KR20140011978A

KR20140011978A - Compositions for treatment of bone disease comprising inhibitor against nod2

Info

Publication number: KR20140011978A
Application number: KR1020130084920A
Authority: KR
Inventors: 최혜선; 정수월; 김운기; 케케
Original assignee: 울산대학교 산학협력단
Priority date: 2012-07-19
Filing date: 2013-07-18
Publication date: 2014-01-29

Abstract

The present invention relates to about a pharmaceutical composition containing nucleotide-binding oligomerization domain 2 (NOD2) as an active ingredient for bone disease prevention or treatment due to estrogen deficiency. The composition can effectively inhibit bone density reduction by inhibiting expression or function of NOD2 gene or protein of patients who have anticipated or progressing estrogen decrease or deficiency due to ovariectomy.

Description

Composition for the treatment of bone disease containing inhibitor of nucleotide-binding oligomerization domain 2 as an active ingredient {Compositions for treatment of bone disease comprising inhibitor against NOD2}

본 발명은 뉴클레오티드-결합 올리고머화 도메인 2(NOD2)의 발현 억제제를 유효성분으로 함유하는 에스트로겐 결핍으로 유도된 골질환 예방 또는 치료용 약학적 조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for preventing or treating bone diseases induced by estrogen deficiency containing an inhibitor of expression of nucleotide-binding oligomerization domain 2 (NOD2) as an active ingredient.

골(bone)은 골 전환(bone turnover)을 통해 노화된 골을 신규한 골로 대체하는 과정을 수행하는 역동적 조직이다. 이러한 골 재형성(bone remodeling)은 조골세포(osteoblast)와 파골세포(osteoclast, OC)의 균형 잡힌 작용을 통해 이루어진다. 에스트로겐(estrogen) 결핍은 이러한 재형성의 공간을 넓혀 골소주 표면(trabecular surface) 상에 피질의 공극(cortical porosity) 및 재흡수 영역(resorption area)을 증대시킨다. 증가된 파골세포생성(osteoclastogenesis)은 이러한 현상의 일차적인 원인이다.A bone is a dynamic organization that performs the process of replacing aged bone with new bone through bone turnover. This bone remodeling is achieved through the balanced action of osteoblasts and osteoclasts (osteoclast, OC). Estrogen deficiency widens the space for this remodeling to increase the cortical porosity and resorption area on the trabecular surface. Increased osteoclastogenesis is the primary cause of this phenomenon.

에스트로겐의 주된 효과는 파골세포 형성을 감소시키는 것이다 [Weitzmann M., et al., J Clin Invest (2006), 116, 1186-94]. 골흡수에서 중요한 역할을 하는 다핵거대세포(multinucleated giant cells)인 파골세포는, 일부 형태학적 및 기능적 특성을 공유하는 단핵구세포/대식세포 계통(monocyte/macrophage lineage)의 조혈세포(hematopoietic cells)로부터 분화된다. 이들 세포는 물리적 골 재형성 뿐 아니라 만성 염증성 질환과 관련된 골 파괴(bone destruction)의 원인이 된다 [Roodman GD, Endocrinol Rev (1996), 17, 308-332].The main effect of estrogens is to reduce osteoclast formation (Weitzmann M., et al., J Clin Invest (2006), 116, 1186-94). Osteoclasts, multinucleated giant cells that play an important role in bone resorption, differentiate from hematopoietic cells of the monocyte / macrophage lineage that share some morphological and functional properties. do. These cells are responsible for bone destruction associated with chronic inflammatory diseases as well as physical bone remodeling [Roodman GD, Endocrinol Rev (1996), 17, 308-332.

파골세포 생성을 촉진하기 위해서는 두 분자가 필수적이다. 이들 인자는 골수 중간엽 세포로부터 생성되는데, M-CSF(macrophage-colony stimulating factor)와 TNF 패밀리의 하나인 RANKL(receptor activator of nuclear factor-κB ligand)이다. [Suda T. et al., Endocrinol Rev (1999), 20, 345-357; Kong YY, et al., Nature (1999), 397, 315-323]. RANKL이 그 수용체인 RANK에 결합하는 것은 NF-κB, c-Fos 및 NFAT2을 포함하는 필수적인 전사인자를 유도하고 활성화시켜 파골세포 분화의 신호를 개시한다 [Cappellen D, et al., J Biol Chem (2002), 277, 21971-82], 활성화된 전사인자들은 TRACP 및 칼시토닌 수용체(calcitonin receptor)를 포함하는 표적 유전자의 발현을 유도한다. 파골세포와 조골세포는 TLR 및 IL-1R을 발현하지만, 조골세포는 파골세포의 분화 및 활성에 영향을 미치는 전염증성 사이토카인(proinflammatory cytokines)을 생산한다.Two molecules are essential for promoting osteoclast production. These factors are produced from myeloid mesenchymal cells, which are macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), one of the TNF families. Suda T. et al., Endocrinol Rev (1999), 20, 345-357; Kong YY, et al., Nature (1999), 397, 315-323]. Binding of RANKL to its receptor, RANK, induces and activates essential transcription factors, including NF-κB, c-Fos and NFAT2, to initiate a signal of osteoclast differentiation [Cappellen D, et al., J Biol Chem (2002), 277, 21971-82, activated transcription factors induce expression of target genes, including TRACP and calcitonin receptor. Osteoclasts and osteoblasts express TLR and IL-1R, but osteoblasts produce proinflammatory cytokines that affect the differentiation and activity of osteoclasts.

한편, 뉴클레오티드-결합 올리고머화 도메인 2(nucleotide-binding oligomerization domain 2, NOD2)는 선천성 면역시스템을 구성하는 패턴 인식 수용체(pattern recognition receptors, PRR) 중 하나이다. PRR은 플레젤린(flagellin), 고유 핵산 구조(unique nucleic acid structure) 및 박테리아 세포벽 구성물질(bacterial cell wall components)과 같이 미생물에 중요한, 병원체-관련 분자 패턴(pathogen-associated molecular patterns, PAMP)을 감지한다. 톨-유사 수용체(toll-like receptors)는 세포 표면 및 엔도좀 내에서 PAMP들을 감지하는 반면, NOD2는 세포내 구획(intracellular compartments)에서 이들을 인식하여 회피하는 미생물에 대한 다른 인지 라인(recognition line)을 제공한다. PAMP의 탐지는 다중 전염증성 신호전달경로를 활성화시켜 칩임 미생물을 표적한다.On the other hand, nucleotide-binding oligomerization domain 2 (NOD2) is one of the pattern recognition receptors (PRR) constituting the innate immune system. PRR detects pathogen-associated molecular patterns (PAMPs) that are important to microorganisms such as flagellin, unique nucleic acid structures, and bacterial cell wall components. do. Toll-like receptors detect PAMPs on the cell surface and in endosomes, while NOD2 blocks other recognition lines for microorganisms that recognize and avoid them in intracellular compartments. to provide. The detection of PAMP activates multiple proinflammatory signaling pathways to target chipmeal microorganisms.

NOD는 뉴클레오티드 결합(nucleotide binding) 및 자가-올리고머화(self-oligomerization)에 필수적인 N-말단 작용기 결합 부위(N-terminal effector biding region), 및 미생물의 패턴을 감지하고 그 활성을 조절하는 C-말단 루신-풍부 반복 모티프(C-terminal leucine-rich repeat motifs)로 구성된다. NOD2의 돌연변이는 만성 염증성 장질환인 크론병(Crohn's disease)과 관련되어 있으며, 이는 NOD2가 여러 만성 염증 질환에서 중요한 역할을 한다는 것을 의미한다. NOD is an N-terminal effector biding region essential for nucleotide binding and self-oligomerization, and a C-terminal that senses and regulates the pattern of microorganisms. It consists of C-terminal leucine-rich repeat motifs. Mutations in NOD2 are associated with Crohn's disease, a chronic inflammatory bowel disease, meaning that NOD2 plays an important role in many chronic inflammatory diseases.

본 발명자들은 난소절제술과 같은 에스트로겐의 감소 또는 결핍 상황에서의 골 소실을 저해하기 위해 예의 연구한 결과, NOD2 유전자의 억제를 통해 에스트로겐 결핍 상황에서의 골소실에 따른 골 질환의 치료가 가능할 것으로 판단하고, 본 발명을 완성하였다.
The present inventors have studied diligently to reduce bone loss in the estrogen reduction or deficiency situation, such as ovarian resection, it is determined that the bone disease caused by bone loss in the estrogen deficiency situation through the inhibition of the NOD2 gene The present invention has been completed.

따라서 본 발명의 목적은 에스트로겐 결핍 상황에서의 골소실에 따른 골 질환의 치료를 위한 NOD2 유전자의 발현 억제제를 유효성분으로 함유하는 골 질환 예방 또는 치료용 약학적 조성물에 관한 것이다.
Accordingly, an object of the present invention relates to a pharmaceutical composition for preventing or treating bone diseases containing an inhibitor of NOD2 gene expression for the treatment of bone diseases caused by bone loss in an estrogen deficiency situation.

상기 목적을 달성하기 위하여, 본 발명은 뉴클레오티드-결합 올리고머화 도메인 2(nucleotide-binding oligomerization domain 2, NOD2) 유전자의 억제제를 유효성분으로 함유하는, 에스트로겐 결핍으로 유도된 골 질환 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention contains an inhibitor of the nucleotide-binding oligomerization domain 2 (NOD2) gene as an active ingredient, the pharmaceutical composition for preventing or treating bone disease induced by estrogen deficiency To provide a composition.

본 발명의 일실시예에 있어서, 상기 NOD2 유전자는 서열번호 2의 아미노산 서열을 코딩하는 염기서열을 가질 수 있다.In one embodiment of the present invention, the NOD2 gene may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

본 발명의 일실시예에 있어서, 상기 NOD2 유전자는 서열번호 1의 염기서열을 가질 수 있다.In one embodiment of the present invention, the NOD2 gene may have a nucleotide sequence of SEQ ID NO: 1.

본 발명의 일실시예에 있어서, 상기 억제제는 NOD2 유전자의 전사를 억제하는 물질, 전사된 NOD2 mRNA의 번역을 억제하는 물질, 또는 NOD2 단백질의 기능을 억제하는 물질일 수 있다.In one embodiment of the present invention, the inhibitor may be a substance that inhibits the transcription of NOD2 gene, a substance that inhibits translation of transcribed NOD2 mRNA, or a substance that inhibits the function of NOD2 protein.

본 발명의 일실시예에 있어서, 상기 NOD2 유전자의 전사를 억제하는 물질은 프로모터(promoter), 인핸서(enhancer) 또는 프로모터에 결합하는 전사조절인자(transcription factor)에 결합하는 단백질 또는 화합물일 수 있다.In one embodiment of the present invention, the agent that inhibits transcription of the NOD2 gene may be a protein or compound that binds to a promoter, enhancer or transcription factor that binds to the promoter.

본 발명의 일실시예에 있어서, 상기 전사된 NOD2 mRNA의 번역을 억제하는 물질은 상기 mRNA에 특이적으로 결합하는 안티센스 분자일 수 있다.In one embodiment of the invention, the substance that inhibits translation of the transcribed NOD2 mRNA may be an antisense molecule that specifically binds to the mRNA.

본 발명의 일실시예에 있어서, 상기 NOD2 단백질의 기능을 억제하는 물질은 상기 단백질에 특이적으로 결합하는 항체일 수 있다.In one embodiment of the invention, the substance that inhibits the function of the NOD2 protein may be an antibody that specifically binds to the protein.

