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Yeast recombinant strain and IFN alpha-2b interferon purifying method

A purification method and interferon technology are applied in the purification field of yeast recombinant strains and IFNα-2b interferon, which can solve the problems of large side effects, low specific activity and high production cost, and achieve cost reduction, shortening purification time and good economic benefits. Effect

Inactive Publication Date: 2003-03-26
海南海梁生物高科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, most of the α-2b 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 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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  • Yeast recombinant strain and IFN alpha-2b interferon purifying method
  • Yeast recombinant strain and IFN alpha-2b interferon purifying method

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Experimental program
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Effect test

Embodiment 1

[0027] 1. Construction of IFNα-2b interferon yeast engineering bacteria

[0028] (1) Materials:

[0029] 1. IFNα-2b gene

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

[0031] Invitrogen Corporation.

[0032] (2) Method:

[0033] 1. Gene PCR amplification:

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

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

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

[0037] remove)

[0038] XhoI Lys-Arg (Kex2 site)

[0039] P2-5'G TCTAGA TCATTCCTTACTTCTTAAACT3'

[0040] wxya

[0041] (2) Thermal cycle: 95°C, 5'; 95°C, 30"→49°C, 30"→72°C, 60"

[0042] 72°C, 10'; 30 cycles of amplification.

[0043] 2. PCR amplification product recovery and cloning:

[0044] (1) The IFNα-2b gene was amplified and electrophoresed to obtain a DNA band of about 521 bp;

[0045] (2) The target fragmen...

Embodiment 2

[0050] Purification method of α-2b interferon: After constructing α-2b interferon yeast engineered bacteria (IFNα-2b / pGAPZα-A / GS115), store the strain at -80°C. Before fermentation, inoculate a plate to activate the strains, 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 through Sephacryl S-200 column chromatography to obtain a stock solution of α-2b interferon with a purity greater than 95%.

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Abstract

The invention refers to the constructive key technology, construction result and the purifying method for producing a-2b interferon of yeast project bacterial strain IFN a-2b / pGAPZa-A / GS115 which can excrete a-2b interferon. The invention uses yeast secretion expressing system as a substitute for colibacillus expressing system, the purpose albumen a-2b interferon needn't repetitiveness, the biological rate activity reaches to 1.0X10 to the 9th power unit / mg albumen, it has no side or noxious effect, and the purification can reach to 95%.

Description

[technical field] [0001] The invention relates to an engineering bacterial strain expressing α-2b interferon and its purification method for producing α-2b interferon. [Background technique] [0002] At present, most of the α-2b interferon produced in China is the expression product of Escherichia coli, and the main process defect is: the expression system of Escherichia coli is an 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-mentioned defects, the present invention adopts the yeast se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K14/56C12N1/19C12N15/21C12N15/81
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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