KR100877152B1 - Cell permeable p66shc fusion protein having anti-cancer activity - Google Patents
Cell permeable p66shc fusion protein having anti-cancer activity Download PDFInfo
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Abstract
Description
본 발명은 활성산소의 생성 및 세포의 사망을 유도하여 암세포의 발생을 억제하는 기능 및 세포 투과 기능을 함께 갖는 융합단백질에 관한 것으로, 보다 상세하게는 인간의 p66shc 단백질과 Tat (transactivator of transcription) 단백질의 형질도입부위가 융합된 Tat-p66shc 융합단백질에 관한 것이다. The present invention relates to a fusion protein having both a function of inhibiting the generation of cancer cells and a cell permeation function by inducing the generation of free radicals and cell death, and more particularly, a human p66shc protein and a Tat (transactivator of transcription) protein. It relates to a Tat-p66shc fusion protein fused to the transduction site of.
p66shc 단백질은 어뎁터 단백질의 일종인 ShcA족에 포함된 단백질로 포유동물의 수명을 조절하는 데 중요한 역할을 하는 것으로 알려져 있다. p66shc 단백질이 제거된 동물은 수명이 약 30%정도 길어지고, 산화적 자극에 저항한다고 알려져 있다(Migliaccio E et al, The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature. 1999 Nov 18;402(6759):309-13.) The p66shc protein is a member of the ShcA family of adapter proteins and is known to play an important role in controlling mammal life. Animals from which the p66shc protein has been removed are known to have a lifespan of about 30% longer and resist oxidative stimuli (Migliaccio E et al, The p66shc adapter protein controls oxidative stress response and life span in mammals. Nature. 1999 Nov 18; 402 (6759): 309-13.)
p66shc 단백질은 미토콘드리아의 활성산소 생성 증가를 유도하며(Trinei et al, A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis. Oncogene. 2002 May 30;21(24):3872-8.), 이러한 활성산소의 생성 증가는 p66shc 단백질의 36번째 아미노산인 세린기의 인산화와 밀접한 관련이 있다(Nemoto S & Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science. 2002 Mar 29;295(5564):2450-2). p66shc protein induces increased mitochondrial free radical production (Trinei et al, A p53-p66Shc signaling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.Oncogene. 2002 May 30; 21 (24) This increased oxygen production is closely related to the phosphorylation of serine, the 36th amino acid of the p66shc protein (Nemoto S & Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway). Science 2002 2002 29; 295 (5564): 2450-2).
최근 p66shc 단백질의 발현감소는 내피세포형 일산화질소생성단백의 활성화를 증가시키며 일산화질소의 생성을 증가시킨다는 보고가 있다(Yamamori et al, P66shc regulates endothelial NO production and endothelium-dependent vasorelaxation: implications for age-associated vascular dysfunction. J Mol Cell Cardiol. 2005 Dec;39(6):992-5). p66shc단백의 과발현은 T-세포에서 세포의 고사를 유도하고 발암신호전달을 억제한다는 사실이 보고되고 있다. (Pacini S et al, p66shc promotes apoptosis and antagonizes mitogenic signaling in T cells. Molecular and Cellular Biology, 24(4), 1747-1757, 2004)Recently, the decrease in the expression of p66shc protein increases the activation of endothelial cell type nitric oxide protein and increases the production of nitric oxide (Yamamori et al, P66shc regulates endothelial NO production and endothelium-dependent vasorelaxation: implications for age-associated vascular dysfunction.J Mol Cell Cardiol. 2005 Dec; 39 (6): 992-5). Overexpression of p66shc protein has been reported to induce cell death in T-cells and inhibit carcinogenic signaling. (Pacini S et al, p66shc promotes apoptosis and antagonizes mitogenic signaling in T cells.Molecular and Cellular Biology, 24 (4), 1747-1757, 2004)
신호전달연구에 있어 단백질을 과발현시키는 방법으로 아데노바이러스 및 지방체를 이용한 유전인자의 과발현기법이 이용되고 있으나 발현시간이 장시간 필요한 단점이 있다. 최근에는 단백질을 세포내로 도입하는 단백질 형질도입부위들이 알려지면서 이러한 도입부위에 단백질을 융합하여 세포내로 도입시킴으로서 생물학적 변화를 연구하고 있다. Overexpression of genetic factors using adenoviruses and fats is used as a method of overexpressing proteins in signal transduction research, but there is a disadvantage in that expression time is required for a long time. Recently, as protein transduction sites for introducing proteins into cells are known, biological changes have been studied by fusing proteins into these introduction sites and introducing them into cells.
아직까지 정확한 세포내 도입기전이 밝혀져 있지는 않으나 Tat (transactivator of transcription) 단백질은 이 단백질 중 단백질 형질도입부위 (protein transduciton domain)가 존재하기 때문에 효율적으로 세포막을 통과하여 세포내로 이동하는 것으로 알려져 있다(Murriel CL, Dowdy SF. Influence of protein transduction domains on intracellular delivery of macromolecules. Expert Opin Drug Deliv. 2006 Nov;3(6):739-46). Tat 단백질의 단백질 형질도입부위를 이용하여 단백질을 융합시키는 경우 세포내로 단백질이 용이하게 도입 되며 단백질의 기능 및 활성을 나타내는 것이 보고되고 있으며 최근 동물실험에서도 그 효과가 보고되고 있다 (Cai SR, Xu G, Becker-Hapak M, Ma M, Dowdy SF, McLeod HL. The kinetics and tissue distribution of protein transduction in mice. Eur J Pharm Sci. 2006 Mar;27(4):311-9).Although the exact cellular transduction mechanism is not known yet, Tat (transactivator of transcription) protein is known to efficiently move through the cell membrane and into the cell due to the presence of the protein transduciton domain (Murriel). CL, Dowdy SF.Influence of protein transduction domains on intracellular delivery of macromolecules.Expert Opin Drug Deliv. 2006 Nov; 3 (6): 739-46). In the case of fusion of protein using the protein transduction site of Tat protein, it is reported that the protein is easily introduced into the cell and shows the function and activity of the protein, and the effects have been reported in recent animal experiments (Cai SR, Xu G). , Becker-Hapak M, Ma M, Dowdy SF, McLeod HL.The kinetics and tissue distribution of protein transduction in mice.Eur J Pharm Sci. 2006 Mar; 27 (4): 311-9).
p66shc 단백은 활성산소의 생성 및 세포의 사망을 유도하여 암세포의 발생을 억제하는 것으로 알려져 있으며, 따라서 세포투과형 p66shc단백은 종양생성을 억제하는 데 활용될 수 있을 것으로 생각된다. 또한 세포의 성장을 억제하는 기능을 가진 p66shc 단백질의 특성을 이용하여 항암제 개발 및 활용물질로 의료산업적으로 유용하게 응용될 수 있다. The p66shc protein is known to inhibit the development of cancer cells by inducing the production of free radicals and the death of cells. Therefore, it is thought that the cell-permeable p66shc protein can be utilized to suppress tumorigenesis. In addition, by using the characteristics of the p66shc protein having a function of inhibiting the growth of cells can be usefully applied to the medical industry as an anticancer drug development and utilization material.
