HUMAN ANTIOXIDATION OR AGING_ASSOCIATED GENE AND

PROTEIN ENCODED THEREIN

[Technical Field]

The present invention relates to a human antioxidant gene, a protein encoded therein, an expression vector including the same, and a cell transformed by the vector.

[Background Art]

In the present era of aging societies all over the world, degenerative diseases associated with aging have become a deep concern. It is not only the hope of individuals but a matter of great social and national importance and the desire of all mankind to live in health in their old age, the last 1/3 of their lives. However, the unfortunate reality is that many people endure a great deal of suffering in their old age. Due to increase in average life span, 30% of the total population are in their old age, infectious diseases, tuberculosis, and other illnesses that were prominent in the past have declined, and chronic illnesses like geriatric diseases, cerebrovascula diseases, and cancer have increased. Such illnesses also place a heavy burden on patients' families and incur considerable medical expenses. This problem is causing growing social and economic losses .

By and large, there are two hypotheses to explain the mechanism of aging so far. One is DNA program theory (programmed aging theory) , according to which there is a senescence gene causing degenerative changes including menopause, gray hair, decline in athletic ability, etc. with age. The other is the free radical theory of aging, according to which aging is the result of the accumulation of biological tissue damage by various harmful factors such as oxidants . With the discovery of single-gene [age-1 (hx54β)] mutation which extends the life of Caenorhabditis elegans from 65% up to 110% (Kenyon, C. et al . , Nature, 366 (6454) : 461-464 (1993)), evidence of the genetic nature of aging has been confirmed, and numerous studies have been conducted recently with the aim of finding the genetic factors that cause aging with molecular biological methods (Lithgow, G. J. and Kirkwood, T. B., Science, 273 (5271) : 80(1996) ) .

Investigation of genes such as age-1 (hx-546) , daf-2, daf-23, daf-28, spe-26, etc., which are the aging-related genes that are known so far, reveals that their functions are mostly related to stress-related systems which maintain a somatic cell or control its repair process (Gem, D. and Riddle, D. L., Nature, 379 (6567) : 723-725 (1996) ; Friedman, D.

B. and Johnson, T. E., Genetics, 118 :75 (1988)). It has been found that transformed age-1 strain (hx-546) is stronger against extrinsic stimulus than the natural type. It has been reported that the age-1 strain (hx-54β) has high resistance to hydrogen peroxide or paraquat which produces a hydroxy radical, has a low deletion frequency of a mitochondrial genome created due to the damage by the hydroxyl radical, and has high activity of anti-oxidant enzymes such as Cu, Zn-superoxide dismutase, catalase, etc. (Murakami, S. and Johnson, T. E., Genetics, 143 (3) :1207- 1218 (1996)). It has also been reported that aging mutations have high resistance to intrinsic thermotolerance or ultraviolet radiation, as well as to oxidants (Murakami, S. and Johnson, T. E., Genetics, 143 (3) ; 1207-1218 (1996); Kale, S. P. and Jazwinski, S. M., Developmental Genetics, 18 (2) :154-160 (1996)), and this resistance is connected directly with the extension of lifetime. In consideration of the research to date, it can be concluded that aging is induced by the combined interaction of the senescence gene and natural oxidants. Free radicals created in a living body can cause damage to cells, called oxidation stress, which brings about the necrosis of cells or apoptosis, and it is presumed that an anti-apoptosis gene in a cell makes a substance that removes free radicals. Free radicals are

molecules having unpaired electrons that may be created in biological reactions, such as OH, O ₂ ^' , NO etc. Such free radicals react with one another or with normal cells and cause harm inside of living bodies. In particular, the mitochondrial genome is very vulnerable to damage from oxidation, which causes deletion in the aging process. To prevent this, enzymes such as superoxide, dismutase, catalase, glutathione, peroxidase, etc. serve to remove free radicals from a living body, and it is reported that caeruloplasmin, haemopexin, haptoblobin, albumin, etc. are related to anti-oxidation as well.

Even, though it is already known that chronic illnesses like cancer, heart disorders and cataracts are caused by oxidant stimulus, this is still only part of the picture. Research into antioxidants has until now mostly focused on general biological effect rather than anti-oxidizing effect itself. Therefore, the answer to the basic question about what degree of oxidant stimulus causes aging or chronic illness is not known yet. The purpose of the present study is to measure the effect on cell growth of administering Korean traditional phytochemicals and ginseng extract known as oxidants or anti-oxidants to HUVECs (human umbilical vein endothelial cells), rapidly proliferating cells. This is done in order

to find out novel oxidation and anti-oxidation-associated genes by examining the change of genes in the cell damaged by the oxidant and medicated by the anti-oxidant using an mRNA differential display (DD) method (Liang, P. and Pardee, A. B., Science, 257 , 967-971 (1992); and Liang, P. et al . , Cancer Res., 52_, 6966-6968 (1993)), and ultimately to use them in gene therapy and medication to slow the progress of aging.

[Disclosure]

[Technical Subject]

An object of the present invention is to provide a novel human anti-oxidation associated gene.

Another object of the present invention is to provide an anti-oxidation protein encoded in the gene. Still another object of the present invention is to provide a novel human aging associated gene.

Yet another object of the present invention is to provide an anti-oxidation protein encoded in the gene.

[Technical Solution]

According to an object, a human anti-oxidation or aging-associated gene 1 (AAGl) having a base sequence of SEQ ID No. 1 is provided.

According to an object, a human anti-oxidation or aging-associated gene 2 (AAG2) having a base sequence of SEQ ID No. 5 is provided.

According to an object, a human anti-oxidation or aging-associated gene 3 (AAG3) having a base sequence of SEQ ID No. 9 is provided.

According to an object, a human anti-oxidation or aging-associated gene 4 (AAG4) having a base sequence of SEQ ID No. 13 is provided. According to an object, a human anti-oxidation or aging-associated gene 5 (AAG5) having a base sequence of SEQ ID No. 17 is provided.

According to an object, a human anti-oxidation or aging-associated gene 6 (AAG6) having a base sequence of SEQ ID No. 21 is provided.

According to an object, a human anti-oxidation or aging-associated gene 7 (AAG7) having a base sequence of SEQ ID No. 25 is provided.

According to an object, a human anti-oxidation or aging-associated gene 8 (AAG8) having a base sequence of SEQ ID No. 29 is provided.

According to an object, a human anti-oxidation or aging-associated gene 9 (AAG9) having a base sequence of SEQ ID No. 33 is provided.

According to an object, a human anti-oxidation or aging-associated gene 10 (AAGlO) having a base sequence of SEQ ID No. 37 is provided.

According to an object, a human anti-oxidation or aging-associated gene 11 (AAGlI) having a base sequence of SEQ ID No. 41 is provided.

According to an object, a human anti-oxidation or aging-associated gene 12 (AAGl2) having a base sequence of SEQ ID No. 45 is provided. According to an object, a human anti-oxidation or aging-associated gene 13 (AAG13) having a base sequence of SEQ ID No. 49 is provided.

According to an object, human anti-oxidation or aging- associated genes 14a and 14b (AAGl4a and AAGl4b) having a base sequence of SEQ ID No. 53 are provided.

According to another object, a human anti-oxidation or aging associated protein having an amino acid sequence of SEQ ID No. 2; SEQ ID No. 6; SEQ ID No. 10; SEQ ID No. 14; SEQ ID No. 18; SEQ ID No. 22; SEQ ID No. 26; SEQ ID No. 30; SEQ ID NO. 34; SEQ ID No . 38; SEQ ID No. 42; SEQ ID No. 46; SEQ ID No. 50; or SEQ ID Nos . 54 and 55, each of which is encoded in the gene of the present invention, is provided.

According to still another object, an expression vector including the gene is provided.

According to yet another object, a cell transformed by the vector is provided.

[Advantageous Effects]

The AAG genes of the present invention relate to oxidation of human cells and aging effects caused thereby, and may be useful for diagnosis, prevention and cure of the effects of aging.