본 발명의 일실시예에 있어서, 상기 에스트로겐 결핍으로 유도된 골 질환은 골다공증(osteoporosis), 골연화증(osteomalacia), 골감소증(osteopenia), 골위축(bone atrophy) 및 골관절염(osteoarthritis)으로 구성된 질환에서 선택될 수 있다.
In one embodiment of the present invention, the bone disease induced by estrogen deficiency may be selected from diseases consisting of osteoporosis, osteomalacia, osteoopenia, bone atrophy and osteoarthritis. Can be.

본 발명은 NOD2 유전자의 억제제를 유효성분으로 함유하는, 에스트로겐 결핍으로 유도된 골 질환 예방 또는 치료용 약학적 조성물에 관한 것으로, 본 발명의 조성물은 NOD2 유전자 또는 단백질의 기능을 억제할 수 있는 물질을 유효성분으로 포함함으로써, 난소절제술, 약물복용, 노화와 같은 에스트로겐의 감소 또는 결핍으로 인한 골 질환의 치료에 유용하게 이용될 수 있다.
The present invention relates to a pharmaceutical composition for preventing or treating bone diseases induced by estrogen deficiency, which contains an inhibitor of the NOD2 gene as an active ingredient. The composition of the present invention provides a substance capable of inhibiting the function of the NOD2 gene or protein. By including it as an active ingredient, it can be usefully used for the treatment of bone diseases due to the reduction or lack of estrogens such as ovarian resection, drug use, and aging.

도 1은 마우스에서 OVA 유도된 골소실에 대한 NOD2의 영향을 나타낸 것이다.
도 2는 NOD2가 파골세포 증가에 미치는 영향을 나타낸 것이다.
도 3은 NOD2가 조골세포생성에 미치는 영향을 나타낸 것이다.1 shows the effect of NOD2 on OVA induced bone loss in mice.
Figure 2 shows the effect of NOD2 on osteoclast growth.
Figure 3 shows the effect of NOD2 on osteoblast production.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 뉴클레오티드-결합 올리고머화 도메인 2(nucleotide-binding oligomerization domain 2, NOD2) 유전자의 억제제를 유효성분으로 함유하는, 에스트로겐 결핍으로 유도된 골 질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating bone disease induced by estrogen deficiency, containing an inhibitor of nucleotide-binding oligomerization domain 2 (NOD2) gene as an active ingredient.

본 발명의 일실시예에 있어서, 상기 NOD2 유전자는 서열번호 2의 아미노산 서열을 코딩하는 어떠한 염기서열로도 구성될 수 있다.In one embodiment of the invention, the NOD2 gene may be composed of any base sequence encoding the amino acid sequence of SEQ ID NO: 2.

본 발명의 일실시예에 있어서, 상기 NOD2 유전자는 서열번호 1의 염기서열로 표시될 수 있다.In one embodiment of the present invention, the NOD2 gene may be represented by the nucleotide sequence of SEQ ID NO: 1.

본 발명의 구현예에 따르면, 난소절제술 후 NOD2 유전자를 억제시킨 NOD2-KO 마우스의 대퇴골 골밀도는 NOD2 유전자를 갖는 야생형 마우스와 비교하여 현저히 높은 것을 확인하였다 (도 1 참조). 이는 난소절제술과 같이 에스트로겐 감소 또는 결핍이 예상되는 상황에서 NOD2 유전자를 억제시키면 에스트로겐의 감소 또는 결핍으로 인한 골소실로부터 골을 효과적으로 보호 및 예방할 수 있음을 의미하는 것이다. 따라서 본 발명에 따른 NOD2 억제제를 유효성분으로 포함하는 조성물은 약물복용, 노화(aging) 또는 수술 등으로 인해 에스트로겐의 감소 또는 결핍이 예상되거나 진행중인 환자에게 투여하여 NOD2의 발현을 억제함으로써 NOD2의 발현으로 유도된 골밀도 감소를 억제시킬 수 있으므로, 골밀도 감소로 유발되는 골 질환의 치료용도로 유용하게 사용될 수 있다.According to the embodiment of the present invention, the femoral bone mineral density of NOD2-KO mice that inhibited the NOD2 gene after ovarian resection was confirmed to be significantly higher than that of wild-type mice having the NOD2 gene (see FIG. 1). This means that inhibiting the NOD2 gene in situations where estrogen reduction or deficiency is expected, such as ovarian resection, can effectively protect and prevent bone from bone loss due to the reduction or deficiency of estrogen. Therefore, the composition comprising the NOD2 inhibitor according to the present invention as an active ingredient is administered to a patient in anticipation or progression of estrogen reduction or deficiency due to drug use, aging, or surgery, thereby suppressing the expression of NOD2. Since it can suppress the decrease in bone density, it can be usefully used for the treatment of bone diseases caused by the decrease in bone density.

본 발명에서, 상기 “NOD2 유전자의 억제제”라 함은 NOD2 유전자의 발현을 억제하거나, NOD2 유전자의 발현으로 생성된 단백질의 기능을 억제하는 모든 물질을 뜻한다.In the present invention, the "inhibitor of NOD2 gene" refers to any substance that inhibits the expression of the NOD2 gene or inhibits the function of a protein produced by the expression of the NOD2 gene.

본 발명의 일실시예에 있어서, 상기 억제제는 NOD2 유전자의 전사를 억제하는 물질, 전사된 NOD2 mRNA의 번역을 억제하는 물질 또는 NOD2 단백질의 기능을 억제하는 물질이 모두 포함된다. 이때, NOD2 유전자의 전사를 억제하는 물질로는 NOD2 유전자의 전사를 조절한다고 알려져 있는 프로모터(promoter), 인핸서(enhancer), 프로모터에 결합하는 전사 조절 인자(transcription factor)에 결합하는 단백질 또는 화합물이 될 수 있으나 반드시 이에 한정되는 것은 아니다. 또한, mRNA의 번역을 억제하는 물질로는 상기 mRNA에 특이적으로 결합하는 안티센스 분자가 있으나 반드시 여기에 한정되지는 아니며, NOD2 단백질의 기능을 억제하는 물질로는 상기 단백질에 특이적으로 결합하는 단일클론 또는 다클론 항체가 있으나 여기에 한정되는 것은 아니다. 상기와 같은 안티센스 또는 단일클론 또는 다클론 항체는 공지된 통상적인 방법을 사용하여 제조될 수 있다.In one embodiment of the present invention, the inhibitor includes a substance that inhibits transcription of NOD2 gene, a substance that inhibits translation of transcribed NOD2 mRNA, or a substance that inhibits the function of NOD2 protein. At this time, a substance that inhibits the transcription of NOD2 gene may be a protein or compound that binds to a promoter, enhancer, or transcriptional transcription factor that binds to the promoter. But it is not necessarily limited thereto. In addition, a substance that inhibits the translation of mRNA includes an antisense molecule that specifically binds to the mRNA, but is not limited thereto. A substance that inhibits the function of the NOD2 protein may be a single substance that specifically binds to the protein. There are clone or polyclonal antibodies, but are not limited thereto. Such antisense or monoclonal or polyclonal antibodies can be prepared using known conventional methods.

본 발명의 일구현예에서, 상기 NOD2 유전자의 전사를 억제하는 물질의 하나의 예는 RNAi이다. 상기 ‘RNAi’는 많은 진핵생물에서 보존된 전사후 유전자 조절의 한 방법이다. RNAi는 세포에 존재하는 짧은 이중나선 RNA(dsRNA)분자에 의해 유도된다 (Fire 등 (1998), Nature 391: 806-811). siRNA 라고도 불리우는 이러한 짧은 dsRNA 분자는 단일가닥으로 분리된 후 RNA-유도 침묵 복합체(RNA induced silencing, RISC)에 결합하여 표적화된 mRNA를 절단하거나 번역을 저해한다 (Elbashir SM 등 (2001), Genes Dev, 15: 188-200).In one embodiment of the invention, one example of a substance that inhibits transcription of the NOD2 gene is RNAi. The term 'RNAi' is a method of post-transcriptional gene regulation conserved in many eukaryotes. RNAi is induced by short double-stranded RNA (dsRNA) molecules present in cells (Fire et al. (1998), Nature 391: 806-811). These short dsRNA molecules, also called siRNAs, separate into single strands and then bind to RNA-induced silencing complexes (RISCs) to cleave targeted mRNAs or inhibit translation (Elbashir SM et al. (2001), Genes Dev, 15: 188-200).

또한 NOD2 유전자의 발현 억제제가 siRNA일 경우, siRNA 폴리뉴클레오티드 서열은 발현 조절 서열에 작동 가능하게 연결될 수 있으며, 상기 작동 가능하게 연결된 유전자 서열과 발현 조절 서열은 선택 마커 및 복제 개시점(replication origin)을 같이 포함하고 있는 하나의 발현벡터 내에 포함될 수 있다. 상기 “작동 가능하게 연결(operably linked)” 된다는 것은 적절한 분자가 발현 조절 서열에 결합될 때 유전자 발현을 가능하게 하는 방식으로 연결된 유전자 및 발현 조절 서열일 수 있다. 상기 “발현 조절 서열(expression control sequence)”이란 특정한 숙주 세포에서 작동 가능하게 연결된 폴리뉴클레오티드 서열의 발현을 조절하는 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 실시하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열, 전사 및 해독의 종결을 조절하는 서열을 포함한다. 상기 NOD2 유전자의 siRNA를 포함하는 발현벡터는 당해 기술분야에서 잘 알려진 형질전환 기술을 이용하여 숙주세포에서 발현시키거나 목적으로 하는 부위에서 발현시킴으로써 NOD2 유전자의 발현을 억제시킬 수 있다. In addition, when the expression inhibitor of the NOD2 gene is siRNA, the siRNA polynucleotide sequence may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence may have a selection marker and replication origin. It can be included in one expression vector included together. The term “operably linked” may be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. The expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. The expression vector comprising the siRNA of the NOD2 gene can be expressed in a host cell using a transformation technique well known in the art or at the site of interest to inhibit the expression of the NOD2 gene.

상기 siRNAs의 이용은 질병의 동물 모델에 있어서 유전자 발현을 저해하는 데 대중성을 나타내고 있다. siRNA의 전달 및 siRNA를 포함한 발현 벡터를 제조하는 기술은 당업자에게 알려져 있다. 예를 들어, 미국 출원 제2004/106567 및 2004/0086884는 바이러스성 벡터, 비바이러스성 벡터, 리포솜 전달운반체, 플라스미드 주입 시스템, 인공 바이러스 엔벨로프 및 폴리라이신 컨쥬게이트를 포함한 전달 메커니즘뿐 만 아니라 많은 발현 벡터를 제공하고 있다.The use of siRNAs has shown popularity in inhibiting gene expression in animal models of disease. Techniques for the delivery of siRNAs and for preparing expression vectors comprising siRNAs are known to those skilled in the art. For example, US applications 2004/106567 and 2004/0086884 describe many expression vectors as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. Providing.

