeated amino acid sequences of the truncated protein. Namely, the present invention includes: 1. a truncated protein of type II collagen has the activity of promoting chondrocyte proliferation, and the amino acid sequence of the truncated protein is shown as SEQ ID NO. 1. SEQ ID NO:1 GLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGER 2. A recombinant type II collagen is constructed by repeating the amino acid sequence shown in SEQ ID NO. 1 for a plurality of times, wherein the number of times of repetition is more than or equal to 2 and less than or equal to 20. 3. The recombinant type II collagen according to item 2, wherein the number of repetitions is 4, and the amino acid sequence of the recombinant type II collagen is shown as SEQ ID NO. 2. SEQ ID NO:2 GLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGERGLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGERGLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGERGLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGER 4. A nucleic acid encoding the truncated protein of item 1 or the recombinant type II collagen of item 2. The nucleotide sequence of the nucleic acid encoding the truncated protein of item 1 may be, for example, as shown in SEQ ID NO. 3, and the nucleotide sequence of the nucleic acid encoding the recombinant type II collagen of item 3 may be, for example, as shown in SEQ ID NO. 4; SEQ ID NO:3 GGTTTGGTCGGACCTAGAGGTGAGCGTGGATTTCCAGGCGAACGTGGATCACCT