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Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor

A technology of colony-stimulating factor and macrophage, applied in chemical instruments and methods, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems of gene loss, unstable expression, low renaturation rate, etc., and achieve The effect of reducing separation and purification steps, reducing clinical side effects, and reducing production costs

Inactive Publication Date: 2006-08-09
海南国栋药物研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] At present, most of the domestically produced rhGM-CSF is the expression product of Escherichia coli, and the main process disadvantages are: the expression system of Escherichia coli is a containment body type, and renaturation is required during the production process, and the renaturation rate is low, with a maximum of no more than 40%. 60% are non-refolding substances and cannot be removed, so the specific activity is low (the highest is only 1.0×10 7 unit / mg protein), with large side effects; the expression of S.

Method used

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  • Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor
  • Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor
  • Purification method of recombinant yeast strain and rhGM-CSF to express human granulocyte-macrophage colony stimulating factor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Construction of rhGM-CSF yeast engineering bacteria

[0034] (1) Materials:

[0035] 1. rhGM-CSF gene

[0036] 2. pGAPZα-A and P. pastoris Strain GS115 (his4) were purchased from Invitrogen, Australia.

[0037] (2) Method:

[0038] 1. Gene PCR amplification:

[0039] (1) Primer design: delete the Ste13 site (Glu-Ala-Glu-A1a) and the initiation codon ATG after the Kex2 (Lys-Arg) site in the 5' end primer.

[0040] P1-5'G CTCGAG AAAAGA ATG GCTCCAGCCCGT3' ("ATG" is removed)

[0041] Xho I Lys-Arg (Kex2 site)

[0042] P2-5'G TCTAGA TTACTCCTGGACTGGCTC3'

[0043] wxya

[0044] (2) Thermal cycle: 95°C, 5'; 95°C, 30"→43°C, 30"→72°C, 40"; 72°C, 10'; 30 cycles of amplification.

[0045] 2. PCR amplification product recovery and cloning:

[0046] (1) The rhGM-CSF gene was amplified and electrophoresed to obtain a DNA band of about 400 bp;

[0047] (2) The target fragment was recovered with a high-purity kit for PCR products produced by Böhringmann, Germany, and ...

Embodiment 2

[0052] Purification method of rhGM-CSF:

[0053] 1. Strain preservation

[0054] After constructing highly efficient rhGM-CSF-expressing yeast engineered bacteria (rhGM-CSF / pGAPZα-A / GS115), the strains were stored at -80°C, or the skim milk was freeze-dried and then sealed.

[0055] 2. Activation of strains

[0056] Plate medium formula (%):

[0057] Yeast powder 1.0 Peptone 2.0 Glucose 2.0 Agar 2.0

[0058] Take a bacterial strain, pick the bacterial liquid to scratch the plate, and then culture it at 30°C for 24 hours

[0059] 3. First-class seed liquid

[0060] YPD medium formula (%):

[0061] Yeast powder 1.0 peptone 2.0 glucose 2.0

[0062] Add 100mg / Lzeocin to the YPD medium, then take the activated bacterial solution to inoculate at 1% of the inoculum size, and incubate at 30°C for 12 hours.

[0063] 4. Secondary seed solution

[0064] YPD medium formula (%):

[0065] Yeast powder 1.0 peptone 2.0 glucose 2.0

[0066] Inoculate at 1% of the inoculum size and in...

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Abstract

The present invention discloses one kind of secretion vector of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and its simple purification method, host cell transformed with the secretion vector, as well as the method of fermentation with the recombinant pichia yeast engineering strain of the said expression vector to express rhGM-CSF. The present inention replaces colibacillus occlusion body expression system with yeast secreting expression sytem and has no need of renaturation of target protein rhGM-CSF, biological specific activity up to 3.4E7 unit / mg protein and less toxic side effect. Therefore, the rhGM-CSF of the present invention has high yield, simple purification process, low cost and less toxic side effect.

Description

[technical field] [0001] The present invention relates to the field of producing medical protein or polypeptide medicine by using recombinant DNA technology, more specifically, the present invention relates to the purification method of secretory expression human granulocyte macrophage colony-stimulating factor recombinant vector, yeast recombinant engineering strain and rhGM-CSF . [Background technique] [0002] At present, most of the domestically produced rhGM-CSF is the expression product of Escherichia coli, and the main process disadvantages are: the expression system of Escherichia coli is a containment body type, and renaturation is required during the production process, and the renaturation rate is low, with a maximum of no more than 40%. 60% are non-refolding substances and cannot be removed, so the specific activity is low (the highest is only 1.0×10 7 unit / mg protein), with large side effects; the expression of Saccharomyces cerevisiae engineering strains is a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/81C12N1/19C12N15/12C12N15/27C12P21/02C07K14/535C07K1/36C12R1/84
Inventor 梁国栋周鹏夏中宁蒲广西陈海宁
Owner 海南国栋药物研究所有限公司
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