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Novel use of ec-sod and method for preparing thereof

a technology of ecsod and ecsod protein, which is applied in the field of new ecsod protein use, can solve the problems of insufficient examination of the relationship between sods, angiogenesis, and removal of reactive oxygen species, and achieve the effects of inhibiting angiogenesis, preventing or inhibiting the over-differentiation of th2 cells

Inactive Publication Date: 2012-04-19
THE CATHOLIC UNIV OF KOREA IND ACADEMIC COOP FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0073]As mentioned above, an EC-SOD protein or a vector having a polynucleotide encoding the EC-SOD protein has the effect on inhibiting angiogenesis by inhibiting the expression of VEGF and MMP-9 which induce angiogenesis. Therefore, the EC-SOD protein or the vector may be useful for preventing or treating angiogenesis-mediated diseases. And the EC-SOD protein or the vector having a polynucleotide encoding said EC-SOD protein also has the effect on inhibiting over-differentiation of Th2 cells which cause allergic diseases, inhibiting transcription factor (NF-κ B), and reducing degranulation of human mast cells. Accordingly, the EC-SOD protein or the vector may be useful for preventing or treating allergy diseases. In addition, the inventive method for preparing an EC-SOD protein may be used in preparing an EC-SOD protein having the activity on a large scale. Therefore, the inventive method may be useful industrially.

Problems solved by technology

However, when angiogenesis is not regulated correctly, diseases such as cancer may be induced.
Reactive oxygen species have been reported to be major regulators of angiogenesis (Jolanta Grzenkowicz-Wydra, et. al., Mol Cell Biochem., 264(1-2):169-81, 2004), but the relationship between SODs, which remove reactive oxygen species, and angiogenesis, has not yet been sufficiently examined.
Meanwhile, with the development of industry, allergy-causing factors have increased due to environmental pollution, an increase in new synthetic substances and a change in residential environments, and thus the number of people suffering from asthma and allergies has gradually increased.
However, these drugs mostly have temporary effects, and in many cases, cause serious side effects.
However, whether EC-SOD has the effect of treating allergic diseases is not yet known.
Also, even if the EC-SOD has various activities, when a method for producing the EC-SOD in large amounts is not developed, there is a problem in that the EC-SOD cannot be efficiently used in industry.
However, mass-production methods for proteins, developed to date, comprise transforming host cells, such as E. coli cells, with expression vectors, and inducing the expression of proteins in the transformed cells, and the mass production of proteins has problems in that it often causes the formation of inclusion bodies, and thus the original activities of the proteins are eliminated or reduced.

Method used

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  • Novel use of ec-sod and method for preparing thereof
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  • Novel use of ec-sod and method for preparing thereof

Examples

Experimental program
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Effect test

example 1

Preparation of Recombinant EC-SOD Protein Having Biological Activity

Cloning of Fusion EC-SOD Gene for Expression of Recombinant EC-SOD Protein

[0097]In order to prepare a recombinant EC-SOD protein having biological activity, recombinant EC-SOD, consisting of 209 amino acids and containing deletion of the signal peptide at the N-terminal end of human EC-SOD and deletion of 13 amino acids at the C-terminal end, was cloned in the following manner. The base sequence and amino acid sequence of the recombinant EC-SOD are set forth in SEQ ID NO: 2 and SEQ ID NO: 1, respectively, and the base sequence and amino acid sequence of the human EC-SOD are set forth in SEQ ID NO: 4 and SEQ ID NO: 3, respectively.

[0098]A pUC 18-hEC-SOD vector (provided by Professor Marklund, Clinical Chemistry, Sweden) containing human EC-SOD cDNA (SEQ ID NO: 4), asa template, was amplified by PCR using the following primers, thus preparing human EC-SOD cDNA:

Sense primer(SEQ ID NO: 9)5′-tagattctggacgggcgagga-3′Anti...