[Description of Drawings]

FIGS. 1 to 13 are gel photographs illustrating PCR results using 5' 13mer random primer H-AP29 of SEQ ID No. 3; H-AP46 of SEQ ID No. 7; H-AP29 of SEQ ID No. 11; H-AP32 Of SEQ ID No. 15; H-AP28 of SEQ ID No. 19; H-AP38 of SEQ ID No. 23; H-AP31 of SEQ ID No. 27; H-AP31 of SEQ ID No. 31; H-AP36 of SEQ ID No . 35; H-AP29 of SEQ ID No . 39; H-AP2 of SEQ ID No. 43; H-AP2 of SEQ ID No. 47; and H-AP29 of SEQ ID No. 51, respectively. FIG. 14 is a gel photograph illustrating a PCR result using H-AP46 of SEQ ID No. 56, and a fixed oligo-dT primer of SEQ ID No. 4 (FIG. 1); SEQ ID No. 8 (FIG. 2); SEQ ID No. 12 (FIG. 3); SEQ ID No. 16 (FIG. 4); SEQ ID No. 20 (FIG. 5); SEQ ID No. 24 (FIG. 6); SEQ ID NO. 28 (FIG. 7); SEQ ID No. 32 (FIG. 8); SEQ ID No. 36 (FIG. 9); SEQ ID No. 40 (FIG. 10); SEQ ID No. 44 (FIG. 11); SEQ ID No. 48 (FIG. 12); SEQ ID No. 52 (FIG. 13); or SEQ ID No. 57 (FIG. 14) .

FIGS. 15 to 28 are photographs illustrating SDS-PAGE analysis results of gene products of AAGl, AAG2 , AAG3 , AAG4,

AAG5, AAG6, AAG7 , AAG8 , AAG9 , AAGlO, AAGlI, AAG12, AAG13 and AAG14, respectively. FIGS. 29 to 42 show northern blot results representing a differential expression profiling of AAGl(FIG. 29);

AAG2(FIG. 30); AAG3(FIG. 31); AAG4(FIG. 32); AAG5(FIG. 33);

AAGβ(FIG. 34); AAG7(FIG. 35); AAG8(FIG. 36); AAG9(FIG. 37);

AAGlO(FIG. 38); AAGlI(FIG. 39); AAG12(FIG. 40); AAG13(FIG. 41); and AAG14(FIG. 42) genes in several human normal tissues, respectively, b in the bottom of each drawing being northern blot results obtained by hybridizing identical blots with a β-actin probe.

FIGS. 43 and 56 are growth curves of a wild-type human umbilical vein endothelial cell, a human umbilical vein endothelial cell transfected with AAGl(FIG. 43); AAG2(FIG.

44); AAG3(FIG. 45); AAG4(FIG. 46); AAG5(FIG. 47); AAG6(FIG.

48); AAG7(FIG. 49); AAG8(FIG. 50); AAG9(FIG. 51); AAGlO(FIG.

52); AAGIl(FIG. 53); AAG12(FIG. 54); AAG13(FIG. 55); and AAG14(FIG. 56) genes, and a human umbilical vein endothelial cell transfected with expression vector pcDNA3.1.

FIGS. 57 and 70 show results indicating cell viabilities in treating AAGl (FIG. 57); AAG2 (FIG. 58);

AAG3 (FIG. 59); AAG4 (FIG. 60); AAG5 (FIG. 61); AAG6 (FIG.

62); AAG7 (FIG. 63); AAG8 (FIG. 64); AAG9 (FIG. 65); AAGlO

(FIG. 66); AAGIl (FIG. 67); AAG12 (FIG. 68); AAG13 (FIG.

69); and AAG14 (FIG. 70) to human umbilical vein endothelial cells after pre-treatment of the cells by an oxidant, hydrogen peroxide.

[Best Mode]

Hereinafter, exemplary embodiments of the present invention will be described in detail. However, the present invention is not limited to the exemplary embodiments disclosed below, but can be implemented in various forms . The present exemplary embodiments are provided so that this disclosure will be complete and enabling of practice of the invention by those of ordinary skill in the art. 1. AAGl :

A gene of the present invention is a human antioxidant gene 1 (AAGl) having a base sequence of SEQ ID No. 1, the sequence is registered as registration No. AY513274 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens eukaryotic translation initiation factor 4 gamma, 2 (EIF4G2) gene that is registered as registration No. NM 001418 in the database.

However, the research found that the AAGl gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No . 1 includes one open reading frame corresponding to are Nos . 920 to 3025 (wherein, base Nos. 3023 - 3025 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 701 amino acid residues, has an amino acid sequence of SEQ ID No. 2, and has a size of about 8OkDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 1. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by reverse transcription- polymerase chain reaction (RT-PCR) using a random primer H- AP29 of SEQ ID No. 3, (5'-AAGCTTACGAGCA-S') and an anchored oligo-dT primer of SEQ ID No. 4, (5'-AAGCTTTTTTTTTTTC-S') to obtain a 237 bp cDNA fragment that is selectively expressed only in an umbilical vein endothelial cell

treated with 1-0-caffeoγl-3-0-β-glucopγranoside, not expressed or weakly expressed in an umbilical vein endothelial cell that is not chemically treated, and a full-length cDNA clone may be obtained by plaque- hybridization with a cDNA library using this fragment as a probe. 1-0-caffeoyl-3-0-β-glucopyranoside is extracted from Rumex gmelini, Polygonaceae growing naturally in Kyungbuk province, Korea (Nishibe S. et al . , Chem. Pharm. Bull., 30(3), 1408-1050 (1982); Warashina T. et al . , Phytochemistry, 31(3) , 961-965(1992)). 2. AAG2 :

A gene of the present invention is the human antioxidant gene 2 (AAG2) having a base sequence of SEQ ID No. 5, which is registered as registration No. AY513286 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens suppression of tumorigenicity 13 (colon carcinoma, Hsp70 interacting protein, ST13) gene, which is registered as registration No. NM_003932 in the database.

However, the research found that the AAG2 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 5 includes one open reading frame corresponding to base Nos . 67 to 1176 (wherein, base Nos. 1174 - 1176 constitute a termination codon) . A protein expressed by the gene of the present invention is formed of 369 amino acid residues, has an amino acid sequence of SEQ ID No. 6, and has a size of about 4IkDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No . 5. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP46 of SEQ ID No. 7 (5'-AAGCTTCGGTCCT-S') and an anchored oligo-dT primer of SEQ ID No. 8 (5'-AAGCTTTTTTTTTTTC-S') to obtain a 321 bp cDNA fragment that is selectively expressed only in an umbilical vein endothelial cell treated with fraxin or 2, 3-bis [4- (β-D-glucopyranosyloxy) benzyl] citrate, not expressed or weakly expressed in an umbilical vein endothelial cell that is not chemically treated, and a full-length cDNA clone may be obtained by plaque-

hybridization with a cDNA library using this fragment as a probe.

Fraxin is extracted from Weigela florida var. glabra, Carprifoliaceae (Hahn, D. and Lee M., Kor. J. Pharmacog. , 14(1) ;1-3(1983) ) .

2 , 3-bis [4- ( β-D-glucopyranosyloxy) benzyl] citrate is extracted from Gastrodia elata, Orchidaceae (LIN, J. et al . , Pytochemistry, 42:549-551, (1996)).

3. AAG3 : A gene of the present invention is the human antioxidant gene 3 (AAG3) having a base sequence of SEQ ID

No. 9, which is registered as registration No. AY513287 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens slicing factor, arginine/serine-rich 7, 35kDa, transcript variant 1 gene, which is registered as registration No. BC022328, and Homo sapiens splicing factor, arginine/serine-rich 7, 35kDa

(SFRS7) gene, which is registered as registration No. NM_001031684 in the database.

However, the research found that the AAG3 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No . 9 includes one open reading frame corresponding to base Nos . 82 to 798 (wherein, base Nos. 796 - 798 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 238 amino acid residues, has an amino acid sequence of SEQ ID No. 10, and has a size of about 27kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 9. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP29 of SEQ ID No. 11 (5'-AAGCTTACGAGCA-S') and an anchored oligo-dT primer of SEQ ID No. 12 (δ'-AAGCTTTTTTTTTTTG-S') to obtain a 283 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with quercetin-3-O-β-D-glucuronopyranoside, 1-0-caffeoyl-3- O-β-glucopyranoside, fraxin, methyl gallate, rhamnosylvitexin, acetoside or 2 , 3-bis [4- (β-D- glucopyranosyloxy) benzyl] citrate, not expressed in a umbilical vein endothelial cell that is not chemically

treated, a cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe. Quercetin-3-O-β-D-glucuronopyranoside is extracted from Rumex aquatics Polygonaceae (Hasan, A. et al . , Phytochemistry, 39 (5) : 1211-1213 (1995)).

1-0-caffeoyl-3-0-β-glucopyranoside is extracted from Rumex gmelini Polygonaceae, which grows naturally in Kyungbuk province, Korea (Nishibe S. et al . , Chem. Pharm. Bull., 30(3) , 1408-1050 (1982); Waeashina T. et al . , Phytochemistry, 31(3) , 961-965 (1992)).

Fraxin is extracted from Weigela florida var. glabra Caprifoliaceae (Hahn, D., and Lee M., Kor. J. Pharmacog. , 14(1) : 1-3 (1983) ) .

