Human interferon alpha 1b and preparation method thereof

A technology of interferon alpha and seeds, applied in the field of biomedicine, can solve the problems of low purification efficiency, increase purification process and the like, and achieve the effects of simplifying purification steps, increasing expression amount, increasing expression amount and biological activity

Pending Publication Date: 2022-01-14
SHENZHEN KEXING PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] During the implementation process, the inventor found that in the prior art, most of the preparation of recombinant human interferon α1b is to express recombinant human interferon α1b in the cell membrane of Escherichia coli, which leads to the need to break the cells and release a large amount of Host protein, increase the purification process, so that the purification efficiency is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Preparation of recombinant E. coli

[0067] 1.1 Recombinant human interferon α1b mutant C86S gene

[0068] It is already very mature using gene synthesis, and can entrust third-party biotechnology service companies for synthesis, and the program is fixed. It can be directly synthesized according to the specified gene sequence, and the purpose of the fixed point mutation can be achieved.

[0069] The amino acid sequence of the target recombinant human interferon α1b mutant is SEQ ID NO: 1, which encodes the gene as SEQ IDNO: 2, entrusting a third-party biotechnology service company direct synthesis target encoding gene.

[0070] 1.2 Recombinant human interferon α1b mutant C86S expression strain construction, conversion, and identification.

[0071] In this example, Hydlobacterium is selected from the selection of E. coli BL21 (DE3).

[0072] The fragment of the Pelb-IFNA1B-86SER gene, Hind III and NDE I-enzyme digestion were synthesized, and the recombinant human ...

Embodiment 2

[0073] Example 2: Inspection of induced temperature (15 ° C)

[0074] Level 1 Seed medium: lb medium.

[0075]Secondary seed medium: the total mass of the secondary seed medium, a 0.1% by weight of glucose, 0.1% by weight of yeast powder, 0.05 wt% sodium chloride and 0.01 wt% of potassium dihydrogen phosphate.

[0076] Fermentation medium: Based on the total mass of fermentation medium, 0.1% by weight of glucose, 0.1% by weight of yeast powder, 0.05% sodium chloride, 0.01 wt% phosphate, 0.005 wt% magnesium sulfate and 0.0005 Wt% copper sulfate.

[0077] Fermentation strain: Example 1 was recombinant E. coli.

[0078] The first stage seed culture: The fermentation strain was seeded into the primary medium, 150 rpm, 37 ° C for 8 h at a volume of inoculation, 150 rpm, 37 ° C for 8 h, and a grade seed liquid;

[0079] Secondary seed culture: Type a seed liquid inoculated into a secondary seed medium in a volume percentage of 2.0%, in the agitating speed of 150 rpm, 50% dissolved in ox...

Embodiment 3

[0084] Example 3: Inspection of induced temperature (30 ° C)

[0085] The primary seed medium, secondary seed medium, fermentation medium, fermentation strain: in Example 2.

[0086] The first stage seed culture: The fermentation strain was seeded into the primary medium, 150 rpm, 37 ° C for 8 h at a volume of inoculation, 150 rpm, 37 ° C for 8 h, and a grade seed liquid;

[0087] Secondary seed culture: Type a seed liquid inoculated into a secondary seed medium in a volume percentage of 2.0%, in the agitating speed of 150 rpm, 50% dissolved in oxygen, 37 ° C, culture for 3 h, resulting in secondary seed fluid ;

[0088] Fermentation culture: The secondary seed liquid is seeded into the fermentation medium in a vaccination of 7.5% by volume percentage, and cultured in the stirred speed of 200 rpm, the amount of oxygen is 20%, and 37 ° C. 1.0 600 After reaching 5.0, it was cooled to 30 ° C, and 1 mmol / L (based on fermented medium) was added to induce and cultured for 15 h, and t...

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PUM

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Abstract

The invention relates to a recombinant human interferon alpha 1b and a preparation method thereof, and belongs to the field of biological medicines. The method is simple to operate, escherichia coli can express the human interferon alpha 1b in a periplasm cell space, cells can be captured without being crushed, host protein release is reduced, purification steps are simplified, and the obtained human interferon alpha 1b is high in purity and activity and beneficial to industrial production of the human interferon alpha 1b.

Description

Technical field [0001] The present invention relates to the field of biomedicine, and more particularly to one human interferon α1b and a method thereof. Background technique [0002] Interferon (IFN) is a broad-spectrum antiviral agent that does not directly kill or inhibits viruses, which mainly causes cell surface receptor to cause anti-viral proteins, thereby inhibiting the replication of hepatitis B viruses; The viability of killing cells (NK cells), macrophages and T lymphocytes, thereby acting as immunoassay, and enhancing antiviral ability. [0003] Recombinant human interferon-α is cloning the interferon-alpha gene from human white blood cells, by recombinant gene technology, connecting human interferon genes into carriers (such as bacterial plasmid), transfer to E. coli, to express interferon, and then High purity, highly active formulations are obtained by production and purification techniques. Depending on the different amino acids, it can be divided into IFNα-2a and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/56C12N15/70C12N15/21C12N1/21C12R1/19
CPCC12P21/02C07K14/56C12N15/70C12N1/20C12N2800/101
Inventor 周新荣鄢成伟秦锁富
Owner SHENZHEN KEXING PHARM CO LTD
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