阅读说明：本技术 类胶原融合蛋白组合物及制备方法 (Collagen-like fusion protein composition and preparation method thereof ) 是由 许洋 李慧 潘仁杰 司武阳 于 2020-06-24 设计创作，主要内容包括：本发明涉及类胶原融合蛋白组合物,类胶原融合蛋白包括依次连接的结构单元,基础结构和活性单元,符合结构通式nR<Sub>1</Sub>-Cg<Sub>1</Sub>-mR<Sub>2</Sub>,其中,R<Sub>1</Sub>为结构单元,R<Sub>2</Sub>为活性单元,Cg<Sub>1</Sub>为为基础结构,m和n均为0-5的自然数；其中,R<Sub>1</Sub>和R<Sub>2</Sub>均为弹性蛋白E和功能蛋白的组合物；这种类胶原融合蛋白能够用于医学美容材料,软骨重建材料,辐射防护材料或皮肤创面修复材料。本发明的有益效果是：类胶原融合蛋白序列中的弹性蛋白结构除了在生物学功能上可以发生网状交联形成稳定的力学结构以外,在融合蛋白的结构中可以起到柔性链接的作用,便于各个基本结构表现出独立的生物活性；增加类胶原融合蛋白的稳定性,也能够提高蛋白活性。(The invention relates to a collagen-like fusion protein composition, wherein the collagen-like fusion protein comprises a structural unit, a basic structure and an active unit which are sequentially connected and conform to a structural general formula nR 1 ‑Cg 1 ‑mR 2 Wherein R is 1 Is a structural unit, R 2 Is an active unit, Cg 1 M and n are both natural numbers of 0-5 for the basic structure; wherein，R 1 And R 2 A composition that is elastin E and a functional protein; the collagen fusion protein can be used for medical beauty materials, cartilage reconstruction materials, radiation protection materials or skin wound repair materials. The invention has the beneficial effects that: the elastin structure in the collagen-like fusion protein sequence can generate net-shaped cross-linking to form a stable mechanical structure in biological function, and can play a role of flexible linkage in the structure of the fusion protein, so that each basic structure can show independent biological activity conveniently; the stability of the collagen-like fusion protein is increased, and the protein activity can also be improved.)

1. A collagen-like fusion protein composition characterized by: the collagen-like fusion protein comprises a structural unit, a basic structure and an active unit which are sequentially connected, and accords with the structural general formula of a formula 1;

nR₁-Cg₁-mR₂formula 1;

wherein R is₁Is a structural unit, R₂Is an active unit, Cg₁For the basic structure, Cg₁Is a truncated protein sequence of type I collagen, and the sequence is shown as SEQ No: 1, m and n are both natural numbers of 0-5.

2. The collagen-like fusion protein composition according to claim 1, wherein: r₁And R₂A composition that is elastin E and a functional protein;

the sequence E is shown as SEQ No: 4, respectively.

3. The collagen-like fusion protein composition according to claim 2, wherein: r₁Is Cg₂-E or Cg₃-E；

Cg₂And Cg₃Truncated protein sequences, Cg, of type II and type III collagen, respectively₂The sequence is shown as SEQ No: 2, Cg is₃The sequence is shown as SEQ No: 3, respectively.

4. The collagen-like fusion protein composition according to claim 2, wherein: r₂Is E-Cg₂、E-Cg₃One of E-SOD and E-EGF;

Cg₂and Cg₃Truncated protein sequences, Cg, of type II and type III collagen, respectively₂The sequence is shown as SEQ No: 2, Cg is₃The sequence is shown as SEQ No: 3 is shown in the specification;

the SOD is a truncated protein sequence of superoxide dismutase, and the sequence is shown as SEQ No: 5 is shown in the specification;

EGF is an epidermal cell factor truncated protein sequence, and the sequence is shown as SEQ No: and 6.

5. A method of preparing a collagen-like fusion protein composition according to any one of claims 1 to 4, wherein: the collagen-like fusion protein sequence is generated by a sequence splicing technology, directionally inserted into a prokaryotic expression vector, and expressed and purified to obtain the collagen-like fusion protein.

6. Use of the collagen-like fusion protein composition according to any one of claims 1 to 4 in a medical prosthetic material.

7. The application of a collagen-like fusion protein composition in medical cosmetic materials is characterized in that: the structural general formula of the collagen-like fusion protein is shown as a formula 2,

m(Cg₃-E)-Cg₁-n(E-Cg₃) Formula 2;

wherein m: n is 2: 1;

preferably, m is 2, n is 1, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown at 7.