Methyl gallate is extracted from Cercis Chinensis (M. Nishizawa, M., and T. Yamagishi, T., J. CHEM. PERKIN TRANS., I: 26-31(1982) ) .

Rhamnosylvitexin is extracted from Crataegus pinnatifida var. psilosa ( (Bykov, V. I., and Glyzin, V. I. Khim. Prir. Soedin. , 9 (4) :557 (1973) ; Nikolov, N. Farmatsiya, 23(3) , 25(1974) ) .

Acetoside is extracted from Cloesodendron trichotomum Verbnaceae (Andray, C. et al . , Pytochemistry, 21 :1123 (1982) ) .

2, 3-bis [4- (β-D-glucopyranosyloxy) benzyl] citrate is extracted from Gastrodia elata Orchidaceae (LIN, J. et al . , Phytochemistry, 42: 549-551, (1996)). 4. AAG4:

A gene of the present invention is the human antioxidant gene 4 (AAG4) having a base sequence of SEQ ID No. 13, which is registered as registration No. AY513288 in the NIH ^GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens clusterin (complement lysis inhibitor, SP-40, 40, sulfate glycoprotein 2, testosterone- repressed prostate message 2, apolipoprotein J) (CLU), transcript variant 1 gene, which is registered as registration No. NM_001831, and the Homo sapiens clusterin (complement lysis inhibitor, SP-40, 40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) (CLU) , transcript variant 2 gene, which is registered as registration No. NM_001831 in the database. However, the research found that the AAG4 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 13 includes one open reading frame corresponding to base Nos . 66 to 1415 (wherein, base Nos. 1413 - 1415 constitute a termination codon) . A protein expressed by the gene of the present invention is formed of 449 amino acid residues, has an amino acid sequence of SEQ ID No. 14, and has a size of about 52kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 13. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP32 of SEQ ID No. 15 (5'-AAGCTTACTTGCAA-S') and an anchored oligo-dT primer of SEQ ID No. 16 (5'- AAGCTTTTTTTTTTTC-3' ) to obtain a 276 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with fraxin, not expressed in a umbilical vein endothelial cell that is not chemically treated, a cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be

obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Fraxin is extracted from Weigela florida var. glabra Caprifoliaceae (Hahn, D., and Lee M., Kor. J. Pharmacog. , 14(1) : 1-3 (1983)) . 5. AAG5:

A gene of the present invention is the human antioxidant gene 5 (AAG5) having a base sequence of SEQ ID No. 17, which is registered as registration No. AY544121 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a partially similar sequence to the Homo sapiens alkylglycerone phosphate synthase (AGPS) gene, which is registered as registration No. NM_003659 in the database. However, the research found that the AAG5 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 17 includes one open reading frame corresponding to base Nos . 1 to 1977 (wherein, base Nos. 1975 - 1977 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 658 amino acid residues, has an amino acid sequence of SEQ ID No. 18, and has a size of about 73kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 17. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP28 of SEQ ID No. 19 (5'-AAGCTTACGATGC-B') and an anchored oligo-dT primer of SEQ ID No. 20 (5' -AAGCTTTTTTTTTTTC-3' ) to obtain a 332 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with rhamnosylvitexin, not expressed in a umbilical vein endothelial cell that is not chemically treated, a cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Rhamnosylvitexin is extracted from Crataegus pinnatifida var. psilosa (Bykov, V.I., and Glyzin, V. I. Khim. Prir. Soedin. , 9 (4) :557 (1973) ; Nikolov, N. Farmatsiya, 23 (3) , 25(1974) ) . 6. AAG6:

A gene of the present invention is the human antioxidant gene 6 (AAG6) having a base sequence of SEQ ID No. 21, which is registered as registration No. AY633609 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a partially similar sequence to the Homo sapiens protein kinase C, alpha gene, which is registered as registration No. BC109274, and a Homo sapiens protein kinase C, alpha gene, which is registered as registration No. BC109273 in the database.

However, the research found that the AAG6 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 21 includes one open reading frame corresponding to base Nos . 6 to 2024 (wherein, base Nos. 2022 - 2024 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 672 amino acid residues, has an amino acid sequence of SEQ ID No. 22, and has a size of about 77kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening

and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 21. Alternatively, total R-XfA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP38 of SEQ ID No. 23, (5'-AAGCTTCCAGTGC-B') and an anchored oligo-dT primer of SEQ ID No. 24, (5'- AAGCTTTTTTTTTTTC-3' ) to obtain a 265 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with ginseng (Panax ginseng CA. Meyer) including Ginsenoside-Rbl , Ginsenoside-Rb2 and Ginsenoside-Rgl , not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with a chemical and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Ginseng, an herbal remedy, derived from a plant's root, is commonly called Panax ginseng, Asian ginseng or Korean ginseng. It is reported that the principal active element is ginsenoside, and its main effects are anti-inflammatory, anti-oxidation, and anticancer effects. 7. AAG7:

A gene of the present invention is the human antioxidant gene 7 (AAG7) having a base sequence of SEQ ID

No. 25, which is registered as registration No. AY633610 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens TIAI cytotoxic granule- associated RNA binding protein-like 1 (TIALl), transcript variant 1 gene, which is registered as registration No. NM_003252, and the Homo sapiens nucleolysin TIAR mRNA gene, which is registered as registration No. M96954 in the database. However, the research found that the AAG7 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 25 includes one open reading frame corresponding to base Nos . 9 to 1133 (wherein, base Nos. 1131 - 1133 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 374 amino acid residues, has an amino acid sequence of SEQ ID No. 26, and has a size of about 4IkDa. The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on

data of the base sequence of SEQ ID No. 25. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP31 of SEQ ID No. 27 (5'-AAGCTTGGTGAAC-B') and an anchored oligo-dT primer of SEQ ID No. 28 (5'-AAGCTTTTTTTTTTTA-B') to obtain a 276 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with quercetin-3-O-β-D-glucuronopyranoside, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque- hybridization with a cDNA library using this fragment as a probe . Quercetin-3-O-β-D-glucuronopyranoside is extracted from Rumex aguarica Polygonaceae (Hasan, A. et al . , Phytochemistry, 39(5) : 1211-1213 (1995)). 8. AAG8: A gene of the present invention is the human antioxidant gene 8 (AAG8) having a base sequence of SEQ ID No. 29, which is registered as registration No. AY633611 in the NIH GenBank database in the United States (expected date of publication: Dec. 31 2005), and has a similar sequence to the Homo sapiens opioid receptor, sigma 1

(OPRSl) , transcript variant 1 gene, which is registered as registration No. NM_005866 in the database.

However, the research found that the AAG8 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 29 includes one open reading frame corresponding to base Nos . 65 to 736 (wherein, base Nos. 734 - 736 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 223 amino acid residues, has an amino acid sequence of SEQ ID No . 30, and has a size of about 25kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 29. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP31 of SEQ ID No. 31 (5'-AAGCTTGGTGAAC-S') and an anchored oligo-dT primer of SEQ ID No. 32 (5'-AAGCTTTTTTTTTTTA-S') to obtain a 312 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated

with methyl gallate or rhamnosylvitexin, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque- hybridization with a cDNA library using this fragment as a probe .

Methyl gallate is extracted from Cercis chinensis ( .M. Nishizawa, M., and T. Yamagishi, T., J ^" . CHEM. PERKIN TRANS., _^ 1:26-31(1982)).

Rhamnosylvitexin is extracted from Crataegus pinnatifida var. psilosa (Bykov, V. I., and Glyzin, V.I. Khim. Prir. Soedin. , 9 (4) :557 (1973) ; Nikolov, N. Farmatsiya, 23 (3) , 25(1974) ) . 9. AAG9:

A gene of the present invention is the human antioxidant gene 9 (AAG9) having a base sequence of SEQ ID No. 33, which is registered as registration No. AY633612 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens glyceraldehyde-3-phosphate dehydrogenase gene, which is registered as registration No. BC083511, and a Homo sapiens glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) gene, which is registered as registration No. NM_002046 in the database.

However, the research found that the AAG9 gene is deeply associated with cell growth and anti-oxidation resisting oxidation stimulus (increase in a cell when hydrogen peroxide is treated) in humans .