본 발명은 p66shc 단백질을 세포내로 도입하기 위하여 단백질형질도입부위인 tat과 인간의 p66shc 단백질을 융합시킨 융합단백질을 제공하고자 하는 것을 목적 으로 한다. It is an object of the present invention to provide a fusion protein in which tat, a protein transduction site, and human p66shc protein are fused to introduce p66shc protein into cells.
또한 본 발명의 또 다른 목적은 상기 Tat-p66shc 융합단백질을 유효성분으로 함유하는 암세포 성장억제용 조성물을 제공하는 것이다.Another object of the present invention to provide a composition for inhibiting cancer cell growth containing the Tat-p66shc fusion protein as an active ingredient.
전술한 목적을 달성하기 위한 본 발명의 융합단백질은 p66shc 단백질과 Tat 단백질의 단백질 형질 도입부위가 융합된 서열번호 1의 세포 투과성 융합단백질 Tat-p66shc인 것을 특징으로 한다.The fusion protein of the present invention for achieving the above object is characterized in that the cell permeable fusion protein Tat-p66shc of SEQ ID NO: 1, wherein the protein transduction site of the p66shc protein and Tat protein is fused.
Tat-p66shc 융합단백질을 만들기 위하여 먼저 p66shc의 시작코돈에 EcoRI 제한효소 사이트를 연결한 primer와 정지코돈에 HindIII 제한효소 사이트를 연결한 primer를 제작하여 인간 p66shc cDNA를 분리하였다. 분리한 p66shc cNDA(1752bp)를 pTAT2.1에 삽입하여 pTat-p66shc 발현벡터(도 1)를 제작하고 대장균 BL21(DE3)에 형질전환한 후 그 형질전환체 BL21(DE3)/pTat-p66shc를 2007년 6월 19일자로 한국생명공학연구원 생물자원센타에 기탁하였다(기탁번호 KCTC 11138BP). 상기 형질전환체를 이용하여 Tat-p66shc 융합단백질을 과발현 시킨 후 변성상태(denature condition)에서 분리하고 정제하였다. 상기 Tat-p66shc 융합단백질은 서열번호 1의 아미노산 서열을 갖는 것을 특징으로 한다. In order to make tat-p66shc fusion protein, human p66shc cDNA was isolated by constructing primer that connects EcoRI restriction enzyme site to start codon of p66shc and HindIII restriction enzyme site to stop codon. PTat-p66shc expression vector (FIG. 1) was prepared by inserting the isolated p66shc cNDA (1752bp) into pTAT2.1, transformed into E. coli BL21 (DE3), and the transformant BL21 (DE3) / pTat-p66shc 2007 It was deposited on June 19, 19th, at the Korea Research Institute of Bioscience and Biotechnology (BRC) (Accession No. KCTC 11138BP). Using the transformant, the Tat-p66shc fusion protein was overexpressed, isolated and denatured in a denatured condition. The Tat-p66shc fusion protein is characterized by having the amino acid sequence of SEQ ID NO: 1.
Tat-p66shc 융합단백질을 코딩하는 유전자는 단백질정제를 위한 6개의 히스 티딘(histidine) 및 TAT형질도입부위의 9개 아미노산 RKKRRQRRR을 코딩하는 유전인자, 그리고 인간의 p66shc 유전인자를 포함하고 있는 것을 특징으로 한다. 상기 유전자는 특히 서열번호 2의 서열을 갖는 것이 바람직하다. The gene encoding the Tat-p66shc fusion protein includes six histidines for protein purification, a gene encoding 9 amino acids RKKRRQRRR of the TAT transduction site, and a human p66shc gene. do. It is particularly preferred that the gene has a sequence of SEQ ID NO: 2.
본 발명에 의한 Tat-p66shc 융합단백질은 1nM 농도에서부터 세포내 투과가 일어나는 것을 관측할 수 있었다. 상기 세포내 투과는 혈관내피세포에서 Tat-p66shc 융합단백질을 1시간 배양액에 처리한 군에서 최대의 발현을 보였으며 그 이후 서서히 감소하는 경향을 보였다. 이러한 결과는 세포내로 유입한 Tat-p66shc은 세포내의 단백질분해 효소에 의하여 서서히 제거된다는 사실을 암시하고 있다. In the Tat-p66shc fusion protein according to the present invention, it was observed that intracellular permeation occurred at a concentration of 1 nM. The intracellular permeation showed the maximum expression in the group treated with Tat-p66shc fusion protein in 1 hour culture in vascular endothelial cells and then gradually decreased. These results suggest that Tat-p66shc introduced into cells is slowly removed by intracellular proteolytic enzymes.
혈관내피세포내로 투과된 Tat-p66shc 융합단백질은 TNF-α를 매개로 하는 eNOS의 인산화를 효과적으로 억제시키는 것을 알 수 있었다. 이러한 현상은 p66shc의 인산화 (특히 36번째 세린기의 인산화) 증가가 중요한 매개체로 관여했다고 보여진다. Tat-p66shc의 이러한 효과는 혈관염증신호물질들로 인해 야기되는 혈관염증성 반응 및 혈관신생과 같은 혈관질환의 치료기술개발에 유용하게 사용될 수 있음을 알 수 있다. 또한 p66shc의 발현은 종양발생을 억제할 수 있어 항암제개발에 활용될 수 있다.Tat-p66shc fusion protein permeated into vascular endothelial cells was found to effectively inhibit phosphorylation of eNOS mediated by TNF-α. This phenomenon seems to be due to the increased phosphorylation of p66shc (especially the phosphorylation of the 36 th serine group) as an important mediator. This effect of Tat-p66shc may be useful in the development of treatment technology for vascular diseases such as angiogenesis and angiogenesis caused by vascular signaling materials. In addition, the expression of p66shc can suppress tumorigenesis and can be used for anticancer drug development.
Tat-p66shc 융합단백질을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에 통상적으로 허용되는 담체와 함께 배합하여 피부도말제제, 경구투여제제, 정맥주사 및 근육주사형태로 제형화할 수 있다. Tat-p66shc 융합단백질을 유효성분으로하는 약제학적 조성물은 혈관질환의 치료기술개발 및 종양질환의 치료법 에 응용될 수 있으며 그 기법은 향후 세포내 유전질환의 치료 및 새로운 질환치료법으로 활용될 수 있다. Pharmaceutical compositions containing the Tat-p66shc fusion protein as an active ingredient can be formulated in the form of skin smears, oral administrations, intravenous injections and intramuscular injections, in combination with carriers commonly accepted in the pharmaceutical art. The pharmaceutical composition using Tat-p66shc fusion protein as an active ingredient can be applied to the treatment of vascular diseases and the treatment of tumor diseases, and the technique can be used for the treatment of intracellular genetic diseases and new diseases.
또한 본 발명에 의한 Tat-p66shc 융합단백질은 약제학적 조성물로서 뿐만 아니라, 상기 융합단백질을 유효성분으로 하는 화장료 조성물로서 사용될 수 있다. 상기 화장료 조성물은 그 제형에 있어서 특별히 한정되지 않으며, 구체적으로 유연화장수, 영양화장수, 영양크림 또는 마사지크림 등의 제형을 가질 수 있다. 또한 그 구체적인 조성물의 성분은 제형에 따라, 목적하는 효과에 따라서 당업자가 적절하게 선정하여 배합할 수 있을 것이다. In addition, the Tat-p66shc fusion protein according to the present invention can be used not only as a pharmaceutical composition, but also as a cosmetic composition containing the fusion protein as an active ingredient. The cosmetic composition is not particularly limited in its formulation, and may specifically have a formulation such as supple cosmetics, nourishing cosmetics, nourishing cream or massage cream. In addition, the components of the specific composition may be appropriately selected and blended by those skilled in the art according to the dosage form and the desired effect.