8. The application of a collagen-like fusion protein composition in a cartilage reconstruction material is characterized in that: the structural general formula of the collagen-like fusion protein is shown as a formula 3,

m(Cg₂-E)-Cg₁-n(E-Cg₂) Formula 3;

wherein m: n is 1: 1;

preferably, m is 3, n is 3, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown in fig. 8.

9. The application of a collagen-like fusion protein composition in a radiation protection material is characterized in that: the structural general formula of the collagen-like fusion protein is shown as a formula 4,

m(Cg₃-E)-Cg₁-n (E-SOD) formula 4;

wherein m: n is 3: 1;

preferably, m is 3, n is 1, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown at 9.

10. The application of the collagen-like fusion protein composition in the skin wound repair material is characterized in that: the structural general formula of the collagen-like fusion protein is shown as a formula 5,

m(Cg₃-E)-Cg₁-n (E-EGF) formula 5;

wherein m: n is 3: 1;

preferably, m is 3, n is 1, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown at 10.

Technical Field

The invention belongs to the fields of molecular biology and transformation medicine, and particularly relates to a collagen-like fusion protein composition.

Background

Collagen is a biological macromolecule, a main component in the connective tissue of animals, and is also a functional protein with the largest content and the widest distribution in mammals, accounting for 25-30 percent of the total protein, and even reaching more than 80 percent of certain organisms. There are many kinds of collagens, and at least 30 kinds of genes encoding collagen chains have been found, and 16 or more kinds of collagen molecules can be formed, and they are classified into fibrous collagens, basement membrane collagens, microfibril collagens, anchored collagens, hexagonal network collagens, non-fibrous collagens, transmembrane collagens, and the like, depending on their structures. Among them, the collagen I, II and III are widely used. The main functions of collagen in the body are represented in the aspects of maintaining the extracellular environment, maintaining the normal physiological functions of tissues and organs, repairing the injury of the body and the like. Compared with other high molecular materials, the collagen has stronger biological histocompatibility, supporting elasticity and degradability to cells no matter being used as a repairing biological scaffold material or a protective agent, so the collagen can be widely applied to industries such as medicines, cosmetics and the like.

The collagen used in large scale at present is mainly extracted from the skin or bone of animals by acid, alkali or enzyme methods, the main source of the collagen is animal connective tissue, but the collagen extracted from animal tissue has risks of animal-derived diseases and the like. Along with the large-scale application of the genetic engineering technology, the bottleneck of large-scale preparation of collagen is successfully solved by using a mode of expressing exogenous protein by using a modeling expression host through genetic engineering recombinant expression collagen. For example, patent 201210482543.2 discloses a fusion recombinant protein of type II, III collagen; the Xian Gemini biological gene technology corporation limited publishes a hydroxylation method of recombinant human-derived collagen, and the recombinant collagen is further subjected to structural modification, so that the use scene of the product is further improved. Patent 201910088754.X published by Jiangsu Yuzhi biomedicine Limited company relates to a recombinant humanized III type collagen a1 chain and application thereof. The collagen related to the patents is designed in a monomer mode, and the requirements of different types of collagen on the aspects of medical cosmetology, cartilage repair, radiation protection, chronic wound surfaces and the like cannot be considered.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a collagen-like fusion protein composition.

The technical scheme adopted by the invention is as follows: the collagen-like fusion protein composition comprises a structural unit, a basic structure and an active unit which are sequentially connected, and accords with the structural general formula of a formula 1;

nR₁-Cg₁-mR₂formula 1;

wherein R is₁Is a structural unit, R₂Is an active unit, Cg₁For the basic structure, Cg₁Is a truncated protein sequence of type I collagen, and the sequence is shown as SEQ No: 1, m and n are both natural numbers of 0-5.

Preferably, R₁And R₂A composition that is elastin E and a functional protein;

the sequence E is shown as SEQ No: 4, respectively.

Preferably, R₁Is Cg₂-E or Cg₃-E；

Cg₂And Cg₃Truncated protein sequences, Cg, of type II and type III collagen, respectively₂The sequence is shown as SEQINO: 2, Cg is₃The sequence is shown as SEQ No: 3, respectively.

Preferably, R₂Is E-Cg₂、E-Cg₃One of E-SOD and E-EGF;

Cg₂and Cg₃Truncated protein sequences, Cg, of type II and type III collagen, respectively₂The sequence is shown as SEQINO: 2, Cg is₃The sequence is shown as SEQ No: 3 is shown in the specification;

the SOD is a truncated protein sequence of superoxide dismutase, and the sequence is shown as SEQ No: 5 is shown in the specification;

EGF is an epidermal cell factor truncated protein sequence, and the sequence is shown as SEQ No: and 6.

