KR101439203B1 - Pharmaceutical composition containing FK506 binding protein fusion protein and fenobam for treating brain ischemic damage - Google Patents

Pharmaceutical composition containing FK506 binding protein fusion protein and fenobam for treating brain ischemic damage Download PDF

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KR101439203B1
KR101439203B1 KR1020130034213A KR20130034213A KR101439203B1 KR 101439203 B1 KR101439203 B1 KR 101439203B1 KR 1020130034213 A KR1020130034213 A KR 1020130034213A KR 20130034213 A KR20130034213 A KR 20130034213A KR 101439203 B1 KR101439203 B1 KR 101439203B1
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최수영
박진서
김대원
음원식
박종훈
박은영
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Abstract

Although various methods for increasing an intracellular level of a therapeutic material have been tried, most of them are filed to pass a blood brain barrier and cell membranes. It has been known that a fusion protein of FK506BP, to which a protein transport domain binds, effectively infiltrates into cells and tissue. The present invention has found that fenobam facilitates introduction of a FK506BP fusion protein into cells and tissue and also enhances the neuroprotective effect of FK506BP against ischemic damage. In C6 cells, fenobam strongly enhances the protective effect of a FK506BP fusion protein against toxicity or DNA fragmentation induced by H_2O_2. In addition, by treating with fenobam and FK506BP in combination, Akt and MAPK activation induced by H_2O_2 is significantly inhibited when compared to treatment with a FK506BP fusion protein alone. Interestingly, in vitro and in vivo cell introduction data shows that fenobam treatment significantly increases cell introduction of a FK506BP hydraulic protein into C6 cells and gerbil brain hippocampus. Also, transient global ischemia gerbil model shows that fenobam treatment in a CA1 portion of hippocampus increases the neuroprotective effect of FK506BP. Thus, these results indicates that fenobam increases an introduction rate of a fusion protein of a protein transport domain into cells and tissue and thus fenobam is useful as an agent for promoting their neuroprotective effect.

Description

ＦＫ506 결합단백질 융합단백질과 피노밤을 포함하는 뇌 허혈손상 치료용 약제학적 조성물 {Pharmaceutical composition containing FK506 binding protein fusion protein and fenobam for treating brain ischemic damage}FIELD OF THE INVENTION [0001] The present invention relates to a pharmaceutical composition for treating cerebral ischemic damage comprising a FK506 binding protein fusion protein and finoBam for treating brain ischemic damage,

ＦＫ506 결합단백질 융합단백질과 피노밤을 포함하는 뇌 허혈손상 치료용 약제학적 조성물 및 치료용 단백질 융합단백질의 세포 또는 조직 침투율을 높이는 피노밤의 용도에 관한 것이다.A pharmaceutical composition for the treatment of brain ischemic injury comprising an FK506 binding protein fusion protein and a finoBarm, and a use of a PinoBak for enhancing cell or tissue permeability of a therapeutic protein fusion protein.

FK506 결합 단백질 (FK506-binding protein; FK506BP)은 FK506, 싸이클로스포린 A 및 라파마이신과 같은 다양한 면역억제제와 높은 친화력으로 결합하는 이뮤노필린 단백질이다 (Kang, C.B., et al., Neurosignals, 2008. 16(4): 318-25, Kino, T., et al., J Antibiot (Tokyo), 1987. 40(9): 1256-65). FK506BP 패밀리는 12~59 kDa에 이르는 분자량 크기에 따라 구분되며, 세포 내에서 다양한 기능을 수행하는 것으로 알려져 있었다. FK506BP는 단백질 폴딩 단계와 관련되어 PPIase (peptidylprolylcis/trans isomerase) 활성을 나타낸다 (Kang, C.B., et al., Neurosignals, 2008. 16(4): 318-25). 또한, 분자량 12 kDa인 FK506BP12는 FK506 또는 라파마이신과 복합체를 형성할 수 있고, 이 결과로 얻어지는 FK506과 FK506BP12 복합체는 칼슘-의존적 포스파테이즈 칼시뉴린 (calcineurin; CaN)의 조절 B 서브유닛과 상호작용하여 칼시뉴린 활성을 저해하고, 그리하여 인터류킨-2 (IL-2) 및 T-세포의 활성을 저해한다 (Harrar, Y., C. Bellini, and J.D. Faure, Trends Plant Sci, 2001. 6(9): 426-31). FK506이 없을 때 FK506BP12는 칼슘 채널의 TGF β 수용체 또는 IP3 {inositol-(1,4,5)-triphosphate} 수용체와 상호작용한다 (Brillantes, A.B., et al., Cell, 1994. 77(4): 513-23, Cameron, A.M., et al., J Biol Chem, 1997. 272(44): 27582-8, Chen, Y.G., F. Liu, and J. Massague, EMBO J, 1997. 16(13): 3866-76). 또한, FK506BP12 녹아웃 마우스는 칼슘 채널 기능장애 및 세포 사이클 조절장애로 인해 심장 결함을 나타낸다는 사실이 보고된바 있다 (Aghdasi, B., et al., Proc Natl Acad Sci U S A, 2001. 98(5): 2425-30, Shou, W., et al., Nature, 1998. 391(6666): 489-92).FK506-binding protein (FK506BP) is an immunophilin protein that binds with high affinity to a variety of immunosuppressants such as FK506, cyclosporin A and rapamycin (Kang, CB, et al., Neurosignals, 2008. 16 (4): 318-25, Kino, T., et al., J Antibiot (Tokyo), 1987. 40 (9): 1256-65). The FK506BP family is distinguished by its molecular size ranging from 12 to 59 kDa and has been known to perform a variety of functions in cells. FK506BP exhibits PPIase (peptidylprolylcis / trans isomerase) activity in association with the protein folding step (Kang, CB, et al., Neurosignals, 2008. 16 (4): 318-25). In addition, FK506BP12 with a molecular weight of 12 kDa can form a complex with FK506 or rapamycin, and the resulting FK506 and FK506BP12 complexes interact with the regulatory B subunit of calcium-dependent phosphatidic calcineurin (CaN) (Harris, Y., C. Bellini, and JD Faure, Trends Plant Sci, 2001. 6 (9)), : 426-31). In the absence of FK506 FK506BP12 interacts with TGF β receptor or IP 3 {inositol- (1,4,5) -triphosphate } receptor calcium channel (Brillantes, AB, et al. , Cell, 1994. 77 (4) JG Biol Chem, 1997. 272 (44): 27582-8, Chen, YG, F. Liu, and J. Massague, EMBO J, 1997. 16 (13): 513-23, Cameron, AM, et al. : 3866-76). In addition, FK506BP12 knockout mice have been reported to exhibit heart defects due to calcium channel dysfunction and cell cycle control disorders (Aghdasi, B., et al., Proc Natl Acad Sci USA, 2001. 98 (5) : 2425-30, Shou, W., et al., Nature, 1998. 391 (6666): 489-92).

불안 완화제인 피노밤 [fenobam; 1-(3-chlorophenyl)-3-(3-methyl-5-oxo-4H-imidazol-2-yl) urea]은 대사성 글루타메이트 (metabotropic glutamate; mGlu) 5 수용체의 강력한 길항제로 알려져 있으며, 뇌조직 특히 해마의 CA1 및 CA3 부분에서 풍부하게 발현되는데, 우울증과 불안을 포함하는 감정 변화에 중요한 역할을 수행한다 (Palucha, A. and A. Pilc, Pharmacol Ther, 2007. 115(1): 116-47, Porter, R.H., et al., J Pharmacol Exp Ther, 2005. 315(2): 711-21).
An anxiolytic agent, fenobam; It has been known as a potent antagonist of the metabotropic glutamate (mGlu) 5 receptor and has been shown to be a potent antagonist of brain tissue (Palucha, A. and A. Pilc, Pharmacol Ther, 2007. 115 (1): 116-47, 1996), which is abundantly expressed in the CA1 and CA3 portions of hippocampus and plays an important role in emotional changes including depression and anxiety Porter, RH, et al., J Pharmacol Exp Ther, 2005. 315 (2): 711-21).

본 발명은 뇌-혈관 장벽을 좀더 효율적으로 투과하고, 치료효과가 높은 뇌허혈 치료용 약학 조성물을 제공하려는 것을 목적으로 한다. It is an object of the present invention to provide a pharmaceutical composition for treating cerebral ischemia that more effectively penetrates the brain-vascular barrier and has a therapeutic effect.

본 발명자들은 피노밤이 불안해소기능 외에도 저빌 뇌에서 산화 스트레스에 대한 FK506BP 융합단백질의 신경보호효과 및 C6 세포에서 산화스트레스에 의해 유도되는 Akt와 MAPK (mitogen activating protein kinase)의 활성화를 저해하는 효과에 긍정적인 영향을 미침을 알게 되었다. 또한, 본 발명자들은 비록 피노밤이 세포내 FK506BP 융합단백질 수준을 어떤 기작으로 상승시키는지는 정확하게 이해하지 못하였지만, 피노밤이 C6 세포 및 뇌조직 내로 FK506BP 융합단백질 도입 증대를 매개함을 입증하였다.
In addition to the anxiolytic function, the present inventors have also found that the neuroprotective effect of the FK506BP fusion protein on oxidative stress and the activation of Akt and mitogen activating protein kinase (MAPK) induced by oxidative stress in C6 cells, I found that it had a positive effect. In addition, the present inventors have demonstrated that PinoBam mediates increased FK506BP fusion protein introduction into C6 cells and brain tissue, although we did not exactly understand how pinotubic elevates intracellular FK506BP fusion protein levels.

본 발명자들은 피노밤이 산화 스트레스에 대한 FK506BP12 융합단백질의 신경보호 활성을 강화하고 인비보 및 인비트로에서 FK506BP 융합단백질의 세포도입을 현저히 증대함을 밝혔다. 따라서, 피노밤은 세포 및 조직 내로 치료용 단백질과 단백질 수송도메인이 융합된 융합단백질의 수송을 강화하는 제제로 이용할 수 있다.The present inventors have found that pinobut enhances the neuroprotective activity of the FK506BP12 fusion protein against oxidative stress and significantly increases the cellular uptake of the FK506BP fusion protein in Invivo and Invitro. Therefore, it can be used as an agent for enhancing the transport of a fusion protein in which a therapeutic protein and a protein transport domain are fused into cells and tissues.