이상과 같이 본 발명에 의한 Tat-p66shc 융합단백질은 Tat와 인간 p66shc의 성질을 동시에 갖고 있으므로, 인간 p66shc를 단백질 수준에서 세포내로 직접 도입시키는 효과를 얻을 수 있어 단백질 치료에 활용될 수 있다.As described above, since the Tat-p66shc fusion protein has the properties of Tat and human p66shc at the same time, it is possible to obtain the effect of directly introducing human p66shc into the cell at the protein level, which can be used for protein treatment.
본 발명에 의한 Tat-p66shc 융합단백질은 활성산소의 생성 및 eNOS의 활성을 효율적으로 억제할 수 있어, 활성산소 및 eNOS와 관련한 혈관질환치료법 개발및 항암제개발에 효과적으로 활용 및 응용될 수 있다. Tat-p66shc fusion protein according to the present invention can effectively inhibit the production of free radicals and the activity of eNOS, it can be effectively utilized and applied in the development of anti-cancer drugs and the treatment of vascular diseases related to free radicals and eNOS .
또한 본 발명의 Tat-p66shc 융합단백질은 질병의 치료 이외에도 화장품 및 건강식품등의 제품개발에 활용될 수 있다. In addition, Tat-p66shc fusion protein of the present invention can be used in the development of products such as cosmetics and health food in addition to the treatment of diseases.
이하 실시예를 통하여 본 발명을 상세하게 설명한다. 그러나, 이들 실시예는 예시적인 목적일 뿐 본 발명이 이에 한정되는 것은 아니다.The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.
실시예Example
실시예 1 : Tat-p66shc 발현벡터의 제조 및 형질전환Example 1 Preparation and Transformation of Tat-p66shc Expression Vector
Tat-p66shc 융합단백질을 과발현시키기 위하여 pTat2.1벡터를 이용하여 6개의 히스티딘의 유전인자와 Tat 형질도입부위(RKKRRQRRR)의 유전인자 및 인간의 p66shc cDNA가 포함되어 있는 Tat-p66shc 발현벡터를 제조하였다. 도 1은 Tat-p66shc 발현벡터의 개열지도(cleavage map)을 나타낸다. To overexpress the Tat-p66shc fusion protein, a Tat-p66shc expression vector was prepared using the pTat2.1 vector containing six histidine genes, the Tat transduction site (RKKRRQRRR), and the human p66shc cDNA. . 1 shows a cleavage map of the Tat-p66shc expression vector.
먼저, p66shc의 시작코돈에 ecoRI제한효소사이트를 연결한 하기 서열번호 3의 프라이머(primer)와 정지코돈에 HindIII제한효소사이트를 연결한 하기 서열번호 4의 프라이머를 제작하여 인간의 p66shc 유전인자가 포함되어 있는 p66shc-pcDNA(피츠버그의대 Kaikobad Irani교수로부터 기증받음)로부터 중합효소연쇄반응을 이용하여(중합효소연쇄반응조조건; 95℃, 30초, 62℃ 1분, 72℃ 2분, 30사이클) 분리하였다. 분리한 p66shc cDNA는 1752bp로 그 서열은 서열번호 5로 서열목록에 기재된 바와 같다. First, primers of SEQ ID NO: 3 linking the ecoRI restriction enzyme site to the start codon of p66shc and primers of SEQ ID NO: 4 linking the HindIII restriction enzyme site to the stop codon include human p66shc genes. Separation of p66shc-pcDNA (Provided by Professor Kaikobad Irani, Pittsburgh University) using polymerase chain reaction (polymerase chain reaction conditions; 95 ° C, 30 seconds, 62 °
서열번호 3 : 5'-CGG AAT TCG AAG GAT CTC CTG CCC CCC-3' (센스프라이머 (sense primer)) SEQ ID NO: 5'-CGG AAT TCG AAG GAT CTC CTG CCC CCC-3 '(sense primer)
서열번호 4 : 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3' (안티센스프라이머 (anti-sense primer)) SEQ ID NO: 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3 '(anti-sense primer)
EcoRI, HindIII 제한효소를 사용하여 pTAT-2.1 벡터(5343 bp, 미국 캘리포니아대학 Steven Dowdy교수로부터 기증받음)를 절단한 다음, p66shc cDNA를 pTat2.1 백터에 삽입하여 Tat-p66shc 벡터를 완성하였다. pTat-p66shc은 단백질정제를 위한 6개의 히스티딘(histidine) 유전인자와, TAT형질도입부위의 9개 아미노산 RKKRRQRRR의 유전인자, 그리고 인간의 p66shc 유전인자를 포함하고 있는 것을 특징으로 한다.The pTAT-2.1 vector (5343 bp, donated by Professor Steven Dowdy, University of California, USA) was digested using EcoRI, HindIII restriction enzymes, and p66shc cDNA was inserted into the pTat2.1 vector to complete the Tat-p66shc vector. pTat-p66shc is characterized by including six histidine genes for protein purification, 9 amino acids RKKRRQRRR in the TAT transduction region, and human p66shc genes.
상기 벡터를 대장균 BL21(DE3)에 형질전환시켜 대장균 형질전환체 BL21(DE3)/pTat-p66shc를 제조하고, 2007년 6월 19일자로 한국생명공학연구원 생물자원센타에 기탁하였다(기탁번호 KCTC 11138BP).The vector was transformed into Escherichia coli BL21 (DE3) to prepare Escherichia coli transformant BL21 (DE3) / pTat-p66shc, and deposited on June 19, 2007 at the Korea Institute of Bioscience and Biotechnology Center (Accession No. KCTC 11138BP). ).
실시예 2 : 재조합 Tat-p66shc 융합단백질의 분리 및 정제Example 2 Isolation and Purification of Recombinant Tat-p66shc Fusion Proteins
실시예 1에서 제조한 대장균 형질전환체 BL21(DE3)/pTat-p66shc를 50mg/ml의 kanamycin이 첨가된 LB배지에 도말하여 37℃에서 200-250 rpm으로 교반하면서 18시간 배양하였다. 형질전환된 콜로니를 선택하여 배지 1 L에 첨가하여 37℃에서 200-250 rpm으로 교반하면서 O.D.값이 0.4-0.6이 될 때까지 37℃에서 200-250rpm으로 배양하였다. O.D.값이 0.4-0.6이 되면, IPTG (isopropylthio-β-D-galactoside) 1 mM을 투여하고 37℃에서 4시간 동안 추가로 배양하여 Tat-p66shc의 발현을 유도하였다. E. coli transformant BL21 (DE3) / pTat-p66shc prepared in Example 1 was purified by 50mg / ml of kanamycin. Plates were added to the added LB medium and incubated for 18 hours with stirring at 200-250 rpm at 37 ° C. Transformed colonies were selected and added to 1 L of medium and incubated at 37-200 rpm at 37-200 rpm with an OD value of 0.4-0.6 while stirring at 200-250 rpm at 37 ° C. When the OD value was 0.4-0.6, 1 mM of IPTG (isopropylthio-β-D-galactoside) was administered and further cultured at 37 ° C. for 4 hours to induce the expression of Tat-p66shc.
