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Method for producing recombinant human epidermal growth factor by using gene engineering colibacillus

A technology of epidermal growth factor and Escherichia coli, which is applied in the direction of epidermal growth factor, growth factor/inducing factor, bacteria, etc., can solve the problems of low separation and purification efficiency, complicated extraction process, high operating cost, etc., and achieve high production automation level, separation The effect of simple process and short production cycle

Inactive Publication Date: 2003-03-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are three ways to produce hEGF, chemical synthesis: in 1988, Marchi et al. first completed the total synthesis of EGF. Due to the presence of disulfide bonds and other functional groups in the hEGF molecule, the purity and yield of the synthesized products cannot meet industrial production. Requirements; biological source extraction: mainly separated and recovered from human urine, the concentration of hEGF in natural sources is low, generally less than 1 μg / L, so the separation and purification efficiency is low; genetic engineering technology: the production of hEGF is currently focused on the research of genetic engineering technology
[0004] Although there are currently some genetic engineering production methods for hEGF, these methods all have low product concentration in the fermentation broth (generally less than 100mg / L), complex extraction process (3-6 steps of chromatographic operations are often required), and high operating costs. And the disadvantages such as low yield (20~30%), therefore, the price of human epidermal growth factor is still as high as US$210~1020 / mg hEGF at present
So far, there has not been a relatively mature industrial fermentation production process report

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The steps of the method for producing recombinant human epidermal growth factor with genetic engineering escherichia coli are as follows:

[0049] 1) Engineering bacteria fermentation

[0050] Pick a single colony from the solid medium and insert it into 50ml of seed medium (500ml Erlenmeyer flask), add ampicillin at the same time, make it reach 100mg / l in the medium concentration, 32 ℃, 220rpm shaker culture 24 After 1 hour, insert the previous culture solution into 50ml seed culture medium again by 1% inoculum size, add ampicillin simultaneously, make it reach 100mg / l in the culture medium concentration, 32 ℃, 220rpm shaking table culture 8 hours, again The activated seed liquid was added to 50 ml of fermentation medium according to the inoculation amount of 1%, and ampicillin was added to make the concentration reach 100 mg / ml at the same time, and cultivated at 32° C. and 220 rpm for 8 hours. Then add the placebo inducer isopropyl-β-D-thiogalactoside for induction,...

Embodiment 2

[0058] The steps of the method for producing recombinant human epidermal growth factor with genetic engineering escherichia coli are as follows:

[0059] 1) Engineering bacteria fermentation

[0060] Pick a single bacterium colony from the solid medium and insert it into 25ml seed medium (500ml Erlenmeyer flask), add ampicillin at the same time, make it reach 120mg / l in the medium concentration, 33 ℃, 200rpm shaker culture 22 After 2 hours, the previous culture solution was inserted into the 25ml seed culture medium again by 2% inoculum size, and ampicillin was added simultaneously to make its concentration in the culture medium reach 120mg / l, 33°C, 200rpm shaker culture for 7.5 hours, and then The activated seed solution was added to 50 ml of fermentation medium according to the inoculum size of 2%, and ampicillin was added to make the concentration reach 120 mg / ml at the same time, and cultured at 33° C. and 200 rpm for 7.5 hours. Then add the placebo inducer isopropyl-β-D-...

Embodiment 3

[0068] The steps of the method for producing recombinant human epidermal growth factor with genetic engineering escherichia coli are as follows:

[0069] 1) Engineering bacteria fermentation

[0070] Pick a single bacterium colony from the solid medium and insert it into 100ml seed medium (500ml Erlenmeyer flask), add ampicillin at the same time, make it reach 80mg / l in the medium concentration, 31 ℃, 240rpm shaker culture 26 After 1 hour, insert the previous culture solution in the seed culture medium of 100ml again by 0.5% inoculum size, add ampicillin simultaneously, make it reach 80mg / l in the culture medium concentration, 31 ℃, 240rpm shaking table culture 8.5 hours, again The activated seed solution was added to 100 ml of fermentation medium according to the inoculation amount of 0.5%, and ampicillin was added to make the concentration reach 80 mg / ml at the same time, and cultured at 31° C. and 240 rpm for 8.5 hours. Then add the placebo inducer isopropyl-β-D-thiogalact...

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Abstract

The method for producing recombinant human epidermal growth factor by using genetically-engineered colibacillus includes the following steps: engineering bacterium fermentation: activating stored colibacillus by means of culture medium, fermenting main medium and introducing the human epidermal growth factor into fermentation cutlure liquor; expanding bed adsorption; centrifugalizing fermentationculture liquor, adding supernatant fluid into the expanding bed to make adsorption and collecting elution peak containing human epidermal growth factor; gel separation; adding collected eluent into gel chromatographic column, separating and collecting human epidermal growth factor elution peak; freezing, sublimating and drying; freezing collected liquor at -40 deg.C, sublimating and drying so as to obtain the invented product.

Description

technical field [0001] The invention relates to a method for producing recombinant human epidermal growth factor by using genetically engineered Escherichia coli. Background technique [0002] In the early 1940s, a substance that could inhibit gastric acid secretion was found in the urine of pregnant women, called urogastrone (UG). In 1962, when Cohen used a carboxymethyl cellulose column to separate and purify Nerve Growth Factor (NGF) from the submandibular gland of mice, he discovered another active substance. At that time, it was named according to its function of promoting teething and eye opening in newborn mice. "Tooth-Lid Factor", later renamed "Epidermal Growth Factor". In 1975, Cohen and Carpenter reported the amino acid residue composition of human epidermal growth factor (hEGF). In the same year, Gregory described a UG isolated from human urine. Studies have shown that UG and hEGF are the same substance. Because hEGF can stimulate mult...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/485C12N1/21C12P21/02
Inventor 姚善泾朱自强童望宇梅乐和关怡新
Owner ZHEJIANG UNIV
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