혈액공급 제한 이후 허혈손상 부위에 산소가 포함된 혈액을 다시 돌리는 허혈/재관류 조건은 ATP 고갈, 이온펌프 실패, Ca2+를 포함하는 이온 불균형을 유발하고 또 활성산소종 수준을 높인다 (Koenitzer, J.R. et al., Ann N Y Acad Sci. 1203: 45-52). 활성산소종은 세포에 지질 과산화, 단백질 변성, DNA 손상 및 최종적으로 세포자기사멸 및 세포괴사를 일으킨다 (Zweier, J.L. and M.A. Talukder, Cardiovasc Res, 2006. 70(2): p. 181-90). 뇌의 주요 세포타입인 성상세포는 뉴런에 에너지 기질을 제공하고 혈관-뇌 장벽을 지지한다 (Sofroniew, M.V. and H.V. Vinters, Acta Neuropathol. 119(1): 7-35). 또한, 뇌 손상 이후 활성 성상세포는 신경영양인자를 생산함으로써 뉴런을 보호하고 염증반응과 같은 다양한 세포 신호, Akt 및 MAPK를 활성화함으로써 뉴런을 보호한다 (Hausenloy, D.J. and D.M. Yellon, Cardiovasc Res, 2004. 61(3): 448-60, Sofroniew, M.V. and H.V. Vinters, Acta Neuropathol. 119(1): 7-35). 따라서, 산화 스트레스에 반응하는 성상세포 활성화의 억제는 일반적으로 뉴런 보호를 위한 치료 타겟으로서 인식된다. 본 발명 데이타들은 FK506BP12 융합단백질이 H2O2로 유도되는 독성 및 DNA 손상 (도 1D, 1E)뿐 아니라 H2O2로 유도되는 Akt 및 p38과 Erk1/2을 포함하는 MAPK의 인산화 (도 2A 및 2B)를 현저히 억제함을 보여준다. 반면, 흥미롭게도 FK506BP 융합단백질 처리전 피노밤 처리는 FK506BP 융합단백질 단독 처리한 경우와 비교할 때 H2O2에 대한 반응으로 일어나는 DNA 손상, 세포독성, Akt 및 MAPK 인산화를 현저히 억제하였다. Ischemia / reperfusion conditions that re-circulate oxygen-containing blood to the ischemic lesion after blood supply restriction lead to ion imbalance including ATP depletion, ion pump failure, Ca 2+ , and elevated levels of reactive oxygen species (Koenitzer, JR et al., Ann NY Acad Sci. 1203: 45-52). Activated oxygen species cause lipid peroxidation, protein denaturation, DNA damage, and cell apoptosis and cell necrosis in cells (Zweier, JL and MA Talukder, Cardiovasc Res, 2006. 70 (2): 181-90). The main cell type of the brain, astrocytes, provides an energy substrate for neurons and supports vascular-brain barrier (Sofroniew, MV and HV Vinters, Acta Neuropathol. 119 (1): 7-35). In addition, after brain injury, active astrocytes protect neurons by producing neurotrophins and protect neurons by activating various cell signals, such as inflammatory responses, Akt and MAPK (Hausenloy, DJ and DM Yellon, Cardiovasc Res, 2004. 61 (3): 448-60, Sofroniew, MV and HV Vinters, Acta Neuropathol. 119 (1): 7-35). Thus, inhibition of astrocytic activation in response to oxidative stress is generally recognized as a therapeutic target for neuronal protection. The present invention data are FK506BP12 fusion protein H 2 toxicity and DNA damage induced by O 2 (Fig. 1D, 1E) as well as the phosphorylation of MAPK, including Akt, and p38 and Erk1 / 2 induced by H 2 O 2 (Fig. 2A And 2B). ≪ / RTI > On the other hand, interestingly, the treatment with FO506BP fusion protein inhibited DNA damage, cytotoxicity, Akt and MAPK phosphorylation caused by the reaction with H 2 O 2 compared with FK506BP fusion protein alone.

단백질 수송 도메인은 Tat 및 PEP-1과 같은 작은 크기의 펩타이드 조각이다. 비록 단백질 수송 도메인 융합단백질의 세포내 침투 기작은 분명히 밝혀지지 않았지만, 이들이 세포나 조직에 운반되기 어려운 다른 단백질과 결합하면 침투력을 증강시킨다고 보고되어 있다 (van den Berg, A. and S.F. Dowdy, Curr Opin Biotechnol. 22(6): p. 888-93). 이어서 단백질 수송 도메인을 이용한 치료용 생분자의 세포내 도입 증가는 약제로서 높은 잠재력은 가지고 있지만 침투력이 낮은 다양한 거대분자들의 이용을 용이하게 해줄 것이라고 제안되었다. 본 발명자들은 세포 및 조직 내로의 증강된 침투의 결과로서 PEP-1-rpS3 융합단백질, Tat-카탈레이즈 융합단백질 및 Tat-아넥신 융합단백질이 산화 스트레스에 대해 세포 또는 조직을 현저히 보호함을 밝힌바 있다 (Ahn, E.H., et al., Toxicology. 276(3): p. 192-7, Kim, D.W., et al., Free Radic Biol Med, 2009. 47(7): 941-52, Lee, S.H., et al., BMB Rep. 45(6): 354-9). 또, 본 발명자들은 PEP-1-FK506BP 융합단백질이 세포 및 조직으로 높은 침투능을 나타내며 Raw264.7 세포와 마우스 귀에서 염증반응을 억제하고 아토피 피부염을 완화함을 밝혔다 (Kim, S.Y., et al., J Invest Dermatol. 131(7): 1477-85, Kim, S.Y., et al., Immunobiology. 216(7): 771-81). 따라서, 도 3 및 도 4와 같이, 피노밤의 존재가 세포독성을 나타내지 않으면서 FK506BP 융합단백질의 성상세포 및 뇌조직으로의 세포도입을 증가시키고, 이어 세포보호효과를 이끌 수 있음을 밝힌 것은 매우 의미있다. The protein transport domain is a small sized peptide fragment such as Tat and PEP-1. Although intracellular penetration mechanisms of protein transport domain fusion proteins have not been elucidated, they have been reported to enhance penetration by binding to other proteins that are difficult to deliver to cells or tissues (van den Berg, A. and SF Dowdy, Curr Opin Biotechnol., 22 (6): 888-93). Subsequently, it was suggested that the intracellular introduction of therapeutic biomolecules using the protein transport domain would facilitate the use of various macromolecules with high potential as drugs but low permeability. The present inventors have found that PEP-1-rpS3 fusion protein, Tat-catalase fusion protein and Tat-annexin fusion protein significantly protect cells or tissues against oxidative stress as a result of enhanced penetration into cells and tissues (7): 941-52, Lee, SH (Kim, DW, et al., Free Radic Biol Med, , et al., BMB Rep 45 (6): 354-9). In addition, the present inventors have found that the PEP-1-FK506BP fusion protein exhibits high infiltration ability into cells and tissues and inhibits inflammatory reaction and alleviates atopic dermatitis in Raw264.7 cells and mouse ear (Kim, SY, et al. J Invest Dermatol, 131 (7): 1477-85, Kim, SY, et al., Immunobiology, 216 (7): 771-81). Therefore, as shown in FIG. 3 and FIG. 4, it has been found that the presence of the pinonuclease does not exhibit cytotoxicity, and that FK506BP fusion protein can increase cell introduction into astrocytes and brain tissue, Meaning.

피노밤은 글루타메이트에 의해 활성화되는 G-단백질과 짝을 이루는 대사성 글루타메이트 5 (mGlu5) 수용체 길항제로서 알려져 있다 (Porter, R.H., et al., J Pharmacol Exp Ther, 2005. 315(2): p. 711-21). mGlu5 수용체는 해마를 비롯하여 감정 처리에 관여하는 대뇌 변연계 부분에서 많이 발현된다. 전시냅스 뉴런 및 후시냅스 뉴런에서 mGlu5 수용체 활성화는 포스포라이페이즈 C (phospholipase C)에 의해 G 단백질과 상호작용을 거쳐 포스포이노시타이드 (phosphoinositide) 인지질을 1,4,5-삼인산 및 다이아실글리세롤로 가수분해하고, 이어 다양한 이온채널과 조절분자 및 신호분자의 조절을 포함하여 여러 가지 생물학적 효과를 일으킨다 (Palucha, A. and A. Pilc, Pharmacol Ther, 2007. 115(1): p. 116-47, Anwyl, R., Brain Res Brain Res Rev, 1999. 29(1): p. 83-120, Niswender, C.M. and P.J. Conn, Annu Rev Pharmacol Toxicol. 50: p. 295-322). 그러므로, 피노밤을 포함하여 불안 및 우울증, 파킨슨병, 통증, 중독 및 취약 X-염색체 증후군 (fragile X syndrome)에 대한 치료 타겟으로 생각되는 mGlu5 수용체에 대한 몇몇 길항제들이 mGlu5 수용체의 활성을 조절하기 위해 개발되고 있다 (Lindemann, L., et al., J Pharmacol Exp Ther. 339(2): p. 474-86). 예를 들어, 피노밤이 절차기억 형성 및 Fmr1 녹아웃 마우스에서 회피행동의 강화에 효과적일 수 있다고 보고된바 있다 (Veloz, M.F., et al., Genes Brain Behav.). 또한, 피노밤은 염증성 및 비염증성 방광 통증을 완화한다 (Crock, L.W., et al., Mol Pain. 8: p. 20). 실제로, 피노밤에 대한 선행 연구들은 기억 및 통증을 포함하여 다양한 중추신경계에 대한 치료잠재력에 주로 촛점을 맞추었다. 반면, 본 발명은 정확한 기작은 밝혀지지 않았지만, 피노밤이 단백질 수송 도메인과 결합한 융합단백질의 인비트로 및 인비보 투과와 관련되어 있다는 점에서 매우 흥미롭다.Pino bam is known as a metabotropic glutamate 5 (mGlu5) receptor antagonist that is paired with a G-protein activated by glutamate (Porter, RH, et al., J Pharmacol Exp Ther, 2005. 315 -21). The mGlu5 receptor is expressed in the hippocampus and the cerebral marginal area involved in emotion processing. MGlu5 receptor activation in pre-synaptic neurons and post-synaptic neurons is mediated by phospholipase C, interacting with the G protein to convert phosphoinositide phospholipids to 1,4,5-triphosphate and diacylglycerol (Palucha, A. and A. Pilc, Pharmacol Ther, 2007. 115 (1): p. 116-47), including the regulation of various ion channels and regulatory molecules and signal molecules , Anwyl, R., Brain Res Brain Res Rev, 1999. 29 (1): 83-120, Niswender, CM and PJ Conn, Annu Rev Pharmacol Toxicol., 50: 295-322). Therefore, several antagonists to the mGlu5 receptor, which are thought to be therapeutic targets for anxiety and depression including Parkinson's disease, Parkinson's disease, pain, addiction and fragile X syndrome, have been used to modulate the activity of the mGlu5 receptor (Lindemann, L., et al., J Pharmacol Exp Ther. 339 (2): 474-86). For example, it has been reported that pinot nocturnal may be effective in enhancing procedural memory formation and avoidance behavior in Fmr 1 knockout mice (Veloz, MF, et al., Genes Brain Behav.). In addition, PinoBall alleviates inflammatory and non-inflammatory bladder pain (Crock, LW, et al., Mol Pain. 8: p. Indeed, previous studies on pinot nocturnes have focused primarily on the therapeutic potential of various central nervous systems, including memory and pain. On the other hand, although the exact mechanism is not disclosed, the present invention is very interesting in that it is related to the invitro and the in vivo transmission of the fusion protein bound to the protein transport domain.

종합하면, 본 발명은 피노밤이 FK506BP 융합단백질의 세포 또는 뇌조직 내로의 도입을 현저히 개선시킬 수 있고, 산화 스트레스에 의해 유도되는 세포독성 및 신호 활성화에 대한 FK506BP 융합단백질의 저해효과를 강화하며, 따라서 FK506BP 융합단백질의 신경보호효과에 기여함을 밝혔다. 그러므로, 피노밤은 다양한 세포 및 조직에서 단백질 수송 도메인과 결합한 치료용 융합단백질의 수준을 높이는 용도로 매우 유용하다고 할 수 있다.
Taken together, the present invention demonstrates that pinotubim can significantly improve the introduction of FK506BP fusion protein into cells or brain tissue, enhance the cytotoxicity induced by oxidative stress and the inhibitory effect of FK506BP fusion protein on signal activation, Thus contributing to the neuroprotective effect of the FK506BP fusion protein. Therefore, it can be said that pino bam is very useful for increasing the level of therapeutic fusion proteins bound to protein transport domains in various cells and tissues.

본 발명은 치료단백질의 N-말단 및 C-말단 중 어느 한 곳 이상에 단백질 수송 도메인이 공유결합된 치료단백질 융합단백질의 세포 또는 조직 침투율을 높이는 피노밤의 용도에 관한 것이다.The present invention relates to the use of PinoBall to enhance the cell or tissue penetration rate of a therapeutic protein fusion protein in which the protein transport domain is covalently bound to at least one of the N-terminal and C-terminal of the therapeutic protein.