Tat-p66shc 단백질의 발현이 유도되면, 4℃에서 5,000rpm으로 5분 동안 원심분리한 후 인산완충생리식염수로 세척하고 다시 원심분리하여 침전분획을 얻는다. 상기 침전 분획에 10 ml의 완충용액 Z (8M Urea, 100mM NaCl, 20mM HEPES pH8.0)을 첨가한 후 초음파 처리하였다. 상기 혼합물을 다시 원심분리한 후 상층액을 취해서 2~3ml의 Ni-NTA (Qiagen, Cat No : 30210)가 첨가된 단백질컬럼 (Bio-rad, Cat No : 731-1550)에 부하하였다. 10 mM 이미다졸(Imidazole)이 포함되어 있는 완충용액 Z로 세척한 다음 각 용출완충액(Imidazole이 100mM, 250mM, 500mM 또는 1M이 포함되어 있는 완충용액 Z)으로 융합단백질을 각각 용출하였다. 도 2는 각 용출완충액을 사용하여 발현된 Tat-p66shc 융합단백질을 정제한 결과를 보여주는 전기영동 사진으로, 이미다졸 250mM이 포함되어 있는 완충용액 Z에서 가장 많은 Tat-p66shc 융합단백질이 용출되어 이미다졸 농도 250mM인 완충용액 Z가 본 융합단백질의 용출에 가장 적합한 용출완충액인 것을 확인할 수 있었다. 또한 Tat-p66shc 융합단백질의 분자량은 약 71 kDa인 것을 알 수 있었다. When the expression of the Tat-p66shc protein is induced, centrifugation at 5,000 rpm at 4 ° C. for 5 minutes, followed by washing with phosphate buffered saline and centrifugation again yields a precipitate fraction. 10 ml of buffer Z (8M Urea, 100 mM NaCl, 20 mM HEPES pH8.0) was added to the precipitate fraction, followed by sonication. The mixture was centrifuged again and the supernatant was taken and loaded into a protein column (Bio-rad, Cat No: 731-1550) to which 2-3 ml of Ni-NTA (Qiagen, Cat No: 30210) was added. After washing with buffer Z containing 10 mM imidazole, the fusion protein was eluted with each elution buffer (buffer Z containing 100 mM, 250 mM, 500 mM or 1M of Imidazole). Figure 2 is an electrophoresis picture showing the results of purification of the expressed Tat-p66shc fusion protein using each elution buffer, the most Tat-p66shc fusion protein eluted in the buffer Z containing imidazole 250mM imidazole It was confirmed that the buffer solution Z having a concentration of 250 mM was the most suitable elution buffer for elution of the fusion protein. It was also found that the molecular weight of the Tat-p66shc fusion protein was about 71 kDa.
용출된 융합단백질은 PD10 column kit (GE Healthcare Bio Sciences AB, Cat No : 17-0851-01)를 이용하여 염분을 제거하였다. 염분이 제거된 정제된 융합단백질은 10% 글리세롤를 첨가하여 -70℃에 보관하여 사용하였으며, SDS-PAGE를 이용하여 코마씨 블루 염색(coomassie blue staining)을 하여 우혈청 알부빈과 비교함으로써 단백질의 농도를 계산한 후 하기 실시예에 사용되는 농도에 따라 희석하여 사용하였다.The eluted fusion protein was desalted using a PD10 column kit (GE Healthcare Bio Sciences AB, Cat No: 17-0851-01). The purified fusion protein without salt was stored at -70 ° C by adding 10% glycerol, and the concentration of protein was compared with bovine serum albumin by coomassie blue staining using SDS-PAGE. After dilution was used according to the concentration used in the following examples.
실시예 3 : 혈관내피세포에서 Tat-p66shc 융합단백질의 세포내 투과Example 3 Intracellular Permeation of Tat-p66shc Fusion Proteins in Vascular Endothelial Cells
본 발명의 하기 실시예에서 웨스턴블럿 분석(western blot analysis)은 다음같이 행하였다. 세포균질액은 10% SDS폴리아크릴아마이드 젤 전기영동법으로 분리하여 젤에 니크로셀룰로스막으로 전기이동시켰다. 단백질이 이동된 막을 5% 스킴우유로 처리한 후 인간 Shc항체(1:1000희석), phospho-eNOS(1:1000희석), 또는 phospho-p66shc(1:1000희석)로 18시간 처리하였다. Horseradish peroxidase- conjugated 2차항체(1:2000희석)로 1시간 반응시킨 후 enhanced chemilumine- scence kit를 이용하여 단백질의 발현량을 확인하였다. Western blot analysis was performed in the following examples of the present invention as follows. Cell homogenates were separated by 10% SDS polyacrylamide gel electrophoresis and electrophoresed to the gel with a microcellulose membrane. The protein-transferred membrane was treated with 5% skim milk and then treated with human Shc antibody (1: 1000 dilution), phospho-eNOS (1: 1000 dilution), or phospho-p66shc (1: 1000 dilution). After the reaction with horseradish peroxidase-conjugated secondary antibody (1: 2000 dilution) for 1 hour, the expression level of the protein was confirmed using the enhanced chemiluminescence-scence kit.
1) Tat-p66shc 융합단백질의 혈관내피세포 투과 1) Tat-p66shc fusion protein permeation of vascular endothelial cells
혈관내피세포에서 Tat-p66shc단백이 세포내로 유입되는 정도를 고찰하였다. 혈관내피세포로는 American Type Culture Collection(Rockvile, MD)에서 분주받은 쥐의 내피세포주 MS(MILE SVEN)-1을 사용하였다. 쥐의 췌장내피세포주인 MS-1 세포주를 5%의 이산화탄소와 95%의 공기가 혼합된 37℃ 수분 공급형 인큐베이터 환경에서 유지·배양하였다. 배양액으로는 10% Fetal Bovine Serum, Penicillin 100U/ml을 보충한 Dulbecco's Modified Eagle's Medium(Gibco, Grand Island, NY)을 사용하였다.The degree of Tat-p66shc protein influx into vascular endothelial cells was investigated. As a vascular endothelial cell, the rat endothelial cell line MS (MILE SVEN) -1 dispensed from American Type Culture Collection (Rockvile, MD) was used. The mouse pancreatic endothelial cell line, MS-1 cell line, was maintained and cultured in a 37 ° C. water supply incubator with 5% carbon dioxide and 95% air. Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum and Penicillin 100 U / ml was used as a culture.
배양된 세포는 105개 세포/ml의 농도로 배양액에 희석하여 사용하였다.The cultured cells were used diluted in the culture solution at a concentration of 10 5 cells / ml.
