EP-4741417-A1 - KERATIN CF2, PREPARATION METHOD THEREFOR, PHARMACEUTICAL COMPOSITION CONTAINING SAME, AND USE THEREOF

Abstract

The present invention relates to the technical field of medicines. Disclosed are a keratin CF2, a preparation method therefor, a pharmaceutical composition containing same, and a use thereof, and the present invention specifically relates to a keratin CF2, a nucleic acid molecule encoding the keratin CF2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or agenome integrated with the nucleic acid molecule, a preparation method for the keratin CF2, a pharmaceutical composition containing the keratin CF2, and a use of the keratin CF2, the nucleic acid molecule, the expression vector, the host cell or the pharmaceutical composition in preparation of a medicament for antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, or antiviral purpose.

Inventors

• YU, SHISHAN
• LI, Mi
• CAI, JIE
• LI, YONG
• WANG, WEIPING
• ZHANG, Mi
• WANG, XIAOLIANG
• WANG, XIAOJING
• MA, SHUANGGANG
• WANG, LING
• QU, JING
• FENG, NAN
• LIU, Yunbao
• XU, SHAOFENG

Assignees

• Institute of Materia Medica, Chinese Academy of Medical Sciences

Dates

Publication Date

20260513

Application Date

20240521

Claims (15)

1. A keratin CF2, wherein the amino acid sequence of the keratin CF2 is: (1) the amino acid sequence shown as SEQ ID NO:1 in the sequence listing; or (2) an amino acid sequence that substantially maintains the same biological function formed by substitution, deletion or addition of 1-45 amino acids to the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing.
2. The keratin CF2 according to claim 1, wherein the keratin CF2 may have a conventional modification; or the keratin CF2 is further linked with a tag for detection or purification, or the keratin CF2 is a homologous protein thereof.
3. The keratin CF2 according to claim 2, wherein theconventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorophore group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamidation, disulfide bond formation, or disulfide bond breakage; and the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.
4. A nucleic acid molecule encoding the keratin CF2 according to any one of claims 1-3.
5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence of the nucleic acid molecule is: (1) the nucleotide sequence shown as SEQ ID NO:2 in the sequence listing; (2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown as SEQ ID NO: 2; or (3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2) above.
6. An expression vector, wherein the expression vector comprises the nucleic acid molecule according to any one of claims 4-5.
7. A host cell, wherein the host cell comprises the expression vector according to claim 6, or has the nucleic acid molecule according to any one of claims 4-5 integrated into its genome.
8. The host cell according to claim 7, wherein the host cell is selected from the group consisting of bacterium, yeast, Aspergillus, plant cell, and insect cell.
9. The host cell according to claim 8, wherein the bacterium is Escherichia coli.
10. A method for producing the keratin CF2 according to any one of claims 1-3, wherein the method comprises the following steps: A. synthesizing the nucleic acid molecule corresponding to the keratin CF2 according to any one of claims 1-3, ligating the nucleic acid molecule into a suitable expression vector, transforming the expression vector into a host cell, culturing the host cell harboring the expression vector under specific conditions in a fermentation device, and inducing expression of the keratin CF2 to obtain a crude protein solution containing the keratin CF2; B. subjecting the crude protein solution obtained in step A to separation and purification, and drying to obtain the keratin CF2.
11. The method according to claim 10, wherein in step A: the host cell is primarily selected from Escherichia coli ; the keratin CF2 is expressed in the inclusion bodies of Escherichia coli ; and the fermentation device includes a shake flask or a fermenter.
12. The method according to claim 10, wherein in step A, after inducing the expression of the keratin CF2, a washing solution may be used to wash away impurities, and then the product is dissolved with a solution to obtain the crude protein solution.
13. The method according to claim 10, wherein in step B, the separation and purification method is selected from the group consisting of ultrafiltration microfiltration membrane technology purification method, column chromatography purification method, salting out method, and dialysis method.
14. A pharmaceutical composition, wherein the pharmaceutical composition comprises the keratin CF2 according to any one of claims 1-3 and a pharmaceutically acceptable carrier or excipient.
15. Use of the keratin CF2 according to any one of claims 1-3, or the nucleic acid molecule according to any one of claims 4-5, or the expression vector according to claim 6, or the host cell according to any one of claims 7-9, or the pharmaceutical composition according to claim 14 in the manufacture of a medicament for antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, or antiviral.

Description

TECHNICAL FIELD The present invention relates to the technical field of medicines, and relates to a keratin CF2, a nucleic acid molecule encoding keratin CF2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or with the nucleic acid molecule genomically integrated, preparation methods for keratin CF2 and pharmaceutical compositions containing such keratin, and uses of said keratin and the pharmaceutical composition in the preparation of medicaments for antipyretic, analgesic, antitussive expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral purposes. BACKGROUND OF THE INVENTION Keratin is a type of protein, which is widely found in the epidermis of humans and animals, and is the main component of hair, feathers, hoofs, shells, claws, horns, etc. It is a highly important structural protein in connective tissues and plays a role in protecting the body. Keratin is widely present in organisms and is a renewable resource with great utilization value, but it has not been widely and effectively utilized. The main reason is that keratin is insoluble in various solvents, and keratin is generally more protease-resistant than other proteins. Therefore, it is very difficult to extract and prepare natural keratin. With the rapid development of modern biotechnology such as genomics, proteomics, genetic engineering, and microbial engineering, an increasing number of genes have been identified, and the use of protein expression systems to prepare and produce target proteins is an important method for studying the biological functions of genes or proteins. No literature has reported the use of a protein expression system to prepare the target keratin and subsequently study its structure and function, demonstrating novelty and inventiveness. SUMMARY OF THE INVENTION The technical problem solved by the present invention is to provide a keratin CF2, a nucleic acid molecule encoding the keratin CF2, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or genome integrating the nucleic acid molecule, and a preparation method of the keratin CF2, a pharmaceutical composition containing the keratin CF2, and use of the above-mentioned keratin CF2, nucleic acid molecule, expression vector, host cell, or pharmaceutical compositions in the preparation medicament for antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory or antiviral. To solve the technical problems of the present invention, the present invention provides the following technical solutions: The first aspect of the technical solution of the present invention is to provide a keratin CF2, characterized in that the amino acid sequence of the keratin CF2 is: (1) the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.(2) an amino acid sequence that substantially maintains the same biological function as the keratin CF2 formed by substitution, deletion or addition of 1-35 amino acids to the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing. Further, the keratin CF2 may have a conventional modification; or the keratin CF2 is further linked with a tag for detection or purification. Furthermore, the conventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, formation of disulfide bond or breakage of disulfide bond; the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact. The second aspect of the technical solution of the present invention provides a nucleic acid molecule encoding the keratin CF2 of the first aspect. Further, the nucleotide sequence of the nucleic acid molecule is: (1) the nucleotide sequence shown in SEQ ID NO:2 in the sequence listing.(2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO:2.(3) a nucleotide sequence complementary to the nucleotide sequence of (1) or (2) above. The third aspect of the technical solution of the present invention provides an expression vector, which is characterized in that the expression vector contains the nucleic acid molecule described in the second aspect. Further, the expression vector may be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα/A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+). The fourth aspect of the technical solution of the present invention provides a host cell, characterized in that the host cell contains the expression vector of the third aspect or the nucleic acid molecule of the se