The base sequence of SEQ ID No. 33 includes one open reading frame corresponding to base Nos . 25 to 1032 (wherein, base Nos. 1030 - 1032 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 335 amino acid residues, has an amino acid sequence of SEQ ID No. 34, and has a size of about 36kDa. The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 33. Alternatively, total RJSIA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP36 of SEQ ID No. 35 (5'-AAGCTTCGACGCT-S') and an anchored oligo-dT primer of SEQ ID No. 36 (5'-AAGCTTTTTTTTTTTG-S')

to obtain a 255 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with hydrogen peroxide, not expressed in a umbilical vein endothelial cell that is not chemically treated or with a chemical, and using a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe. 10. AAGlO: A gene of the present invention is the human antioxidant gene 10 (AAGlO) having a base sequence of SEQ ID No. 37, which is registered as registration No. AY633613 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a similar sequence to the Homo sapiens coiled-coil-helix-coiled-coil- helix domain containing 2 gene, which is registered as registration No. BC100275, and the Homo sapiens coiled- coil-helix-coiled-coil-helix domain containing 2 gene, which is registered as registration No. BC015639 and the Homo sapiens 16.7Kd protein mRNA, which is registered as registration No. AF078845 in the database.

However, the research found that the AAGlO gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 37 includes one open reading frame corresponding to base Nos . 22 to 477 (wherein, base Nos. 475 - 477 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 151 amino acid residues, has an amino acid sequence of SEQ ID No. 38, and has a size of about 15kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 37. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP29 of SEQ ID No. 39 (5'-AAGCTTACGAGCA-S') and an anchored oligo-dT primer of SEQ ID No. 40 (5'-AAGCTTTTTTTTTTTA-S') to obtain a 227 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with methyl gallate, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length

cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Methyl gallate is extracted from Cercis chinensis ( .M. Nishizawa, M. and T. Yamagishi, T., J ^" . CHEM. PERKIN TRANS., 3!:26-31(1982) ) . 11. AAGIl:

A gene of the present invention is the human antioxidant gene 11 (AAGIl) having a base sequence of SEQ ID No. 41, which is registered as registration No. AY634685 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a partially similar sequence to the Homo sapiens four and a half LIM domains 2, transcript variant 2 gene, which is registered as registration No. BC014397, the Homo sapiens four and a half LIM domains 2, transcript variant 2 gene, which is registered as registration No. BC012742, and the Homo sapiens heart protein (FHL-2) mRNA gene, which is registered as registration No. U29332 in the database.

However, the research found that the AAGlI gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 41 includes one open reading frame corresponding to base Nos . 1 to 840 (wherein, base Nos. 838 - 840 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 279 amino acid residues, has an amino acid sequence of SEQ ID No. 42, and has a size of about 32kDa. The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 41. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP2 of SEQ ID No. 43 (5'-AAGCTTCGACTGT-S') and an anchored oligo-dT primer of SEQ ID No. 44 (5'-AAGCTTTTTTTTTTTG-S') to obtain a 323 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with ginseng (Panax ginseng CA. Meyer) including Ginsenoside-Rbl, Ginsenoside-Rb2 and Ginsenoside-Rgl, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with a chemical and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Ginseng, an herbal remedy, derived from a plant's root, is common called Panax ginseng, Asian ginseng or Korean ginseng. It is reported that the principal active element is ginsenoside, and its main effects are anti-inflammatory, anti-oxidation, and anticancer effects (Attele, A. S. et al . , Biochem. Pharmacol., 58 (11) , 1685-1693(1999); Kiefer, D. and Pantuso, T., Am. Fam. Physician. , 68 (8) , 1539-1542 (2003) ) .

12. AAG12: A gene of the present invention is the human antioxidant gene 12 (AAG12) having a base sequence of SEQ ID No. 45, which is registered as registration No. AY634686 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a partially similar sequence to the Homo sapiens migration-related gene 1 protein mRNA gene, which is registered as registration No. AY423731, and the Homo sapiens membrane protein, palmitoylated 1, 55kDa (MPPl) gene, which is registered as registration No. NM_002436 in the database. However, the research found that the AAGl2 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 45 includes one open reading frame corresponding to base Nos . 25 to 1365

(wherein, base Nos . 1363 - 1365 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 446 amino acid residues, has an amino acid sequence of SEQ ID No. 46, and has a size of about 5OkDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 45. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP2 of SEQ ID No. 47 (5'-AAGCTTCGACTGT-B') and an anchored oligo-dT primer of SEQ ID No. 48 (5'-AAGCTTTTTTTTTTTA-B') to obtain a 337 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with ginseng (Panax ginseng CA. Meyer) including Ginsenoside-Rbl, Ginsenoside-Rb2 and Ginsenoside-Rgl, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with a chemical and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by

plaque-hybridization with a cDNA library using this fragment as a probe. 13. AAG13:

A gene of the present invention is the human antioxidant gene 13 (AAG13) having a base sequence of SEQ

ID No. 49, which is registered as registration No. AY762098 in the NIH GenBank database in the United States (expected date of publication: Dec. 31, 2005), and has a partially similar sequence to the Homo sapiens SlOO calcium binding protein Al6 gene, which is registered as registration No.

BC019099, and the Homo sapiens SlOO calcium binding protein

Al6 gene (S100A16) , which is registered as registration No.

BC019099 in the database.

However, the research found that the AAG13 gene is closely associated with cell growth and anti-oxidation in humans .

The base sequence of SEQ ID No. 49 includes one open reading frame corresponding to base Nos . 159 to 470 (wherein, base Nos. 468 - 470 constitute a termination codon) .

A protein expressed by the gene of the present invention is formed of 103 amino acid residues, has an amino acid sequence of SEQ ID No. 50, and has a size of about 12kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 49. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP29 of SEQ ID No. 51 (5'-AAGCTTACGAGCA-S') and an anchored oligo-dT primer of SEQ ID No. 52 (5'-AAGCTTTTTTTTTTTC-S') to obtain a 267 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with 2, 3-bis [4- (β-D-glucopyranosyloxy) benzyl] citrate, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with ginseng and cells treated with hydrogen peroxide, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe. 2, 3-bis [4- (β-D-glucopyranosyloxy) benzyl] citrate is extracted from Gastrodia elata Orchidaceae (LIN, J. et al . , Phytochemistry, 42:549-551, (1996)). 14. AAG14:

A gene of the present invention is the human antioxidant gene 14a (AAGl4a) having a base sequence of SEQ ID No. 53, which is registered as registration No. AY826824 in the NIH GenBank database in the United States (expected date of publication: Mar. 31, 2006), and human antioxidant gene 14b (AAGl4b) having a base sequence of SEQ ID No. 53, which is registered as registration No. AY826825 in the database (expected date of publication: Mar. 31, 2006) . The research found that the AAG14a and AAGl4b are deeply associated with cell growth and anti-oxidation resisting oxidation stimulus (increase in a cell when hydrogen peroxide is treated) in humans .

The base sequence of SEQ ID No. 53 includes two open reading frames corresponding to base Nos . 77 to 445 (wherein, base Nos. 443 - 445 constitute a termination codon) and base Nos. 420 to 1187 (wherein, base Nos. 1185 -

1187 constitute a termination codon) .

A protein that expressed from AAGl4a gene of the present invention is formed of 122 amino acid residues, has an amino acid sequence of SEQ ID No. 54, and is a size of about 14kDa.

A protein that expressed AAGl4b gene of the present invention is formed of 255 amino acid residues, has an

amino acid sequence of SEQ ID No. 55, and is a size of about 28kDa.

The gene and protein of the present invention may be separated from human tissue or synthesized by a known method of synthesizing DNA or peptide. For example, the gene of the present invention may be obtained by screening and cloning according to a conventional method based on data of the base sequence of SEQ ID No. 53. Alternatively, total RNA extracted from normal human cord blood endodermic cell tissue is amplified by RT-PCR using a random primer H- AP46 of SEQ ID No. 56 (5'-AAGCTTCGGTCCT-B') and an anchored oligo-dT primer of SEQ ID No. 57 (5'-AAGCTTTTTTTTTTTC-S') to obtain a 255 bp cDNA fragment that is selectively expressed only in a umbilical vein endothelial cell treated with hydrogen peroxide, not expressed in a umbilical vein endothelial cell that is not chemically treated, a umbilical vein endothelial cell treated with a chemical and cells treated with ginseng, and a full-length cDNA clone may be obtained by plaque-hybridization with a cDNA library using this fragment as a probe.

Meanwhile, various changes in encoding region in the AAG series genes may be made without changing an amino acid sequence of a protein expressed from the encoding region, and various changes or modifications in other parts except

the encoding region may be made without effecting the expression of the gene due to degeneracy of codons or codons preferred in an organism willing to express the gene. Thus, such transformed genes are also included in the scope of the present invention. Therefore, the present invention also includes poly nucleotides having substantially the same base sequence as the gene and fragments of the gene. The substantially same polynucleotide has more than 80%, preferably 90%, and more preferably 95%, similarity of sequence to the gene.

Moreover, substitution, addition or deletion in the amino acid sequence of the AAG series protein may be made as long as it does not affect the function of the protein, and a part of the protein may be used according to the purpose. However, transformed amino acid sequence is also included in the scope of the present invention. Thus, the present invention includes polypeptides having substantially the same amino acid sequence as the protein and its fragments, and the substantially same polypeptide has more than 80%, preferably 90%, and more preferably 95% , similarity of the sequence.