또한, 본 발명은 FK506 결합단백질의 N-말단 및 C-말단 중 어느 한 곳 이상에 단백질 수송 도메인이 공유결합된 FK506 결합단백질 융합단백질과 피노밤을 포함하는 뇌 허혈손상 치료용 약제학적 조성물에 관한 것이다. The present invention also relates to a pharmaceutical composition for the treatment of brain ischemic injury comprising an FK506 binding protein fusion protein in which a protein transport domain is covalently bonded to at least one of the N-terminal and C-terminal of a FK506 binding protein, will be.

뿐만 아니라, 본 발명은 FK506 결합단백질의 N-말단 및 C-말단 중 어느 한 곳 이상에 단백질 수송 도메인이 공유결합된 FK506 결합단백질 융합단백질 투여 전 피노밤을 투여하여 FK506 결합단백질 융합단백질의 세포 또는 조직내 도입율을 높이는 방법에 관한 것이다.In addition, the present invention relates to a method for producing a FK506 binding protein fusion protein, which comprises administering a Fuk506 binding protein fusion protein in which the protein transport domain is covalently bound to at least one of the N-terminal and C-terminal of the FK506 binding protein, And a method for enhancing the introduction rate in the tissue.

이때 상기 단백질 수송 도메인은 Wherein the protein transport domain is

a) 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인(hydrophobic domain), 라이신을 4개 이상 다수 포함하는 친수성 도메인(hydrophilic domain) 및 상기 두 도메인을 분리시켜 주는 스페이서(spacer)로 구성된 단백질 수송 도메인, a) a hydrophilic domain consisting of 15 to 30 amino acids and containing at least 5 tryptophan, a hydrophilic domain containing 4 or more lysines, and a spacer for separating the two domains spacer), < / RTI >

b) 6 내지 12개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 단백질 수송 도메인, b) a protein transport domain consisting of 6 to 12 amino acid residues and containing 3/4 or more arginine or lysine residues,

c) 6 내지 12개의 라이신으로 구성되는 올리고라이신 단백질 수송 도메인, c) an oligo lysine protein transport domain consisting of 6-12 lysines,

d) 6 내지 12개의 아르기닌으로 구성되는 올리고아르기닌 단백질 수송 도메인 및 d) an oligoarginine protein transport domain consisting of 6 to 12 arginines and

e) 6 내지 12개의 라이신 또는 아르기닌으로 구성되는 올리고(라이신,아르기닌) 단백질 수송 도메인 중 선택된 1종 이상임을 특징으로 한다.e) an oligo (lysine, arginine) protein transport domain consisting of from 6 to 12 lysine or arginine.

또한, 상기 융합단백질은 서열번호 4, 서열번호 6, 서열번호 8, 서열번호 10, 서열번호 12 및 서열번호 14 중 선택된 1종인 것을 특징으로 한다.
Also, the fusion protein is one selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO:

FK506BP 융합단백질 및 피노밤을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에서 통상적으로 허용되는 담체와 함께 배합하여 통상적인 방법에 의해 경구 또는 주사 형태로 제형화할 수 있다. 경구용 조성물로는 예를 들면 정제 및 젤라틴 캡슐이 있으며, 이들은 활성 성분 이외에도 희석제(예: 락토스, 덱스트로스, 수크로스, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활탁제(예: 실리카, 탤크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)을 함유하고, 정제는 또한 결합제(예: 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로스, 나트륨 카복시메틸셀룰로스 및/또는 폴리비닐피롤리돈)를 함유하며, 경우에 따라서 붕해제(예: 전분, 한천, 알긴산 또는 그의 나트륨염) 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유하는 것이 바람직하다. 주사용 조성물은 등장성 수용액 또는 현탁액이 바람직하고, 언급한 조성물은 멸균되고/되거나 보조제(예: 방부제, 안정화제, 습윤제 또는 유화제 용액 촉진제, 삼투압 조절을 위함 염/또는 완충제)를 함유한다. 또한 이들은 기타 치료적으로 유용한 물질을 함유할 수 있다.The pharmaceutical composition containing the FK506BP fusion protein and the pinonuclide as an active ingredient can be formulated together with a carrier that is conventionally accepted in the pharmaceutical field and can be formulated orally or injected by a conventional method. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

이와 같이 제조된 약제학적 제제는 목적하는 바에 따라 경구로 투여하거나, 비경구 방식 즉, 정맥 내 , 피하, 복강 내 투여 또는 국소적용할 수 있다. 용량은 일일 투여량 0.0001~100㎎/㎏을 1 내지 수회에 나누어 투여할 수 있다. 특정 환자에 대한 투여용량 수준은 환자의 체중, 연령, 성별, 건강상태, 투여시간, 투여방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single daily dose of 0.0001 to 100 mg / kg dividedly in several doses. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

나아가, 본 발명은 상기 FK506BP 융합단백질 및 피노밤을 유효성분으로 하고 약학적으로 허용되는 담체를 포함하는 것을 특징으로 하는, 뇌허혈과 같은 신경질환의 예방과 치료에 유용한 약제학적 조성물을 제공한다.
Further, the present invention provides a pharmaceutical composition useful for the prophylaxis and treatment of neurological diseases such as cerebral ischemia, which comprises the above FK506BP fusion protein and a pharmaceutically acceptable carrier containing as an active ingredient the above-described FN506BP fusion protein.

본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다.The definitions of the main terms used in the description of the present invention and the like are as follows.

"FK506BP 융합단백질"이란 단백질 수송 도메인과 FK506BP를 포함하며, 단백질 수송 도메인과 화물 분자(cargo molecule, 즉 본 발명에서는 FK506BP를 의미함)의 유전적 융합이나 화학 결합으로 형성된 공유결합 복합체를 의미한다. 본 명세서와 도면에서는 "FK506 결합단백질 융합단백질"과 혼용하였고, 구체적인 일 실시예로서 "PEP-1-FK506BP"는 FK506BP 융합단백질 중 FK506BP의 N-말단에 PEP-1 단백질 수송 도메인이 결합된 것을 말한다."FK506BP fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and FK506BP and a protein transport domain and a cargo molecule (i.e., FK506BP in the present invention). As used herein, the term "PEP-1-FK506BP" refers to FK506BP fusion protein in which the PEP-1 protein transport domain is bound to the N-terminus of FK506BP .

또한, 상기 "유전적 융합"이란 단백질을 코딩하는 DNA 서열의 유전적 발현을 통해서 형성된 선형, 공유결합으로 이루어진 연결을 의미한다.In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

또한, "표적 세포"란 수송 도메인에 의해 화물 분자가 전달되는 세포를 의미하는 것으로서 즉, 표적 세포는 체내 세포, 다시 말하여 살아있는 동물 또는 인간의 장기 또는 조직을 구성하는 세포 또는 살아있는 동물 또는 인간에서 발견되는 미생물을 포함하는 의미이다. 또한, 표적 세포는 체외 세포, 즉 배양된 동물 세포, 인체 세포 또는 미생물을 포함하는 의미이다. 구체적으로 본 명세서에서 표적 세포는 뇌세포 등을 의미한다.The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living animal or a cell or living animal It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in the present specification, the target cell means a brain cell or the like.

본 명세서의 "단백질 수송 도메인"은 화물 분자(목표 단백질) 펩타이드 또는 단백질과 공유결합을 이루어 별도의 수용체나 운반체, 에너지를 필요로 하지 않고 상기 펩타이드나 단백질을 세포 내로 도입시킬 수 있는 것을 말하며, 예를 들면 PEP-1 펩타이드(서열번호 1)를 말한다.As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, PEP-1 peptide (SEQ ID NO: 1).

본 명세서의 "목표 단백질"은 PEP-1 단백질 수송 도메인과 공유결합을 이루어 세포 내로 도입되어 활성을 나타내는 분자를 의미한다. "화물 분자"와 동일한 의미이다. 또한, 본 명세서에서는 "치료용 단백질", "치료단백질"도 동일한 의미로 사용하였다. As used herein, the term "target protein" refers to a molecule that is covalently bound to a PEP-1 protein transduction domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule". In this specification, the terms "therapeutic protein" and "therapeutic protein" are used interchangeably.

본 명세서의 "치료단백질" 또는 "치료용 단백질"이란 질병의 치료를 목적으로 체내, 세포내, 조직내로 투여되는 단백질을 말한다.As used herein, the term " therapeutic protein "or" therapeutic protein "refers to a protein administered into the body, intracellularly, or tissues for the purpose of treatment of diseases.

또한, 본 명세서에서는 단백질 또는 펩타이드를 세포 내로 "도입"시키는 것에 대하여 "형질도입", "운반", "침투", "수송", "전달", "투과", "통과"한다는 표현들과 혼용하였다.
Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

본 발명에서 단백질 수송 도메인으로는 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인, 라이신을 다수 포함하는 친수성 도메인 및 상기 두 도메인을 분리시켜 주는 스페이서로 구성된 단백질 수송 도메인, 6 내지 12개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인, 6 내지 12개의 라이신으로 구성되는 올리고라이신 수송 도메인, 6 내지 12개의 아르기닌으로 구성되는 올리고아르기닌 수송 도메인 또는 6 내지 12개의 라이신 또는 아르기닌으로 구성되는 올리고(라이신,아르기닌) 수송 도메인을 들 수 있다. 또한, 목표 단백질(화물 분자)는 FK506BP이다. 상기 단백질 수송 도메인 및 목표 단백질은 silent change에 따라 서열 내에서 하나 이상의 아미노산이 기능적으로 동등하게 작용하는 유사한 극성의 다른 아미노산(들)로 치환될 수 있다. 서열 내 아미노산 치환은 그 아미노산이 속하는 클래스의 다른 구성원들로부터 선택될 수 있다. 예컨대, 소수성 아미노산 분류는 알라닌, 발린, 류이신, 이소류이신, 페닐알라닌, 발린, 트립토판, 프롤린 및 메티오닌을 포함한다. 극성 중성 아미노산은 글리신, 세린, 트레오닌, 시스테인, 티로신, 아스파라긴 및 글루타민을 포함한다. 양성 염기성 아미노산은 아르기닌, 라이신 및 히스티딘을 포함한다. 음성전하를 띤 산성 아미노산은 아스파르트산 및 글루탐산을 포함한다. 또한, 본 발명의 융합단백질과 아미노산 서열간의 일정 범위의 상동성 예컨대 85-100% 범위 내에서 동일·유사한 생물학적 활성을 갖는 절편 또는 이들의 유도체들도 본 발명의 권리범위에 포함된다.In the present invention, the protein transport domain is a protein transport domain composed of 15 to 30 amino acids and composed of a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing many lysines, and a spacer separating the two domains, A transport domain comprising 6 to 12 amino acid residues and comprising ¾ or more arginine or a lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoroginalin transport domain comprising 6 to 12 arginines, Or an oligo (lysine, arginine) transport domain consisting of 12 lysine or arginine. In addition, the target protein (cargo molecule) is FK506BP. The protein transport domain and the target protein may be replaced by other amino acid (s) of similar polarity in which one or more amino acids functionally equivalently function in the sequence according to the silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, such as 85-100%, between the fusion protein of the present invention and the amino acid sequence are included in the scope of the present invention.

또한, 본 발명은 상기 세포 도입성 FK506BP 융합단백질이 서열번호 4, 서열번호 6, 서열번호 8, 서열번호 10, 서열번호 12 또는 서열번호 14의 아미노산 서열을 갖는 것을 특징으로 한다.In addition, the present invention is characterized in that the cell-inducing FK506BP fusion protein has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO:

또한, 본 발명은 상기 단백질 수송도메인(protein transducing domain; "PTD")이 FK506BP 단백질의 카복시 말단과 아미노 말단의 일측 또는 양측에 공유결합된 것을 특징으로 한다.
Also, the present invention is characterized in that the protein transducing domain ("PTD") is covalently bonded to one or both of the carboxy terminal and the amino terminal of the FK506BP protein.