상기 희석된 배양세포를 2 ml씩 6 well에 분주하고, Tat-p66shc을 0, 1, 5, 10 및 20nM 농도로 각각 추가하여 2시간 동안 37 ℃ 수분공급형 배양기에서 배양하였다. 이후 인산화완충식염수로 3회 세척하고 세포분쇄기로 분쇄하였으며 12000rpm에서 15분 동안 원심분리하여 상층액을 획득하였다. 단백질량은 Bio-rad assay를 이용하여 정량하였다. 정량된 단백질 30 ㎍을 sampling하여 100℃에서 5분간 열을 가한 후 10분 동안 급랭시켰다. 세포내로 투과된 Tat-p66shc 융합단백질의 양을 ShcA 단일클론항체를 이용한 웨스턴블럿으로 측정하여 그 결과를 도 3에 나타내었다. 도 3에서 확인할 수 있듯이 Tat-p66shc 융합단백질은 1nM 농도부터 세포내 투과가 일어나고, 농도가 진해짐에 따라 농도의존적으로 세포내로 유입되는 것을 알 수 있었다.The diluted culture cells were dispensed into 6 wells of 2 ml, and Tat-p66shc was added at 0, 1, 5, 10 and 20 nM concentrations, respectively, and incubated in a 37 ° C. water incubator for 2 hours. After washing three times with phosphorylated buffered saline and pulverized with a cell crusher and centrifuged for 15 minutes at 12000rpm to obtain a supernatant. Protein amount was quantified using a Bio-rad assay. 30 μg of the quantified protein was sampled, heated at 100 ° C. for 5 minutes, and quenched for 10 minutes. The amount of Tat-p66shc fusion protein permeated into cells was measured by Western blot using ShcA monoclonal antibody, and the results are shown in FIG. 3. As can be seen in FIG. 3, the Tat-p66shc fusion protein was found to be intracellularly permeated from 1 nM concentration, and as the concentration increased, it was introduced into the cell in a concentration-dependent manner.
2) Tat-p66shc 융합단백질의 혈관내피세포 투과 후 지속 시간2) Duration after vascular endothelial cell penetration of Tat-p66shc fusion protein
혈관내피세포에서 유입된 Tat-p66shc가 어느 시간 동안 세포 내에 존재하는지를 알기 위하여 20 nM의 Tat-p66shc 융합단백질을 시간별로 배양액에 처리하여 세포 내의 지속시간을 조사하였다. 세포내의 유입된 Tat-p66shc의 지속정도는 웨스턴블럿 분석방법을 이용하였으며 그 결과를 도 4에 나타내었다. 단 sampling과정은 실시예 3의 1)과 동일하다.In order to know how long the Tat-p66shc introduced from vascular endothelial cells existed in the cells, the duration of the cells was investigated by treating 20 nM Tat-p66shc fusion protein to the culture medium by time. The persistence of Tat-p66shc introduced into the cells was measured by Western blotting, and the results are shown in FIG. 4. However, the sampling process is the same as in Example 1).
도 4에서 확인할 수 있듯이, 혈관내피세포에서 Tat-p66shc을 1시간 배양액에 처리한 군에서 최대의 발현을 보였으며 그 이후 서서히 감소하는 경향을 보였다. 이러한 결과는 세포내로 유입한 Tat-p66shc은 세포내의 단백질분해 효소에 의하여 서서히 제거된다는 사실을 암시하고 있다. As can be seen in Figure 4, in the vascular endothelial cells treated with Tat-p66shc for 1 hour culture showed the maximum expression and then showed a tendency to decrease gradually. These results suggest that Tat-p66shc introduced into cells is slowly removed by intracellular proteolytic enzymes.
본 실시예를 통해 본 발명에 의한 Tat-p66shc 융합단백질이 성공적으로 혈관내피세포에 도입됨을 확인 할 수 있었다.Through this example, it was confirmed that the Tat-p66shc fusion protein according to the present invention was successfully introduced into vascular endothelial cells.
실시예 4 : Tat-p66shc 융합단백질의 eNOS 단백질 인산화 억제 효과 측정Example 4 Determination of eNOS Protein Phosphorylation Inhibitory Effect of Tat-p66shc Fusion Protein
세포내로 유입된 Tat-p66shc 단백질이 정상정인 생리학적 기능을 갖는지를 조사하기 위하여 혈관내피세포에서 TNF-α에 의해 유도되는 eNOS단백의 인산화에 미치는 영향을 측정하였다. In order to investigate whether the Tat-p66shc protein introduced into cells has normal physiological function, the effect on the phosphorylation of eNOS protein induced by TNF-α in vascular endothelial cells was measured.
실시예 3의 1) 내피세포 배양 희석액에 20nM의 Tat-p66shc를 2시간동안 전처리한 군과 처리하지 않은 대조군에서, 15 ng/ml의 TNF-α를 5 ~ 60분동안 처리한 후 eNOS의 인산화 및 p66shc의 인산화의 정도를 웨스턴블럿분석으로 측정하여 그 결과를 도 5에 도시하였다. 단 sampling과정은 실시예 3의 1)과 동일하다. Example 1 1) In the endothelial cell culture dilution, 20 nM Tat-p66shc for 2 hours and in the untreated control group, 15 ng / ml of TNF-α for 5 to 60 minutes after phosphorylation of eNOS And the degree of phosphorylation of p66shc was measured by Western blot analysis, and the results are shown in FIG. 5. However, the sampling process is the same as in Example 1).
도 5로부터 Tat-p66shc 융합단백질 20 nM을 2시간 동안 세포배양액에 처리하는 경우 Tat-p66shc 융합단백질이 세포 내에서 안정적으로 과발현됨을 확인하였다. 유입된 Tat-p66shc 융합단백질은 세포내에서 TNF-α에 의하여 증가되는 eNOS 인산화를 효과적으로 억제시키는 것을 알 수 있었다.From FIG. 5, it was confirmed that Tat-p66shc fusion protein was stably overexpressed in cells when 20 nM of Tat-p66shc fusion protein was treated in the cell culture medium for 2 hours. The introduced Tat-p66shc fusion protein effectively inhibited eNOS phosphorylation increased by TNF-α in cells.
도 1은 Tat-p66shc 발현벡터의 개열지도.1 is a cleavage map of a Tat-p66shc expression vector.
도 2는 여러 가지 농도의 imidazole을 포함한 용출완충액에 의해 Tat-p66shc 융합단백질을 정제한 결과를 보여주는 전기영동 사진. Figure 2 is an electrophoresis picture showing the results of the purification of Tat-p66shc fusion protein by elution buffer containing various concentrations of imidazole.
도 3은 Tat-p66shc 융합단백질의 농도별 혈관내피 세포내 투과 정도를 웨스턴블럿으로 확인한 결과를 보여주는 사진.Figure 3 is a photograph showing the results confirmed by Western blotting the degree of vascular endothelial cell permeation by concentration of Tat-p66shc fusion protein.
도 4는 Tat-p66shc 융합단백질의 혈관내피 세포내 투과 후 지속 시간을 웨스턴블럿으로 확인한 결과를 보여주는 사진. Figure 4 is a photograph showing the results confirmed by Western blot after the intravascular endothelial cell permeation of Tat-p66shc fusion protein.
도 5는 Tat-p66shc를 농도별로 혈관내피세포에 처리한 후 혈관내피세포에서 eNOS의 인산화정도를 웨스턴블럿을 이용하여 확인한 결과를 보여주는 사진.Figure 5 is a photograph showing the results of confirming the degree of phosphorylation of eNOS in vascular endothelial cells after treatment with vascular endothelial cells Tat-p66shc by concentration using Western blot.
