EP-4741418-A1 - KERATIN CF1, PREPARATION METHOD THEREFOR, PHARMACEUTICAL COMPOSITION COMPRISING SAME, AND USE THEREOF

Abstract

The present application relates to the technical field of medicines, and relates to keratin CF1, a preparation method therefor, a pharmaceutical composition comprising same, and a use thereof, and in particular to keratin CF1, a nucleic acid molecule encoding keratin CF1, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or a genome into which the nucleic acid molecule is integrated, a preparation method for keratin CF1, a pharmaceutical composition containing keratin CF1, and a use of keratin CF1, the nucleic acid molecule, the expression vector, the host cell or the pharmaceutical composition in preparation of medications for relieving fever, easing pain, relieving cough, eliminating phlegm, resisting convulsion, resisting epilepsy, reducing blood pressure, resisting inflammation, and resisting viruses.

Inventors

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

Assignees

• Institute of Materia Medica, Chinese Academy of Medical Sciences

Dates

Publication Date

20260513

Application Date

20240521

Claims (15)

1. A keratin CF1, characterized in that the amino acid sequence of the keratin CF1 is: (1) the amino acid sequence set forth in SEQ ID NO.1 in the sequence listing; or (2) an amino acid sequence formed by substitution, deletion or addition of 1-45 amino acids in the amino acid sequence set forth in SEQ ID NO.1 in the sequence listing that basically maintains the same biological function.
2. The keratin CF1 according to claim 1, characterized in that the keratin CF1 can comprise a conventional modification; or be linked with a tag for detection or purification; or be homologous to keratin CF1.
3. The keratin CF1 according to claim 2, characterized in that the conventional modification comprises acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, immobilization modification, sulfation, oxidation, methylation, deamination, formation of disulfide bond(s) or breakage of disulfide bond(s); the tag comprises His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, Profinity eXact.
4. A nucleic acid molecule encoding the keratin CF1 of any one of claims 1 to 3.
5. The nucleic acid molecule according to claim 4, characterized in that the nucleotide sequence of the nucleic acid molecule is: (1) the nucleotide sequence set forth in SEQ ID NO. 2 in the sequence listing; (2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence set forth in SEQ ID NO.2; or (3) a nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above-mentioned.
6. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule of any one of claims 4-5.
7. A host cell, characterized in that the host cell comprises the expression vector of claim 6 or the nucleic acid molecule of any one of claims 4-5 integrated in the genome.
8. The host cell according to claim 7, characterized in that the host cell comprises bacteria, yeast, Aspergillus, plant cell, or insect cell.
9. The host cell according to claim 8, characterized in that the bacteria comprises Escherichia coli.
10. A method for preparing the keratin CF1 according to any one of claims 1 to 3, characterized in that it comprises the following steps: A. synthesizing a nucleic acid molecule corresponding to the keratin CF1 according to any one of claims 1 to 3, ligating the nucleic acid molecule to a corresponding expression vector, transforming the expression vector into a host cell, culturing the host cell with the expression vector in a fermentation equipment under certain conditions and inducing the expression of keratin CF1 to obtain a crude protein solution comprising keratin CF1; B. separating, purifying and drying the crude protein solution expressed in step A to obtain keratin CF1.
11. The method according to claim 10, characterized in that , in step A, the host cell is mainly selected from Escherichia coli, the keratin CF1 is expressed in inclusion bodies of Escherichia coli, and the fermentation equipment includes shake flask or fermentation tank.
12. The method according to claim 10, characterized in that , in step A, after inducing the expression of keratin CF1, removing impurities by using a detergent, dissolving keratin CF1 in a solution to obtain a crude protein solution.
13. The method according to claim 10, characterized in that , in step B, the method of separating and purifying comprises ultrafiltration or microfiltration membrane technology purification method, column chromatography purification method, salting out method, and dialysis method.
14. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the keratin CF1 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.
15. Use of the keratin CF1 according to any one of claims 1 to 3 or the nucleic acid molecule according to any one of claims 4-5 or the expression vector according to claims 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, analgesia, antitussive, expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and antiviral.

Description

Technical field The present invention pertains to the field of medical technology, relates to a keratin CF1, a nucleic acid molecule encoding the keratin CF1, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector or the nucleic acid molecule integrated in the genome, and preparation methods of the keratin CF1 and pharmaceutical compositions comprising the keratin and use of the keratin and the pharmaceutical composition in the manufacture of medicaments for antipyretic, analgesia, antitussive, expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and antiviral. Background Art Keratin is a kind of protein that is widely found in the epidermis of humans and animals, and it is the main component of hair, feathers, hoofs, shells, claws, horns, etc. It is an extremely important structural protein for connective tissue and plays a role in protecting the body. Keratin exists widely in organisms and is a kind of renewable resource with great utilization value, but it has not been widely and effectively utilized. The main reasons are that keratin is not easy to dissolve in various solvents, and keratin is generally more resistant to protease hydrolysis 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, microbial engineering, etc., more and more genes are discovered. It is an important means to produce target proteins by utilizing the protein expression system for studying the biological function of genes or proteins. It is novel and innovative and is not reported in other literatures to prepare the target keratin by utilizing the protein expression system, and further to study its structure and function. Summary of the invention The technical problem solved by the present invention is to provide a keratin CF1, a nucleic acid molecule encoding keratin CF1, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector or the nucleic acid molecule integrated in the genome, and preparation methods of keratin CF1, pharmaceutical compositions comprising the keratin CF1, and use of the above-mentioned keratin CF1, nucleic acid molecule, expression vector, host cell, or pharmaceutical compositions in the manufacture of medicaments for antipyretic, analgesia, antitussive, expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and antiviral. In order 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 CF1, characterized in that the amino acid sequence of the keratin CF1 is as follows: (1) the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; or(2) the amino acid sequence formed by substitution, deletion or addition of 1-35 amino acids in the amino acid sequence shown in SEQ ID NO.1 in the sequence listing that basically maintains the same biological function. Further, conventional modification can be performed on keratin CF1; or a tag for detection or purification can be attached to keratin CF1. 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 bonds or disulfide bonds breakage; The tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, Profinity eXact. The second aspect of the technical solution of the present invention provides a nucleic acid molecule encoding the keratin CF1 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 in (1) or (2) above-mentioned. The third aspect of the technical solution of the present invention provides an expression vector, characterized in that the expression vector comprises the nucleic acid molecule described in the second aspect. Further, the expression vector can 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 comprises the expression vector of the third aspect or the nucleic acid molecule of the second aspect inte