BR-112024022558-B1 - Polypeptide, polynucleotide, nucleic acid, vector, host cell, method for producing the polypeptide, composition, and in vitro method for increasing cell adhesion.

BR112024022558B1BR 112024022558 B1BR112024022558 B1BR 112024022558B1BR-112024022558-B1

Abstract

POLYPEPTIDE, NUCLEIC ACID, VECTOR, HOST CELL, METHOD FOR PRODUCING THE POLYPEPTIDE, AND COMPOSITION. A collagen and its components are provided. The collagen includes an N-terminal sequence and a C-terminal sequence, wherein the N-terminal sequence includes one or more repeating units, and the repeating unit includes an amino acid sequence shown in SEQ ID NO. 15. The recombinant type II humanized collagen prepared by the present invention has high activity in promoting cell adhesion, does not produce an immune response when applied to the human body, can be obtained on a large scale, and is expected to be widely used in the field of cartilage repair.

Inventors

Xia Yang
Xiaobin Lan
Lingling Wang
Yongjian Zhang
Xin Liu
Zengyao Liu
Wenfei Yang

Assignees

Shanxi Jinbo Bio-Pharmaceutical Co., Ltd

Dates

Publication Date: 20260317
Application Date: 20231127
Priority Date: 20230512

Claims (17)

1. Polypeptide, characterized in that the amino acid sequence of the polypeptide is SEQ ID NO: 10 and the polypeptide is a recombinant humanized type II collagen.
2. Polynucleotide, characterized in that it encodes the polypeptide as defined in claim 1, wherein is the nucleotide sequence as shown in SEQ ID NO: 25.
3. Nucleic acid, characterized in that it comprises the polynucleotide as defined in claim 2 and nucleotides encoding a purification tag.
4. Nucleic acid according to claim 3, characterized in that the purification label is a His label, a GST label, an MBP label, a SUMO label or a NusA label.
5. Nucleic acid according to claim 3, characterized in that it further comprises nucleotides encoding a leader sequence.
6. Vector, characterized in that it comprises the polynucleotide as defined in claim 2 or the nucleic acid as defined in any one of claims 3 to 5.
7. Vector according to claim 6, characterized in that the vector is an expression vector.
8. Vector according to claim 6 or 7, characterized in that the vector comprises an expression control element operationally linked to the polynucleotide or nucleic acid.
9. Vector according to claim 8, characterized in that the expression control element is a promoter, a terminator and/or an intensifier.
10. Host cell, characterized in that it comprises the polynucleotide as defined in claim 2, the nucleic acid as defined in any one of claims 3 to 5, or the vector as defined in any one of claims 6 to 9, wherein the host cell is a bacterium or a fungus.
11. Host cell according to claim 10, characterized in that the bacterium is E. coli; and/or the fungus is yeast.
12. Host cell according to claim 11, characterized in that the yeast is Saccharomyces cerevisiae.
13. Method for producing the polypeptide as defined in claim 1, characterized in that it comprises: (1) cultivating a host cell as defined in any of claims 10 to 12 under suitable culture conditions; (2) harvesting the host cells and/or the culture medium containing the polypeptide; and (3) purifying the polypeptide.
14. Composition, characterized in that it comprises the polypeptide as defined in claim 1.
15. Composition according to claim 14, characterized in that it is a composition for cartilage repair.
16. Composition according to claim 15, characterized in that it is an injectable composition for cartilage repair.
17. In vitro method for increasing cell adhesion, characterized in that it comprises bringing cells into contact with the polypeptide as defined in claim 1, wherein the method is a method for non-therapeutic or non-diagnostic purposes.

Description

[001] This application claims priority benefit from Chinese Patent Application No. 202310537499.9, filed on May 12, 2023, entitled “POLYPEPTIDE AND USE THEREOF”. The content of the aforementioned application is incorporated herein by reference. Technical Field [002] The present description belongs to the field of synthetic biotechnology and refers specifically to structural materials of the human body and methods of biosynthetic preparation thereof. Fundamentals of the Invention [003] Collagen is a type of protein widely distributed in human connective tissue and is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. It has been found that there are at least 28 subtypes of collagen in the human body, which are located in different tissues and organs. [004] Type II collagen, a high molecular weight protein, is found primarily in cartilage tissue, the vitreous body, and the cornea, where its filamentous collagen fibers are interwoven with elastin and polysaccharide proteins to form a network structure, also known as complex bone collagen. Type II collagen is an essential component for cartilage and bone formation, bone growth, and the maintenance of mature cartilage; therefore, undenatured collagen can be used as a structural and functional component of cartilage. [005] Type II collagen, as the main component of the articular cartilage matrix, along with lubricating components such as hyaluronic acid and proteoglycans, protects the cartilage from wear and tear. With increasing age, the rate of collagen synthesis gradually decreases, collagen loss intensifies, articular cartilage degenerates, and friction between bones intensifies, which can trigger inflammation in the joints. Currently, the common treatment method for osteoarthritis is collagen supplementation, but its protein utilization rate is low, resulting in slow cartilage regeneration, which is not beneficial for patient recovery and affects the treatment process and efficiency. Due to the unsatisfactory effectiveness of current treatment methods, patients often need to choose artificial joint replacement, which is not only expensive but can also lead to catastrophic complications such as postoperative thrombosis and infection after replacement, leading to amputation. [006] However, the collagen material currently used as cartilage is mainly derived from the extraction of animal cartilage. The immunogenicity of collagen of animal origin cannot be eliminated, and the processes for removing and extracting impurities are still relatively complicated; therefore, it is difficult to guarantee the triple helix structure of the collagen and it cannot be used for large-scale production. [007] At the same time, the animal-derived immune response is also an important reason that limits the application of collagen. With the growing collagen industry in the country, the use of biosynthetic pathways for obtaining collagen has become increasingly mature, especially humanized collagen, which has been at the forefront worldwide. In 2021, the National Medical Products Administration (NMPA) established a nomenclature and classification of biosynthetic collagen, in which recombinant humanized collagen refers to the functional region of the total or partial amino acid sequence encoded by the specific type of human collagen gene prepared by recombinant DNA technology, or the combination of functional regions containing the function of human collagen. [008] The traditional method of collagen production is to use acid, alkaline, and enzymatic hydrolysis to process animal tissues and extract collagen derivatives. Collagen extracted by these methods has lost its original biological activity and therefore cannot be applied in the biomedical field to play a real role. With the development of modern technology, some methods of impurity removal and enzymatic extraction of animal cartilage to obtain undenatured type II collagen have emerged both domestically and internationally. Although collagen prepared by these methods can be used for cartilage repair, the time for impurity removal and extraction is long, the purity of the product is low, and the stability of the product is low, therefore it is not beneficial for large-scale production. Given the shortcomings of existing technology, some companies have proposed using Pichia pastoris as a host strain to prepare type II collagen for cartilage repair. However, this collagen is not humanized collagen and therefore has a certain immunogenic response. [009] Therefore, there is an urgent need for a recombinant humanized type II collagen that can be injected directly into the human body and not cause an immunogenic response, to be used as a structural material for the human body applied to cartilage repair. Summary of the Invention [0010] The structural materials of the human body are primarily structural proteins, including collagen, which have complex structures and precise functions and ar