The gene created like this is inserted in a vector for expression of a microorganism or animal cell, thereby creating an expression vector, and the expression vector is

induced into an appropriate host cell, for example, E. coli or a MCF-7 cell line, such that a bulk of DNA of the gene may be created or a bulk of protein may be produced. In the creation of the vector, an expression regulating sequence such as a promoter and a terminator, a self- replication sequence and a secretion signal may be properly selected and combined depending on the kind of the host cell which will produce the gene or protein.

The gene of the present invention is overexpressed in a normal cell, preferably, brain, heart, muscle, placenta and lungs, and also expressed in large intestine, thymus, spleen, kidney, small intestine and leukocyte tissues to stimulate cell growth. In these tissues, the gene of the present invention may be mostly overexpressed as about 4.0 kb mRNA transcript (AAGl); about 2.2 kb mRNA transcript

(AAG2); about 2.4 kb mRNA transcript (AAG3); about 2.2 kb mRNA transcript (AAG4) ; about 3.0 kb mRNA transcript

(AAG5) ; about 2.0 kb mRNA transcript (AAG6); about 1.8 kb mRNA transcript (AAG7); about 2.0 kb mRNA transcript (AAG8); about 1.4 kb mRNA transcript (AAG9) ; about 1.0 kb mRNA transcript (AAGlO); about 2.0 kb mRNA transcript

(AAGIl); about 1.4 kb mRNA transcript (AAG12); about 1.0 kb mRNA transcript (AAG13); and about 3.0 kb mRNA transcript

(AAG14) . Particularly, a human umbilical vein endothelial

cell into which the gene is induced has high cell growth rate and an anti-oxidation ability, so the gene of the present invention may be useful for aging diagnosis, prevention and cure. [Mode for Invention]

Hereinafter, the present invention is shown and described with reference to examples in more detail. However, the scope of the present invention in not limited thereto. Example: Isolation of Total RNA

Total RNA was isolated from a fresh tissue or cultured cell using a total RNA isolation kit (RNeasy total RNA kit, Quagen Inc., Germany), and DNA contaminated with RNA was removed using a message clean kit (GenHunter Corp., MA, U.S.A. ) .

Example 1: Isolation of Total RNA and mRNA differential display result

A differential pattern of gene expression in a normal embryonic human umbilical vein endothelial cell (HUVEC) was investigated as below.

The HUVEC was isolated from an umbilical cord. A vein was cannulated and flushed with phosphate-buffered saline (PBS, 10 mM NaCl, 1OmM Na ₂ HPO ₄ , 3.3 mM KCl and 1.8 iti KH ₂ PO ₄ ,

pH 7.4), and then filled with 0.2% (v/v) collagenase type II (Sigma Corp. St. Louis, MO, U.S.A.). The cell was

cultured for 10 minutes at 37 ^° C (5% CO ₂ ), and the

collagenase was removed by being flushed with M-199 medium and centrifuged for 10 minutes at 1000 rpm to pellet the cell suspension. The cell was re-suspended in a growth medium (M-199) , and seeded on a 25cm ² cell culture flask previously coated with 1% (v/v) liquid gelatin (Sigma®) PBS. The cell was cultured up until the beginning of the experiment.

Total RNA was isolated from these tissues and cells by performing the same method as in the referential example.

The RT-PCR was performed using the total RNA samples of the tissues and cells by partially changing the method disclosed in the literature (Liang, P. and Pardee, A. B.,

Science, 257, 967-971 (1992); and Liang, P. et al . , Cancer

Res., 5J2, 6966-6968 (1993)). 0.2μg of total RNA is reverse-transcribed using a kit (RNAimage kit, GenHunter) together with a SEQ ID No. 4 (AAGl); SEQ ID No. 8 (AAG2); SEQ ID No. 12 (AAG3); SEQ ID No. 16 (AAG4); SEQ ID No. 20

(AAG5); SEQ ID No. 24 (AAG6); SEQ ID No. 28 (AAG7); SEQ ID

No. 32 (AAG8); SEQ ID No. 36 (AAG9); SEQ ID No. 40 (AAGlO);

SEQ ID No. 44 (AAGlI); SEQ ID No. 48 (AAG12); SEQ ID No. 52

(AAG13); or SEQ ID No. 57 (AAGl4) anchored oligo-dT primer,

and the PCR was performed in the presence of 0.5mM [α- ³⁵ S]- labeled dATP (1,200 Ci/mmol) using the same anchored oligo- dT primer and a 5'13mer random primer such as SEQ ID No . 3 H-AP29 (AAGl) ; SEQ ID No. 7 H-AP46 (AAG2 ) ; SEQ ID No. 11 H- AP29(AAG3); SEQ ID No. 15 H-AP29; SEQ ID No. 19 H-AP28; SEQ ID NO. 23 H-AP38; SEQ ID No. 27 H-AP31; SEQ ID No. 31 H- AP31; SEQ ID No. 35 H-AP36(i); SEQ ID No. 39 H-AP29(j); SEQ ID No. 43 H-AP2(k); SEQ ID No. 47 H-AP2(1); SEQ ID No. 51 H-AP29(m); SEQ ID No. 56 H-AP46 (n) (RNAimage primer set 5, GenHunter Corporation, U.S.A.). The PCR consisting of cycles performed for 40 seconds at 95 ^° C 2 minutes at 40 ^° C and 40 seconds at 72 ^° Cwas repeated 40 times, and then held for 5 minutes at 72 ^° C again. Amplified fragments were electrophoresed in a 6% polyacrylamide base sequence gel and then analyzed by autoradiography.

FIGS. 1 to 14 show PCR results using a 5' 13mer random primer and an anchored oligo-dT primer. 7 kinds of natural products (phytochemicals) extracted from domestic naturally occurring plants, RbI, Rb2 and RgI extracted from ginseng, and hydrogen peroxides were applied to the HUVEC, and a novel gene candidate for inhibiting anti-oxidation and inducing oxidation was searched for using the differential display reverse transcription polymerase chain reaction

(DDRT-PCR) .

In FIG. 1, a first column is a HUVEC that is not chemically treated as a control group, and a second column Al is a HUVEC treated with quercetin-3-O-β-D- glucuronopyranoside. Quercetin-3-O-β-D-glucuronopyranoside was extracted from Rumex aquatica Polygonaceae, and its chemical formula is as follows (Hasan, A. et al . , Phytochemistry, 39(5) : 1211-1213 (1995) ) .

A third column A2 is a HUVEC treated with 1-0- caffeoyl-3-0-β-glucopyranoside extracted from Rumex gmelini Polygonaceae naturally occurring in Kyungbuk province, Korea, and its chemical formula is as follows (Nishibe S. et al., Chem. Pharm. Bull., 30(3) , 1408-1050 (1982); Warashina T. et al . , Phytochemistry, 31(3) , 961-965 (1992))

A fourth column A3 is a HUVEC treated with fraxin extracted from Weigela florida var. glabra Caprifoliaceae, and its chemical formula is as follows (Hahn D. and Lee M., Kor. J. Pharmacog., 14(1) :l-3 (1983) ) .

A fifth column A4 is a HUVEC treated with methyl gallate extracted from Cercis chinensis, and its chemical formula is as follows (.M. Nishizawa, M. and T. Yamagishi, T., J. CHEM. PERKIN TRANS., IC : 26-31 (1982) ).

A sixth column A5 is a HUVEC treated with rhamnosylvitexin extracted from Crataegus pinnatifida var. psilosa, and its chemical formula is as follows (Bykov, V.

I., and Glyzin, V. I. Khim. Prir. Soedin. , 9 (4) : 557(1973); Nikolov, N. Farmatsiya, 23(3), 25(1974)).

A seventh column A6 is a HUVEC treated with acetoside extracted from Cleorodendron trichotomum Verbnaceae, and its chemical formula is as follows (Andray, C. et al . , Phytochemistry, 21:1123 (1982)).

An eighth column A7 is a HUVEC treated with 2,3-bis[4- ( β-D-glucopyranosyloxy) benzyl] citrate extracted from Gastrodia elata Orchidaceae, and its chemical formula is as follows (LIN, J. et al., Phytochemistry, 42:549-551 (1996)).

A ninth column ginseng is a HUVEC treated with ginsenosides RbI, Rb2 , and RgI (50μM) extracted from Panax ginseng CA. Meyer grown in Korea, and A tenth column is a HUVEC treated with lOOμM hydride peroxide.