또한 당업자는 당해 기술 분야에 공지된 방법을 이용하여 원하는 방식대로 상기 siRNA를 합성하고 변형시킬 수 있다 (Andreas Henschel, Frank Buchholz1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32(Web Server Issue):W113-W120). 본 발명에서 유전공학적 기술과 관련된 사항은 샘브룩 등의 문헌 (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N. Y. (2001)) 및 프레드릭 등의 문헌 (Frederick M. Ausubel et al., Current protocols in Molecular Biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994))에 개시되어 있는 내용에 의해 보다 명확하게 설명된다.One skilled in the art can also synthesize and modify the siRNA in a desired manner using methods known in the art (Andreas Henschel, Frank Buchholz 1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs.Nucleic Acids Research 32 (Web Server Issue): W113-W120). Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in Molecular Biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994).

본 발명에서, 상기 NOD2 단백질의 활성을 억제하는 물질의 다른 예로는, NOD2 단백질의 뉴클레오티드 결합(nucleotide binding) 및 자가-올리고머화(self-oligomerization)에 필수적인 N-말단 작용기 결합 부위(N-terminal effector biding region) 또는 미생물의 패턴을 감지하고 그 활성을 조절하는 C-말단 루신-풍부 반복 모티프(C-terminal leucine-rich repeat motifs)를 포함하는 활성부위에 직접적으로 결합하여 활성을 저해하는 경쟁적 저해제(competitive inhibitor), 또는 상기 활성부위가 아닌 부위에 결합하여 활성부위의 기능을 방해하는 비경쟁적 저해제(noncompetitive inhibitor), NOD2 단백질의 세포 내 특정 위치로의 이동(localization)을 방해하는 물질, NOD2 단백질을 분해(degradation)하거나 분해를 유도하는 물질, 및 NOD2의 활성 기작(mechanism)에 대한 차단제(blocking agent)로서의 활성을 나타내는 것으로서, 구체적으로 단백질(proteins), 항체(antibodies), 폴리펩티드(polypeptides), 펩티드(peptides), 백신(vaccines), 유전자(oligonucleotides), 호르몬(hormones), 효소(enzymes), 리보자임(ribozyme), 사이토카인(cytokines) 또는 케모카인(chemokines) 등이 사용될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, another example of a substance that inhibits the activity of the NOD2 protein, an N-terminal effector binding site (N-terminal effector) essential for nucleotide binding and self-oligomerization of the NOD2 protein biding region) or competitive inhibitors that directly inhibit the activity by binding to active sites, including C-terminal leucine-rich repeat motifs, which detect patterns of microorganisms and regulate their activity. competitive inhibitor), a noncompetitive inhibitor that binds to a non-active site and interferes with the function of the active site, a substance that blocks the localization of NOD2 protein to a specific location in the cell, a NOD2 protein Exhibiting activity as a blocking agent for the degradation mechanism or inducing degradation, and the mechanism of activity of NOD2. Specifically, proteins, antibodies, polypeptides, peptides, vaccines, oligonucleotides, hormones, enzymes, ribozymes, Cytokines or chemokines may be used, but the present invention is not limited thereto.

본 발명의 일실시예에 있어서, 상기 에스트로겐 결핍으로 유도된 골 질환은 골다공증(osteoporosis), 골연화증(osteomalacia), 골감소증(osteopenia), 골위축(bone atrophy) 및 골관절염(osteoarthritis)으로 구성된 군에서 선택된 질환이다.In one embodiment of the present invention, the bone disease induced by estrogen deficiency is a disease selected from the group consisting of osteoporosis (osteoporosis), osteomalacia (osteomalacia), osteopenia, bone atrophy and osteoarthritis (osteoarthritis) to be.

본 발명에서 사용되는 용어 “골 질환”은 골밀도가 감소되어 일어나는 모든 질환을 의미한다. 상기 골 질환은 골다공증, 골연화증, 골감소증, 골위축, 골관절염 등을 모두 포함하는 의미이다.As used herein, the term "bone disease" refers to any disease caused by reduced bone density. The bone disease is meant to include all osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis and the like.

상기 골다공증은 골다공증의 증상인 골밀도의 감소는 뼈(bone)내의 파골세포와 조골세포의 불균형에 의한 결과이다. 과도한 파골세포의 생성은 골다공증과 같은 골밀도를 감소시키는 질병을 유도한다. 뼈 흡수를 수행하는 파골세포는 크고 다핵의 세포로써 골 기질을 무너뜨리고 뼈 미네랄을 분해하는 기능을 한다. 활성화된 파골세포는 세 개 이상의 핵을 가지고 있는데, 파골세포는 파골세포 전구세포(precusor cell)로부터 성숙한 다핵의 파골세포로의 분화를 유도하기 위해 다양한 호르몬들과 인자들을 요구한다. 그 인자들 중 가장 중요한 두 가지 인자는 조골세포(osteoblast)로부터 생산되는 MCSF(macrophage colony stimulating factor)와 RANKL(receptor activator of nuclear factor-kB ligand)이다 (Mojtaba A. et al., Cancer Biol Ther., 7:1,3-9;1 (2008)). 따라서 골밀도 감소에 따른 골 질환은 본 발명의 조성물을 이용하여 골밀도 감소를 억제함으로써 예방하거나 치료될 수 있다.The osteoporosis is a symptom of osteoporosis, the decrease in bone density is a result of the imbalance of osteoclasts and osteoblasts in the bone (bone). Excessive osteoclast production leads to diseases that reduce bone density, such as osteoporosis. Osteoclasts that perform bone resorption are large, multinucleated cells that break down bone matrices and break down bone minerals. Activated osteoclasts have three or more nuclei, which require various hormones and factors to induce differentiation from osteoclast precursor cells to mature multinucleated osteoclasts. The two most important factors are the macrophage colony stimulating factor (MCSF) and receptor activator of nuclear factor-kB ligand (RANKL) produced from osteoblasts (Mojtaba A. et al., Cancer Biol Ther. , 7: 1,3-9; 1 (2008)). Therefore, bone diseases caused by bone mineral density can be prevented or treated by inhibiting bone mineral density reduction using the composition of the present invention.

본 발명의 약학적 조성물은 약물복용, 노화, 수술 등으로 인해 에스트로겐의 감소 또는 결핍이 예상되거나 진행중인 대상, 예를 들면 난소절제술(ovariectomy), 폐경, 갱년기, 아스피린 등의 비스테로이드성 소염진통제(NSAID)를 포함하는 약물 복용 등으로 인해 에스트로겐의 감소 또는 결핍이 예상되거나 진행중인 환자에 전신 또는 국소적으로 처리될 수 있으며, 유효성분인 NOD2 유전자에 대한 억제제의 투여형태 및 투여량은 연령, 성별, 체중, 증상, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 달라질 수 있다.The pharmaceutical composition of the present invention is a nonsteroidal anti-inflammatory drug (NSAID), such as an ovariectomy, menopause, menopause, aspirin, or the like, which is expected or undergoing a reduction or deficiency of estrogen due to drug use, aging, surgery, or the like. May be administered systemically or locally to patients in anticipation or progression of estrogen reduction or deficiency due to the use of drugs, and the dosage form and dosage of the inhibitor for the active ingredient NOD2 gene may be age, sex, or weight. , Symptoms, extent of disease, drug form, route of administration, and duration.

본 발명의 약학적 조성물은 통상적인 방법에 따라 약제학적 제제에 적합한 어떠한 형태로든 제형화(formulation)하여 사용될 수 있으며, 예를 들면 정제, 환제, 산제, 캅셀(capsule)제, 시럽, 에멀젼, 마이크로에멀젼 등을 포함하는 다양한 경구 투여 형태로, 또는 근육내, 정맥내 또는 피하 주사를 위한 멸균 주사용액 형태 또는 좌제 등을 포함하는 다양한 비경구 투여 형태로 제형화할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical compositions of the present invention can be used in any form suitable for pharmaceutical formulations according to conventional methods, for example tablets, pills, powders, capsules, syrups, emulsions, micro It may be formulated in various oral dosage forms, including emulsions, or in various parenteral dosage forms, including, but not limited to, sterile injectable solutions or suppositories for intramuscular, intravenous or subcutaneous injection.

또한, 본 발명의 약학적 조성물은 그 투여 형태에 따라 조성물의 제형화에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있으며, 예를 들면 본 발명의 조성물이 경구제형의 형태로 제조되는 경우에는 사용되는 담체의 예로 셀룰로오스, 규산칼슘, 옥수수전분, 락토오스, 수크로스, 덱스트로스, 인산칼슘 및 스테아르산, 스테아르산 마그네슘, 스테아르산 칼슘, 젤라틴, 탈크, 계면활성제, 현탁제, 유화제, 희석제 등을 들 수 있고, 주사제의 형태로 제조되는 경우에는 담체의 예로 물, 식염수, 포도당 수용액, 유사 당수용액, 알콜, 글리콜, 에테르(예: 폴리에틸렌글리콜 200), 오일, 지방산, 지방산에스테르, 글리세라이드, 계면활성제, 현탁제, 유화제 등을 들 수 있다.In addition, the pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in formulating the composition according to the dosage form thereof, for example, the composition of the present invention is prepared in the form of oral dosage form. Examples of carriers used may include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate and stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifiers, Diluents; and the like, when prepared in the form of injectables, examples of the carrier include water, saline, aqueous glucose solution, pseudoglucose solution, alcohol, glycol, ether (e.g. polyethylene glycol 200), oil, fatty acid, fatty acid ester, glycerol. Ride, surfactant, a suspending agent, an emulsifier, etc. are mentioned.

본 발명의 조성물의 약학적 투여 형태는 이들의 약학적 허용가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타약학적 활성 약물과 결합하거나 적당한 조합의 형태로 사용될 수 있다.Pharmaceutical dosage forms of the compositions of the invention may also be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active drugs or in the form of a suitable combination.

본 발명의 투여에 있어서, 본 발명의 조성물은 투여의 모든 방식이 가능하며, 예를 들면, 경구, 직장, 정맥, 복강, 근육 또는 피하 주사에 의해 투여될 수 있다.In the administration of the present invention, the composition of the present invention is possible in any manner of administration, and may be administered by oral, rectal, intravenous, intraperitoneal, intramuscular or subcutaneous injection, for example.

본 발명의 투여에 있어서, 본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 1일 0.0001 내지 100 ㎎/kg으로, 바람직하게는 0.001 내지 100 ㎎/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.
In the administration of the present invention, the preferred dosage of the composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. It may be administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
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 merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

실시예Example 1: 실험동물의 준비 1: Preparation of Laboratory Animals

C57BL/6J 계통의 6-주령 NOD2^+/+(야생형, WT) 및 NOD2^-/-(NOD2 유전자 넉아웃, NOD2-KO) 마우스를 잭슨 연구소(Jackson Laboratory)로부터 구입하였다.
Six-week old NOD2 ^{+ / +} (wild type, WT) and NOD2 ^{− / −} (NOD2 gene knockout, NOD2-KO) mice of the C57BL / 6J line were purchased from the Jackson Laboratory.

실시예Example 2: 2: NOD2NOD2 유전자 억제로 인한 Due to gene suppression OVXOVX -유도 골 밀도 감소 방지 효과-Prevents induced bone density reduction

NOD2 유전자를 갖는 야생형 WT 마우스, 및 상기 실시예 1에서 제조한 NOD2 유전자를 억제한 NOD2-KO 마우스를 대상으로 난소절제술(OVX) 또는 겉보기수술(sham)을 시행하였으며, 수술 8주 후 각 실험 마우스로부터 대퇴골을 적출하여 방사선사진 및 골 밀도를 비교 분석하였다.Ovarian excision (OVX) or apparent surgery (sham) were performed on wild-type WT mice bearing NOD2 gene and NOD2-KO mice that inhibited the NOD2 gene prepared in Example 1, and each experimental mouse was 8 weeks after surgery. The femurs were extracted from and analyzed for radiograph and bone density.