피노밤과 FK506BP 융합단백질을 조합하여 처리하면 인비보 및 인비트로에서 뇌세포 또는 뇌 조직으로 FK506BP 융합단백질 침투효율이 높아졌다.The combination of FinoBO and FK506BP fusion protein increased the penetration efficiency of FK506BP fusion protein into brain cells or brain tissue in Invivo and Invitro.

또한, 피노밤과 FK506BP 융합단백질을 조합하여 처리하면 FK506BP 융합단백질의 뇌허혈 손상에 대한 보호효과가 높아졌다.In addition, the combination treatment of the FO506BP fusion protein with the pinotain enhances the protective effect of the FK506BP fusion protein against the cerebral ischemic injury.

도 1은 C6 세포에서 과산화수소로 유도되는 세포독성 및 DNA 손상에 대한 피노밤과 FK506BP 융합단백질의 효과를 나타낸다. (A) 피노밤의 화학구조, (B) 일 실시예에 의한 FK506BP의 모식도. His 서열은 FK506BP 융합단백질과 Tat-GFP의 정제 및 탐지에 이용된다. (C) C6 세포 생존에 미치는 피노밤의 영향. C6 세포를 다양한 농도의 피노밤 (50~250 ng/㎖)으로 18시간 동안 처리한 후 세포 생존율은 MTT 분석법으로 평가하였다. (D) 과산화수소로 유도된 세포독성에 대해 피노밤으로 선처리한 후 FK506BP 융합단백질의 효과. C6 세포는 FK506BP 융합단백질 처리하기 한 시간 전 피노밤 (150 ng/㎖)으로 처리한 후 과산화수소 (1 mM)로 18시간 동안 처리하였다. 세포 생존율은 MTT 분석법으로 평가하였다. (E) 피노밤 존재하에서 H2O2로 유도되는 DNA 손상에 대한 FK506BP 융합단백질의 효과. C6 세포는 피노밤 (150 ng/㎖)으로 한 시간 선처리한 후 FK506BP 융합단백질 (2.0 μM)로 처리하였다. 세포를 H2O2 (1mM)로 6.5시간 처리한 후 TUNEL 염색한 다음 모든 시료의 DNA 손상을 비교하였다.
도 2는 H2O2로 유도되는 Akt와 MAPK 활성화에 대한 피노밤과 FK506BP 융합단백질 공동처리의 효과를 나타낸다. C6 세포는 피노밤 (150 ng/㎖)으로 한 시간 선처리한 후 FK506BP 융합단백질 (2.0 μM)로 처리하였다. 그 후 세포내 Akt/p-Akt, p38/p-p38 및 Erk1/2 /p-Erk1/2 수준을 평가하기 위해 H2O2 처리 후 각각 8분, 25분 및 35분에 세포를 하비스트하였다. (A) p-p38/ p38, p-Akt/Akt 및 p-Erk1/2/Erk1/2 수준을 나타내는 대표적인 웨스턴 블랏팅 데이타. (B) 각 수준은 밀도계로 정량하였다. 막대그래프는 세 번의 독립적 실험에서 얻은 평균 ± 표준편차이다. 데이타는 Student's t test로 분석하였다. *, p<0.05 및 **, p<0.01는 H2O2 처리군과 다른 군 간의 유의한 차이를 나타낸다.
도 3은 C6 악성신경교종 세포 내로 FK506BP 융합단백질의 도입에 대한 피노밤의 효과를 나타낸다. C6 세포는 피노밤 (150 ng/㎖)을 넣고 또는 넣지 않고 한 시간 선처리한 후 FK506BP 융합단백질에 노출하였다. (A) FK506BP 융합단백질 세포도입의 투여량 의존도를 조사하기 위해 처리하는 두 단백질 농도를 0.5, 1.0, 1.5 및 2.0 μM로 변화시켰다. (B) 또한 FK506BP 융합단백질 세포도입의 시간 의존성을 알아보기 위해 처리 시간을 5~30분으로 변화시켰다. FK506BP 융합단백질과 함께 배양한 후 세포는 트립신 처리하고 PBS로 세 번 세척하였다. 세포 용균액은 His 항체 및 2차 항체를 이용하여 웨스턴 블랏 분석하였다. 웨스턴 블랏의 상대적 띠 강도는 밀도계로 측정하였다. (C, D) 막대그래프는 세 번의 독립적 실험에서 얻은 평균 ± 표준편차를 나타낸다. 데이타는 Student's t test로 분석하였다. *, p<0.05 및 **, p<0.01는 H2O2 처리군과 다른 군 간의 유의한 차이를 나타낸다. (E) 피노밤 존재 하에 C6 세포에서 FK506BP 융합단백질의 분포. C6 세포는 피노밤 (150 ng/㎖)으로 한 시간 선처리한 후 FK506BP 융합단백질 (2.0 μM)로 한 시간 처리하였다. 세포는 His 및 Alexafluor 488-결합된 2차 항체로 면역염색하였다.
도 4는 피노밤이 뇌조직으로 FK506BP 융합단백질 인비보 도입을 증가시킴으로써 CA1 부분의 FK506BP 융합단백질의 신경보호효과를 높임을 보여준다. (A) 뇌조직 내로의 FK506BP 세포도입에 미치는 피노밤의 영향. 피노밤 (150 ㎍/㎏)은 몽골리안 저빌 (n=7/군)에 피하주사하였고 한 시간 후 FK506BP 융합단백질을 200 ㎍/㎏ 주사하였다. 여섯 시간 후 각 군의 저빌 뇌를 모았다. His 항체 및 2차 항체를 이용하여 해마를 조직학적으로 분석하였다. (B) 일시적 대뇌 전뇌 허혈 모델의 CA1 부분에 대한 FK506BP 융합단백질의 신경보호효과. 피노밤 (150 ㎍/㎏)은 몽골리안 저빌 (n=7/군)에 피하주사하였고 한 시간 후 FK506BP 융합단백질을 200 ㎍/㎏ 주사하였다. 저빌에 일시적 전뇌허혈 손상을 유도한 후 여섯 시간 지나 각 군의 저빌 뇌를 모았다. 저빌의 뇌 절편은 크레실 바이올렛으로 염색하여 해마 CA1 부분의 신경생존율을 평가하였다.Figure 1 shows the effect of pinotubic and FK506BP fusion protein on cytotoxicity and DNA damage induced by hydrogen peroxide in C6 cells. (A) the chemical structure of the pinot nois, (B) a schematic diagram of FK506BP according to one embodiment. His sequence is used for the purification and detection of FK506BP fusion protein and Tat-GFP. (C) Effect of Pinotubemia on C6 cell survival. C6 cells were treated with various concentrations of pinot (50-250 ng / ml) for 18 hours, and cell viability was evaluated by MTT assay. (D) Effect of FK506BP fusion protein after pre-treatment with PinoBum for hydrogen peroxide-induced cytotoxicity. C6 cells were treated with FN506BP fusion protein one hour before treatment with FN506BP fusion protein (150 ng / ml) and then treated with hydrogen peroxide (1 mM) for 18 hours. Cell viability was assessed by MTT assay. (E) Effect of FK506BP fusion protein on DNA damage induced by H 2 O 2 in the presence of pinonobyte. C6 cells were pretreated with pinonut (150 ng / ml) for one hour and then treated with FK506BP fusion protein (2.0 μM). Cells were treated with H 2 O 2 (1 mM) for 6.5 h and then stained with TUNEL.
Fig. 2 shows the effect of co-treatment of finoBak and FK506BP fusion protein on Akt and MAPK activation induced by H 2 O 2 . C6 cells were pretreated with pinonut (150 ng / ml) for one hour and then treated with FK506BP fusion protein (2.0 μM). Cells were harvested at 8, 25 and 35 minutes after H 2 O 2 treatment to assess Akt / p-Akt, p38 / p-p38 and Erk1 / 2 / p-Erk1 / . (A) Representative Western blotting data representing p-p38 / p38, p-Akt / Akt and p-Erk1 / 2 / Erk1 / 2 levels. (B) Each level was quantified as a density meter. The bar graph is the mean ± standard deviation from three independent experiments. Data were analyzed by Student's t test. *, p <0.05 and **, p <0.01 show significant differences between the H 2 O 2 treated group and the other groups.
Figure 3 shows the effect of pinotubic on the introduction of FK506BP fusion protein into C6 malignant glioma cells. C6 cells were exposed to FK506BP fusion protein after pre-treatment with and without pinotubicin (150 ng / ml) for one hour. (A) To investigate the dose dependence of FK506BP fusion protein cell introduction, the two protein concentrations to be treated were varied to 0.5, 1.0, 1.5 and 2.0 μM. (B) In order to examine the time dependence of FK506BP fusion protein cell introduction, the treatment time was changed from 5 to 30 minutes. After incubation with the FK506BP fusion protein, cells were trypsinized and washed three times with PBS. The cell lysate was subjected to Western blot analysis using His antibody and secondary antibody. The relative band strength of the western blot was measured by density meter. (C, D) The bar graph shows the mean ± standard deviation from three independent experiments. Data were analyzed by Student's t test. *, p <0.05 and **, p <0.01 show significant differences between the H 2 O 2 treated group and the other groups. (E) Distribution of FK506BP fusion protein in C6 cells in the presence of PinoBall. C6 cells were pretreated for one hour with PinoBy (150 ng / ml) and treated with FK506BP fusion protein (2.0 μM) for one hour. Cells were immunostained with His and Alexafluor 488-conjugated secondary antibodies.
Figure 4 shows that PinoBy enhances the neuroprotective effect of the FK506BP fusion protein of the CA1 portion by increasing the FK506BP fusion protein invivo integration into brain tissue. (A) Influence of pinotonin on the induction of FK506BP cells into brain tissue. Pine nodule (150 ㎍ / ㎏) was subcutaneously injected into Mongolian zebilla (n = 7 / group) and injected with 200 ㎍ / kg of FK506BP fusion protein one hour later. Six hours later, the lower brain of each group was collected. His and secondary antibodies were used to analyze the hippocampus histologically. (B) The neuroprotective effect of the FK506BP fusion protein on the CA1 portion of the transient cerebral frontal ischemic model. Pine nodule (150 ㎍ / ㎏) was subcutaneously injected into Mongolian zebilla (n = 7 / group) and injected with 200 ㎍ / kg of FK506BP fusion protein one hour later. Six hours after inducing transient total cerebral ischemic injury, the lower brain of each group was collected. The brain slices of Zvil were stained with cresyl violet to evaluate the nerve survival rate of hippocampal CA1 portion.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 본 발명을 좀 더 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예의 기재 범위 내로 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 자명하다. 특히, 각 실험예의 결과로서 구체적인 실시예에서 제조한 여러 가지 융합단백질 중 PEP-1-FK506BP 융합단백질을 시료로 한 데이터를 기재하였으나, 그 외의 융합단백질들 또한 PEP-1-FK506BP 융합단백질을 시료로 한 결과와 유사한 정도의 결과를 나타내었음을 밝힌다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are intended to further illustrate the present invention and that the scope of the present invention is not limited by the scope of these embodiments. Particularly, as a result of each experimental example, data on the PEP-1-FK506BP fusion protein among the various fusion proteins prepared in the specific examples have been described, but other fusion proteins have also been used as the samples for the PEP-1-FK506BP fusion protein The results are similar to those of the one result.