example 2

Angiogenesis Inhibitory Effect of Natural EC-SOD Protein

[0115] Morphological Comparison of EC-SOD-Expressing Mice with Wild-Type Mice

[0116]The back skin of each of the mouse EC-SOD-overexpressing mice, prepared in Test Example 1, and wild-type BDF1 mice, was irradiated four times with UVB at a dose of 2 kJ / m2 at 24-hr intervals. Specifically, the back skin of each mouse was shaved at 2 days before UVB irradiation, and twenty-four 8-week-old mice were irradiated with UVB at a dose of 200 mJ / cm2 using 6 UVB lamps (FS24T12 / UVB / HO, 290-320 nm, Voltare, Fairfield, Conn., USA) having the highest energy at 305 nm in a wavelength range from 280 μm to 340 nm. During the UVB irradiation, the height of the lamps was controlled such that the surface of the back skin of each of the anesthetized mice was irradiated with a dose of 0.3 mW / cm2. The mice of each group (n=6) were irradiated four times with UVB at 24-hr intervals, and at 1 hour after the 4th UVB irradiation, the mice were sacrificed fo...

example 3

Angiogenesis Inhibitory Effect of Recombinant EC-SOD Protein

[0129]Whether the recombinant EC-SOD protein, separated and purified in Example 1, has the effect of inhibiting angiogenesis was examined. Keratinocyte HaCaT cells were plated on 18-mm cover slips at a concentration of 1×104 cells / cover slip. The cells were cultured in DME medium (Dulbecco's minimum essential medium, GIBCO), containing 10% fetal bovine serum (FBS, GIBCO) and 100 units penicillin and 100 g streptomycin / ml of medium, in 5% CO2 at 37° C. When the adhered cells reached a confluence of 30-40%, the fetal bovine serum was removed, and then cultured for 24 hours. When the cells reached a confluence of 50-60%, the cells were pretreated with 10 μg of EC-SOD. After the cells were treated with the purified recombinant EC-SOD protein for 30 minutes, the cells were treated with UV light at a dose of 100 J, and after 24 hours, the skin keratinocyte cells were fixed with paraformaldehyde and immunostained. The fixed cells ...

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Abstract

The present invention relates to compositions for preventing or treating angiogenesis-mediated diseases or allergic diseases which contain, as an active ingredient, an EC-SOD protein or a vector having a polynucleotide encoding thereof. The EC-SOD protein or the vector having a polynucleotide encoding the EC-SOD protein has the effect on inhibiting angiogenesis by inhibiting the expression of VEGF and MMP-9 which induce angiogenesis. Therefore, the EC-SOD protein or the vector may be useful for preventing or treating angiogenesis-mediated diseases. And the EC-SOD protein or the vector having a polynucleotide encoding said EC-SOD protein also has the effect on inhibiting over-differentiation of Th2 cells which cause allergic diseases, inhibiting transcription factor (NF-κ B), and reducing degranulation of human mast cells. Accordingly, the EC-SOD protein or the vector may be useful for preventing or treating allergy diseases. In addition, the inventive method for preparing an EC-SOD protein may be used in preparing an EC-SOD protein having the activity on a large scale. Therefore, the inventive method may be useful industrially.

Description

TECHNICAL FIELD[0001]The present invention relates to a novel use of an EC-SOD protein and a method for preparing thereof. More particularly, the present invention relates to compositions for preventing or treating angiogenesis-mediated disease or allergic diseases which contain, an EC-SOD protein or a vector having a polynucleotide encoding said EC-SOD proteins an active ingredient.BACKGROUND ART[0002]Superoxide dismutases (SODs) function to protect cells by removing reactive oxygen species and enabling other antioxidant enzymes, and SODs known to date include Cu / Zn SOD (SOD 1) containing copper and zinc atoms, Mn SOD (SOD 2) containing a manganese atom, and extracellular superoxide dismutase (EC-SOD) present on the cell surface or in the extracellular fluid.[0003]Particularly, EC-SOD contains copper and zinc atoms as in Cu / Zn SOD, but is characterized in that a heparin binding domain is present in the C-terminal end. Since EC-SOD has the heparin-binding domain, it is assumed that ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/44A61P3/10A61P29/00A61P27/06A61P9/10A61P27/02A61P27/14A61P17/10A61P37/08A61P17/06A61P11/06C12N9/02C12N15/63A61K48/00A61P11/02A61P17/00A61P35/00
CPCA61K38/00C12Y115/01001A61K38/446C12N9/0089A61P11/02A61P11/06A61P17/00A61P17/02A61P17/04A61P17/06A61P17/10A61P19/02A61P27/02A61P27/06A61P27/14A61P27/16A61P29/00A61P3/10A61P35/00A61P37/08A61P43/00A61P9/10
Inventor SHIN, DONG HEONKIM, TAE YOON
Owner THE CATHOLIC UNIV OF KOREA IND ACADEMIC COOP FOUND
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