As seen from FIG. 1, a 237 bp cDNA fragment definitely increasing in differential expression only in the column where the HUVEC was treated with 1-0-caffeoyl-3-0-β- glucopyranoside was found (from 3404bp to 3640bp of the full-length AAGl gene sequence) . The cDNA fragment was named N20.

After the 237 bp band, the N20 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 2, a 321 bp cDNA fragment definitely increasing in differential expression in the column where

the HUVEC was treated with fraxin or 2 , 3-bis [4- ( β-D- glucopyranosyloxy) benzyl] citrate was found (from 2769bp to 3089bp of the full-length AAG2 gene sequence) . The cDNA fragment was named N2. After the 321 bp band, the N2 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]~ labeled dATP and 20μM dNTP. As seen from FIG. 3, a 283 bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with quercetin-3-O-β-D- glucruronopyranoside, 1-0-caffeoy1-3-0-β-glucopyranoside, fraxin, methyl gallate, rhamnosylvitexin, acetoside or 2,3- bis [4- (β-D-glucopyranosyloxy) benzyl] citrate was found (from 2017bp to 2299bp of the full-length AAG3 gene sequence) . The cDNA fragment was named N21.

After the 283 bp band, the N21 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 4, a 276 bp cDNA fragment definitely increasing in differential expression in the column where

the HUVEC was treated with fraxin was found (from 1524bp to 1799bp of the full-length AAG4 gene sequence) . The cDNA fragment was named N38.

After the 276 bp band, the N38 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 5, a 332bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with rhamnosylvitexin was found (from

2268 bp to 2599 bp of the full-length AAG5 gene sequence) .

The cDNA fragment was named Nl8.

After the 332 bp band, the Nl8 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 6, a 265 bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with ginseng was found (from 1723 bp to 1987 bp of the full-length AAG6 gene sequence) . The cDNA fragment was named A38.

After the 265 bp band, the A38 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 7, a 276 bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with quercetin-3-O-β-D- glucruronopyranoside was found (from 824 bp to 1099 bp of the full-length AAG7 gene sequence) . The cDNA fragment was named N28.

After the 276 bp band, the N28 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 8, a 312 bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with methyl gallate or rhamnosylvitexin was found (from 477 bp to 788 bp of the full-length AAG8 gene sequence) . The cDNA fragment was named N29.

After the 312 bp band, the N29 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA,

and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 9, a 255bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with hydrogen peroxide was found

(from 735 bp to 989 bp of the full-length AAG9 gene sequence) . The cDNA fragment was named A44.

After the 255 bp band, the A44 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 10, a 227bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with methyl gallate was found (from

233 bp to 459 bp of the full-length AAGlO gene sequence) .

The cDNA fragment was named N48.

After the 227 bp band, the N48 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 11, a 323bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with ginseng was found (AAGlI, from

454 bp to 776 bp of full-length gene sequence) . The cDNA fragment was named All.

After the 323 bp band, the All fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 12, a 337bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with ginseng was found (from 963 bp to 1299 bp of the full-length AAG12 gene sequence) . The cDNA fragment was named A3.

After the 337 bp band, the A3 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 13, a 267bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with 2 , 3-bis [4- ( β-D- glucopyranosyloxy) benzyl] citrate was found (from 722 bp to

988 bp of the full-length AAG13 gene sequence) . The cDNA fragment was named N23.

After the 267 bp band, the N23 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

As seen from FIG. 14, a 363bp cDNA fragment definitely increasing in differential expression in the column where the HUVEC was treated with hydrogen peroxide was found

(from 2417 bp to 2779 bp of the full-length AAG14a gene sequence) . The cDNA fragment was named N4.

After the 363 bp band, the N4 fragment, was cut from a dried gel and boiled for 15 minutes to elute the cDNA, and the eluted cDNA was re-amplified by PCR using the same primer pair under the same conditions without [α- ³⁵ S]- labeled dATP and 20μM dNTP.

The re-amplified cDNA fragment was cloned in an expression vector, pGEM-T Easy, using a cloning system (TA cloning system, Promega) , and its base sequence was determined using a base sequence determination kit (Sequenase Version 2.0 DNA Sequencing System, United States Biochemical Co.).

Example 2 : cDNA Library Screening

The cDNA fragment obtained from Example 1 was labeled according to the method described in the literature (Feinberg, A. P. and Vogelstein, B., Anal. Biochem. , 132, 6-13 (1983)), thereby obtaining ³² P-labeled N20 cDNA probe. And, the probe was plaque-hybridized with a bacteriophage λgtll human lung embryonic fibroblast cDNA library (Miki, T. et al., Gene, 83, 137-146 (1989)) according to the method described in the literature (Sambrook, J. et al . , Molecular Cloning: A Laboratory manual, New York: Cold Spring Harbor Laboratory (1989)). Thus, a full-length cDNA clone of a human antioxidant gene such as AAGl; AAG2 ; AAG3 ; AAG4; AAG5; AAG6; AAG7 ; AAG8 ; AAG9 ; AAGlO; AAGlI; AAGl2 ; AAG13 or AAGl4 was obtained. The base sequences of the full-length cDNAs were determined, and the results were SEQ ID No. 1 (AAGl); SEQ ID No. 5(AAG2); SEQ ID No. 9(AAG3); SEQ ID No. 13(AAG4); SEQ ID No. 17(AAG5); SEQ ID No. 21(AAG6); SEQ ID No. 25(AAG7); SEQ ID No. 29(AAG8); SEQ ID No. 33(AAG9); SEQ ID No. 37(AAGlO); SEQ ID No. 41(AAGlI); SEQ ID No. 45(AAG12); SEQ ID No. 49(AAG13); and SEQ ID No. 53 (AAGl4a and 14b). The base sequence includes an opening reading frame encoding 701 (AAGl); 369 (AAG2); 238 (AAG3); 449 (AAG4); 658 (AAG5); 672 (AAG6); 374 (AAG7); 223 (AAG8); 335 (AAG9); 151

(AAGlO); 279 (AAGlI); 446 (AAG12); 103 (AAG13); or 122 (AAG14a) and 255 (AAGl4b) amino acid residues, and the amino acid sequence inferred therefrom was SEQ ID No. 2(AAGl); SEQ ID No. β(AAG2); SEQ ID No. 10(AAG3); SEQ ID No. 14(AAG4); SEQ ID No. 18(AAG5); SEQ ID No. 22(AAG6); SEQ ID No. 26(AAG7); SEQ ID No. 30(AAG8); SEQ ID No. 34(AAG9); SEQ ID NO. 38(AAGlO); SEQ ID No. 42(AAGlI); SEQ ID No. 46(AAG12); SEQ ID No. 5O(AAG13); or SEQ ID Nos . 54 and 55 (AAGl4a and 14b) . Also, molecular weight of the inferred protein was about 8OkDa(AAGl); 41kDa(AAG2); 27kDa(AAG3); 52kDa(AAG4); 73kDa (AAG5 ) ; 77kDa(AAG6); 41kDa(AAG7); 25kDa(AAG8); 36kDa (AAG9 ) ; 15kDa (AAGlO) ; 32kDa (AAGlI ); 50kDa(AAGl2) ; 12kDa (AAG13 ) ; or 14 kDa and 28kDa(AAGl4a and 14b) . The AAGl gene was expressed by culturing transformed E. coli in a LB broth with ImM isopropyl-β-D-

thiogalactopyranoside (IPTG) for 3 hours at 37 ^° C. The

protein sample was obtained from the culture solution according to the method in the literature (Sambrook, J. et al . , Molecular Cloning: A Laboratory manual, New York: Cold Spring Harbor Laboratory (1989)), and then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run to separate the protein from the sample.

FIGS. 15 to 28 are photographs showing SDS-PAGE analysis results of the respective AAG proteins. In FIG. 2, the first column is a protein sample before induction by IPTG, the second column is a protein sample after induction to expression of an AAG gene by IPTG.

Referring to FIG. 15, the size of the expressed AAGl protein is about 80 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 16, the size of the expressed AAG2 protein is about 41 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 17, the size of the expressed AAG3 protein is about 27 kDa, which is the same as inferred from the base sequence. Referring to FIG. 18, the size of the expressed AAG4 protein is about 52 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 19, the size of the expressed AAG5 protein is about 73 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 20, the size of the expressed AAG6 protein is about 77 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 21, the size of the expressed AAG7 protein is about 41 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 22, the size of the expressed AAG8 protein is about 25 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 23, the size of the expressed AAG9 protein is about 36 kDa, which is the same as inferred from the base sequence. Referring to FIG. 24, the size of the expressed AAGlO protein is about 15 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 25, the size of the expressed AAGIl protein is about 32 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 26, the size of the expressed AAG12 protein is about 50 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 27, the size of the expressed AAG13 protein is about 12 kDa, which is the same as inferred from the base sequence.