구체적으로, 상기 마우스들을 대상으로 난소절제술(OVX)(13 마리) 또는 겉보기수술(sham operation)(11 마리)을 실시하였다. 수술 8주 후에 골 밀도(bone density)를 분석하였다. 모든 마우스들은 IRC의 무균시설(specific pathogen-free animal facility)에서 사육되었으며, IRC의 실험동물운영위원회의 지침에 따라 관리되었다. 사육 및 동물실험 기준은 동 위원회의 승인을 받았다(2010-020).Specifically, ovarian resection (OVX) (13 animals) or sham operation (11 animals) were performed on the mice. Bone density was analyzed after 8 weeks of surgery. All mice were bred in IRC's specific pathogen-free animal facility and managed according to the guidelines of the IRC's Experimental Animal Steering Committee. The criteria for breeding and animal testing were approved by the Committee (2010-020).

대퇴골(femora)의 방사선사진 분석(radiographic analysis)은 X-선 시스템(soft X-ray system, 모델명: CMB-2)을 통해 수행되었으며, 대퇴골의 원위 골간단(distal metaphysis)의 상대적 골 밀도(relative bone density)를 실시간 영상 프로세싱(real time image processing) 및 측정 시스템(measurement system, ZET-1)을 이용한 X-선 방사선사진 분석으로 측정하였다. 이러한 측정치의 정확도 및 정밀도는 이전에 보고된 방법(Kyung TW, et al., Mice. J Nutr. 2009 :139, 502-506)에 따라 산출되었으며, 각 동물의 대퇴골은 회수 후 10% 중성 완충 포르말린에서 보관하였다.Radiographic analysis of the femur was performed by means of a soft X-ray system (model name: CMB-2) and the relative bone density of the distal metaphysis of the femur. Bone density was measured by X-ray radiographic analysis using real time image processing and a measurement system (ZET-1). The accuracy and precision of these measurements were calculated according to previously reported methods (Kyung TW, et al., Mice. J Nutr. 2009: 139, 502-506), and the femurs of each animal were 10% neutral buffered formalin after recovery. Stored at.

통계분석은 모든 결과 값을 평균±표준편차(SEM)로 표현하였다. 각 군간의 분석은 two-way ANOVA에 의해 추정하였으며, 본페로니 사후 검정(Bonferroni posttests)을 실시하였다. 통계적 P 값이 0.05 미만일 때 통계적 유의성이 있는 것으로 판단하였다.Statistical analysis expressed all results as mean ± standard deviation (SEM). The analysis between groups was estimated by two-way ANOVA and Bonferroni posttests were performed. Statistical significance was determined when the statistical P value was less than 0.05.

도 1은 NOD2의 결핍 시 마우스에서 OVX-유도된 골 소실에 대한 효과를 나타낸다. OVX (17 주령) 및 겉보기수술(9 주령) 8주 후, 야생형(OVX, n = 5; sham, n = 6) 및 NOD2-KO 마우스(OVX, n = 5; sham, n = 6)의 X-선 사진(도 1a)으로부터 Image J program을 이용하여 대퇴골의 원위 골간단(distal metaphysis)의 상대적인 강도(도 1b)를 측정하였다. 또한 sandwich ELISA를 이용하여 혈청 M-CSF를 측정 (도 1c)하였다.1 shows the effect on OVX-induced bone loss in mice in the absence of NOD2. After 8 weeks of OVX (17 weeks) and apparent surgery (9 weeks), X of wild-type (OVX, n = 5; sham, n = 6) and NOD2-KO mice (OVX, n = 5; sham, n = 6) The relative intensity (distal metaphysis) of the distal metaphysis of the femur was measured using the Image J program from the line photograph (FIG. 1A). Serum M-CSF was also measured using sandwich ELISA (FIG. 1C).

그 결과, 도 1에 나타난 바와 같이, 수술 8주 후 마우스의 대퇴골 밀도는 겉보기 수술군(난소 미절제군)에서 NOD2 유전자 억제 유무에 따른 유의한 차이가 나타나지 않았으나, OVA 수술군(난소 절제군)에서는 NOD2 유전자가 억제된 NOD2-KO 마우스의 실험군에서 야생형 마우스와 비교하여 현저하게 높게 나타났다 (도 1a, 1b; P < 0.05). 또한 NOD2-KO 마우스에서 혈청 M-CSF도 감소한 것으로 나타났다 (도 1c, P < 0.001). As a result, as shown in FIG. 1, the femoral density of the mice after 8 weeks of surgery was not significantly different depending on the NOD2 gene suppression in the apparent surgery group (non-ovarian resection group), but in the OVA surgery group (ovarian resection group). The experimental group of NOD2-KO mice with NOD2 gene inhibition were significantly higher than wild type mice (FIGS. 1A, 1B; P <0.05). In addition, serum M-CSF was also reduced in NOD2-KO mice (FIG. 1C, P <0.001).

이러한 결과는 NOD2 유전자의 억제를 통해 난소 기능의 상실로 유도되는 골 소실을 완화시키고 파골세포의 생성을 촉진시키는 M-CSF의 발현을 감소시켜 파골세포에 의한 골 밀도의 감소를 억제할 수 있음을 의미한다. 따라서 NOD2 유전자의 억제제는 난소 기능의 상실, 노화, 약물 복용 등 에스트로겐 결핍 상황으로 인한 골 질환의 치료에 효과적으로 사용될 수 있다.
These results suggest that inhibition of NOD2 gene can alleviate bone loss induced by loss of ovarian function and reduce the expression of M-CSF, which promotes the production of osteoclasts, thereby inhibiting the decrease in bone density by osteoclasts. it means. Therefore, inhibitors of the NOD2 gene can be effectively used for the treatment of bone diseases caused by estrogen deficiency, such as loss of ovarian function, aging, drug use.

실시예Example 3: 3: NOD2NOD2 가 파골세포의 증가에 미치는 영향Effect on the increase of osteoclasts

먼저, BMMs(bone marrow-derived macrophage)을 RANKL에 노출시켜 파골세포형성 시 NOD2 발현이 조절되는지를 확인하였다. 총 RNA는 제조사의 지시대로 올리고(dT) 프라이머 및 AMV 역전사 효소(M5101; Promega, Madison, WI)를 이용하여 역전사 시켰다. qPCR은 QuantiTect SYBR Green PCR kit (204143; Qiagen, Hilden, Germany) 및 제조사의 지시에 따른 프라이머를 이용하여 DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc., Waltham, MA)에서 수행하였다. 각 프라이머쌍의 특이성은 멜팅커브(melting curve) 분석 및 아가로스겔 전기영동으로 확인하였다. GAPDH 유전자도 동일하게 증폭하였다. GAPDH와 비교하여 상대적인 카피수는 2^-t.를 이용하여 계산하였다. 상기 프라이머 서열은 다음과 같다: 5’-ggcaccctgaagttgacattttgc-3’ 및 5’-acatctcccacagagttgtaatcc-3’ (NOD2); 5’-acccagaagactgtggatgg-3’ 및 5’-cacattgggggtaggaacac-3’ (GAPDH).First, BMMs (bone marrow-derived macrophage) was exposed to RANKL to determine whether NOD2 expression is regulated during osteoclast formation. Total RNA was reverse transcribed using oligo (dT) primers and AMV reverse transcriptase (M5101; Promega, Madison, Wis.) As directed by the manufacturer. qPCR was performed on a DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc., Waltham, MA) using QuantiTect SYBR Green PCR kit (204143; Qiagen, Hilden, Germany) and primers according to the manufacturer's instructions. The specificity of each primer pair was confirmed by melting curve analysis and agarose gel electrophoresis. The GAPDH gene was also amplified in the same way. Relative copy numbers compared to GAPDH were calculated using 2 ^-t . The primer sequences are as follows: 5'-ggcaccctgaagttgacattttgc-3 'and 5'-acatctcccacagagttgtaatcc-3'(NOD2);5'-acccagaagactgtggatgg-3'and5'-cacattgggggtaggaacac-3' (GAPDH).

실험 결과, NOD2는 BMM에서 구성적으로(constitutively) 발현되고 RANKL에 노출 후 현저하게 감소되었다 (도 2a).
As a result, NOD2 was constitutively expressed in BMM and significantly decreased after exposure to RANKL (FIG. 2A).

다음으로, NOD2가 파골세포생성에 영향을 미치는지 평가하기 위해, WT 및 NOD2-KO 마우스로부터 분리된 BMM의 배양에서 파골세포 형성을 측정하였다. 구체적으로, 대퇴골과 정강이뼈를 무균상태로 제거하고 부착된 연조직이 없는 상태로 절개하였다. 골 말단을 잘라내고, 골수강을 뼈의 한쪽 말단으로부터 α-MEM으로 멸균된 21 게이지 바늘을 이용하여 플러싱하였다. 골수강을 파스퇴르 피펫을 이용하여 휘저어서 단일세포 현탁액을 얻었으며, 이를 두 번 세척하고 M-CSF (20 ng/ml) (416-ML; R&D Systems, Minneapolis, MN)가 있는 플레이트 상에서 16시간 동안 배양하였다. 이후 비-부착성 세포들을 수집하고, Ficoll-Hypaque gradient로 놓아서, 2일 이상 배양하였으며, 이전에 보고된 바와 같이 (Lee JE, et al., Exp Hematol 35; 1100-1108, 2007.), 부착성 단핵구/대식세포 유사 세포의 대규모 집단이 M-CSF로 처리된 배양 플레이트의 바닥에 형성되었다. 일부 비부착성 세포들을 PBS로 세척하여 제거하고, 부착성 세포(BMMs)를 0.02% EDTA를 이용하여 수집하여, 8×10³ cells/well로 씨딩하였다. 부착성 세포들은 FACs로 분석하여 CD3와 CD45R에 대해 음성, 그리고 CD11b와 F4/80에 대해 양성임을 확인하였다. 오염된 기질세포가 없음은 M-CSF 없는 상태에서 세포 성장의 결핍으로 확인되었다. M-CSF 및 RANKL (40 ng/ml) (462-TR; R&D Systems)이 포함된 추가 배지를 3일째에 첨가여 신선하게 하였다. 지시된 배양시간 이후, 세포들은 10% 포르말린으로 10분간 고정하고 TRAP(tartrate-resistant acid phosphatase)으로 염색하였다 (Suda T, et al., Endocrinol Rev 20: 345-357, 1992.). TRAP-양성 다중핵 세포(MNC)의 수 (예. OC with ≥3 nuclei)를 확인하였다.Next, to assess whether NOD2 affects osteoclastogenesis, osteoclast formation was measured in culture of BMM isolated from WT and NOD2-KO mice. Specifically, the femur and tibia were removed aseptically and dissected without attached soft tissue. The bone ends were cut off and the bone marrow cavity was flushed from one end of the bone using a 21 gauge needle sterilized with α-MEM. The intramedullary canal was agitated using a Pasteur pipette to obtain a single cell suspension, which was washed twice and for 16 hours on a plate with M-CSF (20 ng / ml) (416-ML; R & D Systems, Minneapolis, MN). Incubated. The non-adherent cells were then collected, placed in a Ficoll-Hypaque gradient, incubated for 2 days or more, as previously reported (Lee JE, et al., Exp Hematol 35; 1100-1108, 2007.), a large population of adherent monocyte / macrophage like cells was formed at the bottom of the culture plate treated with M-CSF. Some non-adherent cells were removed by washing with PBS and adherent cells (BMMs) were collected using 0.02% EDTA and seeded at 8 × 10 ³ cells / well. Adherent cells were analyzed by FACs to confirm they were negative for CD3 and CD45R and positive for CD11b and F4 / 80. The absence of contaminated stromal cells was confirmed by a lack of cell growth in the absence of M-CSF. Additional medium containing M-CSF and RANKL (40 ng / ml) (462-TR; R & D Systems) was added fresh on day 3. After the indicated incubation time, cells were fixed with 10% formalin for 10 minutes and stained with tartrate-resistant acid phosphatase (TRAP) (Suda T, et al., Endocrinol Rev 20: 345-357, 1992.). The number of TRAP-positive multinucleated cells (MNC) (eg OC with ≧ 3 nuclei) was identified.