<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Cell permeable p66shc fusion protein having anti-cancer activity <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 619 <212> PRT <213> Artificial Sequence <220> <223> Tat-p66shc <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30 Asp Pro Asn Ser Met Asp Leu Leu Pro Pro Lys Pro Lys Tyr Asn Pro 35 40 45 Leu Arg Asn Glu Ser Leu Ser Ser Met Glu Glu Gly Ala Ser Gly Ser 50 55 60 Thr Pro Pro Glu Glu Leu Pro Ser Pro Pro Ala Ser Ser Leu Gly Pro 65 70 75 80 Ile Leu Pro Pro Leu Pro Gly Asp Asp Ser Pro Thr Thr Leu Cys Ser 85 90 95 Phe Phe Pro Arg Met Ser Asn Leu Arg Leu Ala Asn Pro Ala Gly Gly 100 105 110 Arg Pro Gly Ser Lys Gly Glu Pro Gly Arg Ala Ala Asp Asp Gly Glu 115 120 125 Gly Ile Val Gly Ala Ala Met Pro Asp Ser Gly Pro Leu Pro Leu Leu 130 135 140 Gln Asp Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Arg Thr Arg Val 145 150 155 160 Glu Gly Gly Gln Leu Gly Gly Glu Glu Trp Thr Arg His Gly Ser Phe 165 170 175 Val Asn Lys Pro Thr Arg Gly Trp Leu His Pro Asn Asp Lys Val Met 180 185 190 Gly Pro Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cys Val Glu Val 195 200 205 Leu Gln Ser Met Arg Ala Leu Asp Phe Asn Thr Arg Thr Gln Val Thr 210 215 220 Arg Glu Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gly Ala Lys Gly 225 230 235 240 Ala Thr Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Ser Ser Ile Leu 245 250 255 Gly Arg Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Thr Leu Thr Val 260 265 270 Ser Thr Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Lys Gln Ile Ile 275 280 285 Ala Asn His His Met Gln Ser Ile Ser Phe Ala Ser Gly Gly Asp Pro 290 295 300 Asp Thr Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pro Val Asn Gln 305 310 315 320 Arg Ala Cys His Ile Leu Glu Cys Pro Glu Gly Leu Ala Gln Asp Val 325 330 335 Ile Ser Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Lys Gln Tyr Leu 340 345 350 Arg Asn Pro Pro Lys Leu Val Thr Pro His Asp Arg Met Ala Gly Phe 355 360 365 Asp Gly Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pro Asp His Gln 370 375 380 Tyr Tyr Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gly Gly Val Val 385 390 395 400 Asp Met Arg Leu Arg Glu Gly Ala Ala Pro Gly Ala Ala Arg Pro Thr 405 410 415 Ala Pro Asn Ala Gln Thr Pro Ser His Leu Gly Ala Thr Leu Pro Val 420 425 430 Gly Gln Pro Val Gly Gly Asp Pro Glu Val Arg Lys Gln Met Pro Pro 435 440 445 Pro Pro Pro Cys Pro Gly Arg Glu Leu Phe Asp Asp Pro Ser Tyr Val 450 455 460 Asn Val Gln Asn Leu Asp Lys Ala Arg Gln Ala Val Gly Gly Ala Gly 465 470 475 480 Pro Pro Asn Pro Ala Ile Asn Gly Ser Ala Pro Arg Asp Leu Phe Asp 485 490 495 Met Lys Pro Phe Glu Asp Ala Leu Arg Val Pro Pro Pro Pro Gln Ser 500 505 510 Val Ser Met Ala Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gly Lys 515 520 525 Leu Ser Arg Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gly Asp Phe 530 535 540 Leu Val Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Val Leu Thr Gly 545 550 555 560 Leu Gln Ser Gly Gln Pro Lys His Leu Leu Leu Val Asp Pro Glu Gly 565 570 575 Val Val Arg Thr Lys Asp His Arg Phe Glu Ser Val Ser His Leu Ile 580 585 590 Ser Tyr His Met Asp Asn His Leu Pro Ile Ile Ser Ala Gly Ser Glu 595 600 605 Leu Cys Leu Gln Gln Pro Val Glu Arg Lys Leu 610 615 <210> 2 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> gene coding Tat-p66shc <400> 2 atgaggaaga agcggagaca gcgacgaaga ggctcggatc cgaattcgat ggatctcctg 60 ccccccaagc ccaagtacaa tccactccgg aatgagtctc tgtcatcgat ggaggaaggg 120 gcttctgggt ccaccccccc ggaggagctg ccttccccac cagcttcatc cctggggccc 180 atcctgcctc ctctgcctgg ggacgatagt cccactaccc tgtgctcctt cttcccccgg 240 atgagcaacc tgaggctggc caacccggct ggggggcgcc cagggtctaa gggggagcca 300 ggaagggcag ctgatgatgg ggaggggatc gtaggggcag ccatgccaga ctcaggcccc 360 ctacccctcc tccaggacat gaacaagctg agtggaggcg gcgggcgcag gactcgggtg 420 gaagggggcc agcttggggg cgaggagtgg acccgccacg ggagctttgt caataagccc 480 acgcggggct ggctgcatcc caacgacaaa gtcatgggac ccggggtttc ctacttggtt 540 cggtacatgg gttgtgtgga ggtcctccag tcaatgcgtg ccctggactt caacacccgg 600 actcaggtca ccagggaggc catcagtctg gtgtgtgagg ctgtgccggg tgctaagggg 660 gcgacaagga ggagaaagcc ctgtagccgc ccgctcagct ctatcctggg gaggagtaac 720 ctgaaatttg ctggaatgcc aatcactctc accgtctcca ccagcagcct caacctcatg 780 gccgcagact gcaaacagat catcgccaac caccacatgc aatctatctc atttgcatcc 840 ggcggggatc cggacacagc cgagtatgtc gcctatgttg ccaaagaccc tgtgaatcag 900 agagcctgcc acattctgga gtgtcccgaa gggcttgccc aggatgtcat cagcaccatt 960 ggccaggcct tcgagttgcg cttcaaacaa tacctcagga acccacccaa actggtcacc 1020 cctcatgaca ggatggctgg ctttgatggc tcagcatggg atgaggagga ggaagagcca 1080 cctgaccatc agtactataa tgacttcccg gggaaggaac cccccttggg gggggtggta 1140 gacatgaggc ttcgggaagg agccgctcca ggggctgctc gacccactgc acccaatgcc 1200 cagaccccca gccacttggg agctacattg cctgtaggac agcctgttgg gggagatcca 1260 gaagtccgca aacagatgcc acctccacca ccctgtccag gcagagagct ttttgatgat 1320 ccctcctatg tcaacgtcca gaacctagac aaggcccggc aagcagtggg tggtgctggg 1380 ccccccaatc ctgctatcaa tggcagtgca ccccgggacc tgtttgacat gaagcccttc 1440 gaagatgctc ttcgggtgcc tccacctccc cagtcggtgt ccatggctga gcagctccga 1500 ggggagccct ggttccatgg gaagctgagc cggcgggagg ctgaggcact gctgcagctc 1560 aatggggact tcttggtacg