Referring to FIG. 28, the size of the expressed AAG14b protein is about 28 kDa, which is the same as inferred from the base sequence.

Example 3 : Northern Blot of AAG Gene

In order to investigate expression profiling of AAG genes, northern blot was executed as follows: The northern blot was performed using the blots to which total RNA samples were transferred (available from Clontech, U.S.A.) and hybridized with one another in Example 1. Then, using Rediprime II random prime labeling system (Amersham, U.K.), a nylon layer was hybridized with ³² P-labeled random prime probe manufactured from N20; N2 ; N21; N38; N18; A38; N28; N29; A44; ;N48; All; A3; or N23, which are partial sequences of the full-length cDNA such as AAGl; AAG2; AAG3 ; AAG4 ; AAG5 ; AAG6 ; AAG7 ; AAG8 ; AAG9 ; AAGlO; AAGIl; AAGl2 ; AAGl3 ; or AAG14, or N4 cDNA over night at 42 ^° C The same northern blot procedure was repeated twice, one northern blot result was analyzed using a dencitometer, and the other result was hybridized with a β- actin probe to verify the total amount of mRNA.

The northern blot was performed on the normal human multiple tissue (Clontech) . That is, blots to which total RNA samples extracted from normal tissues such as brain, heart, muscle, colon, thymus, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocyte

tissues were transferred (Clontech, U.S.A.) were hybridized with one another to start the northern blot.

In FIG. 29, a shows northern blot results of differential expression profiling of the AAGl gene in several normal tissues, and b shows northern blot results of β-actin probe hybridization of identical blots. As seen from FIG. 29, it seems that the gene is overexpressed in the heart and placenta, and also expressed in the brain, muscle, large intestine, thymus, colon, kidney, liver, small intestine, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 4.0 kb mRNA transcripts in theses tissues .

In FIG. 30, a shows northern blot results of differential expression profiling of AAG2 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 30, it seems that the gene is overexpressed in the placenta and liver to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.2 kb mRNA transcripts in theses tissues .

In FIG. 31, a shows northern blot results of differential expression profiling of AAG3 gene in several normal tissues, and b shows northern blot results of β-

actin probe hybridization of identical blots. As seen from FIG. 31, it seems that the gene is overexpressed in the heart, colon, kidney and liver to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.4 kb mRNA transcripts in theses tissues. Also, 1.35 kb transcripts are weakly expressed.

In FIG. 32, a shows northern blot results of differential expression profiling of AAG4 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 32, it seems that the gene is overexpressed in the brain, heart and liver, and also expressed in the muscle, large intestine, thymus, colon, kidney, small intestine, placenta, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.2 kb mRNA transcripts in theses tissues .

In FIG. 33, a shows northern blot results of differential expression profiling of AAG5 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots.

As seen from FIG. 33, it seems that the gene is overexpressed in the brain, heart, muscle, liver, placenta and lung, and also expressed in the large intestine, thymus,

colon, kidney, small intestine and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 3.0 kb mRNA transcripts in these tissues. From such results, it can be noted that the AAG5 gene of the present invention promotes cell growth in brain, heart, muscle, placental, lung, large intestine, thymus, kidney, small intestine and leukocyte tissues.

In FIG. 34, a shows northern blot results of differential expression profiling of AAG6 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 34, it seems that the gene is overexpressed in the brain, heart, muscle, liver, placenta and lung, and also expressed in the large intestine, thymus, colon, kidney and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.0 kb mRNA transcripts in theses tissues.

In FIG. 35, a shows northern blot results of differential expression profiling of AAG7 gene in several normal tissue, and b shows northern blot results of β-actin probe hybridization of identical blots. As seen from FIG. 35, it seems that the gene is overexpressed in the heart and muscle, and also expressed in the liver and placenta to stimulate cell growth. The gene of the present invention

is mostly overexpressed in about 1.8 kb mRNA transcripts in these tissues.

In FIG. 36, a shows northern blot results of differential expression profiling of AAG8 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots . As seen from FIG. 36, it seems that the gene is overexpressed in the heart, muscle, liver, placenta and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.0 kb mRNA transcripts in these tissues. From such results, it can be noted that the AAG8 gene of the present invention promotes cell growth in the normal muscle, liver, placenta and leukocyte tissues.

In FIG. 37, a shows northern blot results of differential expression profiling of AAG9 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 37, it seems that the gene is overexpressed in the heart and muscle, and also expressed in the brain, large intestine, thymus, colon, kidney, liver, small intestine, placenta, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 1.4 kb mRNA transcripts in these tissues .

In FIG. 38, a shows northern blot results of differential expression profiling of AAGlO gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 38, it seems that the gene is overexpressed in the heart, muscle and liver to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 1.0 kb mRNA transcripts in these tissues. Also, 1.4 kb transcripts are weakly expressed. In FIG. 39, a shows northern blot results of differential expression profiling of AAGlI gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. As seen from FIG. 39, it seems that the gene is overexpressed in the heart, and also expressed in the brain, muscle, large intestine, thymus, colon, kidney, liver, small intestine, placenta, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 2.0 kb mRNA transcripts in these tissues .

In FIG. 40, a shows northern blot results of differential expression profiling of AAG12 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. Referring to

FIG. 40, it seems that the gene is overexpressed in the heart and muscle, and also expressed in the brain, large intestine, thymus, colon, kidney, liver, small intestine, placenta, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 1.4 kb mRNA transcripts in these tissues. Also, about 2.0 kb mRNA transcripts are expressed. In FIG. 41, a shows northern blot results of differential expression profiling of AAG13 gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. Referring to FIG. 41, it seems that the gene is overexpressed in the heart, muscle and liver to stimulate cell growth. The gene of the present invention is mostly overexpressed in about 1.0 kb mRNA transcripts in these tissues.

In FIG. 42, a shows northern blot results of differential expression profiling of AAGl4a gene in several normal tissues, and b shows northern blot results of β- actin probe hybridization of identical blots. Referring to FIG. 42, it seems that the gene is overexpressed in the heart, muscle, liver and placenta, and also expressed in the brain, large intestine, thymus, colon, kidney, small intestine, lung and leukocyte tissues to stimulate cell growth. The gene of the present invention is mostly

overexpressed in about 3.0 kb mRNA transcripts in these tissues .

According to such results, it can be noted that the

AAG genes of the present invention promotes cell growth in the brain, heart, muscle, liver, placenta, lung, large intestine, thymus, colon, kidney, small intestine and leukocyte tissues .

Example 4: Creation of Expression Vector and Transfection

An expression vector including an encoding region of an AAG gene was created as follows:

First, the full-length cDNA clones obtained from Example 2, such as AAGl; AAG2 ; AAG3 ; AAG4 ; AAG5 ; AAG6 ; AAG7; AAG8; AAG9 ; AAGlO; AAGlI; AAGl2; AAGl3; or AAGl4, were inserted into expression vector pcDNA3.1 (Invitrogen, U.S.A.) for eukaryote to obtain an expression vector PCDNA3.1/AAG1; pcDNA3.1/AAG2 ; pcDNA3.1/AAG3 ; pcDNA3.1/AAG4; pcDNA3.1/AAG5 ; pcDNA3.1/AAG6 ; pcDNA3.1/AAG7 ; pcDNA3.1/AAG8 ; pcDNA3.1/AAG9 ; pcDNA3.1/AAGl0 ; pcDNA3.1/AAGl1 ; PCDNA3.1/AAG12; pcDNA3.1/AAG13 ; or pcDNA3.1/AAG14. The HUVECs were transfected with the expression vector using lipofectamine (Gibco BRL) , and cultured in a DMEM medium containing 0.6 mg/ml, and then the transfected cells were

selected. Here, a HUVEC transfected with expression vector PCDNA3.1, which does not include AAGl; AAG2 ; AAG3 ; AAG4 ; AAG5; AAG6; AAG7 ; AAG8 ; AAG9 ; AAGlO; AAGIl; AAGl2 ; AAGl3 or AAG14 cDNA , was used as a control group.

Example 5: Growth Curve of HUVEC Transfected with AAG Gene

To investigate effects of the AAG gene on HUVEC growth, 1 * 10 ⁵ wild-type HUVECs, a HUVEC transfected with vector pcDNA3.1/AAGl ; pcDNA3.1/AAG2 ; pcDNA3.1/AAG3 ; pcDNA3.1/AAG4 ; PCDNA3.1/AAG5 ; pcDNA3.1/AAG6 ; pcDNA3.1/AAG7 ; pcDNA3.1/AAG8 ; pcDNA3.1/AAG9 ; pcDNA3.1/AAGl0 ; pcDNA3.1/AAGl1 ; PCDNA3.1/AAGl2 ; pcDNA3.1/AAGl3 ; pcDNA3.1/AAGl4a; or pcDNA3. l/AAGl4b, and a HUVEC transfected only with vector pcDNA3.1 were cultured for 9 days in the respective DMEM medium. After a cell sticking on a flask was treated with trypsin (Sigma) and isolated from each culture solution, the number of living cells was counted after 1, 3, 5, 7 and 9 days according to trypan blue dye exclusion (Freshney, I. R., Culture of Animal Cells, 2 ^nd Ed. A. R. Liss, New York (1987)) .

FIGS. 43 to 56 are growth curves of a wild-type HUVEC, HUVECs transfected with vector pcDNA3.1/AAGl; pcDNA3.1/AAG2 ; pcDNA3.1/AAG3 ; pcDNA3.1/AAG4 ; pcDNA3.1/AAG5 ;

PCDNA3.1/AAG6 ; pcDNA3.1/AAG7 ; pcDNA3.1/AAG8 ; pcDNA3.1/AAG9 ;

PCDNA3.1/AAGl0 ; pcDNA3.1/AAGl1 ; pcDNA3.1/AAG12 ; pcDNA3.1/AAGl3 ; pcDNA3. l/AAG14a; and pcDNA3.1/AAGl4b, which were obtained from Example 4, and a HUVEC transfected only with vector pcDNA3.1 , respectively. Referring to FIGS. 43 to 56, it can be noted that a death rate of the HUVEC transfected with vector pcDNA3.1/AAGl; pcDNA3.1/AAG2 ;

PCDNA3.1/AAG3 ; pcDNA3.1/AAG4 ; pcDNA3.1/AAG5 ; pcDNA3.1/AAG6 ;

PCDNA3.1/AAG7 ; pcDNA3.1/AAG8 ; pcDNA3.1/AAG9 ; pcDNA3.1/AAGlO ; pcDNA3.1/AAGIl ; pcDNA3.1/AAG12 ;

PCDNA3.1/AAG13; pcDNA3. l/AAG14a; or pcDNA3. l/AAG14b is lower than those of the HUVEC transfected with vector pcDNA3.1 and wild-type HUVEC.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAGl was about

155% higher than that of the wild-type HUVEC, about 140% higher than that of the HUVEC transfected only with vector pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcHNA3.1/AAG2 was about

130% higher than that of the wild-type HUVEC.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG3 was about

180% higher than that of the wild-type HUVEC, and about

140% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG4 was about 132% higher than that of the wild-type HUVEC, and about

115% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG5 was about 137% higher than that of the wild-type HUVEC, and about

116% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG6 was about 150% higher than that of the wild-type HUVEC, and about

119% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG7 was about 155% higher than that of the wild-type HUVEC, and about

131% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG8 was about

137% higher than that of the wild-type HUVEC, and about 116% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG9 was about

133% higher than that of the wild-type HUVEC, and about

113% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAGlO was about

137% higher than that of the wild-type HUVEC, and about

116% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAGlI was about

144% higher than that of the wild-type HUVEC, and about

122% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG12 was about

144% higher than that of the wild-type HUVEC, and about

122% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vector pcDNA3.1/AAG13 was about

155% higher than that of the wild-type HUVEC, and about

131% higher than that of the HUVEC transfected only with pcDNA3.1.

On the 9 ^th day after the culture, the survival rate of the HUVEC transfected with vectors pcDNA3. l/AAG14a and pcDNA3.1/AAGl4b was about 137% higher than that of the wild-type HUVEC, and about 116% higher than that of the HUVEC transfected only with pcDNA3.1.

From these results, it can be noted that the AAG genes promote the growth of the HUVEC cells .

Example 6: Evaluation of Anti-oxidation Effect of AAG Protein After Treating HUVEC With Oxidant

Viability changes of a HUVEC exposed to an oxidant stress, hydrogen peroxide, were preliminarily measured. The cell viability was measured after culturing for 0.5, 1 or 2 hours in the presence of 0.2 to 5mM hydrogen peroxide. The viability versus that of a control group gone through the same process except application of hydrogen peroxide was shown as a percentage. There is no significant difference between the results depending on culture time, and the cell viabilities were 60%, 40% and 10% in the

presence of 0.2, 1 and 5mM hydrogen peroxide, respectively. In the following experiment, 0.2 mM hydrogen peroxide was used as the oxidant stress to obtain more information on relationship between a test sample and its activity. For cell viability analysis, 10 ⁵ HUVECs per a well were finally plated and cultured on a 6-well micro plate. The cells were grown to confluence, and test samples and hydrogen peroxide were applied to the cells at regular intervals. First, the cells were cultured for 1 hour in the presence of 200μM hydrogen peroxide. Then, the cells were treated with AAG proteins at a concentration of 4, 20 and 100 μg/ml to evaluate the anti-oxidation effect of the AAG protein. After that, a XTT analysis kit (R&D System Inc., Minneapolis, MN, U.S.A.) was used according to the method described in a manual to measure the cell viability.

In the evaluation of the anti-oxidation effect of the

AAG protein, the comparative group represented increase in cell viability as a percentage compared with that of the

HUVEC treated only with hydrogen peroxide, and the evaluation process was repeated three times per test sample to get an average .

On the 2 ^nd day of the culture after treating the AAGl protein, it can be noted that the HUVEC treated with the AAGl greatly increased in cell viability according to

treating concentration of the AAGl protein compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% cell viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAGl protein were recovered up to about 90%, 95% and 98 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively. On the 2 ^nd day of the cultivation of the cell treated with the AAG2 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG2 protein in the HUVEC treated with AAG2 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG2 protein were recovered up to about 90%, 92% and 94 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG3 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG3 protein in the HUVEC treated with AAG3 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type

HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG3 protein were recovered up to about 92%, 96% and 99 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG4 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG4 protein in the HUVEC treated with AAG4 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG4 protein were recovered up to about 91%, 90% and 50 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG5 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG5 protein in the HUVEC treated with AAG5 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG5 protein were recovered

up to about 89%, 93% and 93 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG6 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG6 protein in the HUVEC treated with AAGβ compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAGβ protein were recovered up to about 90%, 92% and 94% according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively. On the 2 ^nd day of the cultivation of the cell treated with the AAG7 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG7 protein in the HUVEC treated with AAG7 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG7 protein were recovered up to about 88%, 95% and 98 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20

μg/ml and 100 μg/ml , respectively. From such results, it can be noted that the AAG7 protein promotes the cell growth by recovering the viability of the HUVEC decreasing due to hydrogen peroxide. On the 2 ^nd day of the cultivation of the cell treated with the AAG8 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG8 protein in the HUVEC treated with AAG8 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG8 protein were recovered up to about 88%, 92% and 93 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG9 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG9 protein in the HUVEC treated with AAG9 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG9 protein were recovered up to about 90%, 92% and 94% according to the

concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAGlO protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAGlO protein in the HUVEC treated with AAGlO compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAGlO protein were recovered up to about 90%, 92% and 92 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAGlI protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAGIl protein in the HUVEC treated with AAGIl compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAGIl protein were recovered up to about 88%, 89% and 90 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG12 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG12 protein in the HUVEC treated with AAG12 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG12 protein were recovered up to about 88%, 89% and 90 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAG13 protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAG13 protein in the HUVEC treated with AAG13 compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG13 protein were recovered up to about 89%, 95% and 98 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

On the 2 ^nd day of the cultivation of the cell treated with the AAGl4a protein, it can be noted that the cell

viability was greatly increased according to the concentration of the AAGl4a protein in the HUVEC treated with AAGl4a compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAG14a protein were recovered up to about 88%, 90% and 92 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively. From such results, it can be noted that the AAG14a protein promotes the cell growth by recovering the viability of the cell decreasing due to hydrogen peroxide .

On the 2 ^nd day of the cultivation of the cell treated with the AAGl4b protein, it can be noted that the cell viability was greatly increased according to the concentration of the AAGl4b protein in the HUVEC treated with AAGl4b compared to the HUVEC treated only with hydrogen peroxide. Compared to the 60% viability of the wild-type HUVEC treated only with hydrogen peroxide, the viabilities of the HUVEC treated with the AAGl4b protein were recovered up to about 88%, 90% and 92 % according to the concentrations of the AAC protein, for example, 4 μg/ml, 20 μg/ml and 100 μg/ml, respectively.

These results are illustrated in FIGS. 57 to 70, respectively, and therefrom it can be noted that AAG proteins promote cell growth by recovering viabilities of HUVECs decreasing due to hydrogen peroxide.

[industrial Applicability]

The AAG genes of the present invention relate to oxidation of human cells and aging effects caused thereby, and may be useful for diagnosis, prevention and cure of the effects of aging.