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Construction and expression method of recombinant G-CSF (15-75) polypeptide vector

A GS115 and carrier technology, applied in the field of biopharmaceuticals, can solve the problems of easy gene loss, low product expression, low renaturation rate, etc., and achieve the effect of easy purification, simple purification method and short fermentation cycle

Active Publication Date: 2015-08-19
SHANDONG NEWTIME PHARMA
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Problems solved by technology

[0005] Chinese patent CN103233053A discloses a production method of recombinant human granulocyte-stimulating factor, using recombinant plasmid pKG931 as the expression vector, and Escherichia coli HB101 as the recipient bacterium to improve production efficiency. Only 50% of human granulocyte-stimulating factor is expressed
[0006] Chinese patent CN103114115 discloses a method for preparing recombinant human granulocyte colony-stimulating growth factor by screening with microorganisms, and its pharmaceutical composition and preparation use engineering bacteria of recombinant human granulocyte colony-stimulating factor for fermentation and culture; Ultrasonic crushing of the obtained bacteria, collection of inclusion bodies, washing with inclusion body washing solution and centrifugation at low temperature; inclusion body dissolving solution was added to the inclusion bodies, and after 8 to 12 hours at 4°C, centrifugation to obtain inclusion body extracts; SephacrylS -200 gel column to separate the extract, collect the refolded recombinant human granulocyte colony-stimulating factor fraction; then use SephadexG25 column to separate, and finally obtain high-purity, high-activity G-CSF, but the recovery rate is only 47.5 %
[0008] At present, most of the G-CSF produced in China is the expression product of Escherichia coli. The use of E. coli requires renaturation during the production process, and the renaturation rate is low, and the specific activity is relatively low; the use of polyethylene glycol-modified G-CSF , its biological activity will decrease; the expression of Saccharomyces cerevisiae engineered strains belongs to non-integrated expression, the expression process is unstable, the product expression level is low and the gene is easily lost.

Method used

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  • Construction and expression method of recombinant G-CSF (15-75) polypeptide vector
  • Construction and expression method of recombinant G-CSF (15-75) polypeptide vector
  • Construction and expression method of recombinant G-CSF (15-75) polypeptide vector

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, recombinant plasmid construction

[0038] 1. Gene Design

[0039]The base sequence of the 15th to 75th amino acids of the unoptimized G-CSF sequence is:

[0040] CT AAGTG TT GA CAAGT G AAGAT CA GG GATGG GC GC CT CA GA AAA CT TGTGC AC TACAAG TGTG CACCC GA GA TGGT CTGCT GG CAC TCTGGG ATCCC TGGGCTCC CTGAG AG TGCCC AG CA GC CTGCA TGGCAGG TG TTG.

[0041] According to the full human G-CSF sequence, the G-CSF gene from the 15th to the 75th amino acid was optimized by using common yeast codons (marked in bold black), and an EcoR Ⅰ site and kex2 enzyme were added at the 5′ end Cutting site, TAA stop codon and Not I site are added to the 3′ end, as shown in the underlined part, the full-length sequence of the synthesized gene is:

[0042] 5' -GAATTCGAAAAACGC CT AAGTG TT GA CAAGT G AAGAT CA GG GATGG GC GC CT CA GA AAA CT TGTGC AC TACAAG TGTG ...

Embodiment 2

[0073] Example 2. Electroporation of recombinant plasmids integrated into Pichia pastoris cells

[0074] 1. Preparation of Pichia Competent Cells:

[0075] 1. Pick a single colony of yeast GS115 and inoculate it into a 5ml YPD test tube, incubate at 200rpm at 30°C for 6 hours, then transfer it to a 50ml YPD shaker flask at 1% inoculum size, and incubate overnight at 200rpm at 30°C;

[0076] 2. Pre-cool the centrifuge tube for standby, pre-cool the bacteria solution at 4°C for 30 minutes, and collect the bacteria by centrifugation at 5000 rpm for 10 minutes;

[0077] 3. Add 40ml 4℃ pre-cooled sterilized water to wash twice;

[0078] 4. Add 20ml of 1mol / L sorbitol pre-cooled at 4℃ and wash once;

[0079] 5. Add 500 μL of 1mol / L sorbitol pre-cooled at 4°C to the mixed cells, and aliquot 80 μL / cartridge.

[0080] 2. Plasmid linearization:

[0081] 1. Plasmid linearization enzyme digestion:

[0082]

[0083] Digest overnight at 37°C.

[0084] 2. Heat up the digested sample...

Embodiment 3

[0105] Embodiment 3: G-CSF (15-75) strain shake flask expression

[0106] In order to verify the expression level of recombinant G-CSF (15-75) polypeptide, Pichia pastoris (pPIC9K-G-CSF(15-75)-GS115) (A1-A5), Pichia pastoris (pPIC9K-G- CSF-GS115)(B1-B5) and negative control strain GS115(C) were used for preliminary expression studies on shake flasks;

[0107] 1. Inoculate each bacterial strain in BMGY medium (1% yeast powder, 2% peptone, 100mM potassium phosphate pH6.0, 1.34% YNB4×10 -5 % biotin, 1% glycerol), inoculate three bottles per plant, culture at 30°C for 24h, OD 600 reach 4;

[0108] 2. Centrifuge the bacterial solution at 1200rpm for 30min to collect the cells, remove the supernatant, and use BMMY medium (1% yeast powder, 2% peptone, 100mM potassium phosphate pH6.0, 1.34%YNB4×10 -5 % biotin, 0.5% methanol) to resuspend the bacteria, induce expression, and induce 80h to end the culture;

[0109] 3. After centrifuging the fermentation broth at 12000 rpm for 10 min...

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Abstract

The invention discloses a construction method of a recombinant G-CSF polypeptide (15-75) vector and a method for efficiently expressing the recombinant G-CSF polypeptide (15-75) in Pichia pastoris. The recombinant vector is formed by cloning a G-CSF polypeptide (15-75) gene into a pPIC9K expression vector, and transforms Pichia pastoris GS115, and methanol is utilized to induce the expression of the recombinant G-CSF polypeptide. The pPIC9K-G-CSF-GS115 recombinant Pichia pastoris constructed by the method can efficiently and stably express the recombinant G-CSF polypeptide (15-75). The expression level of the target protein is 2.8 g / L above, the purity of the purified recombinant G-CSF polypeptide (15-75) is up to 99.8%, and the biological specific activity is 1.5*10<8>IU / mg.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to the construction of an expression vector of G-CSF (15-75) polypeptide and its expression in Pichia pastoris. Background technique [0002] The main function of granulocyte stimulating factor (granulocyte colony stimulating factor, G-CSF) is to stimulate the proliferation and differentiation of hematopoietic cells of the neutrophil lineage, increase the number of neutrophils in peripheral blood, activate the function of neutrophils, and prevent tumors after chemotherapy. Patients injected with G-CSF can increase the level of circulating neutrophils, which may be related to shortening the time for some bone marrow cells to enter the S phase and increasing the number of progenitor cells that produce granulocytes. [0003] G-CSF was put on the market by the US FDA in 1991 for the treatment of neutropenia after radiotherapy. Now there are many companies in production and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C07K14/535C12R1/84
Inventor 张贵民刘忠张苏
Owner SHANDONG NEWTIME PHARMA
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