ggagagcacg accacacctg gccagtatgt gctcactggc 1620 ttgcagagtg ggcagcctaa gcatttgcta ctggtggacc ctgagggtgt ggttcggact 1680 aaggatcacc gctttgaaag tgtcagtcac cttatcagct accacatgga caatcacttg 1740 cccatcatct ctgcgggcag cgaactgtgt ctacagcaac ctgtggagcg gaaactgtga 1800 1800 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 3 cggaattcga aggatctcct gcccccc 27 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer <400> 4 cgaagctttc acagtttccg ctccacagg 29 <210> 5 <211> 1752 <212> DNA <213> human <400> 5 atggatctcc tgccccccaa gcccaagtac aatccactcc ggaatgagtc tctgtcatcg 60 atggaggaag gggcttctgg gtccaccccc ccggaggagc tgccttcccc accagcttca 120 tccctggggc ccatcctgcc tcctctgcct ggggacgata gtcccactac cctgtgctcc 180 ttcttccccc ggatgagcaa cctgaggctg gccaacccgg ctggggggcg cccagggtct 240 aagggggagc caggaagggc agctgatgat ggggagggga tcgtaggggc agccatgcca 300 gactcaggcc ccctacccct cctccaggac atgaacaagc tgagtggagg cggcgggcgc 360 aggactcggg tggaaggggg ccagcttggg ggcgaggagt ggacccgcca cgggagcttt 420 gtcaataagc ccacgcgggg ctggctgcat cccaacgaca aagtcatggg acccggggtt 480 tcctacttgg ttcggtacat gggttgtgtg gaggtcctcc agtcaatgcg tgccctggac 540 ttcaacaccc ggactcaggt caccagggag gccatcagtc tggtgtgtga ggctgtgccg 600 ggtgctaagg gggcgacaag gaggagaaag ccctgtagcc gcccgctcag ctctatcctg 660 gggaggagta acctgaaatt tgctggaatg ccaatcactc tcaccgtctc caccagcagc 720 ctcaacctca tggccgcaga ctgcaaacag atcatcgcca accaccacat gcaatctatc 780 tcatttgcat ccggcgggga tccggacaca gccgagtatg tcgcctatgt tgccaaagac 840 cctgtgaatc agagagcctg ccacattctg gagtgtcccg aagggcttgc ccaggatgtc 900 atcagcacca ttggccaggc cttcgagttg cgcttcaaac aatacctcag gaacccaccc 960 aaactggtca cccctcatga caggatggct ggctttgatg gctcagcatg ggatgaggag 1020 gaggaagagc cacctgacca tcagtactat aatgacttcc cggggaagga accccccttg 1080 gggggggtgg tagacatgag gcttcgggaa ggagccgctc caggggctgc tcgacccact 1140 gcacccaatg cccagacccc cagccacttg ggagctacat tgcctgtagg acagcctgtt 1200 gggggagatc cagaagtccg caaacagatg ccacctccac caccctgtcc aggcagagag 1260 ctttttgatg atccctccta tgtcaacgtc cagaacctag acaaggcccg gcaagcagtg 1320 ggtggtgctg ggccccccaa tcctgctatc aatggcagtg caccccggga cctgtttgac 1380 atgaagccct tcgaagatgc tcttcgggtg cctccacctc cccagtcggt gtccatggct 1440 gagcagctcc gaggggagcc ctggttccat gggaagctga gccggcggga ggctgaggca 1500 ctgctgcagc tcaatgggga cttcttggta cgggagagca cgaccacacc tggccagtat 1560 gtgctcactg gcttgcagag tgggcagcct aagcatttgc tactggtgga ccctgagggt 1620 gtggttcgga ctaaggatca ccgctttgaa agtgtcagtc accttatcag ctaccacatg 1680 gacaatcact tgcccatcat ctctgcgggc agcgaactgt gtctacagca acctgtggag 1740 cggaaactgt ga 1752 <110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Cell permeable p66shc fusion protein having anti-cancer activity <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 619 <212> PRT <213> Artificial Sequence <220> <223> Tat-p66shc <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30 Asp Pro Asn Ser Met Asp Leu Leu Pro Pro Lys Pro Lys Tyr Asn Pro 35 40 45 Leu Arg Asn Glu Ser Leu Ser Ser Met Glu Glu Gly Ala Ser Gly Ser 50 55 60 Thr Pro Pro Glu Glu Leu Pro Ser Pro Pro Ala Ser Ser Leu Gly Pro 65 70 75 80 Ile Leu Pro Pro Leu Pro Gly Asp Asp Ser Pro Thr Thr Leu Cys Ser 85 90 95 Phe Phe Pro Arg Met Ser Asn Leu Arg Leu Ala Asn Pro Ala Gly Gly 100 105 110 Arg Pro Gly Ser Lys Gly Glu Pro Gly Arg Ala Ala Asp Asp Gly Glu 115 120 125 Gly Ile Val Gly Ala Ala Met Pro Asp Ser Gly Pro Leu Pro Leu Leu 130 135 140 Gln Asp Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Arg Thr Arg Val 145 150 155 160 Glu Gly Gly Gln Leu Gly Gly Glu Glu Trp Thr Arg His Gly Ser Phe 165 170 175 Val Asn Lys Pro Thr Arg Gly Trp Leu His Pro Asn Asp Lys Val Met 180 185 190 Gly Pro Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cys Val Glu Val 195 200 205 Leu Gln Ser Met Arg Ala Leu Asp Phe Asn Thr Arg Thr Gln Val Thr 210 215 220 Arg Glu Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gly Ala Lys Gly 225 230 235 240 Ala Thr Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Ser Ser Ile Leu 245 250 255 Gly Arg Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Thr Leu Thr Val 260 265 270 Ser Thr Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Lys Gln Ile Ile 275 280 285 Ala Asn His His Met Gln Ser Ile Ser Phe Ala Ser Gly Gly Asp Pro 290 295 300 Asp Thr Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pro Val Asn Gln 305 310 315 320 Arg Ala Cys His Ile Leu Glu Cys Pro Glu Gly Leu Ala Gln Asp Val 325 330 335 Ile Ser Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Lys Gln Tyr Leu 340 345 350 Arg Asn Pro Pro Lys Leu Val Thr Pro His Asp Arg Met Ala Gly Phe 355 360 365 Asp Gly Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pro Asp His Gln 370 375 380 Tyr Tyr Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gly Gly Val Val 385 390 395 400 Asp Met Arg Leu Arg Glu Gly Ala Ala Pro Gly Ala Ala Arg Pro Thr 405 410 415 Ala Pro Asn Ala Gln Thr Pro Ser His Leu Gly Ala Thr Leu Pro Val 420 425 430 Gly Gln Pro Val Gly Gly Asp Pro Glu Val Arg Lys Gln Met Pro Pro 435 440 445 Pro Pro Pro Cys Pro Gly Arg Glu Leu Phe Asp Asp Pro Ser Tyr Val 450 455 460 Asn Val Gln Asn Leu Asp Lys Ala Arg Gln Ala Val Gly Gly Ala Gly 465 470 475 480 Pro Pro Asn Pro Ala Ile Asn Gly Ser Ala Pro Arg Asp Leu Phe Asp 485 