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Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor

An epidermal growth factor and engineering bacteria technology, applied in the field of bioengineering, can solve the problems of high extraction cost, low hEGF content, hEGF limitation, etc., and achieve the effect of shortening fermentation time and increasing yield

Pending Publication Date: 2019-12-13
ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Before the 1980s, the source of hEGF was mainly extracted from tissues and body fluids. Due to the extremely low content of hEGF, the extraction cost was high and the product purity was low, which limited the application of hEGF

Method used

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  • Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor
  • Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor
  • Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] For primary seed cultivation, inoculate 100ul of engineering bacteria frozen in glycerol into 50ml LB liquid medium containing 50ug / ml kanamycin, shake at 35°C and 220rpm for 7 hours (primary seed cultivation time), and measure OD 600 (Level 1 seed OD 600 ) is 2, stop the cultivation, and obtain the primary seed culture solution.

[0092] Secondary seed culture, the primary seed culture liquid is inoculated to 500ml with the inoculum size of 0.2vol.% in the LB liquid culture medium that contains 50ug / ml kanamycin, at 25 ℃, shaking 12 hours under 220rpm (secondary seed Incubation time), measured OD 600 (secondary seed OD 600 ) was 3.5, the cultivation was stopped to obtain the secondary seed culture solution.

[0093] The secondary seed culture solution was inoculated into a 20L fermenter containing 12L medium at an inoculum size of 5vol.% (the inoculum size of the secondary seed culture solution), stirred at 37°C at 200rpm, and the pH was adjusted to 6.8 with ammoni...

Embodiment 2

[0098] Carry out embodiment 2 in the same manner according to above-mentioned embodiment 1, just the OD of primary seed culture liquid 600 changed to 3, and the OD of the secondary seed culture 600 Change to 4. The results of this example, as well as primary and secondary seed incubation times are shown in Table 1 below.

Embodiment 3

[0100] Carry out embodiment 3 in the same manner according to above-mentioned embodiment 1, just the OD of primary seed culture liquid 600 Change to 2.5, and the OD of the secondary seed culture medium 600 Change to 3.75. The results of this example, as well as primary and secondary seed incubation times are shown in Table 1 below.

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Abstract

The invention discloses a method for culturing engineering bacteria for expressing a recombinant human epidermal growth factor. The method comprises the following steps: (1) primary seed culture: inoculating glycerol cryopreserved engineering bacteria into an LB liquid culture medium containing kanamycin, and culturing to obtain a primary seed culture solution; (2) secondary seed culture: inoculating the primary seed culture solution obtained in the step (1) into an LB liquid culture medium containing kanamycin, and culturing to obtain a secondary seed culture solution; (3) inoculating the secondary seed culture solution obtained in the step (2) into a fermentation tank, culturing, and then inducing to obtain a fermentation tank supernatant; and (4) separating and purifying a recombinant human epidermal growth factor from the fermentation tank supernatant obtained in the step (3). The OD600 of the primary seed culture solution is 2 to 3, and the OD600 of the secondary seed culture solution is 3.5 to 4. Through the method, the fermentation time of engineering bacteria is shortened, and the yield of hEGF is increased.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically, the invention relates to a method for producing recombinant human epidermal growth factor by adopting a genetic engineering method. Background technique [0002] Human epidermal growth factor (hEGF) is a small molecular polypeptide composed of 53 amino acids, with a molecular weight of about 6000 Daltons and an isoelectric point of about 4.2-4.6. There are three pairs of disulfide bonds in the molecule, and it is relatively stable to physical and chemical factors such as acid, alkali and heat. [0003] hEGF has a wide range of biological effects and can strongly promote the growth of various epidermal tissues. It has been used in the treatment of burns, ulcers, various wounds and corneal injuries in medicine. hEGF can also promote the metabolism of normal epidermal cells, adding it to beauty and skin care products can achieve the effects of whitening, anti-wrinkle and anti-ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02C12R1/19
CPCC07K14/485C12N1/20C12P21/02
Inventor 钱永常张旭初宋叶华
Owner ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD