Recombinant yeast strain and IFN alpha-la interferon purifying process

A technology of interferon and yeast, which is applied in the field of purification of α-1a interferon, which can solve the problems of high production cost, large side effects, and low specific activity, and achieve good economic benefits, reduce costs, and shorten purification time.

Inactive Publication Date: 2004-11-24
海南海梁生物高科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, most of the α-1a interferon produced in China is the expression product of Escherichia coli, the main process defect is: the expression system of Escherichia coli is a inclusion body type, and renaturation is required in the production process, and the renaturation rate is low, up to 40%. The specific activity is low, only 1.0×10 8 unit / mg of protein, which has relatively large side effects; moreover, expensive monoclonal antibody columns must be used in the separation and purification, and the purity can reach more than 95%. To meet the needs of manufacturers, it takes a lot of money to import, and the production cost is very high

Method used

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  • Recombinant yeast strain and IFN alpha-la interferon purifying process
  • Recombinant yeast strain and IFN alpha-la interferon purifying process

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Effect test

Embodiment 1

[0027] 1. Construction of IFNα-1a interferon yeast engineering bacteria

[0028] (1) Materials:

[0029] 1. IFNα-1a gene

[0030] 2. pGAPZα-A, P. pastoris Strain GS115 (his4) were purchased from

[0031] Invitrogen, Australia.

[0032] (2) Method:

[0033] 1. Gene PCR amplification:

[0034] (1) Primer design: delete the Kex2 (Lys-Arg) site in the 5' end primer

[0035] The following Ste13 site (Glu-Ala-Glu-Ala) and the initiation codon ATG.

[0036] P1-5'G CTCGAG AAAAGA ATG TGTGATCTGCCTCAAACC3' ("ATG" is removed)

[0037] Xho I Lys-Arg (Kex2 site)

[0038] P2-5'G TCTAGA TCATTCCTTACTTCTTAAACT3'

[0039] wxya

[0040] (2) Thermal cycle: 95°C, 5'; 95°C, 30"→49°C, 30"→72°C, 60"72°C, 10' amplification for 30 cycles.

[0041] 2. PCR amplification product recovery and cloning:

[0042] (1) The IFNα-1a gene was amplified and electrophoresed to obtain a DNA band of about 521bp;

[0043] (2) The target fragment was recovered with a high-p...

Embodiment 2

[0048]Purification method of α-1a interferon: After constructing α-1a interferon yeast engineered bacteria (IFNα-1a / pGAPZα-A / GS115), store the strain at -80°C. Before fermentation, inoculate the plate to activate the strain, inoculate a single colony in YPD+Zeocin100 mg / L medium and ferment for 72 hours, collect the fermentation supernatant by centrifugation, adjust the pH to 4.0-5.0 with acetic acid, and pass through the CM Sepharose column layer Analysis, collect 0.4 mol / L sodium chloride elution peak, add ammonium sulfate to 30%, centrifuge to get supernatant, pass PhenylSepharose column chromatography, collect 10% ammonium sulfate elution peak, pass DEAE Sepharose column chromatography, collect The peak was eluted with 0.1 mol / L sodium chloride, and the protein peak was collected by Sephacryl S-200 column chromatography to obtain a stock solution of α-1a interferon with a purity greater than 95%.

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Abstract

The present invention relates to the key technology and result of constructing engineering yeast strain IFN alpha-1a / pGAPZ alpha-A / GS115 secreting alpha-1a interferon and the purification process of produced interferon. The present invention adopts yeast secretion expressing system, rather than traditional colibacillus inclusion body expression system, and the target protein alpha-1a interferon has no needs of renaturation, biological specific activity of 1.0E9 unit / mg protein and low toxic side effect. The purification process can obtain target protein alpha-1a interferon of 95 % purity without needing expensive monoclonal antibody column for lowering production cost.

Description

[technical field] [0001] The invention relates to an engineering bacterial strain secreting and expressing alpha-1a interferon and a purification method for producing alpha-1a interferon using it. [Background technique] [0002] At present, most of the α-1a interferon produced in China is the expression product of Escherichia coli, the main process defect is: the expression system of Escherichia coli is a inclusion body type, and renaturation is required in the production process, and the renaturation rate is low, up to 40%. The specific activity is low, only 1.0×10 8 unit / mg of protein, which has relatively large side effects; moreover, expensive monoclonal antibody columns must be used in the separation and purification, and the purity can reach more than 95%. To meet the needs of manufacturers, it takes a lot of money to import, and the production cost is very high. [Content of the invention] [0003] In order to overcome the above defects, the present invention adopt...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/56C12N1/19C12N15/20C12N15/21C12N15/81
Inventor 梁国栋周鹏夏中宁
Owner 海南海梁生物高科技有限公司
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