그 결과, 두 가지 골형성 사이토카인, M-CSF 및 RANKL의 존재하에서, 최대 파골세포 형성은 3일 후에 나타났다. TRAP-양성 MNC(multinuclear cell)를 카운팅하여 측정한 결과, NOD2-KO BMM에서 파골세포 형성의 정도는 WT BMM 보다 27% 낮았다 (도 2b). 또한 NOD2의 억제는 3개 이상의 핵을 갖는 파골세포와 비교하여, 10개 이상의 핵을 갖는 파골세포를 높은 정도로 감소시켰다 (27% vs. 42%). MDP(muramyl dipeptide)에 의한 NOD2의 자극은 파골세포 형성을 현저하게 증가시켰는데 (도 2c), 이는 파골세포 형성 동안 NOD2가 동격의 신호(appositive signal)로서 작용함을 확인하는 것이다. 이러한 결과와 일치하게, RANKL 자극 48시간 후, TRAP, NFAT2, cathepsin K, calcitonin receptor, ATP6v0d2, 및 DC-STAMP의 전사체들은 WT 마우스로부터 얻은 파골세포 보다 NOD2-KO 마우스로부터 얻은 파골세포에서 현저하게 낮았다 (도 2d).
As a result, in the presence of two osteogenic cytokines, M-CSF and RANKL, maximal osteoclast formation appeared after 3 days. As a result of counting TRAP-positive multinuclear cells (MNC), the degree of osteoclast formation in NOD2-KO BMM was 27% lower than that of WT BMM (FIG. 2B). Inhibition of NOD2 also reduced osteoclasts with at least 10 nuclei to a high degree (27% vs. 42%) compared to osteoclasts with 3 or more nuclei. Stimulation of NOD2 by MDP (muramyl dipeptide) significantly increased osteoclast formation (FIG. 2C), confirming that NOD2 acts as an equivalent signal during osteoclast formation. Consistent with these results, 48 hours after RANKL stimulation, the transcripts of TRAP, NFAT2, cathepsin K, calcitonin receptor, ATP6v0d2, and DC-STAMP were significantly higher in osteoclasts from NOD2-KO mice than in osteoclasts from WT mice. Low (FIG. 2D).

실시예Example 4: 4: NOD2NOD2 가 조골세포생성에 미치는 영향Effect on osteoblast production

NOD2-KO 마우스에서 관찰된 증가된 골소실이 증가된 골형성에 의해 일어나는지를 확인하기 위해, WT 마우스와 비교하여 NOD2-KO의 BMSC(bone marrow mesenchymal stromal cells)의 조골세포생성(osteoblastigenesis)을 평가하였다. To determine if the increased bone loss observed in NOD2-KO mice is caused by increased bone formation, assess osteoblastigenesis of bone marrow mesenchymal stromal cells (BMSCs) of NOD2-KO compared to WT mice. It was.

구체적으로, 대퇴골과 정강이뼈를 무균상태로 제거하고 부착된 연조직이 없는 상태로 절개하였다. 골 말단을 잘라내고, 골수강을 뼈의 한쪽 말단으로부터 RPMI-1640으로 멸균된 21 게이지 바늘을 이용하여 플러싱하였다. 골수 현탁액을 파스퇴르 피펫을 이용하여 휘저어서 단일세포를 얻었으며, 이를 두 번 세척하고 분리배지(RPMI-1640 supplemented with 9% fetal bovine serum, 9% horse serum, 100 u/ml penicilline, 100 ug/ml streptomycin, 12 uM L-glutamine)로 재현탁한 다음, 현탁액을 플레이트 상에서 24시간 동안 배양하였다. 비부착성 세포들을 제거하고, 부착성 세포들을 세척한 다음, 신선한 분리배지를 매 3일 마다 첨가하였다. 4주 후, 세포들을 PBS로 세척하고 37℃에서 0.25% trypsin/1 mM EDTA로 2분간 반응시켰다. 위에 뜬 세포(lifted cells)를 세척하고, 1주일간 더 배양한 다음, 확장배지(expansion medium: α-MEM supplemented with 9% FBS, 9% horse serum, 100 u/ml penicilline, 100 ug/ml streptomycin, 12 uM L-glutamine)에서 10일간 8000 cells/dish로 플레이팅함으로써 확장시켰다. 남은 BMSC를 수집하여 CD44 양성, 및 CD3, CD11b, CD45R 음성임을 FACS로 확인하였다 (데이터 미도시). 골 형성을 위해, 상기 배양물을 50 μM 아스코르브산, 및 10 mM β-글리세로포스페이트를 포함하는 확장배지로 21일간 매 3일 마다 배지를 교환하면서 배양하였다 (Zhang X, et al., J Clin Invest 2002;109:1405-1415.). 세포외기질 석회화는 Alizarin과 S 염색을 이용하여 평가하였다. 골 형성 분화는 이미지 분석 프로그램(Image J)를 이용한 Alizarin red-염색 영역 및 밀도의 측정을 통해 정량화하였다. 배양물에서 ALP(alkaline phosphatase) 수준은 제조사의 설명서에 따라 세포 용해물에 대한 형광분석에 의해 검출되었다 (AtarBright Alkaline Phosphatase Detection kit; MT-1000, Sigma Aldrich).Specifically, the femur and tibia were removed aseptically and dissected without attached soft tissue. The bone ends were cut off and the bone marrow cavity was flushed from one end of the bone using a 21 gauge needle sterilized with RPMI-1640. The bone marrow suspension was agitated using a Pasteur pipette to obtain single cells, which were washed twice and separated (RPMI-1640 supplemented with 9% fetal bovine serum, 9% horse serum, 100 u / ml penicilline, 100 ug / ml). streptomycin, 12 uM L-glutamine), and then the suspension was incubated for 24 hours on a plate. Non-adherent cells were removed, adherent cells were washed, and fresh separation medium was added every 3 days. After 4 weeks, cells were washed with PBS and reacted for 2 minutes with 0.25% trypsin / 1 mM EDTA at 37 ° C. The lifted cells were washed, incubated for one more week, followed by expansion medium: α-MEM supplemented with 9% FBS, 9% horse serum, 100 u / ml penicilline, 100 ug / ml streptomycin, 12 uM L-glutamine) was expanded by plating at 8000 cells / dish for 10 days. The remaining BMSCs were collected and confirmed by FACS to be CD44 positive and CD3, CD11b, CD45R negative (data not shown). For bone formation, the cultures were incubated with medium every 50 days for 21 days with expansion medium containing 50 μM ascorbic acid and 10 mM β-glycerophosphate (Zhang X, et al., J Clin Invest 2002; 109: 1405-1415.). Extracellular matrix calcification was assessed using Alizarin and S staining. Bone formation differentiation was quantified by measuring the Alizarin red-stained area and density using an image analysis program (Image J). Alkaline phosphatase (ALP) levels in culture were detected by fluorescence analysis of cell lysates according to the manufacturer's instructions (AtarBright Alkaline Phosphatase Detection kit; MT-1000, Sigma Aldrich).

도 3에 나타난 바와 같이, NOD2의 억제는 골 형성에 대한 마커인, ALP(alkaline phosphatase)를 증가시켰다. MDP에 의한 NOD2의 자극은 ALP와 Alizarin을 감소시켰는데, 이는 NOD2가 시험관내에서 조골세포생성을 감소시킴을 확인한 것이다.
As shown in FIG. 3, inhibition of NOD2 increased alkaline phosphatase (ALP), a marker for bone formation. Stimulation of NOD2 by MDP reduced ALP and Alizarin, confirming that NOD2 reduced osteoblast production in vitro.