The preparation method of the collagen-like fusion protein composition comprises the steps of generating a collagen-like fusion protein sequence by a sequence splicing technology, directionally inserting the collagen-like fusion protein sequence into a prokaryotic expression vector, and carrying out expression and purification to obtain the collagen-like fusion protein.

Use of a collagen-like fusion protein composition in a medical repair material.

An application of a collagen-like fusion protein composition in medical cosmetic materials, the collagen-like fusion protein has a structural general formula shown in formula 2,

m(Cg₃-E)-Cg₁-n(E-Cg₃) Formula 2;

wherein m: n is 2: 1;

preferably, m is 2, n is 1, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown at 7.

An application of a collagen-like fusion protein composition in cartilage reconstruction materials, the structural general formula of the collagen-like fusion protein is shown as a formula 3,

m(Cg₂-E)-Cg₁-n(E-Cg₂) Formula 3;

wherein m: n is 1: 1;

preferably, m is 3, n is 3, and the sequence of the collagen-like fusion protein is shown in SEQ No: shown in fig. 8.

An application of a collagen-like fusion protein composition in a radiation protection material, the collagen-like fusion protein has a structural general formula shown in a formula 4,

m(Cg₃-E)-Cg₁-n (E-SOD) formula 4;

wherein m: n is 3: 1;

preferably, m is 3, n is 1, and the sequence of the collagen-like fusion protein is shown as SEQ No: shown at 9.

An application of a collagen-like fusion protein composition in a skin wound repair material, the collagen-like fusion protein has a structural general formula shown in formula 5,

m(Cg₃-E)-Cg₁-n (E-EGF) formula 5;

wherein m: n is 3: 1;

preferably, m is 3, n is 1, and the sequence of the collagen-like fusion protein is shown as SEQ No: shown at 10.

The invention has the advantages and positive effects that: the elastin structure in the collagen-like fusion protein sequence can generate net-shaped cross-linking to form a stable mechanical structure in biological function, and can play a role of flexible linkage in the structure of the fusion protein, so that each basic structure can show independent biological activity conveniently; the stability of the collagen-like fusion protein is increased, and the protein activity can also be improved.

Drawings

FIG. 1 is a recombinant human collagen-like fusion protein A-type plasmid;

FIG. 2 is a recombinant human collagen-like fusion protein B-type plasmid;

FIG. 3 is a recombinant human collagen-like fusion protein C-type plasmid;

FIG. 4 is a recombinant human collagen-like fusion protein D-type plasmid.

Detailed Description

The invention simulates the composition mode of various collagens in human tissue structures according to the requirements of the collagens in various fields, and re-analyzes and designs a group of human-derived collagen fusion protein compositions. The collagen-like fusion protein comprises a structural unit, a basic structure and an active unit which are sequentially connected, and accords with the structural general formula of a formula 1;

nR₁-Cg₁-mR₂formula 1;

wherein R is₁Is a structural unit, R₂Is an active unit, Cg₁M and n are both natural numbers of 0-5 for the basic structure;

wherein R is₁And R₂A composition that is elastin E and a functional protein; e is Elastin (Elastin, hereinafter abbreviated asE) An active structural unit of (a); the elastin structure can generate net-shaped cross-linking to form a stable mechanical structure in biological function, and can play a role of flexible linkage in the structure of the fusion protein, so that each basic structure can show independent biological activity conveniently;

wherein R is₁Is Cg₂-E or Cg₃-E；R₂Is E-Cg₂、E-Cg₃One of E-SOD and E-EGF; cg₁、Cg₂And Cg₃Truncated protein sequences of type I collagen, type II collagen and type III collagen respectively are shown in SEQ No: 1. SEQ No: 2 and SEQ No: 3 is shown in the specification; SOD is a truncated protein sequence of superoxide dismutase, EGF is a truncated protein sequence of epidermal cell factor, and the sequences are respectively shown as SEQ No: 5 and SEQ No: 6 is shown in the specification; the sequence of the elastin E is shown as SEQ No: 4, respectively. R1 can also be elastin E and other functional protein composition, such as E-FGF, FGF fibroblast growth factor truncated protein sequence. The structural constituent units related by the invention are obtained by carrying out protein structure and function analysis according to a human protein sequence in GeneBank and Swiss-Prot.