<재료><Material>

제한 효소와 T4 DNA 리가아제(ligase)는 Promega(USA)에서 구입하였고, Pfu 폴리머라아제는 stratagene(USA)에서 구입하였다. Tat 올리고뉴클레오타이드는 Gibco BRL custom primer(USA)에서 합성하였다. IPTG는 Duchefa(Netherland)에서 구입하였다. pET-15b와 BL21(DE3) 플라스미드는 Novagen(USA)에서 구입하였고, Ni-니트릴로-트리아세틱 애시드 세파로즈 슈퍼플로우는 Qiagen(Germany)에서 구입하였다. 사람 FK506 결합 단백질(FK506BP) cDNA는 PCR 방법으로 사람 간 cDNA 라이브러리에서 분리하였다. 이외 모든 시약은 특급 제품을 이용하였다.
Restriction enzymes and T4 DNA ligase were purchased from Promega (USA), and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized with Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilo-triacetic acid Sepharose Superflow was purchased from Qiagen (Germany). Human FK506 binding protein (FK506BP) cDNA was isolated from human liver cDNA library by PCR method. All of the other reagents were made by high-grade products.

<실시예 1: PEP-1-FK506BP 융합단백질의 발현벡터 제조 및 형질변환>&Lt; Example 1: Preparation of expression vector of PEP-1-FK506BP fusion protein and transformation >

기능을 가진 단백질을 세포 내로 침투시키는 기술을 개발하기 위하여 세포 내로 목표 단백질을 전달할 수 있는 융합단백질 발현벡터를 제조하였고, PEP-1 펩타이드가 단백질을 세포 내로 전달하는 능력을 용이하게 분석하기 위하여 사람 FK506BP를 선택하였다.In order to analyze the ability of PEP-1 peptide to transfer the protein into cells, a fusion protein expression vector was constructed which can express the protein FK506BP Respectively.

먼저, PEP-1-FK506BP 융합단백질을 생산하기 위해 PEP-1 펩타이드(KETWWE TWWTEW SQP KKKRKV)(서열번호 1)가 포함된 pET-PEP 발현벡터를 제조하였다. PEP-1 펩타이드에 해당하는 두 종류의 올리고뉴클레오타이드(윗 사슬, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3'(서열번호 15); 아랫 사슬, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3'(서열번호 16))를 NdeⅠ-XhoⅠ 제한효소로 자른 pET-15b에 연결(ligation)하여 삽입하였다. 이어, 사람 FK506BP의 cDNA의 서열을 기본으로 하여 두 종류의 올리고뉴클레오타이드를 합성하였다. 정방향 프라이머는 5’-CTCGAGATGGGAGTGC AGGTGGAAACCATC-3'(서열번호 17)로 XhoⅠ 제한 부위를 지니고 있으며 역방향 프라이머는 5'-GGATCCTCATTCCAGTTTTAGAAGCTCCAC-3'(서열번호 18)로 BamHⅠ 제한 부위를 갖고 있다.First, PEP-1-FK506BP A pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV) (SEQ ID NO: 1) was prepared to produce a fusion protein. Two kinds of oligonucleotides corresponding to PEP-1 peptide nucleotide (top chain, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 '(SEQ ID NO: 15); lower chain, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3' (SEQ ID NO: 16)) the NdeⅠ - XhoⅠ limit And inserted into pET-15b cut with an enzyme. Two kinds of oligonucleotides were synthesized based on the sequence of human FK506BP cDNA. Forward primer has a 5'-CTCGAGATGGGAGTGC AGGTGGAAACCATC-3 '(SEQ ID NO: 17), and to have a restriction site XhoⅠ reverse primer 5'-GGATCCTCATTCCAGTTTTAGAAGCTCCAC-3' BamHⅠ restriction site (SEQ ID NO: 18).

중합효소 연쇄반응(PCR)은 온열 순환반응기(Perkin-Elmer, model 9600)에서 수행하였다. 반응 혼합액을 50㎕ 실리콘 튜브에 넣고 94℃에서 5분간 가열하였다. PCR 반응을 수행하였다. PCR 수행 후, 아가로즈 젤 전기영동으로 분리하여 반응물을 분리하고 이것을 TA 클로닝 벡터(Invitrogen, Sandiego, USA)에 연결(ligation)한 다음, 클로닝이 용이한 컴피턴트 세포(competent cell)에 형질변환시키고 형질변환된 박테리아로부터 플라스미드를 알칼리 용균법(alkaline lysis method)으로 분리하였다. 사람 FK506BP cDNA가 포함된 TA 벡터를 XhoⅠ과 BamHⅠ로 절단한 다음 PEP 발현 벡터에 삽입하였다. PEP-1-FK506BP로 형질변환된 E. coli BL21(DE3)를 선택한 다음, 콜로니를 100ml LB 배지에 접종하고 IPTG(0.5mM)를 배지 내에 첨가하여 재조합된 PEP-1-FK506BP 융합단백질의 과대발현을 유도하였다. 과대발현된 PEP-1-FK506BP 융합단백질은 SDS-PAGE와 웨스턴 블랏 분석으로 확인하였다.Polymerase chain reaction (PCR) was performed in a warm circulation reactor (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 占 퐇 silicone tube and heated at 94 占 폚 for 5 minutes. PCR reaction was performed. After the PCR, the reaction product was separated by agarose gel electrophoresis, and the reaction product was ligated to a TA cloning vector (Invitrogen, Sandiego, USA), transformed into competent cells that were easy to clone Plasmids were isolated from the transformed bacteria by an alkaline lysis method. Cutting the TA vector containing the human cDNA in FK506BP XhoⅠ BamHⅠ and then was inserted into the PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-FK506BP was selected and colonies were inoculated into 100 ml LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of the recombinant PEP-1-FK506BP fusion protein Lt; / RTI &gt; Overexpressed PEP-1-FK506BP fusion protein was confirmed by SDS-PAGE and Western blot analysis.

상기 방법을 변형하여 FK506BP-PEP-1 융합단백질 및 PEP-1-FK506BP-PEP-1 융합단백질을 제조하였다.
The above method was modified to prepare FK506BP-PEP-1 fusion protein and PEP-1-FK506BP-PEP-1 fusion protein.

<실시예 2: PEP-1-FK506BP 융합단백질의 발현 및 정제>&Lt; Example 2: Expression and purification of PEP-1-FK506BP fusion protein &

상기 실시예 1과 같이 제조한 인간 FK506BP cDNA가 PEP-1-FK506BP 형태로 포함되어 있는 E. coli BL21(DE3)세포를 암피실린이 포함된 LB 배지에 넣고 37℃에서 200rpm으로 교반하며 배양하였다. 배양액 내의 박테리아 농도가 O.D600 = 0.5～1.0을 나타낼 때 IPTG를 배지 내에 첨가하여 최종농도가 0.5와 1mM 되게 한 다음 30℃에서 12시간을 더 배양하였다. 배양한 세포를 원심분리하여 모은 뒤 5ml 결합완충액(5mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)를 넣고 초음파 분쇄기로 분쇄(sonication)하였다. 원심분리하여 상층액을 즉시 Ni2+-니트릴로 트리아세틱 애시드 세파로즈 슈퍼 플로우(Ni2+-nitrilotriacetic acid sepharose super flow) 컬럼에 부하하고 10배 부피의 결합 완충액과 6배 부피의 세척 완충액(60mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)으로 세척한 다음 용출 완충액(1M 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)로 융합단백질을 용출하였다. 이어, 융합단백질이 포함된 분획들을 모아 PD-10 컬럼 크로마토그래피를 수행하여 분획 중에 포함된 염분을 제거하였다. E. coli BL21 (DE3) cells containing the human FK506BP cDNA in the form of PEP-1-FK506BP prepared as in Example 1 were placed in LB medium containing ampicillin and cultured at 37 ° C with stirring at 200 rpm. When the bacterial concentration in the culture medium showed OD 600 = 0.5 to 1.0, IPTG was added to the medium to a final concentration of 0.5 and 1 mM, followed by further incubation at 30 ° C for 12 hours. The cultured cells were collected by centrifugation, and then 5 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) was added and sonication was carried out using an ultrasonic grinder. The supernatant was immediately centrifuged Ni 2+ - acrylonitrile triazol setik acid Sepharose Super flow (Ni 2+ -nitrilotriacetic acid sepharose super flow ) load the column and binding buffer of 10 times by volume with 6-fold volume wash buffer (60mM of Imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and then eluted the fusion protein with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Then, the fractions containing the fusion protein were collected and subjected to PD-10 column chromatography to remove the salt contained in the fraction.

정제된 단백질 농도는 우혈청 알부민을 표준물질로 사용하여 브래드포드(Bradford) 방법으로 측정하였다.The purified protein concentration was measured by the Bradford method using bovine serum albumin as a standard.

상기 방법과 동일한 방법으로 FK506BP-PEP-1 융합단백질 및 PEP-1-FK506BP-PEP-1 융합단백질을 과대발현 및 정제하였다.
The FK506BP-PEP-1 fusion protein and the PEP-1-FK506BP-PEP-1 fusion protein were overexpressed and purified in the same manner as described above.

<실시예 3: Tat-FK506BP 융합단백질 발현벡터의 제조 및 형질변환>&Lt; Example 3: Preparation of Tat-FK506BP fusion protein expression vector and transformation >

Tat-FK506BP 융합단백질을 과다 발현시키기 위하여 FK506BP, HIV-1 Tat의 형질 도입부위(Tat49-57) 및 6개의 히스티딘에 대한 cDNA가 연속적으로 포함되어 있는 pET-Tat-FK506BP 발현 벡터를 제조하였다. 먼저, HIV-1 Tat의 기본 도메인(basic domain, 즉 아미노산 49-57)이 포함된 pET-Tat 발현벡터를 만들었다. Tat 기본 도메인에 해당하는 두 종류의 올리고뉴클레오타이드(상위 쇄(top strand), 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3'(서열번호 19); 하위 쇄(bottom strand), 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3'(서열번호 20))를 NdeI-XhoI 제한효소로 자른 pET15b에 결찰(ligation)하여 삽입하였다. 이어 인간 FK506BP의 cDNA의 서열을 기본으로 하여 2종류의 올리고뉴클레오타이드를 합성하였다. 정방향 프라이머(Forward primer)는 위 실시예 1의 서열번호 17로 Xho I 제한부위를 지니고 있으며 역방향 프라이머(reverse primer)는 위 실시예 1의 서열번호 18로 BamH I제한부위를 갖고 있다.To overexpress the Tat-FK506BP fusion protein, a pET-Tat-FK506BP expression vector was prepared in which FK506BP, HIV-1 Tat transduction site (Tat49-57) and cDNA for 6 histidine were consecutively contained. First, a pET-Tat expression vector containing the basic domain of HIV-1 Tat (i.e., amino acids 49-57) was prepared. (Top strand, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 '(SEQ ID NO: 19); bottom strand, 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3' (SEQ ID NO: ) Was ligated to pET15b cut with NdeI-XhoI restriction enzyme and inserted. Two kinds of oligonucleotides were synthesized based on the sequence of human FK506BP cDNA. The forward primer has an Xho I restriction site in SEQ ID NO: 17 in Example 1 and the reverse primer has a BamH I restriction site in SEQ ID NO: 18 in Example 1 above.