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> University of Ulsan Foundation For Industry Cooperation <120> Compositions for treatment of bone disease comprising inhibitor against NOD2 <130> NP13-1188 <150> KR 12/078699 <151> 2012-07-19 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 46720 <212> DNA <213> Homo sapiens <400> 1 catctctggt gccagaggaa agggggcatc tcgaggctgg ggaggcccct ggcaggagtg 60 agaagtcctg aggagcccgg gcttcaggtc tcagaggggg ccactgtttt ttgagtacct 120 tcctctctgt gggactcagt ttccccatct tcaccatgcc agtgagtggg tgggtgtaag 180 tgagcgagtc tgcctggagg atcctactgc tgctctctgc acctcttagg ggaatctcct 240 gcctcctcct tctcccttgg tgggcgtgag atcaaagttc aaatgtgtac ctcagtttct 300 gcatctataa gttagaggca ataatagtca ataccctaga ggtggaagta actgagttac 360 tgtctgtaaa gactagcaca tggtaaacac tttctagatg ccagttattt tcgttatttt 420 taaaaattga cttcacattt tttaaaacaa ccaaatatga gtttcaaaag gaacatttac 480 attaacatgt taaatggaaa accggtgtca tttgctccgg agaacagaag aatgtaaaat 540 aaaatatatt taatggaaac aaaaacaatg ctattaaagc taattagata gtttcagagc 600 caagtaatct aggcagtgct agaaaggtac tcaaacccta ttagtactaa actgagaaat 660 aaactccatt caacataaac ggaaaaactg aaaggaaaat gaaaaaaaag aaccttaaat 720 gaccagtcca ctcttaccta acatgtaacc tgtcacactc tggaaaagtt ttcctagttt 780 tggctgtgtg tgcgtgtgtg tgtgtgtgtg tgttgggaag tgaggcgtgt gttgggaagc 840 gatgtgtgtg ttgggaagcg atgtgtgtgt gttgggaagt ggggtgtgtg tatattggga 900 agcatgtttg tgtgtgttgg gaagcggtgt gtgtgttggg aagtggtgtg tgtggggtgt 960 ggtgtgtgtg tgttgggaag tggtgtgtgt gtggggtgtg tgtatgtgca cgtgtgtgtt 1020 gggaagcagt gtgtgtgggg tgtgtgtgtg ttgggaaagc agggagtgta gtgtgtgttt 1080 gtgttggaaa gtggtgtgtg tgtggggtgt gtgtgtgtgt gcacgtgtgt gttgggaagc 1140 ggtgtgtgtg gggtgtatgt gttgggaaag cagggtgtgt gtgtgttggg aagtggtgtg 1200 tgtgtgagtg ttgggaagca gggtgtgtgt gttggaagta gggggtgtat gtgtatcggg 1260 aagcatgttt gtggtggggg gaagcagggc gtgtgtgtgt gtgtatgtga gtgttgggaa 1320 gcaaggtatg tgtgttgggg gaagcgaggg ctgtgtgtgt attgggaagc atgtttgtgt 1380 gtggggggaa gcgagggctg tgtgtgtgta ttgggaaaca tgtttgcgtg tgggggggaa 1440 gcagggcgtg tgtgtgtgtt gcgaagcggt gtgtgtgtgg tgtgtgtgtg tgcgcgtgtg 1500 tgctgggaag cggtgtgtgt ggtgtgtggt gtgtgtgtgt gtgtatgtgt atgtgttggg 1560 aagcgggttg ttccgaggac tccccgattc cctcctggtt gaaacgtggc ccagaaagag 1620 aggcacactg gctagggcac acagccaggg ccgtccagcc ccgcaggaag ccccggtcct 1680 gccctgtccc aggtaagcct ggccagccct ggagccggcg ctgcggggct gcggggctgc 1740 ggggctgcgg ggccacggga caggagcggg tgctgcggtc ccggcagagc aggggcgggg 1800 tccgggcggg ggcggagccg agcgcccgcg cacccggagc cgcagcccga gccggagccg 1860 gagccgggag tcgtggcccg gagtgggcct tggagtcggc gcgcaggcgg ctcgcggtga 1920 gtgctgtgcc cagcgcctgg gggtgggctg gggggagact gtgggcgccg agggtcccac 1980 ccccgggact tgaacacttg gcttctgagc cgtcctggcc gcagggcttg ggcaagggtt 2040 gcctcggttt ctcctggggg agtcccagaa tgtagatggg aagtgagacg gaaagggggc 2100 atctcgaggt tggggaggcc cctgggccac tcttttgatt tccttcctct ctgtgggact 2160 cagtttcccc agcttcacca tgccagtgag tgggtgggta taagtgagag agtctgcctg 2220 gaggatcctg ctgctgctct ctgtacctct tatgggaatc tcctgcctcc tccttctccc 2280 ttgttgggtg tgagatctaa gttcaaatcc atcccttccc agacagccca caccaggacc 2340 tgcgaggcca catcccccag ctgtttgctc tcctatgctt ctcaggttga tggagcagcc 2400 atctggtcct gctttcattc caacaaactg aaaagccctc caccccaatc cccctgggga 2460 ggacctcctg aagctgcatg agggctgggg gctggacccc tgcttctgag tttgctcttt 2520 ctgagcctct cctgccatgg ccttggtttc cccatgggct gtgacctagc ctcctccagg 2580 gtccttgttc tacagagggg aaacacctgc cctgtcaccc tcccctgacc ccttagtgaa 2640 aaagaccaga gtgacatccc caccacctgt acttgcctgt cccgatcact cagccatggg 2700 gctgctgggg cccctgagtt tgttggctgc tcaaggcccc tttgcctcgc tgcaccctgg 2760 gatctgaggc tggaagcatc ctcatccagc ccacctccct cctacccagt gcagagcccc 2820 tctgtgacac atgcccagtg gtggggagct tattactgat gaggtggctt tttagtgcct 2880 tgcagctctt gtttccttta gctaaaatct gtggcttaca gattctacac cttggttcca 2940 tgtctgtctc ccggagccat gcagagcaag tcagttcttt cattctttcc agcagtcatc 3000 attcatgcac tggatgagac acgagacaga gcagtaggaa aatcagacaa aaccccctgc 3060 cctcatggag tgcacactct agaaaggaat ggacagcaat gaaataagta aatacatggc 3120 gtagcaggtg gcgggaagta ctacgagtaa aacaaagctg ggaagcagga tggggtgctg 3180 gggctgggta ttgcattgta tatagcacaa ccagggaagg cttccccaag accacgatag 3240 ctgggcaaag tcataaggaa ttgaggggtg agccacatga gtatctgggg ggctgggagg 3300 ctttggtttt cactctgagc atgatgggag cacgggtttg gaacagagga gtcacctgct 3360 ttgacttggg gtctggcagg cccatctagc tgtggggtgg agaatggatc atgggggggg 3420 tggggtaagg gcagaaggag agcccgttga gcggatcatt gctgtcattc tcgggagaga 3480 ccatggtgtc ttgcccatgg cccagaggtg gttaaaaaaa agaaaaccag acattcagat 3540 ttgggatata tttcaaaggc cgtgtcgata gggtttgggg tgcaagagaa aggtagagtc 3600 aaagatgatt ctgagtgttt ggctcgagct tctggaggtt tgaagctgcc atgaactgaa 3660 atgggagttg cttagcggag agatcaagag tttggttttg tgcttgtgga gtttgagctg 3720 cccattaggc atcaatgatg agtaagcagt tgggaaaagt taagggcgga ggttggagtt 3780 gaaaataaaa ctttgggcat catcaccata taactggtat ttaaagccac aagagcaggt 3840 gggctcatct agggatggag tgatatggag aagagaaggg gtctaaccat tgagtgctgg 3900 ggcccccagt gttaggaacc agccaagaag acagaaagag tgaaaatcag agagttgggg 3960 tgtcctggag gaaatgaaga aaatgcccca aagaggaagg agggaacaaa tatgaccaat 4020 gcccctggca gagcaagcag gctgagggct gaggattgag caatgggagg tcactggtga 4080 cagtttcact ggagctggat ggggaactag agggaatggg aggggatggg aggacttggg 4140 gacagcagta caggcaacag acaagggggc ctgctgtaaa gggagcagat aaatgggatt 4200 ggagccaaat gaagaagggg agtgtcaaga gagtgcttta cttttacaat ggagaattag 4260 agtgcattgt gcactggtgg ggggatttga tctcttaggg agagaacagt gttagggagg 4320 gagaatgcag gatagctggg ggagggtggg gggcttggcc ccagcagaga ctcaggacac 4380 ttgggaagtt gagcttccct gggcttcccc tcctctcctg tctgcaaggg gtcagtgggc 4440 tgagatttca gcacttaagc aaagcatttg ctcttggccc cagagaaacc gggctggctg 4500 tggtctcagg aaggaaggag gtgtccaggc tcaggcctgg gcctgggttt cagggagggc 4560 ccacgtgggt cgccccttga ccctctcttt cagcaaggaa gtgatccttt ctctacatgg 4620 gcctcacctt ggggaggaca atggtgtctt tgaagttgta gtaactgaag tagagatcaa 4680 aaggcaatgc agatagactg acagatttcg cctgaagagg ggaagcccga ccaggtaata 4740 aaggagtaag aggaaggatg ttaaggacaa ttttaggaaa cagataatga gtgaatattt 4800 tttctctctc tttcccaatt taaactgaag caggagaaac tgaagctaga cataatgatt 4860 aacttcccaa gctggtgagc ttcctgagct ggttagtgag aacagcacta aggccaggtt 4920 ctcctcccca gatgtttaag atgagacagg acaatgcctg ctcagagaca gggcctggct 4980 gaattggccc tcaggattct ctctgctctg aggtttctgg aagaaggcca gggcagaggt 5040 gtggtgatgt agctgctggg aggacagagc tccgagtcac gtggcttggg cgggcctccc 5100 cttcctggtg tccacagaag cccaacgtca ctagctgggg tgtgtatggc tcacacgtag 5160 gccaggctgc cctaggcttg gtgtgcaagg gaggggcccc tacttacttg tggcctgtcc 5220 cctcgtgaat gtgtctcatg tccccagtgg ggtttttcag tgagggtcat ggtctccagg 5280 atgcacaagg ctttgtgcca gaattgcttg gaattgccta gttctggaag gctggttggc 5340 caactctggc ctccggcttt tcctttggga atttcccttg aaggtggggt tggtagacag 5400 atccaggctc accagtcctg tgccactggg cttttggcgt tctgcacaag gcctacccgc 5460 agatgccatg cctgctcccc cagcctaatg ggctttgatg ggggaagagg gtggttcagc 5520 ctctcacgat gaggaggaaa gagcaagtgt cctcctcgga cattctccgg gtaagaggag 5580 caggcattgt cccgtcccag cttgatcctc agccttcttt catccttggc cgcgacatgc 5640 tcccaggcct ggggtcagat ggggagtgct gactctgttt ctgggctgtt ttctggggag 5700 aatgggtcgg cgggtttttt tccccaggac ctgggcaggg tcaatggtgg gggccgctgt 5760 cgcatccttg gctggtgttt ccacagctga gaaccactcc agggccaagc ccagagctta 5820 ttctaccctt ttttgtcctc tcttcccctg tcctcggcca ccccaccctc ttggctcctc 5880 tgcttagatg tgggcacaag gaggagaact ccttggcctg agagaactac cttagatcct 5940 ggcttccagt ggcctctgca ggggggtaca ccctctctcc caagcagcca gacacacaag 6000 taacctcatt gcctcagttt ccccatctga ccagcacagg gccccctgtg ccccagcagc 6060 gttctgagag attggagctt tctccttttg cttaccttgg ctaccgtatg aggacggata 6120 cagagtgttc cccccacccc cagcccaggg gatatttgat tcatgaacat tccctcagtg 6180 tctttgtggg ggacaatgct gtgccaggct cagggatgcc aggacgagta agacccaggc 6240 tcccacgtgg cccaggcagg gagagagaca cataaacaac catcaggaaa gaggtaaaat 6300 ccccaggcca cttggcatct gctcccttga gtgtctggga atgtccctga tttataaaaa 6360 gaagctgacg gccctctttg ttgtccatgc ctacaccctt tcactttcgt ttcttcgggg 6420 cactgcagca gcccttgtcc acagacccca tgacaatcgc agaactgacc atgctgagag 6480 attttcttgg ctgctcaggg accctgccag ggcttgaagc tcctggaggg tcacttgccc 6540 tcaaattccc agaacgcaca gcaggtcact gatgatagca gtggcagcag tctgtgcacg 6600 