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Method for producing recombinant platelet-derived growth factors

A growth factor and platelet technology, applied in the biological field, can solve the problems of weak functions and differences such as disulfide bonds and post-translational modifications, and achieve the effects of good safety and efficacy, good application prospects and simple methods.

Active Publication Date: 2018-12-07
智享生物(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Regarding the expression and production process of PDGF-BB, there are many reports in yeast expression vectors and Escherichia coli. The expression levels of Escherichia coli and yeast expression systems are relatively high, which can effectively reduce production costs. However, the disulfide bonds of these two systems The post-translational modification and other functions are weak, and the post-translational modification of the expressed protein will be different from the target protein

Method used

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  • Method for producing recombinant platelet-derived growth factors
  • Method for producing recombinant platelet-derived growth factors
  • Method for producing recombinant platelet-derived growth factors

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Experimental program
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Embodiment 1

[0029] Construction of embodiment 1 recombinant cell strain

[0030]The rhPDGF-BB gene was correctly placed in the expression vector of CHO cells by conventional DNA recombination technology, transformed into CHO cells, and a recombinant cell line secreting and expressing rhPDGF-BB was established through screening. Reference books include "Molecular Cloning Technical Guide", "Molecular Biology Guide", "Animal Cell Culture".

Embodiment 2

[0031] The recovery of embodiment 2 recombinant cell lines

[0032] Place the seed medium in a water bath, preheat at 37±0.5°C for more than 20 minutes, take a cell from the working bank, melt it quickly in a water bath at 37±0.5°C, control the melting time within 120±30s, and transfer the cells into 8.5ml of seed medium In a 15ml centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant, resuspend the cells with 5ml of seed medium and transfer to a 125ml shake flask, add volume to 30±5ml, mix well, take 0.5ml for counting, and place in 36.5±0.5℃, 6±1%CO 2 , 130rpm shaker for 3±1 days.

Embodiment 3

[0033] The preparation of embodiment 3 primary seeds

[0034] Cells were seeded at 0.5 ± 0.1 × 10 with seed medium pre-warmed to 36.5 ± 0.5 °C 6 Cells / ml density diluted to a new shake flask, after mixing, sample 0.5ml of cell fluid for counting. Shake the flask with a carbon dioxide shaker at 36.5±0.5°C, 6±1% CO 2 , 130r / min, each expansion culture 3±1 days, the density of living cells does not exceed 6.0×10 6 cells / ml, amplified 3 times.

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Abstract

The invention discloses a method for producing recombinant platelet-derived growth factors and belongs to the technical field of biology. The method comprises the following steps: 1) construction of recombinant CHO cell strains containing target genes of the recombinant platelet-derived growth factors; 2) first-stage seed culture; 3) second-stage seed culture; 4) continuous feeding culture in a reactor; 5) collection of supernatant for purifying preparation. The method provided by the invention for expressing and producing rhPDGF-BB in CHO cells is simple in process, can realize large-scale production and is wide in application prospect.

Description

technical field [0001] The invention relates to a method for producing heavy platelet-derived growth factor, which belongs to the field of biotechnology. Background technique [0002] Recombinant platelet derived growth factor (Platelet derived growth factor, PDGF) is a basic protein stored in platelet α granules, and it is a low molecular weight mitogen. It can stimulate fibroblasts, glial cells, smooth muscle cells and other cells stagnant in G0 / G1 phase to enter the cycle of division and proliferation. Platelet-derived growth factor PDGF is a peptide regulator discovered in 1974 that stimulates the growth of connective tissue and other tissue cells. It is named for its origin from platelets. The disintegrated platelets are released and activated, and have the biological activity of stimulating specific cell chemotaxis and promoting specific cell growth. In addition, macrophages, vascular smooth muscle cells, fibroblasts, endothelial cells, embryonic stem cells, etc. can...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N5/10C07K14/49C12R1/91
CPCC07K14/49C12P21/02
Inventor 黄高敏查鑫华徐小芳
Owner 智享生物(苏州)有限公司
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