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Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof

A receptor antagonist, interleukin technology, applied in the field of preparing recombinant interleukin-1 receptor antagonist, can solve the problems of high cost, low eukaryotic expression yield, good protein activity and the like, and achieves high bacterial yield , Good repeatability, fast growth of bacteria

Active Publication Date: 2011-04-06
BEIJING C & N INT SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Eukaryotic expression has low yield and high cost, but good protein activity

Method used

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  • Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof
  • Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof
  • Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, construction IL-1ra engineered bacterium

[0051] Escherichia coli expression vector PET-30a was purchased from Novagen, catalog number: 69909-3;

[0052] Escherichia coli JM109 strain was purchased from Promega;

[0053] Escherichia coli strain BL21 (DE3) was purchased from MERCK;

[0054] The PHA-stimulated human peripheral blood T cell cDNA library was purchased from Clontech Laboratories.Inc, USA.

[0055] 1. Obtain IL-1ra gene

[0056] 1. PCR amplification of IL-1ra cDNA

[0057] Use the PHA-stimulated human peripheral blood T cell cDNA library as a template, add synthetic IL-1ra primers and dNTPs, and the upstream primer is: 5′-GG CATATG CGACCCTCTGGGA-3′, the downstream primer is: 5′-GGAGGACGAGTAA GTC GAC AA-3', the upstream primer introduces the NdeI restriction site, starting from the first amino acid encoding mature human IL-1ra, and the downstream primer introduces the efficient termination codon TAA and SalI restriction site of Escherichi...

Embodiment 2

[0102] Embodiment 2, fermentation process of recombinant interleukin-1 receptor antagonist

[0103] 1. Shake flask fermentation

[0104] 1. Seed cultivation

[0105] Inoculate the IL-1ra engineering bacteria monoclonal into a 250ml Erlenmeyer flask containing 50ml LB medium (the composition of LB medium is: tryptone 10g / L, yeast powder 5g / L and sodium chloride 10g / L), at 37°C, 200rpm, shaking culture for 12-15h, to obtain seeds.

[0106] 2. Fermentation bottle culture

[0107] On the basis of step 1, insert the seeds into 2×YT medium by 1% inoculum size (the composition of 2×YT medium is: tryptone 16g / L, yeast powder 10g / L and sodium chloride 5g / L) Fermentation flask, 37°C, 200rpm, shaking culture for 2-2.5h, OD 600 When it reaches 1.0 or so, add isopropyl-β-D thiogalactopyranoside (IPTG) to induce culture.

[0108] 3. Exploring the concentration of IPTG induction

[0109] Using shake flasks, in the fermentation broth OD 600 When it reaches 1.0, add IPTG respectively, t...

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Abstract

The invention discloses a fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and a fermentation method thereof. The fermentation method comprises the following step of: inoculating recombination strains on the fermentation medium for fermentation cultivation to obtain a fermentation broth, namely obtaining a recombination IL-1ra. The recombination strains are recombination escherichia coli obtained by guiding genes shown in sequence 1 in a sequence table into escherichia coli, and the guided genes which are shown in the sequence 1 in the sequence table are guided into the escherichia coli via the following recombination expression vectors: inserting the genes shown in the sequence 1 of the sequences into multiple clone sites of a PET-30a vector to obtain the recombination expression vectors. The method has good repeatability; the growth of the strains is fast so as to greatly shorten the period of fermentation; the yield of the strain is high, which can meet the high-density fermentation requirements, and target proteins has high expression rate and good solubility.

Description

technical field [0001] The invention relates to a fermentation medium for preparing recombinant interleukin-1 receptor antagonist and a fermentation method thereof. Background technique [0002] Recombinant interleukin-1 receptor antagonist (IL-1ra for short) is a protein cytokine receptor antagonist that exists in the human body. By binding to interleukin-1 (IL-1) receptor, it specifically Blocks the activity of IL-1. [0003] Since Arend and others cloned the cDNA of human IL-1ra in 1990, scholars from various countries have been conducting more comprehensive and in-depth research on IL-1ra. At the same time, many pharmaceutical manufacturers are also working on the development of IL-1ra. In 2003, the US FDA approved the recombinant human IL-1ra (trade name Kineret) produced by Amgen Company for the treatment of rheumatoid arthritis. my country's SFDA approved recombinant human IL-1ra produced by Tibet Huaxi Pharmaceutical Co., Ltd. in 2008 for the treatment of ocular no...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12R1/19
Inventor 揣文华梁伟锋韩普王瑞琳洪海燕
Owner BEIJING C & N INT SCI TECH
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