gtggtttcga gggcgtggga gggaggtgag ggccctaggg caagtgtgtg tgggaagtgt 6660 tgatggggga caaggcacca gaacgctcgg aaacaactta gtttgcaccg taatttttca 6720 cttcgcctag gacaggacct ttagagcaat attctgagtc taccccttgg agtagcagtg 6780 tgcaaaacac acagcacggg cttggggccc ccgtggggaa cccaaatgta agagttagag 6840 acatgcattc cggagtcata catggctcgt gttgaaatcc tgactctgcc tgtctagctg 6900 tgacacatcg tacaaatcac ttagcttctt ggtgcctcag tgtcttcctc tgtagaatgg 6960 gtagatcata ggcactactt cagagtggct gggagggttc agtgaattcc tgcaggagag 7020 cacttagaat ggcacttggt gtgtagttta tgcttaatta atattagccg ttactgaaac 7080 tgctgtagcc tgaatccagc cagcatgaaa gagcccctct caccctgctt cgaagagaat 7140 gaattccctg attgtttgga agatctctct ctctctctct gtcttttttt tttttttttg 7200 agaaacggtc ttgctctctt gcccaggctg gagcgcaatg gtgccatctt ggctcactgc 7260 aacctctgcc tcccgggttc aagtgattct cctgtctcag cctcctgagt agctgggatt 7320 acaggcgctc gccaccacgc ctggctaatt tttgtatttt tagtagagac agcgtttcac 7380 cgtgttggcc gggctggtct agcgctcctg atctcaagtg accttgggag atctcttgct 7440 cctaatatta cctcaagcct ttttaaacgt tttaagccgg agaccaagca tggatatggg 7500 agttaggggt cttgatttaa ttcttggttg cttcaaactc tgtggaacct tgaggtgttt 7560 cttgccttct ctgggtctca attttcacat ctatatggtg gggagcttgg attgggtaat 7620 gtctgaggct agaaccatgg ccaactcggg ttctgctggg gctgacttgc cctggccttc 7680 cctgaccacc ctgcatctgg cttctggaga agtcccgcac tgaccttgtt ctcctcccca 7740 ggttgtgaaa tgtgctcgca ggaggctttt caggcacaga ggagccagct ggtcgagctg 7800 ctggtctcag ggtccctgga aggcttcgag agtgtcctgg actggctgct gtcctgggag 7860 gtcctctcct gggaggacta cgagggcttc cacctcctgg gccagcctct ctcccacttg 7920 gccaggcgcc ttctggacac cgtctggaat aagggtactt gggcctgtca gaagctcatc 7980 gcggctgccc aagaagccca ggccgacagc cagtccccca agctgcatgg ctgctgggac 8040 ccccactcgc tccacccagc ccgagacctg cagagtcacc ggccagccat tgtcaggagg 8100 ctccacagcc atgtggagaa catgctggac ctggcatggg agcggggttt cgtcagccag 8160 tatgaatgtg atgaaatcag gttgccgatc ttcacaccgt cccagagggt gaggcactcc 8220 tggtgtgcat cacagagttc tcaggaaagg ggtgcttagt caccaagact gatttgtcct 8280 catgaagtca gcctgtgggg taacttggtc catgggattt cccctaaaaa ggtagccagg 8340 caggtaaaat ttgctcttga ctcttggcag gaaacgtaca actctttctt tcttcttttc 8400 ttttcttttt ctcactctgt taccctggct agaatgcagt ggcacaatca tagctcactg 8460 tagccttgaa ttcctgcgct caagtgatct tctggcctta gagtagctgg gactacggct 8520 gctgtaccac catgaacagc taattttttt tttttctttt agagatgggg tgttgctatg 8580 ttgcccaggc tggtctccag ctcctggctt taagcaatcc tcccgccttg gcctcccaaa 8640 ctgttgggat tgcaggcatg agccactttg cctggccaac agaacacttc tgccgagagg 8700 aagtgtgtgg tggccaggaa ctcagattct ggagccagaa tggtgcaggc tcaaggtcaa 8760 ccctgtgtga tctcaggctt ccctatggag cctctccagc ctcagtctcc cttgtttcag 8820 tttcctcatc tacaaaacaa tgttaatagt caaatggtgc ctatcctata aggctcttgg 8880 gaggattcag tgagttaatt tgagtaatgc ttaggatagt gtctattacc actggctgct 8940 atttattatt tctgttatga gtgatactct gtacttgtac acttttattt ctgtctgttt 9000 taaattaaca gcacaacaga ccataacact gcagtatatt gaatttattt tataattaac 9060 atagcatatt ataaactaat atagcttaaa tgtttatgta ggatttctga catgaaattg 9120 cattagatca tagatgttca gagttggtat ataacagccc ctgagaatgt agtaactcag 9180 cagagaccag aaggtcagag aaatgaccac tgagtatttt tgaaactctt ttgttttctt 9240 ccaaatagtg attcttaggg ctcctgagag gcagatggaa caatcattaa cattccactt 9300 tataaatcgg gaagttgaga ccaaggaaag tagtttgaat aagctcacag tagttaatga 9360 gggggccagt gctggaccaa ttggccagca ctggtcattg acttattcat ccatcattca 9420 tttattcagc cagaatctat taggtgcttc atacatattt gcttaaagtt tgttgtgttc 9480 atagagcttt gcacacggta ggtactccat aaacatttgt tgatgaaata agtgagttac 9540 tgaatgaatg attgaattag aatgacactg cagtgttaaa atgggctggg ttggggaaca 9600 ttttagtttt tgtttttgtc tgttttccaa aaatgtatgt gttgttcaca tgagtctgga 9660 taaccctaga ttgagattga tgacataaat aaatttgtct tcaaggctgc actaaagctg 9720 gctcacatgg ctaggtattt acagagcaga agtggtgcag tcctctctga ttagttgcac 9780 gtacagaaga catattcgtt attggactga ccttagtttc tcttataatt tgttagggga 9840 attgaatcag cccatctgag aagttacaag attgtgtctt gtcatcttta aaagttcagc 9900 aatgtgatgt ggtacagatg gtctgagggg tttggagaag gtagcctaga tccctagggc 9960 ccagagaaga caggatgtga acagaggaag tacatggatt ggtgaagaaa agaaatggga 10020 taactcatgg gtcaaagaag aaatcatgat ggaaatcaga aaatattcag aaccatacaa 10080 taatgagaat attatttatc aaaatctatt ggatgcagct aaagcaggac atagggggaa 10140 atttacaacc ttaggtgcct agattaggaa agaaggaagg catttgttta tttatttgtt 10200 tatttattta tttgagatgg gggtctcact gtgtcaccca ggctgctgga gtgcagtagc 10260 acgatcataa atcactgaag tctcgaactt ctgggctgaa gtgatcctcc cgcctcagcc 10320 ttccaagtag gtgggacaca ggctagcacc accataccag gctaattttt tttttgtaga 10380 cacagggtct tgctatgttg aggtctcaaa ctcctgggct caagtaatcc tcctccctcg 10440 gcttcccaaa gtgctgggat tacaggcatg agccactgcg cccatctaag gctgaatttt 10500 aatgagctaa gaattcatct taagaaaggg ctaaatagac agcaaaagca aacattgaag 10560 gttgggactg agctgagtgg gtagcaggga tgggagacaa cagatctgag gagagcagga 10620 gattttgaaa ggattgcact gcctgaggtt taagccttta gaatccagct ctctctgagc 10680 tccctttgag ctctgacatt ctgtgactct gatttggtgg ccttccctta gtggccttac 10740 tgatttcatt tggatggtgc ttgtggtata tccaaccaac atgtcttccc aaatggcctt 10800 ttaatttcct ataaagaagt agttgtcatt gattgcaggt tagggacaga aaatgctgtg 10860 gaatgaaaca aaatgcaagt taaagaacta aattccaaaa atacccattg ctactattga 10920 ctgagtgaat tcctactgtg tgccagacac tgtacccagt ccattccctg tattgtttta 10980 tttaagcctc acaagggtat agtgtgacta cactgtttct taacaatgaa gaaactgccc 11040 aaatcgccca tctgggaagc ggcccagcta gaatttgaat ccaggcctgt tttcctccag 11100 agcttgtgct attctctgtc tgtcataaaa tgtgggggct ttgtgtggta aacttgctca 11160 gttgggcata gcagttgtta ggaaacctga ggctggtaac accagctgta ataccagctg 11220 tccgtctgac tcatgcaact gttaaagttg atagggctga ggtgtcagac tgagctctga 11280 attgcctgat tcctataaca atattaactt aaacattttt taaattggga aatgcaccat 11340 gcatacagaa gagtgtgtat atttcatatg tatagtgtaa actgttccca tcacccaggt 11400 taaaaaacag gatgttgcca gtacctgggg ccttctttaa ctgcaactgc tagaggtaaa 11460 cactggcttg acttttgtgt aaatcatctc tttgcctttc tttaatgttt tagcatcttt 11520 taaaataaat ccccaaataa tgtattgttc tattttgaaa aactgagtag caagccaaaa 11580 atagctgtgt aaagaaaggt cacttaaatt aggctgggtg cagtggctca agcctttaat 11640 cccagtactt tgggaggctg aggcaggtgg atcacaaggt caggagatcg agaccatcct 11700 ggccaacatg gagaaacccc gtctctacta aaaatacaaa aaattagcca agaatagtgg 11760 catgtgcctg tagtcccagc tactcgggag gctgaggcag gagaatcgct tgaacccggg 11820 aggcagatgt tgcagtgagc cgagatcgca ctgcttgaac ccgggaggca gaggttgcag 11880 tgagccaaga tcgcaccact gcactctagc ctgggtcaca gagcaagact ctgtctcaaa 11940 aaaaaaaaaa aaaaaaagaa aggttactat tgccttttct tagatgaagg ttcccaaggc 12000 agggaaagct aagtggagtc tcagggactt ggtctggctt ttccttccct gggaatttat 12060 aaggacctct tctgggaagt cagtcggcaa tgccatgaat gagtctgggg aaatattggg 12120 ctcattgcaa ctggagggtc tggtaggact gatgtgaatt aggtgctgtg tccggaggaa 12180 aatggccaga ggaagtgggc tgctttgtac agtcagtggt aaagttgcca aaggctatta 12240 tagctcacag gaatgggcca aggctaaaca ctcctgtgga gtgaaatgaa tgtcctcagc 12300 tgactgaggc agcgggagtt gaaaagaaac gatattagtt catggtgaag acaagtcaaa 12360 tatagataaa ggttagggtc aggcttgcct ggacatctag gagataactg ccctcaactt 12420 gtttgaatct tgagtcactg ctccattttg tttgaactgg tggccatcta cttatagtat 12480 acagccatca acctgagatt tccctacatg gtcttcctgc cttggtctcc tgtatcctga 12540 atcctatggc ctcttcttcc ctggtttact acattttgct agaccgtatc ctccagtcaa 12600 ttccttagaa tgaatgtatg aaagttaaaa tttctgaggt ctcacatgtc ttaaagttcc 12660 ctcatactgg attgatagtt tggctgggta taaaattctg ggctggccat cattttcctt 12720 cagaattttg attgcattat tccattatcc tctcttttca atattgcttc taagaattcc 12780 aaaacctttt tttttttttc tttttgagac agtgtctcac tctgtcaccc aggctggaat 12840 gcagtagtgt gatctcagct cactgcaacc tccacctcct gggtttaagt gattcttctt 12900 cctcagcctc ctgagcagct gggattacag gcacccacca ccacaccctt tagtagagat 12960 ggggttttgc tatgttggcc aggctggtct tgaacttctg actttaggtg atctgcctac 13020 ttcggcctcc caaagtgctg ggattaaagg cgtgagccac cacacccagc ctccaaaacc 13080 attttaaaac tctttctgga agcttttaaa attttctttt agtccccaga attttaaaat 13140 ttcaattatg tgccttggtg ttcttccatt atgttagtca cccaagaggt actttcaatc 13200 tggaaacttc tctatgtttt gggaaatgtt cttgattagt ttacaggtga tttcttcctc 13260 tccattttat ctcttctctt ttcatgaaac tactattaat tcaatgttag aattccttga 13320 ctgatcattt aattttcttc tattttccat ctctgtgtct ttttgctcta cttttctatg 13380 atagtcacag ctctatcttt aaactcttga gtttttcatt tttgatgtca