Korean Diabetes J.  2008 Apr;32(2):131-140. 10.4093/kdj.2008.32.2.131.

Cloning of Novel Epidermal Growth Factor (EGF) Plasmid for Gene Therapy on Diabetic Foot Ulcer

• Chung HS
• Yoon CS
• Kwon MJ
• Kim MK
• Lee SH
• Ko KS
• Rhee BD
• Park JH

• KMID: 2222509
• DOI: http://doi.org/10.4093/kdj.2008.32.2.131

Abstract

BACKGROUND: Epidermal Growth Factor (EGF) is one of the important growth factors involved in the epithelialization during cutaneous wound healing. Peptide EGF has been used for the treatment of diabetic foot ulcer. But the inferiority of cost-effectiveness and the inconvenience of daily application might have restricted its wide clinical usage. EGF gene therapy could dramatically improve the efficacy and inconvenience through long-term expression and bypassing the EGF degradation by hostile non-specific proteinases expressed in the wound bed.
METHODS
EGF DNAs were amplified via PCR. For the more effective secretion from the transfected cell, we inserted furin cleavage site into EGF plasmids. The efficacy of novel plasmid pbeta-EGF was verified by transfection into the various animal cell lines, and the biologic potency of expressed EGF was confirmed via phosphorylation of PI3K and GSK3beta by Western blotting.
RESULTS
We tested various kinds of human EGFs. One of the human EGF isoforms, EGF(828) including a membrane-anchoring domain was successfully released as the mature EGF protein in the cell culture media. Also EGF plasmid including furin cleavage site showed more than 2-fold increased EGF expression compared with the sequence without furin cleavage site.
CONCLUSION
In conclusion, these findings suggest that mature EGF could be released easily out of cells by modifying EGF DNA sequence. Our novel EGF plasmid DNA could markedly increase the efficiency of non-viral gene therapy for diabetic foot ulcer.

Keyword

Diabetes mellitus; Epidermal growth factor; Gene therapy; Wound healing

MeSH Terms

Animals
Base Sequence
Cell Culture Techniques
Cell Line
Clone Cells
Cloning, Organism
Diabetes Mellitus
Diabetic Foot
DNA
Epidermal Growth Factor
Furin
Genetic Therapy
Glycogen Synthase Kinase 3
Humans
Intercellular Signaling Peptides and Proteins
Peptide Hydrolases
Phosphorylation
Plasmids
Polymerase Chain Reaction
Protein Isoforms
Transfection
Ulcer
Wound Healing
DNA
Epidermal Growth Factor
Furin
Glycogen Synthase Kinase 3
Intercellular Signaling Peptides and Proteins
Peptide Hydrolases
Protein Isoforms

Figure

• 

  Fig. 1 EGF structures. Original EGF sequence is composed signal peptide, prepro region, mature EGF, transmembrane domain (TM), and cytoplasmic domain (CP). Only mature EGF sequence (EGF159) (A) and mature EGF included Furin site (Furin-EGF159) (B). EGF excluded prepro region in full-length EGF (EGF828) (C) and EGF828 included Furin site (D).

• 

  Fig. 2 pβ-EGF plasmid cloning. EGFs and pβ vector including chicken β actin promoter was cut by EcoR I and Not I restriction enzyme and ligased with T4 DNA ligase. Completed plasmids re-confirmed by EcoR I and Not I enzymes. pβ-vector size is 4.0 kbp. pβ-EGF159 (A) and pβ-Furin EGF159 (B). pβ-EGF828 (C) and pβ-Furin EGF828 (D).

• 

  Fig. 3 EGF expression in diverse cell lines. EGF plasmid containing furin site in N-terimal and cytoplasmic domain and transmembrane domain in C-terminal highly increased released hEGF protein in culture medium in HEK293 cells (A), CHO cells (B), and NIH3T3 cells (C).

• 

  Fig. 4 Biological function of soluble hEGF protein in A549 cell. Cells were treated with 70 pg/mL EGF for a variety of time prior to extraction. Phosphorylation of GSK3β and PI3K was determined in immunoblot. After treated EGF, phosphorylation of PI3K (A) was maximum at 2 min and GSK3β (B) was at 15 min.

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