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Rapid purification method of recombinant human interferon <alpha>2b

A recombinant human interferon and purification method technology, which is applied in the field of rapid purification of recombinant human interferon α2b, can solve the problems of complex purification process, short production time, and many process steps of recombinant human interferon α2b, and achieve high protein purity, The effect of short production time and simple process

Inactive Publication Date: 2021-02-02
SHENZHEN KEXING PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies in the prior art, the present invention provides a rapid purification method of recombinant human interferon α2b, which mainly solves the complicated purification process of recombinant human interferon α2b, many process steps, long production time, high production cost, etc.; through the present invention, it can Rapid and effective extraction and purification of interferon α2b, the obtained protein has high purity, and has the advantages of simple process, high recovery rate and short production time

Method used

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  • Rapid purification method of recombinant human interferon &lt;alpha&gt;2b
  • Rapid purification method of recombinant human interferon &lt;alpha&gt;2b

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Collect the bacteria produced by fermentation and add 50mM phosphate buffer (PB, pH7.0) at a ratio of 1:15 and stir at room temperature for 2 hours; homogenizer crushed twice, 600bar; use a centrifuge to centrifuge at 14000rpm for 20min, and collect the centrifuged supernatant; The supernatant was filtered using 0.2 μm, and the filtrate was collected.

[0063] Chromatography: flow rate 2ml / min (60cm / hr), CV=35mL, column diameter=26mm

[0064] Chromatography packing material: Ni Sepharose excel (cytiva)

[0065] Buffer: Solution A: 50mM PB+0.15M NaCl (pH 7.5)

[0066] Solution B: 50mM PB+0.15M NaCl+30mM imidazole (pH 7.5)

[0067] Solution C: 50mM PB+0.15M NaCl+100mM imidazole (pH 7.5)

[0068] Solution D: 50mM PB+0.15M NaCl+0.8M imidazole (pH 7.5)

[0069] Solution E: 20% ethanol solution.

[0070] Equilibration: equilibrate 6 times column volume (CV) with solution A,

[0071] Sample loading: Load 400ml of the filtered sample solution,

[0072] Washing: wash 7CV ...

Embodiment 2

[0077] Collect the bacteria produced by fermentation and add 50mM phosphate buffer (PB, pH8.0) at a ratio of 1:10 and stir at room temperature for 2 hours; homogenizer crushed twice, 500bar; use a centrifuge to centrifuge at 14000rpm for 30min, and collect the centrifuged supernatant; The supernatant was filtered using 0.2 μm, and the filtrate was collected.

[0078] Chromatography: flow rate 5ml / min (150cm / hr), CV=35mL, column diameter=26mm

[0079] Chromatography packing material: Ni Sepharose excel (cytiva)

[0080] Buffer: Solution A: 50mM PB+0.15M NaCl (pH 8.0)

[0081] Solution B: 50mM PB+0.15M NaCl+10mM imidazole (pH 8.0)

[0082] Solution C: 50mM PB+0.15M NaCl+60mM imidazole (pH 8.0)

[0083] Solution D: 50mM PB+0.15M NaCl+1M imidazole (pH 8.0)

[0084] Solution E: 20% ethanol solution.

[0085] Equilibration: equilibrate 5 times column volume (CV) with solution A,

[0086] Sample loading: Load 400ml of the filtered sample solution,

[0087] Washing: wash 10CV w...

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Abstract

The invention belongs to the technical field of protein separation and purification, and particularly discloses a rapid purification method of recombinant human interferon <alpha>2b. The rapid purification method comprises the following steps of collecting thalli produced by fermentation, resuspending the thalli by using an extraction buffer solution, crushing the thalli by using a high-pressure homogenizer, then carrying out high-speed centrifugation to remove precipitates, collecting supernatant, filtering, and carrying out chromatographic separation on a filtrate. The method disclosed by the invention solves the problems of complex purification process, multiple process steps, long production time, high production cost and the like of the recombinant human interferon <alpha>2b. The interferon <alpha>2b can be rapidly and effectively extracted and purified, the obtained protein has high purity, and the method has the advantages of simple process, high recovery rate, short productiontime and the like.

Description

technical field [0001] The invention relates to the technical field of protein separation and purification, in particular to a rapid purification method of recombinant human interferon α2b. Background technique [0002] Interferons are a class of cytokines that can be expressed under the induction of bacteria and viruses and play a non-specific antiviral and immune response role. Natural interferon is mainly produced by white blood cells. Its antiviral mechanism is mainly to interfere with virus proliferation and increase the lethality of NK cells on virus-infected cells and tumor cells. Viral, antiproliferative, and immunomodulatory activities. Interferon has been approved for the treatment of many diseases, including chronic hepatitis B, chronic hepatitis C, multiple sclerosis, hairy cell leukemia, and others. [0003] Interferon is generally divided into type I and type II, type I includes α, β, ω interferon, and type II includes γ interferon. Among them, interferon α1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C07K1/14C07K1/36C07K1/34C07K1/22
CPCC07K14/56
Inventor 谢宗旺王向阳潘志友马鸿杰柏江涛秦锁富
Owner SHENZHEN KEXING PHARM CO LTD
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