SEQ No.1:pro-SEQ Cg1

GEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP

SEQ No.2:pro-SEQ Cg2

GPPGPACGGG

SEQ No.3:pro-SEQ Cg3

GERGAPGFRGPAGPNGLPGEKGPAGERGAP

SEQ No.4:pro-SEQ E

VPGVG

SEQ No.5:pro-SEQ SOD

KHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQTALQPALKFNGGGHINHSIFWTNLSPNGGGEPKGELLEAIKRDFGSFDKFKEKLTAASVGVQGSGWGWLGFNKERGHLQIAACPNQDPLQGTTGLIPLLGIDVWEHAYYLQYKNVRPDYLKAIWNVINWENVTER

SEQ No.6:pro-SEQ EGF

NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

In some embodiments of the present invention, the collagen-like fusion protein may be classified into a type a for medical cosmetic materials, a type B for cartilage reconstruction materials, a type C for radiation protection materials, and a type D for skin wound repair materials according to the intended use.

In some embodiments of the present invention, the type a collagen fusion protein is designed according to the distribution and functional requirements of various types of collagen in medical cosmetology and callus, and specifically, the type I collagen (collagen I, abbreviated as Cg)₁) Collagen type III (collagen III, Cg for short)₃) And an Elastin (hereinafter referred to as E) functional unit, wherein the structure of the A type collagen fusion type protein is m (Cg)₃-E)-Cg₁-n(E-CG₃) Wherein m and n are natural numbers of 0-5, and according to the proportion of I, III type proteins in the skin structure, making m: n is 2: 1, further, m is 2, n is 1, and the specific protein sequence is shown in SEQ No. 7.

SEQ No.7：pro-A

GERGAPGFRGPAGPNGLPGEKGPAGERGAPVPGVGGERGAPGFRGPAGPNGLPGEKGPAGERGAPVPGVGGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPVPGVGGERGAPGFRGPAGPNGLPGEKGPAGERGAP

In some embodiments of the invention, according to the requirement of chondrocyte induction and conduction in the cartilage reconstruction process, the biological scaffold should meet the differentiation and basic mechanical action of chondrocytes, and the cartilage reconstruction type B collagen fusion protein is designed to be composed of type I collagen and type II collagen (collagen II, Cg for short)₂) The structure of the type B collagen fusion protein is m (Cg)₂-E)-Cg₁-n(E-CG₂) Wherein m and n are natural numbers of 0-5, and according to the requirement of I, II type collagen in the structure in the process of cartilage reconstruction, m is made to be: n is 1:1, further, m is 3, n is 3, and the specific protein sequence is shown in SEQ No 8.

SEQ No8：pro-B

GPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPVPGVGGPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGPPGPCCGGG

In some embodiments of the present invention, a type C collagen fusion protein for radiation protective materials is designed according to the mechanism of occurrence and tissue repair of radiation damage to skin and mucosa, and specifically comprises type III collagen, type I collagen, elastin E, and superoxide dismutase (SOD) functional units, and the type C collagen fusion protein has a structure of m (Cg)₃-E)-Cg₁N (E-SOD), wherein m and n are natural numbers of 0-5, and the ratio of m: n is 3: 1, further, m is 3, n is 1, and the specific protein sequence is shown in SEQ No. 9.

SEQ No.9：pro-C

GPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPVPGVGKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQTALQPALKFNGGGHINHSIFWTNLSPNGGGEPKGELLEAIKRDFGSFDKFKEKLTAASVGVQGSGWGWLGFNKERGHLQIAACPNQDPLQGTTGLIPLLGIDVWEHAYYLQYKNVRPDYLKAIWNVINWENVTER

In some embodiments of the invention, a type D collagen fusion protein for skin wound repair materials is designed according to a non-healing wound generation mechanism and a tissue repair mode, and specifically comprises type III collagen, type I collagen, elastin E, and epidermal cytokine (EGF) functional units, and the structure of the type D collagen fusion protein is m (Cg)₃-E)-Cg₁-n (E-EGF), wherein m, n are natural numbers 0-5, let m: n is 3: 1, further, m is 3, n is 1, and the specific protein sequence is shown in SEQ No. 10.

SEQ No.10：pro-D

GPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGPPGPCCGGGVPGVGGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPVPGVGNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

The preparation method of the collagen-like fusion protein composition comprises the steps of optimizing prokaryotic expression cell codons, generating a collagen-like fusion protein sequence by a sequence splicing technology, directionally inserting the collagen-like fusion protein sequence into a prokaryotic expression vector pET28a, transferring the collagen-like fusion protein sequence into escherichia coli, and carrying out expression and purification to obtain the collagen-like fusion protein.

The following describes the scheme of the present invention with reference to the accompanying drawings, wherein experimental methods without specific description of operation steps are all performed according to corresponding commercial specifications, and instruments, reagents and consumables used in the examples can be purchased from commercial companies without specific description.