중합효소 연쇄반응(PCR)은 thermal cycler(Perkin-Elmer, model 9600)에서 수행하였으며, 반응 혼합액을 50 ㎕ 실리콘 튜브(siliconized reaction tube)에 넣고 94℃에서 5 분간 가열하였다. PCR 반응은 94℃에서 40초간 30회의 연장(extension), 54℃에서 1분간 변성(denaturation), 70℃에서 3분간 어닐링(annealing), 그리고 72℃에서 10분, 20℃에서 5분간 최종 연장(final extension)을 유도하였다. PCR 수행후 아가로즈 젤 전기이동(agarose gel electrophoresis)으로 분리하여 반응물을 분리하고 이것을 TA 클로닝 벡터(Invitrogen, Sandiego, USA)에 결찰하였다. 이어 이 벡터를 형질변환용 세포(competent cell)에 형질변환(transformation)시키고 형질변환된 박테리아로부터 플라스미드를 알칼리 용균 방법(alkaline lysis method)으로 분리하였다[Sambrook, J., Fritsch, F.E. and Maniatis, T(1989) Molecular cloning, Cold spring harbor laboratory press, Cold spring harbor]. 인간 FK506BP cDNA가 포함된 TA 벡터를Xho I과BamH I로 절단한 다음 pET-15b 및 pET-15b-Tat 발현 벡터에 삽입하였다. 벡터의 발현은 T7 프로모터와 lacO-오퍼레이터의 조절 하에 있다.Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600) and the reaction mixture was placed in a 50 μl siliconized reaction tube and heated at 94 ° C for 5 minutes. The PCR reaction was carried out at 94 ° C for 30 seconds, 30 minutes extension, 54 ° C for 1 minute, 70 ° C for 3 minutes, annealing at 72 ° C for 10 minutes, final extension). After the PCR, the reaction product was separated by agarose gel electrophoresis and ligated to TA cloning vector (Invitrogen, Sandiego, USA). This vector was then transformed into competent cells and the plasmid was isolated from the transformed bacteria by an alkaline lysis method (Sambrook, J., Fritsch, F.E. and Maniatis, T (1989) Molecular cloning, Cold spring harbor laboratory press, Cold spring harbor]. The TA vector containing human FK506BP cDNA was digested with Xho I and BamH I and inserted into the pET-15b and pET-15b-Tat expression vectors. Expression of the vector is under the control of the T7 promoter and lacO-operator.

pET-Tat-FK506BP로 형질변환된 E. coli BL21(DE3)를 선택한 다음, 콜로니(colony)를 100ml LB 배지에 접종하고 IPTG (0.5 mM)를 배지 내에 첨가하여 과다 발현을 유도하였다. IPTG로 융합단백질의 과다 발현을 유도한 대장균 세포를 4℃에서 초음파처리(sonication)로 파쇄한 다음, 원심분리하여 상청액의 단백질을 15% SDS-폴리아크릴아미드 젤 전기이동방법으로 분리하였다. 과다 발현된 SOD와 Tat-SOD는 SDS-폴리아크릴아미드 젤 전기이동과 웨스턴 블랏 분석으로 확인하였다.E. coli BL21 (DE3) transformed with pET-Tat-FK506BP was selected and colony was inoculated into 100 ml of LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression. Escherichia coli cells inducing overexpression of fusion proteins with IPTG were disrupted by sonication at 4 ° C and then centrifuged to separate proteins of the supernatant by 15% SDS-polyacrylamide gel electrophoresis. Overexpressed SOD and Tat-SOD were confirmed by SDS-polyacrylamide gel electrophoresis and Western blot analysis.

상기 방법을 변형하여 FK506BP-Tat 융합단백질 및 Tat-FK506BP-Tat 융합단백질을 제조하였다.
The above method was modified to prepare FK506BP-Tat fusion protein and Tat-FK506BP-Tat fusion protein.

<실시예 4: Tat-FK506BP 융합단백질 과다 발현 및 정제>&Lt; Example 4: Overexpression and purification of Tat-FK506BP fusion protein >

형질전환된 E. coli BL21을 앰피실린이 포함된 LB 배지에 넣고 37℃에서 200rpm으로 교반하며 배양하였다. 배양액 내의 박테리아 농도가 O.D 600 = 0.5∼1.0를 나타낼 때 IPTG를 배지 내에 첨가하여 최종농도가 0.5mM가 되게 한 다음 3시간을 더 배양하였다. 배양한 세포를 원심분리하여 모은 뒤 5ml 결합 완충용액(binding buffer; 5 mM 이미다졸, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)을 넣고 초음파처리(sonication)하였다. The transformed E. coli BL21 was added to LB medium containing ampicillin and cultured at 37 DEG C with stirring at 200 rpm. When the bacterial concentration in the culture medium exhibited OD 600 = 0.5 to 1.0, IPTG was added to the culture medium to a final concentration of 0.5 mM, followed by further incubation for 3 hours. The cultured cells were harvested by centrifugation and sonicated with 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9).

원심분리하여 상청액을 즉시 2.5 ml Ni 2+ -니트릴로트리아세틱 애시드 세파로즈 컬럼(nitrilotriacetic acid sepharose column)에 부하하고 10배 부피의 결합 완충용액과 6배 부피의 세척 완충액(washing buffer; 60mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)으로 세척한 다음 용출 완충액(elution buffer; 1M 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)로 융합단백질을 용출하였다. 이어 융합단백질이 포함된 분획들을 모아 세파덱스 G-15 컬럼 크로마토그래피를 수행하여 분획 중에 포함된 염분을 제거하였다. 융합단백질은 N-말단에 6개의 히스티딘을 포함하고 있기 때문에 고정 금속-킬레이트 친화 크로마토그래피(immobilized metal-chelate affinity chromatography) 단일 단계로 융합단백질을 거의 순수하게(순도 > 90%) 정제하였다. 분획의 단백질 농도는 우혈청 알부민을 표준물질로 사용하여 브래드포드(Bradford) 방법으로 측정하였다[Bradford, M.A. (1976) Anal. Biochem. 72, 248-254].The supernatant was immediately centrifuged and loaded onto a 2.5 ml Ni 2+ -nitrile nitrilotriacetic acid sepharose column and washed with a 10-fold volume of binding buffer and 6-fold volume of wash buffer (60 mM imidazole , 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and eluted the fusion protein with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Fractions containing the fusion protein were then collected and subjected to Sephadex G-15 column chromatography to remove the salt contained in the fraction. Because the fusion protein contains six histidines at the N-terminus, the fusion protein was purified to near pure (purity> 90%) in a single step of immobilized metal-chelate affinity chromatography. The protein concentration of the fraction was determined by the Bradford method using bovine serum albumin as a standard (Bradford, M.A. (1976) Anal. Biochem. 72, 248-254].

상기 방법과 동일한 방법으로 FK506BP-Tat 융합단백질 및 Tat-FK506BP-Tat 융합단백질을 과대발현 및 정제하였다.
The FK506BP-Tat fusion protein and the Tat-FK506BP-Tat fusion protein were overexpressed and purified in the same manner as described above.

<실시예 5: 시료 제조>&Lt; Example 5: Preparation of sample >

상기 실시예 2 및 4에서 정제된 각종 FK506BP 융합단백질을 생리식염수에 5㎍/5㎕로 용해하여 이후 실험에 시료로 사용하였다.
The various FK506BP fusion proteins purified in Examples 2 and 4 were dissolved in physiological saline at 5 占 / / 5 占 퐇 and used as a sample in the following experiment.

세포배양Cell culture

C6 랫트 성상세포종 (astrocytoma) 세포는 10% 가열-불활성화 우태혈청 및 항생제 (100 ㎍/㎖ 스트렙토마이신, 100 Ｕ/㎖ 페니실린; Gibco BRL)이 함유된 DMEM (Gibco BRL, Grand Island, NY, USA)에서 37 ℃ 온도로, 95% 공기 및 5% CO2, 습한 조건으로 유지하였다.
C6 rat astrocytoma cells were cultured in DMEM (Gibco BRL, Grand Island, NY, USA) containing 10% heat-inactivated fetal calf serum and antibiotics (100 ug / ml streptomycin, 100 U / ml penicillin; Gibco BRL) ) as the temperature at 37 ℃, it was kept at 95% air and 5% CO 2, moist conditions.

세포생존율 측정Cell viability measurement

세포생존율을 측정하기 위해 MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide} 분석법을 수행하였다 (Vistica, D.T., et al., Cancer Res, 1991. 51(10): 2515-20). 세포를 피노밤 (150 ng/㎖) 및/또는 FK506BP 융합단백질과 함께 배양한 후 과산화수소 (H2O2, 1mM)로 4시간 동안 처리하여 세포독성을 유도하였다. 그런 다음 각 웰에 MTT 용액을 네 시간 동안 가하고 각 웰의 상층액을 제거하였다. 포마잔 크리스탈을 DMSO (dimethyl sulfoxide)에 용해하고 570 ㎚ 흡광도를 측정하였다. 세포생존율은 과산화수소 무처리 대조군의 백분율로 나타내었다.
(Vistica, DT, et al., Cancer Res., 1991, 51 (10) ): 2515-20). The cells were incubated with FN50 (150 ng / ml) and / or FK506BP fusion protein and treated with hydrogen peroxide (H 2 O 2 , 1 mM) for 4 hours to induce cytotoxicity. The MTT solution was then added to each well for four hours and the supernatant of each well was removed. The formazan crystal was dissolved in dimethyl sulfoxide (DMSO) and the absorbance at 570 nm was measured. The cell viability was expressed as a percentage of the hydrogen peroxide-untreated control group.

TUNEL (Terminal deoxynucletodyltransferase-mediated dUTP nick-end-labeling) 분석법TUNEL (Terminal deoxynucletodyltransferase-mediated dUTP nick-end-labeling) assay

C6 세포의 DNA 손상은 TUNEL 분석법으로 평가하였다. 간단히 설명하면, 피노밤 및/또는 FK506BP 융합단백질로 처리한 C6 세포는 H2O2 (1 mM) 처리 후 6.5시간 동안 DMEM에서 유지하였다. TUNEL 염색은 세포사멸 탐지 킷트 (Roche Applied Science, Basel, Switzerland)를 이용하여 수행하였다. 형광은 Eclipse 80i 형광현미경 (Nikon, Tokyo, Japan)을 이용하여 탐지하였다.
DNA damage of C6 cells was assessed by TUNEL assay. Briefly, C6 cells treated with the FO506BP fusion protein were maintained in DMEM for 6.5 hours after treatment with H 2 O 2 (1 mM). TUNEL staining was performed using a cell death detection kit (Roche Applied Science, Basel, Switzerland). Fluorescence was detected using an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).

웨스턴 블랏 분석Western blot analysis

동량의 세포 용균액을 12% SDS-PAGE에 적용하여 분리하고, 또 나이트로셀룰로스 막에 전기이동하였다. 막은 5% 탈지분유로 블로킹하고 목표 단백질은 특이적 항체 및 호스래디쉬 퍼옥시데이즈-결합된 2차 항체로 탐침하였다. 띠는 증강된 화학형광제 (Amersham, Franklin Lakes, NJ, USA)를 이용하여 탐지하였다.
The same amount of cell lysate was applied to 12% SDS-PAGE and electrophoresed on a nitrocellulose membrane. The membrane was blocked with 5% defatted milk powder and the target protein probed with a specific antibody and a horseradish peroxidase-conjugated secondary antibody. The strips were detected using an enhanced chemiluminescent agent (Amersham, Franklin Lakes, NJ, USA).

형광현미경Fluorescence microscope

글라스 커버슬립 상에 세포를 시딩하고 피노밤 및/또는 FK506BP 융합단백질과 함께 배양하였다. 배양 후 세포는 10분간 4% 파라포름알데하이드로 고정시키고 항-His 항체 및 Alexa Fluor 488-결합된 2차 항체와 순서대로 배양하였다. 핵은 1 ㎍/㎖의 DAPI (4',6-diamidino-2-phenylindole) (Roche Applied Science, Basel, Switzerland)로 30분간 염색하였다. 형광은 Olympus FV-300 공촛점 형광현미경 (Olympus, Tokyo, Japan)으로 분석하였다.
The cells were seeded on a glass cover slip and incubated with the finoBAL and / or FK506BP fusion protein. After incubation, the cells were fixed with 4% paraformaldehyde for 10 minutes and cultured in order with anti-His antibody and Alexa Fluor 488-conjugated secondary antibody. Nuclei were stained with 1 μg / ml of DAPI (4 ', 6-diamidino-2-phenylindole) (Roche Applied Science, Basel, Switzerland) for 30 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

동물실험Animal experiment

웅성 몽고 저빌 (Meriones unguiculatus)은 한림대 실험동물센터에서 입수하였다. 동물은 일정한 온도 (23 ℃)와 상대습도 (60%)로 12시간 밝음: 12시간 어두움 상태로 사육하였으며, 물과 사료는 자유롭게 섭취할 수 있도록 하였다. 모든 동물 및 사육은 국립수의과학검역원의 실험동물 사육과 이용에 관한 안내규정에 적합함을 한림대학교 의과대학 동물 사육 및 이용 위원회가 승인한다. Meriones unguiculatus was obtained from Hallym University Laboratory Animal Center. The animals were kept in a dark condition for 12 hours at a constant temperature (23 ° C) and a relative humidity (60%) for 12 hours, and water and feed were freely available. All animals and breeding are approved by the National Veterinary Research and Quarantine Service for animal breeding and utilization committee of Hallym University Medical School, which is in conformity with the guidelines for the rearing and use of laboratory animals.

