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Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same

A technology for Escherichia coli and epidermal growth factor, which is applied in the field of Escherichia coli and its high-efficiency secretion and expression of human epidermal growth factor, can solve the problem of low expression level, achieve high expression level, and avoid bacterial physiology Effect of state change and plasmid loss, reducing production cost

Active Publication Date: 2013-04-24
ZHEJIANG APELOA KANGYU PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of secreted expression in E. coli also has disadvantages relative to intracellular expression, such as low expression

Method used

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  • Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same
  • Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same
  • Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction and identification of expression plasmid pTEGF9

[0048] For routine molecular experiment operation methods, refer to "Molecular Cloning Experiment Guide" (Science Press, third edition). With EGF-1 (5'-AAA CATATG AAACAAAGCACTATTGCACTG-3', the underlined part is the restriction site introduced) and EGF-2 (5'-AAA GGATCC TTATTAACGCAGTTCCCACC-3') was used as primer, and plasmid pE-5 was used as template to amplify the hEGF gene fragment with phosphatase signal peptide. The amplified gene fragment was digested with BamHI and NdeI. NdeI double-digested plasmid pET30a was connected and transformed into E. coli In TOP10 competent cells, spread on LB plates containing 30 μg / ml kanamycin, culture overnight, pick colonies and use EGF-2 and EGF-3 (5'-TACCATCGACACCACCCACGC-3') as primers for colonization PCR, verified by agarose gel electrophoresis, verified that the correct plasmid was through DNA sequencing, and the correct plasmid was named pTEGF9 (...

Embodiment 2

[0049] Example 2 Construction of expression strains, screening of high expression strains and detection of plasmid stability

[0050] 2.1 Construction of expression strains and screening of high expression strains

[0051] The plasmid pTEGF9 was treated with CaCl 2 Transform method into E. coli BL21(DE3) competent cells were spread on LB plates containing 30 μg / ml kanamycin, cultured overnight at 37°C, and 100 positive clones were picked and inoculated into 10 mL liquid containing 30 μg / ml kanamycin In LB medium, 37°C, 220 rpm, culture for 36 hours, take 0.5 mL of the culture solution, centrifuge to get the supernatant, put it into a vacuum rotary evaporator for concentration, and concentrate to 50 μl. Use the Tricine-SDS-PAGE method for electrophoresis (this gel has three layers, from top to bottom are 5% stacking gel, 10% interlayer gel and 15% separating gel). After electrophoresis, use high-sensitivity Coomassie brilliant blue Staining method Stain and decolorize, sc...

Embodiment 3

[0056] Example 3 Shake flask culture of Escherichia coli (Escherichia coli) BTE-9 (ie E. coli BL21(DE3) / pTEGF9) expressing rhEGF

[0057] Shake flask culture:

[0058] The medium formula and culture method were optimized by single factor analysis and uniform experimental design. Culture in a 250 mL shake flask containing 50 mL of medium, and the ingredients in each 50 mL of medium are:

[0059] Polypeptone 0.5 g Yeast extract 0.4 g

[0060] Glucose 0.4 g Potassium dihydrogen phosphate 0.04 g

[0061] Dipotassium hydrogen phosphate 0.1 g Ammonium sulfate 0.1 g

[0062] Anhydrous magnesium sulfate 0.025 g Glycine 0.05 g

[0063] Saturated phenol red 100 μl Kanamycin 30 μg / mL

[0064] 1% (v / v) of the frozen strains were inoculated in the shake flask culture medium, the culture temperature was 37°C, the rotation speed was 220 rpm, and the culture was continued for 35 hours, during which the pH was adjusted to 7.0.

[0065] During the culture process, samples were taken e...

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Abstract

The invention relates to the construction and expression of a strain by virtue of a genetic engineering method, discloses (escherichia coli) BTE-9 which was collected in the CGMCC (China General Microbiological Culture Collection Center) on April 16, 2012 with the collection number of CGMCC NO. 6014 and provides a method for efficiently secreting and expressing the human epidermal growth factor by using the (escherichia coli) BTE-9. During fermenting and expressing, no inducer is used, so that the change of the physiological state and the loss of plasmids due to an adopted inducer are avoided, the production cost is reduced, and the pollution of the toxic inducer to a target product is avoided, and high expression quantity is achieved.

Description

technical field [0001] The present invention relates to the field of biotechnology, relates to the construction of expression strains by means of genetic engineering, and the fermentative production of human epidermal growth factor (hEGF), more specifically to a kind of Escherichia coli and its high-efficiency secretion and expression of human epidermal growth factor Methods. Background technique [0002] Human epidermal growth factor (Human epidermal growth factor, referred to as hEGF), also known as urogastrone, hEGF is found in almost all body fluids under normal physiological conditions, such as urine, milk, blood, saliva, tears, gastric juice, bone marrow fluid, etc. Both contain EGF, which is generally believed to be more abundant in urine and emulsion in the kidney (Orsinib B, et al. Clin Biochem, 1991, 24:135-137). Cohen S (Cohen S, et al. Proc. Natl. Acad. Sci, USA, 1975,72:1317-1321) and Gregorg H (Gregorg H, et al. Nature, 1975,257:325-327) in 1975 hEGF was isol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P21/02C12R1/19
Inventor 杨圣勇朱冬发李仁宝张辉王莉韩文琦董国秀吕岳寅
Owner ZHEJIANG APELOA KANGYU PHARMA
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