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Keratin BD-13, preparation method, pharmaceutical composition and application thereof

A technology of BD-13 and keratin, applied in the direction of drug combination, keratin, cytokeratin, etc., to achieve the effect of reducing the number of writhing, high sample purity, and prolonging the onset latency

Active Publication Date: 2021-04-30
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using the protein expression system to prepare the target keratin, and then study its structure and function, no other literature reports, novel and creative

Method used

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  • Keratin BD-13, preparation method, pharmaceutical composition and application thereof
  • Keratin BD-13, preparation method, pharmaceutical composition and application thereof
  • Keratin BD-13, preparation method, pharmaceutical composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 shake flask fermentation preparation protein BD-13 crude solution A (TB medium)

[0115] Synthesize the nucleotide sequence shown in SEQ ID No.2, and transfer it into the pET-28a (+) vector; sequencing confirms that the expression vector containing the correct sequence is obtained; the expression vector is transfected into BL21 (DE3) cells, The expression-competent host cells containing the target nucleotide sequence are obtained. Add it to LB medium, culture in a shaker at 37° C. and 220 rpm for 1 hour to obtain a recombinant strain.

[0116] The recombinant strain was dipped and streaked on the LBA plate containing Kanamycin, and the plate was placed upside down in a constant temperature incubator at 37°C for overnight cultivation for 16 hours.

[0117] Prepare 400ml of TB culture medium, divide into 2 bottles, each bottle is 200ml. Add Kanamycin (final concentration 50 μg / ml) to each bottle (200ml) of TB medium, take a single colony on the plate and add i...

Embodiment 2

[0124] Example 2 Shake flask fermentation to prepare protein BD-13 crude solution B (other medium)

[0125] Synthesized and sequenced in Example 1 to obtain an expression vector containing the sequence shown in SEQ ID No.2; the expression vector was transfected into BL21 (DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence.

[0126] Prepare 20 ml of LB medium, take 800 μl, add 50 μl of host cells containing the target coding sequence, and culture in a shaker at 37° C. and 220 rpm for 1 hour.

[0127] Dip the above bacterial solution and streak it on the LBA plate containing Kanamycin, and place the plate upside down in a constant temperature incubator at 37°C for overnight cultivation for 16 hours.

[0128] Take 10 ml of LB medium, add Kanamycin (final concentration 50 μg / ml), take a single colony on the plate and add it to LB medium, in a shaker, under the conditions of 37 ° C and 220 rpm, overnight amplification culture for 15 hours...

Embodiment 3

[0134] Example 3 Fermenter preparation of protein BD-13 crude solution C

[0135] Synthesized and sequenced in Example 1 to obtain an expression vector containing the sequence shown in SEQ ID No.2; the expression vector was transfected into BL21 (DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence. Add it to LB medium, culture in a shaker at 37° C. and 220 rpm for 1 hour to obtain a recombinant strain.

[0136] On the LBA plate containing Kanamycin, add 100 μl of the recombinant strain, spread it with a spreader until it dries evenly, and place the plate upside down in a constant temperature incubator at 37°C for overnight culture. Take three single colonies and streak them on a plate containing Kanamycin, culture the plate overnight, and after three batches of shake flask fermentation and expression verification confirm that it is correct, preserve the strain with 15% glycerol, and pack it into 0.8ml each to obtain Working cell bank...

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Abstract

The invention relates to a keratin BD-13, a nucleic acid molecule encoding the keratin BD-13, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or a genome integrating the nucleic acid molecule, a preparation method of the keratin BD-13, and a pharmaceutical composition containing the keratin BD-13. The invention also discloses an application of the keratin BD-13, the nucleic acid molecule, the expression vector, the host cell or the pharmaceutical composition in preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory and antiviral drugs.

Description

technical field [0001] The present invention relates to a keratin BD-13, a nucleic acid molecule encoding keratin BD-13, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or genome integration of the nucleic acid molecule, and keratin BD-13 The preparation method, the pharmaceutical composition containing this keratin, and this keratin and said pharmaceutical composition are used in the preparation of antipyretic and analgesic, antitussive and expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, anti-inflammatory Application in viral medicine. Background technique [0002] Keratin is a kind of protein, which widely exists in the epidermis of humans and animals, and is the main component of hair, feathers, hooves, shells, claws, horns, etc. It is an extremely important structural protein of connective tissue and plays a role in protecting the body . [0003] Keratin exists widely in living orga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N1/21A61K38/17A61P29/00A61P25/08A61P11/14A61P11/10A61P9/12A61P31/12C12R1/19
CPCC07K14/4741C12N15/70A61P29/00A61P25/08A61P11/14A61P11/10A61P9/12A61P31/12A61K38/00C07K14/47Y02A50/30
Inventor 庾石山王晓良刘云宝屈晶李江冯楠李勇张咪苏国柱
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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