FK506BP 융합단백질이 혈관-뇌 장벽을 침투할 수 있는지를 평가하기 위해 피노밤(mg/kg) 및 PEP-1-FK506BP 융합단백질 (mg/kg)을 저빌 (n=7/군)에 각각 피하주사하였고, 뇌를 모았다. CA1 부분의 신경 생존을 시험하기 위해 허혈손상 유도 30분 전 피노밤, PEP-1-FK506BP 융합단백질 그리고, 피노밤과 PEP-1-FK506BP 융합단백질을 함께 피하주사하였다. 대뇌 전뇌 허혈손상 모델은 종래 방법으로 제조하였다 (J.S. Wadia, S.F. Dowdy, Curr. Opin. Biotechnol. 13. 2002, pp 52-56). 조직학적 분석을 위해 뇌절편은 항-His 또는 항-신경 핵 항체 (1:1,000)로 48시간 동안 4℃에서 배양하였다.
To evaluate the ability of the FK506BP fusion protein to penetrate the blood-brain barrier, subcutaneous injections of pinovirus (mg / kg) and PEP-1-FK506BP fusion protein (mg / kg) And collected the brain. To examine the neuronal survival of the CA1 region, PinoBil, PEP-1-FK506BP fusion protein, and Pino-Bone and PEP-1-FK506BP fusion protein were injected subcutaneously 30 min before induction of ischemic injury. The cerebral forebrain ischemic injury model was prepared by conventional methods (JS Wadia, SF Dowdy, Curr. Opin. Biotechnol. 13, 2002, pp 52-56). For histological analysis, brain slices were incubated with anti-His or anti-neuronal antibody (1: 1,000) for 48 hours at 4 ° C.

결과 1: 피노밤 선처리는 HResult 1: Pinochannel pretreatment is H 22 OO 22 로 유도된 세포독성에 대한 FK506BP 융합단백질의 보호효과를 현저히 증대시켰다.Lt; RTI ID = 0.0 &gt; FK506BP &lt; / RTI &gt;

본 발명자들은 산화 스트레스에 대한 C6 세포 생존율에 관한 FK506BP 융합단백질과 피노밤의 효과를 연구하였다. 피노밤의 화학구조와 FK506BP 융합단백질 모식도를 도 1A와 B에 나타내었다. 피노밤은 50~150 ng/㎖ 농도에서 C6 세포에 대해 세포독성 효과를 거의 나타내지 않았다 (도 1C). 다음으로, 본 발명자들은 FK506BP 융합단백질이 H2O2로 유도되는 C6 세포사를 저감할 수 있는지와 H2O2에 대한 FK506BP 융합단백질의 보호효과에 피노밤이 어떤 방식으로 영향을 미치는지를 시험하였다. H2O2는 대조군에 비해 C6 세포 생존율을 최대 40%까지 낮추었다. 반면, FK506BP 융합단백질은 H2O2로 유도되는 독성을 투여량 의존적으로 억제하였다 (도 1D). 뿐만 아니라, 피노밤 선처리는 H2O2 독성에 대한 FK506BP 융합단백질의 보호효과를 좀더 증대시켰다 (도 1D). 또한, 피노밤 및/또는 FK506BP 융합단백질 존재 하에서 H2O2-처리된 세포의 DNA 손상을 TUNEL 분석법으로 평가하였다. 도 1E와 같이, FK506BP 융합단백질은 H2O2-유도 DNA 손상을 저해하였고, 나아가 FK506BP와 피노밤을 조합하여 처리하면 FK506BP 융합단백질에 의한 H2O2-유도 DNA 손상 억제효과는 현저히 증가하였다. 대조적으로, 피노밤 단독 처리는 DNA 손상에 대해 현저한 보호효과를 나타내지 않았다. 이러한 결과를 볼 때 피노밤은 C6 세포에서 H2O2로 유도되는 산화 스트레스에 대한 FK506BP 융합단백질의 보호효과를 촉진하는 것으로 생각된다.
The present inventors have studied the effect of the FK506BP fusion protein and the pinonuclast on C6 cell viability against oxidative stress. The chemical structure of the pinot nois and the FK506BP fusion protein schematic diagram are shown in FIGS. 1A and B. FIG. PinoBy showed little cytotoxic effect on C6 cells at 50-150 ng / ml concentration (Fig. 1C). Next, the present inventors have tested this Pinot night for protection of the FK506BP fusion protein for FK506BP fusion protein, the H 2 O 2 C6 number that the H 2 O 2 to reduce the cell death induced to affect in any way . H 2 O 2 reduced the viability of C6 cells by up to 40% compared to the control group. On the other hand, the FK506BP fusion protein dose-dependently inhibited H 2 O 2 -induced toxicity (FIG. 1D). In addition, the pre-treatment of the pinothucks increased the protective effect of the FK506BP fusion protein on H 2 O 2 toxicity (FIG. 1D). In addition, DNA damage of H 2 O 2 -treated cells in the presence of the FO506BP fusion protein was assessed by TUNEL assay. As shown in FIG. 1E, the FK506BP fusion protein inhibited H 2 O 2 -induced DNA damage, and furthermore, the combination of FK506BP and pinotubic significantly increased the inhibitory effect of FK506BP fusion protein on H 2 O 2 -induced DNA damage . In contrast, the treatment with pinobay alone did not show a significant protective effect on DNA damage. These results suggest that PinoByme promotes the protective effect of FK506BP fusion protein on oxidative stress induced by H 2 O 2 in C6 cells.

결과 2: HResult 2: H 22 OO 22 로 유도되는 MAPK 및 Akt 활성화는 FK506BP 융합단백질 단독 처리에 비해 피노밤과 함께 처리할 때 현저히 억제할 수 있다.Induced MAPK and Akt activation can be significantly inhibited when treated with PinoBy compared to FK506BP fusion protein alone.

Akt와, p38과 ERK를 포함하는 MAPK 등의 생존 신호전달 카이네이즈 (survival signaling kinase)는 싸이토카인과 UV 조사와 같은 다양한 자극에 대해 반응하여 활성화된다 (Burke, R.E., Pharmacol Ther, 2007. 114(3): 261-77, Hausenloy, D.J. and D.M. Yellon, Cardiovasc Res, 2004. 61(3): 448-60). Akt, p38 및 Erk1/2의 인산화는 H2O2 처리한 다음 각각 8분, 35분 및 25분 후에 증가하였다. 피노밤은 H2O2로 유도되는 Akt와 p38의 활성화를 아주 약간 감소시킨 반면, FK506BP 융합단백질은 Akt, p38 및 Erk1/2 활성화를 강하게 억제하였다 (도 2A 및 2B). 또한, 피노밤과 FK506BP 융합단백질을 조합하여 처리하면 FK506BP 융합단백질을 단독 처리한 것과 비교할 때 p-Akt와 p-p38 수준은 매우 현저히 감소하였고, p-Erk1/2 수준은 약간 감소하였다 (도 2A 및 2B). 도 1D, 1E와 함께 이러한 결과들은 피노밤이 직접 H2O2로부터 C6 세포를 보호한다기 보다는 H2O2로 유도되는 Akt와 MAPK 활성화를 억제함으로써 FK506BP 융합단백질의 보호효과에 기여하는 것으로 설명할 수 있다.
Survival signaling kinases such as Akt and MAPK, including p38 and ERK, are activated in response to a variety of stimuli such as cytokines and UV irradiation (Burke, RE, Pharmacol Ther, 2007. 114 (3) : 261-77, Hausenloy, DJ and DM Yellon, Cardiovasc Res, 2004. 61 (3): 448-60). Phosphorylation of Akt, p38 and Erk1 / 2 increased after 8, 35 and 25 min, respectively, after H 2 O 2 treatment. The FO506BP fusion protein strongly inhibited Akt, p38, and Erk1 / 2 activation (Figs. 2A and 2B), while pino bast slightly reduced the activation of Akt and p38 induced by H 2 O 2 . In addition, treatment of the combination of the FO506BP fusion protein with pinoBM significantly reduced p-Akt and p-p38 levels and slightly decreased levels of p-Erk1 / 2 compared with the single treatment of FK506BP fusion protein (Fig. 2A And 2B). By Fig. 1D, these results with 1E are suppressed Akt and MAPK activation induced by H 2 O 2 rather than protect the C6 cells from the direct H 2 O 2 Pinot night described as contributing to the protection of the FK506BP fusion protein can do.

결과 3: C6 세포로의 FK506BP 융합단백질 도입에 대한 피노밤의 영향Result 3: Influence of pinotonin on the introduction of FK506BP fusion protein into C6 cells

본 발명자들은 앞서 마우스에서 FK506BP 융합단백질이 HaCaT 세포 내로 도입되어 아토피 피부염을 완화할 수 있음을 보고한바 있다 (Kim, S.Y., et al., J Invest Dermatol. 131(7): 1477-85). 세포내 FK506BP 융합단백질 수준을 증대하면 FK506BP 융합단백질의 보호효과를 증대시킬 수 있다 (Ahn, E.H., et al., BMB Rep. 44(5): 329-34, Sohn, E.J., et al., Biochem Biophys Res Commun. 406(3): 336-40). 그리하여 본 발명자들은 피노밤이 C6 세포로의 FK506BP 융합단백질의 투과능에 영향을 미치는지를 평가하였다. FK506BP 융합단백질은 C6 세포에 투여량 의존적으로 (도 3A, 3B), 그리고 처리시간 의존적으로 (도 3C 및 3D) 투과하였고, 피노밤을 선처리한 경우 C6 세포로 FK506BP 융합단백질의 투과도는 현저히 증가하였다 (도 3A~3D). 또한, 피노밤을 선처리하면 세포 내로 Tat-GFP 융합단백질 도입 또한 증가하여 FK506BP 융합단백질 결과와 일치하였다 (데이타 나타내지 않음). 형광현미경 데이타는 FK506BP 융합단백질 단독처리와 비교하여 피노밤이 FK506BP 융합단백질의 세포내 투과를 촉진함을 보여주었다 (도 3E). The present inventors have previously reported that FK506BP fusion protein in mice can be introduced into HaCaT cells to alleviate atopic dermatitis (Kim, S. Y., et al., J Invest Dermatol. 131 (7): 1477-85). Increasing intracellular FK506BP fusion protein levels can increase the protective effect of FK506BP fusion protein (Ahn, EH, et al., BMB Rep. 44 (5): 329-34, Sohn, EJ, et al., Biochem Biophys Res Commun. 406 (3): 336-40). Thus, the present inventors evaluated whether the effect of the finoBam on the permeability of FK506BP fusion protein to C6 cells. FK506BP fusion protein permeated C6 cells in a dose dependent manner (Figures 3A, 3B) and in a time dependent manner (Figures 3C and 3D), and the permeability of FK506BP fusion protein to C6 cells significantly increased when pre- (Figures 3A-3D). In addition, pretreatment of the PinoByte resulted in an increase in Tat-GFP fusion protein introduction into the cells, consistent with the results of the FK506BP fusion protein (data not shown). Fluorescence microscopy data showed that pinotubic promotes intracellular permeation of FK506BP fusion protein compared to FK506BP fusion protein alone (Fig. 3E).

