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Nucleic acid construct of epidermal growth factor, method of production and composition thereof

A technology of epidermal growth factor and nucleic acid constructs, applied in the field of biology, can solve problems such as inability to remove signal peptides or affinity tags

Pending Publication Date: 2022-02-22
DREAMTEC INTPROP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the inability to remove the signal peptide or affinity tag, only fusion proteins can be produced

Method used

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  • Nucleic acid construct of epidermal growth factor, method of production and composition thereof
  • Nucleic acid construct of epidermal growth factor, method of production and composition thereof
  • Nucleic acid construct of epidermal growth factor, method of production and composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1: Construction of EGF expression vector

[0096] The expression construct pET42a(+)-GST-gp41-1-EGF was constructed as follows:

[0097] A synthetic DNA fragment (SEQ ID NO:5) encoding the 5' to 3' end sequence of NotI-stop codon-EGF-gp41-1-thrombin site-SpeI was synthesized by Thermo Fisher Scientific. The synthetic DNA fragment was amplified by PCR extension using the forward primer 5'-AAAAAGCGGCCGCTTAGCGCAGTTC-3' and the reverse primer 5'-AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3'. The amplified PCR product was purified by PCR cleaning kit (Axygen AxyPrepClean-Up Kit) and digested with NotI and SpeI. The digested fragment was purified with 1% agarose and ligated into pET42a(+) digested with the same restriction enzymes.

[0098] The plasmid sequence of pET42a(+)-GST-gp41-1-EGF was confirmed by Sanger sequencing.

Embodiment 2

[0099] Embodiment 2: the expression of EGF fusion protein in shake flask

[0100] The plasmid pET42a(+)-GST-gp41-1-EGF was transformed into T7 Express (T7 Express). A single colony of transformants was supplemented with 40 μg·mL -1 Kanamycin was grown in 1 L of LB medium at 37°C (rotating at 250 rpm). When A 600 When the value reaches 0.5, reduce the growth temperature to 16 °C and add IPTG at a final concentration of 0.1 mM. Cultures were grown overnight. Collect 1 mL of the cell pellet and resuspend it in 200 μL of resuspension buffer (50 mM Tris-Cl, 200 mM EDTA, pH 8.0) supplemented with 1x PMSF, aprotinin, benzamide, and leupeptin, then Incubate for 5 minutes. Then at 37°C, with 120 μL lysozyme solution (1 mg·mL -1 ) to treat the mixture for 20 minutes. Then 80 μL of lysis buffer (10 mM EDTA, 10% Triton X-100 and 50 mM Tris-Cl, pH 8.0) was added. The tube containing the solution was gently inverted and then centrifuged at 14,800 rpm for 5 minutes. Cell lysate samp...

Embodiment 3

[0101] Embodiment 3: the expression of EGF fusion protein in fermentor

[0102] T7 expresses pET42a(+)-GST-gp41-1-EGF transformant, add 40μg·mL-1 Kanamycin was grown in 1 L of LB medium at 37°C (rotating at 250 rpm). When A 600 When the value reached 1, the whole culture was subcultured into a 50 liter fermenter containing 44 liters of LB medium. The culture was grown at 37 °C (rotation at 100 rpm, aeration ratio 1.5 vvm) until A 600 When the value reaches 0.5, reduce the growth temperature to 16 °C and add IPTG at a final concentration of 0.1 mM. 1M H in use 2 SO 4 The culture was grown overnight maintaining a pH of 7.0 with 1 M NaOH. Cell pellets were collected by serial centrifugation and washed twice with Buffer A (1x PBS and 1x PMSF, aprotinin, benzamide, and leupeptin) for long-term storage.

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Abstract

The invention relates to a nucleic acid construct, which comprises an insertion fragment and is characterized in that the insertion fragment comprises a polynucleotide sequence for coding an affinity tag, intein and an epidermal growth factor from the 5' end to the 3' end, the affinity tag is a GST affinity tag, and the intein is gp41-1 protein. The research reveals the ability of the gp41-1 micro intron in the aspects of intracellular expression and C-exon lysis of the EGF fusion protein. The finally purified EGF is proved to have very high activity in the aspect of accelerating the healing speed of bedsore, diabetic foot ulcer and skin rupture.

Description

technical field [0001] The present invention relates to the field of biology, in particular to a nucleic acid construct of epidermal growth factor, an expression vector thereof, a host cell, a production method of the construct, primers used, and the use of the construct or expression vector or host cell Method for producing EGF and EGF produced by the method, and compositions thereof. Background technique [0002] Epidermal growth factor (EGF) is a 53 amino acid oligopeptide with three disulfide bonds, which was discovered 60 years ago. EGF can bind to EGF receptors, thereby activating downstream signaling cascades in triggering epidermal cell proliferation. Studies have shown that EGF not only plays a very good role in the improvement of wound healing (Su,Z.,et al.(2014).Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor.Materials Science and Engineering: C,44,440-448.), but also involved in various phy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N15/67C07K14/485C07K19/00C07K1/36C07K1/34C07K1/22C12N15/11A61K38/18A61P3/10A61P17/02C12R1/19
CPCC12N15/70C12N15/67C07K14/485A61P3/10A61P17/02C07K2319/23C07K2319/92A61K38/00
Inventor 钟树根邝纬阳林庭匡
Owner DREAMTEC INTPROP LTD
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