490 495 Met Lys Pro Phe Glu Asp Ala Leu Arg Val Pro Pro Pro Gln Ser 500 505 510 Val Ser Met Ala Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gly Lys 515 520 525 Leu Ser Arg Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gly Asp Phe 530 535 540 Leu Val Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Val Leu Thr Gly 545 550 555 560 Leu Gln Ser Gly Gln Pro Lys His Leu Leu Leu Val Asp Pro Glu Gly 565 570 575 Val Val Arg Thr Lys Asp His Arg Phe Glu Ser Val Ser His Leu Ile 580 585 590 Ser Tyr His Met Asp Asn His Leu Pro Ile Ile Ser Ala Gly Ser Glu 595 600 605 Leu Cys Leu Gln Gln Pro Val Glu Arg Lys Leu 610 615 <210> 2 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> gene coding Tat-p66shc <400> 2 atgaggaaga agcggagaca gcgacgaaga ggctcggatc cgaattcgat ggatctcctg 60 ccccccaagc ccaagtacaa tccactccgg aatgagtctc tgtcatcgat ggaggaaggg 120 gcttctgggt ccaccccccc ggaggagctg ccttccccac cagcttcatc cctggggccc 180 atcctgcctc ctctgcctgg ggacgatagt cccactaccc tgtgctcctt cttcccccgg 240 atgagcaacc tgaggctggc caacccggct ggggggcgcc cagggtctaa gggggagcca 300 ggaagggcag ctgatgatgg ggaggggatc gtaggggcag ccatgccaga ctcaggcccc 360 ctacccctcc tccaggacat gaacaagctg agtggaggcg gcgggcgcag gactcgggtg 420 gaagggggcc agcttggggg cgaggagtgg acccgccacg ggagctttgt caataagccc 480 acgcggggct ggctgcatcc caacgacaaa gtcatgggac ccggggtttc ctacttggtt 540 cggtacatgg gttgtgtgga ggtcctccag tcaatgcgtg ccctggactt caacacccgg 600 actcaggtca ccagggaggc catcagtctg gtgtgtgagg ctgtgccggg tgctaagggg 660 gcgacaagga ggagaaagcc ctgtagccgc ccgctcagct ctatcctggg gaggagtaac 720 ctgaaatttg ctggaatgcc aatcactctc accgtctcca ccagcagcct caacctcatg 780 gccgcagact gcaaacagat catcgccaac caccacatgc aatctatctc atttgcatcc 840 ggcggggatc cggacacagc cgagtatgtc gcctatgttg ccaaagaccc tgtgaatcag 900 agagcctgcc acattctgga gtgtcccgaa gggcttgccc aggatgtcat cagcaccatt 960 ggccaggcct tcgagttgcg cttcaaacaa tacctcagga acccacccaa actggtcacc 1020 cctcatgaca ggatggctgg ctttgatggc tcagcatggg atgaggagga ggaagagcca 1080 cctgaccatc agtactataa tgacttcccg gggaaggaac cccccttggg gggggtggta 1140 gacatgaggc ttcgggaagg agccgctcca ggggctgctc gacccactgc acccaatgcc 1200 cagaccccca gccacttggg agctacattg cctgtaggac agcctgttgg gggagatcca 1260 gaagtccgca aacagatgcc acctccacca ccctgtccag gcagagagct ttttgatgat 1320 ccctcctatg tcaacgtcca gaacctagac aaggcccggc aagcagtggg tggtgctggg 1380 ccccccaatc ctgctatcaa tggcagtgca ccccgggacc tgtttgacat gaagcccttc 1440 gaagatgctc ttcgggtgcc tccacctccc cagtcggtgt ccatggctga gcagctccga 1500 ggggagccct ggttccatgg gaagctgagc cggcgggagg ctgaggcact gctgcagctc 1560 aatggggact tcttggtacg ggagagcacg accacacctg gccagtatgt gctcactggc 1620 ttgcagagtg ggcagcctaa gcatttgcta ctggtggacc ctgagggtgt ggttcggact 1680 aaggatcacc gctttgaaag tgtcagtcac cttatcagct accacatgga caatcacttg 1740 cccatcatct ctgcgggcag cgaactgtgt ctacagcaac ctgtggagcg gaaactgtga 1800 1800 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 3 cggaattcga aggatctcct gcccccc 27 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer <400> 4 cgaagctttc acagtttccg ctccacagg 29 <210> 5 <211> 1752 <212> DNA <213> human <400> 5 atggatctcc tgccccccaa gcccaagtac aatccactcc ggaatgagtc tctgtcatcg 60 atggaggaag gggcttctgg gtccaccccc ccggaggagc tgccttcccc accagcttca 120 tccctggggc ccatcctgcc tcctctgcct ggggacgata gtcccactac cctgtgctcc 180 ttcttccccc ggatgagcaa cctgaggctg gccaacccgg ctggggggcg cccagggtct 240 aagggggagc caggaagggc agctgatgat ggggagggga tcgtaggggc agccatgcca 300 gactcaggcc ccctacccct cctccaggac atgaacaagc tgagtggagg cggcgggcgc 360 aggactcggg tggaaggggg ccagcttggg ggcgaggagt ggacccgcca cgggagcttt 420 gtcaataagc ccacgcgggg ctggctgcat cccaacgaca aagtcatggg acccggggtt 480 tcctacttgg ttcggtacat gggttgtgtg gaggtcctcc agtcaatgcg tgccctggac 540 ttcaacaccc ggactcaggt caccagggag gccatcagtc tggtgtgtga ggctgtgccg 600 ggtgctaagg gggcgacaag gaggagaaag ccctgtagcc gcccgctcag ctctatcctg 660 gggaggagta acctgaaatt tgctggaatg ccaatcactc tcaccgtctc caccagcagc 720 ctcaacctca tggccgcaga ctgcaaacag atcatcgcca accaccacat gcaatctatc 780 tcatttgcat ccggcgggga tccggacaca gccgagtatg tcgcctatgt tgccaaagac 840 cctgtgaatc agagagcctg ccacattctg gagtgtcccg aagggcttgc ccaggatgtc 900 atcagcacca ttggccaggc cttcgagttg cgcttcaaac aatacctcag gaacccaccc 960 aaactggtca cccctcatga caggatggct ggctttgatg gctcagcatg ggatgaggag 1020 gaggaagagc cacctgacca tcagtactat aatgacttcc cggggaagga accccccttg 1080 gggggggtgg tagacatgag gcttcgggaa ggagccgctc caggggctgc tcgacccact 1140 gcacccaatg cccagacccc cagccacttg ggagctacat tgcctgtagg acagcctgtt 1200 gggggagatc cagaagtccg caaacagatg ccacctccac caccctgtcc aggcagagag 1260 ctttttgatg atccctccta tgtcaacgtc cagaacctag acaaggcccg gcaagcagtg 1320 ggtggtgctg ggccccccaa tcctgctatc aatggcagtg caccccggga cctgtttgac 1380 atgaagccct tcgaagatgc tcttcgggtg cctccacctc cccagtcggt gtccatggct 1440 gagcagctcc gaggggagcc ctggttccat gggaagctga gccggcggga ggctgaggca 1500 ctgctgcagc tcaatgggga cttcttggta cgggagagca cgaccacacc tggccagtat 1560 gtgctcactg gcttgcagag tgggcagcct aagcatttgc tactggtgga ccctgagggt 1620 gtggttcgga ctaaggatca ccgctttgaa agtgtcagtc accttatcag ctaccacatg 1680 gacaatcact tgcccatcat ctctgcgggc agcgaactgt gtctacagca acctgtggag 1740 cggaaactgt ga 1752
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