다음으로 본 발명자들은 피노밤이 저빌 뇌조직으로의 FK506BP 융합단백질 인비보 세포투과를 촉진하는지를 시험하였다. 피노밤은 150 ㎍/㎏의 투여량으로 FK506BP 융합단백질 투여 (200 ㎍/㎏) 한 시간 전 저빌에 피하주사하였다. 여섯 시간 후 뇌를 수집하고 뇌 절편은 His 항체 및 2차 항체로 탐지하였다. 면역조직분석결과 피노밤을 처리하지 않은 경우와 비교하여 피노밤을 처리한 경우 FK506BP 융합단백질이 현저히 많고 강하게 관찰되었다 (도 4A). 따라서, 이 데이타들은 피노밤이 FK506BP 융합단백질의 혈과-뇌 장벽 침투를 현저히 증가시켰음을 말해준다.
Next, the present inventors have examined whether or not PinoByte promotes penetration of the FK506BP fusion protein in vivo to hypervascular brain tissue. PinoBy was subcutaneously injected into the hypothalamus one hour prior to administration of the FK506BP fusion protein (200 占 퐂 / kg) at a dose of 150 占 퐂 / kg. Six hours later, the brain was harvested and the brain slices were detected as His and secondary antibodies. Immunohistochemical analysis revealed that the FO506BP fusion protein was significantly and strongly observed when treated with the PinoBy compared to the case without the PinoBy (Fig. 4A). Thus, these data indicate that pinotubic significantly increased blood-brain barrier penetration of the FK506BP fusion protein.

결과 4: 피노밤은 일시적 대뇌 전뇌 허혈모델에서 CA1 부분에 대한 FK506BP 융합단백질의 보호효과에 긍정적인 효과를 미쳤다.Result 4: PinoBurm had a positive effect on the protective effect of the FK506BP fusion protein on the CA1 region in the transient cerebral frontal ischemia model.

본 발명자들은 허혈손상 저빌 모델의 CA1 부분의 신경보호를 평가함으로써 피노밤에 의한 FK506BP 융합단백질 세포도입 증대가 산화 스트레스에 대한 FK506BP 융합단백질의 신경보호효과에 영향을 미치는지를 연구하였다. 조직학적 분석을 통해 피노밤 처리군이 허혈손상에 대해 CA1 부분에서 매우 약한 신경보호효과를 나타내며 (도 4B, 세 번째 패널), 반면 FK506BP 융합단백질은 같은 부분에서 강력한 신경보호효과를 나타냄을 밝혔다 (도 4B, 네 번째 패널). 가장 중요한 것은 피노밤 선처리가 허혈손상으로부터 CA1 부분에 대한 FK506BP 융합단백질의 신경보호효과를 현저히 강화시켰다는 점이다 (도 4B, 다섯 번째 패널). 종합해 볼 때 피노밤은 산화스트레스에 대해 뉴런에 미치는 FK506BP의 신경보호효과를 현저히 강화하는 약제로 기능할 수 있다고 결론지을 수 있다.The present inventors have evaluated the neuroprotective effect of FK506BP fusion protein on oxidative stress by increasing the introduction of FK506BP fusion protein by pinotubic by evaluating the neuroprotection of the CA1 portion of the low-ischemic model of ischemic injury. Histological analysis revealed that the PinoBM treated group exhibited very weak neuroprotective effects in the CA1 region (FIG. 4B, third panel) against ischemic injury, whereas the FK506BP fusion protein exhibited strong neuroprotective effects in the same region 4B, fourth panel). Most importantly, the PinoBoard pretreatment significantly enhanced the neuroprotective effect of the FK506BP fusion protein on the CA1 region from ischemic injury (Fig. 4B, Fifth panel). Taken together, it can be concluded that pinotassium can function as an agent that significantly enhances the neuroprotective effect of FK506BP on neurons against oxidative stress.

<110> Industry-Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for arthritis containing fk506 binding protein fusion protein <130> sychoi-fk506bp-arthritis <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 1 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 2 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <210> 3 <211> 404 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding PEP-1-FK506BP fusion protein <400> 3 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcc 404 <210> 4 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP fusion protein <400> 4 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser 20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val 35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg 50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile 65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala 85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro 100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu 115 120 125 Lys Leu Glu 130 <210> 5 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-PEP-1 fusion protein <400> 5 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaatgagga tcctaaaaga aacctggtgg gaaacctggt 360 ggaccgaatg gtctcagccg aaaaaaaaac gtaaagtgta g 401 <210> 6 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-PEP-1 fusion protein <400> 6 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Lys Glu Thr Trp 100 105 110 Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys 115 120 125 Val Gly Ser 130 <210> 7 <211> 472 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding PEP-1-FK506BP-PEP-1 fusion protein <400> 7 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcctaaaag aaacctggtg 420 ggaaacctgg tggaccgaat ggtctcagcc gaaaaaaaaa cgtaaagtgt ag 472 <210> 8 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP-PEP-1 fusion protein <400> 8 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser 20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val 35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg 50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile 65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala 85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro 100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu 115 120 125 Lys Leu Glu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu 130 135 140 Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 145 150 <210> 9 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding Tat-FK506BP fusion protein <400> 9 aggaagaagc ggagacagcg acgaagactc gagatgggag tgcaggtgga aaccatctcc 60 ccaggagacg ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg 120 atgcttgaag atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt 180 atgctaggca agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg 240 ggtcagagag ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca 300 ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaatga 360 ggatcc 366 <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Tat-FK506BP fusion protein <400> 10 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val 1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln 20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe 35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys 50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val 65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala 85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp 100 105 110 Val Glu Leu Leu Lys Leu Glu 115 <210> 11 <211> 361 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-Tat fusion protein <400> 11 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaaggatcc taggaagaag cggagacagc gacgaagata 360 g 361 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-Tat fusion protein <400> 12 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg 100 105 110 Arg Gln Arg Arg Arg 115 <210> 13 <211> 395 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding Tat-FK506BP-Tat fusion protein <400> 13 taggaagaag cggagacagc gacgaagact cgagatggga gtgcaggtgg aaaccatctc 60 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg 120 gatgcttgaa gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt 180 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt 240 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc 300 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg 360 atcctaggaa gaagcggaga cagcgacgaa gatag 395 <210> 14 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Tat-FK506BP-Tat fusion protein <400> 14 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val 1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln 20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe 35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys 50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val 65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala 85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp 100 105 110 Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg Arg Gln Arg Arg Arg 115 120 125 <210> 15 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1 <400> 15 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> anticoding sequence for PEP-1 <400> 16 tcgagcactt tacgtttttt tttcggctga caccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctcgagatgg gagtgcaggt ggaaaccatc 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggatcctcat tccagtttta gaagctccac 30 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> top strand for Tat peptide <400> 19 taggaagaag cggagacagc gacgaagac 29 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> bottom strand for Tat peptide <400> 20 tcgagtcttc gtcgctgtct ccgcttcttc c 31 <110> Industry-Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for arthritis containing fk506 binding          protein fusion protein <130> sychoi-fk506bp-arthritis <160> 20 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 1 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 2 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 3 <211> 404 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding PEP-1-FK506BP fusion protein <400> 3 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcc 404 <210> 4 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP fusion protein <400> 4 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser              20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val          35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg      50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile  65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala                  85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro             100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu         115 120 125 Lys Leu Glu     130 <210> 5 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-PEP-1 fusion protein <400> 5 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaatgagga tcctaaaaga aacctggtgg gaaacctggt 360 ggaccgaatg gtctcagccg aaaaaaaaac gtaaagtgta g 401 <210> 6 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-PEP-1 fusion protein <400> 6 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe   1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu              20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys          35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val      50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp  65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala                  85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Lys Glu Thr Trp             100 105 110 Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys         115 120 125 Val Gly Ser     130 <210> 7 <211> 472 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding PEP-1-FK506BP-PEP-1 fusion protein <400> 7 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcctaaaag aaacctggtg 420 ggaaacctgg tggaccgaat ggtctcagcc gaaaaaaaaa cgtaaagtgt ag 472 <210> 8 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP-PEP-1 fusion protein <400> 8 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser              20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val          35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg      50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile  65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala                  85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro             100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu         115 120 125 Lys Leu Glu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu     130 135 140 Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 145 150 <210> 9 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding Tat-FK506BP fusion protein <400> 9 aggaagaagc ggagacagcg acgaagactc gagatgggag tgcaggtgga aaccatctcc 60 ccaggagacg ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg 120 atgcttgaag atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt 180 atgctaggca agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg 240 ggtcagagag ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca 300 ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaatga 360 ggatcc 366 <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Tat-FK506BP fusion protein <400> 10 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val   1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln              20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe          35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys      50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val  65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala                  85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp             100 105 110 Val Glu Leu Leu Lys Leu Glu         115 <210> 11 <211> 361 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-Tat fusion protein <400> 11 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaaggatcc taggaagaag cggagacagc gacgaagata 360 g 361 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-Tat fusion protein <400> 12 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe   1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu              20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys          35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val      50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp  65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala                  85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg             100 105 110 Arg Gln Arg Arg Arg         115 <210> 13 <211> 395 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding Tat-FK506BP-Tat fusion protein <400> 13 taggaagaag cggagacagc gacgaagact cgagatggga gtgcaggtgg aaaccatctc 60 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg 120 gatgcttgaa gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt 180 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt 240 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc 300 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg 360 atcctaggaa gaagcggaga cagcgacgaa gatag 395 <210> 14 <211> 128 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Tat-FK506BP-Tat fusion protein <400> 14 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val   1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln              20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe          35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys      50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val  65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala                  85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp             100 105 110 Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg Arg Gln Arg Arg Arg         115 120 125 <210> 15 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1 <400> 15 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Anticoding sequence for PEP-1 <400> 16 tcgagcactt tacgtttttt tttcggctga caccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctcgagatgg gagtgcaggt ggaaaccatc 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggatcctcat tccagtttta gaagctccac 30 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> top strand for Tat peptide <400> 19 taggaagaag cggagacagc gacgaagac 29 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> bottom strand for Tat peptide <400> 20 tcgagtcttc gtcgctgtct ccgcttcttc c 31

Claims (9)

삭제delete 삭제delete 삭제delete FK506 결합단백질의 N-말단 및 C-말단 중 어느 한 곳 이상에 PEP-1 또는 HIV Tat 단백질 수송 도메인이 공유결합된 FK506 결합단백질 융합단백질과 피노밤을 포함하는 뇌 허혈손상 치료용 약제학적 조성물.
A pharmaceutical composition for treating cerebral ischemic injury comprising an FK506 binding protein fusion protein in which a PEP-1 or HIV Tat protein transport domain is covalently linked to at least one of the N-terminus and the C-terminus of an FK506 binding protein, and a polynucleotide.
삭제delete 청구항 4에 있어서,
상기 융합단백질은 서열번호 4, 서열번호 6, 서열번호 8, 서열번호 10, 서열번호 12 및 서열번호 14 중 선택된 1종인 것을 특징으로 하는 뇌 허혈손상 치료용 약제학적 조성물.
The method of claim 4,
Wherein the fusion protein is one selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.
삭제delete 삭제delete 삭제delete
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US7902346B2 (en) 2006-03-23 2011-03-08 Industry Academic Cooperation Foundation, Hallym University Fusion protein comprising FK506 binding protein and DNA encoding the same
JP2013509450A (en) 2009-11-02 2013-03-14 ジ アドミニストレーターズ オブ ザ トゥレーン エデュケーショナル ファンド Pituitary adenylate cyclase activating polypeptide (PACAP) analogs and methods of use thereof

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JP2009534415A (en) 2006-04-20 2009-09-24 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Pharmacological control of the action of AMPA receptor modulators on neurotrophin expression
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