tgattttaat 13440 ttgcaagggg taggtttgac tgattctttt ttgtagtatc ttactcttgt tttatggatg 13500 caacatcttc tttgacttaa ggatcataag ataggtgggt tctttgtttg tttgtttgac 13560 tgtttttcac cctatgtaaa ctttttctac aagtttcttt ccccttcccc cctttttggc 13620 ttctatctcc cacattagat gctttctctg ggctcatgat actctttggt tttctttctc 13680 aagattgaca ggtaggactt taaaacttgt tgagcatgcg ggtgaaactt gtctaccatg 13740 aatttcactg tagatatttt ggagattgac agtgtttata tctttagatc tcgcctcctg 13800 ggttgatcaa gttatctgag tacaccacag accttttgcc tggggataaa ccagaaatct 13860 gtttcagaaa ccactttgat tcagtcttcc ttgttttagt catttccttc agttccggag 13920 gtccctcatg ctgatcattc cagagccctt tacagatcct agggtacaca ctgcatggtt 13980 ttcaactttc ttgttttggg gttaagattt ggctttcagg agtctcctca gtccgttact 14040 attcattcaa tcagcaagtc cttgagcacc tgatttgtgc cagacattct tctaggtgtt 14100 agggatacct cagtgaacaa aacagacaaa aatctttgtc ttggaaatac acacactcca 14160 gtcaggggag agggacaata agccaaagga aggaaattac agcgtgtgct agaaggtgat 14220 aagtgctgta gaaagtaagt aaagtgggtt tgggagttga gagtttggga aggggataaa 14280 tgatggcaat tgtaaataga gtagtcagag ttctcactta gaaggtgaaa ttcaagtaaa 14340 gacttgaagg aggacaggga attagccaca tggatggcta ggggaaggct tccaagctga 14400 gaggacagcc agagccaagg cccagaggca ggagcatacc tggtagtttt aggaaacagg 14460 aggccaggat gctgagtgga gtaagagggg gcatgaaagg agaaacttgg gtccacgtgg 14520 ttctagacag gtatttttgt ctgttttggg ccctgaaggt tactattgga cttggactct 14580 tactctgagg aaatagggac gctattggga cgtttgtaca ggagcaatgt gacctgagtt 14640 ttgtttgtaa aggattagac tctggctgtg gcattaaggc taggctgtgg gggcaggaac 14700 agaagcaggg ggaccagttt tgcagcctgt gcagctttcc agataagcag ggattgtggc 14760 ttggaggagg atggtataga ggaggtgaca agaaatgact ctatgtctgg tatgtagata 14820 ttggccacag atggcatttg agcactagag acctggctgg tccacatgga gtttccataa 14880 gcacataata cacatcagat ttcaaagact taatatgaaa gaaagaatga aacgtacctt 14940 gctatttctt tctttttttt ttttttttga gacccagtct tgctctgtca cccaggctgg 15000 agtgcagtgg tgtgatctcg gctcactgca acctccgcct cccaggttca agtgattctc 15060 ctgcctcagc ctcctgagta cctgggacta caggcacctg ccaccacgcc tggctaattt 15120 tttgtatttt tagtagtgat ggggtttcac catgttgtcc aggctggtct ggaactccgg 15180 accttagggg atctacccgc cttggcctcc caaattgctg ggattacagg catgagccac 15240 catgctcagc catatcttgc tattttctac atggattaca tgttgaaatg gtaatgtttt 15300 ggctattgtg gattaaatag aatatatgat taaagttgat ttcatctatt tcttttaact 15360 ttaaaaaata tgtctgttag aggatttgaa attccacatg cggcttgcat ttgtgacctg 15420 catttcattt ctgtggaaca gtgccctttt tgggacatgc tttgaaggtg gagtcaacag 15480 gatttggcag attacagacg agaggcttca agggtgactc caagacttcg gggcagagca 15540 cctggaagaa aggggttaat attagccaag atgaggaagg ctgtcggttt ggcaggtgca 15600 tgggcaggtt aggagtttag ttttgaatat gttggaggtg tttatgaaac ttttaagtgg 15660 agatggaaaa taggcagttg gatgtgcaag tccagggttc agggagacag ttcaggctgg 15720 agatgaagat gtgggagtct gaggagagat tgtattcaaa tattcaatcc atgagacttg 15780 atgaaatcac ttctcttcca aatgatttac agcctgcaga atcattttcc ctatctttgt 15840 aggtttatgt cttcattttg tttcatttat ttttcagtta ttcactgttt tagtgagttt 15900 tgagtaggag ccagattgga tgcatgcgtt caattcacca tccaacactg tattaactac 15960 ttgaaactca tgtggttgtt cggttgtttt tttgaccttt tattctggat ggaagagaga 16020 tgcttatgaa gttgcagtaa tcagtaagcc ttcccacatt gctccatcag ccttcctgga 16080 agaataatgt cttctgcctt tcctgtaggc aagaaggctg cttgatcttg ccacggtgaa 16140 agcgaatgga ttggctgcct tccttctaca acatgttcag gaattaccag tcccattggc 16200 cctgcctttg gaaggtaggt gtatgttctc agttaatcag aaagggaagg gcagtcagtg 16260 cagatccatg gttaagagca gaacacacct cggttaacat cccatatgct ggcagtatag 16320 cctccctatg actcaatttc cttgttttaa ggctagcacc accccgtctc attgggattt 16380 tgggagcatt aaaaggacaa aagcgtgtaa tgttagctat tagctttcat tatctcccac 16440 acagtatact gacaattggg ctaccatata ttgagggcta actaaaggtg ttacttacca 16500 tccaaactct cattatctgt accgaaaaga tatggacaca tgttttgagt tagggctggt 16560 atctcttgat ctctgaaatt tagcagctca caatgggaaa ctcaagaacc aagtggatct 16620 agagactctg gtatccctca gtgcccaggg tcaccaccca aactcaggaa caggaggggc 16680 ttggaccgca ccacttgaac ataccaggca tcctgccagg tgctttatgg acaatgtcta 16740 ccctttgcaa caaccctgag aagtaggtgg tgtttttttc caccttatag atgtggaaac 16800 tgggcaggga ggttaagtga cgagggaggg gaagatgggt ctgattgtaa attgtcccca 16860 cctacacttt ctcttttctt gggagaagaa atgtcagttg taaagagaga gtgcaagcct 16920 ggcactcttt agggcttgtt cctacaccac tgtagggaaa gctcattggc actgaagccc 16980 cctgagctgt gtgtggtgct ggcagatggg tctatcaccc tgggctgtgt cctctgggca 17040 gcaagcaagc ctgtgggcgg ggtggctgga agtctgtgcc tggcactcgc gagtgcaccg 17100 tctcattgaa gaacaggatc taaacatcag tgcgccacag cagggtgcgc ggcacggagt 17160 gcaggccctg gtttggccct tggttgaggt ttgctgttga catcatcaag cacagctagt 17220 cactgtaaga ccaggccagg gtgcaagatt ccccacactt ctaaaggtga caattggtgt 17280 gtttatttct ctataaaatg acattttttt tttctggaga attttagtat cattggtgat 17340 gactggaaaa cctgcatcag aaatcaggtc ggaagaggaa gatatatatc tgatatgtac 17400 tggagaggaa gatatctatc ttatggtcta agttcaggga tcctggtata ttcagagggc 17460 agaaagctca gcaataatca tcaactctgg gaacagaggt gacataaaca cagggcgtcc 17520 cctttgtgtg actgcagata gtcatcagtg agctcagagc tctatgaaaa ttacttgcta 17580 gtttttgggt tgaaaatagt gggccagtgt ttggttgggg gcagtgaggc tgtgatggcg 17640 ggggaccatg ccaagctcct accagcctgg gacgctaaac cagcacttcc ccatttcctg 17700 aaaggggaac taaactctga cacaggaaat ggtttgcttg cattactttc aggatgagaa 17760 aggaagagca ctggccttcc aaacacaccc cgtgcatgaa aactctccct gcatggggtg 17820 catggggagg atggggaagt ggaggcagga tcacagactc ttgttcgagt gctcagctgg 17880 ggcaccccgg tgaccccgag gccttccctt gctaggtcca cccagatcaa tcaggatcat 17940 ctccccatct cgaagtttaa ctttatcaca tctcagagtt ccttttgcca cgtaaggtaa 18000 catattcaca ggttctgaga atccggacat ggacatcttt gagggtctat tgttgtgcct 18060 actatatcca tgaataataa tgataataag caccattttt tgagagtttg ccatgtcaga 18120 tattctttta aactgtattt tatctcgctg cctcctgaaa aaatccttcc aggtgtatat 18180 tgtccccatt tttacagatg agagaactga ggcccagaaa ggctaaatgg cttgcccaag 18240 tgtatggtgg acccaggttt tcaaactcag gtgtgtctgg cttcagagac tgggctcctg 18300 agcccttaag ccctttgttc ccctttagaa aaagtcacct gaggctgagt ggtgaaggga 18360 tttatccaaa gccacccggc cactatggca ggacagatat cagaatacag gtcttccgat 18420 cccagcccag agccccttcc cgtcatctag aactcctcct ggtgtcagta atgataacgg 18480 cagtcactga tgtcttttga gcacttactt tgtgttgagc acttacactg tgctaagcac 18540 ttgacatagg tcatcttagt tgatccgtgt aaaactctgt gaggtagtga ccaacatttc 18600 tcccacctta cagaggtgga aactgagggt taggaagttt ccttgactgt cctcaaagtg 18660 cacagcttgt gaatggagga gccaggatgg gcgcccgctg gctctcctat cccttcagtt 18720 atgtcagcgt cccccgcagc agcccattgt ctggttaggt cccgtcttca ccatggtgcc 18780 accttcatct gcctcttctt ctgccttcca gctgccacat gcaagaagta tatggccaag 18840 ctgaggacca cggtgtctgc tcagtctcgc ttcctcagta cctatgatgg agcagagacg 18900 ctctgcctgg aggacatata cacagagaat gtcctggagg tctgggcaga tgtgggcatg 18960 gctggacccc cgcagaagag cccagccacc ctgggcctgg aggagctctt cagcacccct 19020 ggccacctca atgacgatgc ggacactgtg ctggtggtgg gtgaggcggg cagtggcaag 19080 agcacgctcc tgcagcggct gcacttgctg tgggctgcag ggcaagactt ccaggaattt 19140 ctctttgtct tcccattcag ctgccggcag ctgcagtgca tggccaaacc actctctgtg 19200 cggactctac tctttgagca ctgctgttgg cctgatgttg gtcaagaaga catcttccag 19260 ttactccttg accaccctga ccgtgtcctg ttaacctttg atggctttga cgagttcaag 19320 ttcaggttca cggatcgtga acgccactgc tccccgaccg accccacctc tgtccagacc 19380 ctgctcttca accttctgca gggcaacctg ctgaagaatg cccgcaaggt ggtgaccagc 19440 cgtccggccg ctgtgtcggc gttcctcagg aagtacatcc gcaccgagtt caacctcaag 19500 ggcttctctg aacagggcat cgagctgtac ctgaggaagc gccatcatga gcccggggtg 19560 gcggaccgcc tcatccgcct gctccaagag acctcagccc tgcacggttt gtgccacctg 19620 cctgtcttct catggatggt gtccaaatgc caccaggaac tgttgctgca ggaggggggg 19680 tccccaaaga ccactacaga tatgtacctg ctgattctgc agcattttct gctgcatgcc 19740 acccccccag actcagcttc ccaaggtctg ggacccagtc ttcttcgggg ccgcctcccc 19800 accctcctgc acctgggcag actggctctg tggggcctgg gcatgtgctg ctacgtgttc 19860 tcagcccagc agctccaggc agcacaggtc agccctgatg acatttctct tggcttcctg 19920 gtgcgtgcca aaggtgtcgt gccagggagt acggcgcccc tggaattcct tcacatcact 19980 ttccagtgct tctttgccgc gttctacctg gcactcagtg ctgatgtgcc accagctttg 20040 ctcagacacc tcttcaattg tggcaggcca ggcaactcac caatggccag gctcctgccc 20100 acgatgtgca tccaggcctc ggagggaaag gacagcagcg tggcagcttt gctgcagaag 20160 gccgagccgc acaaccttca gatcacagca gccttcctgg cagggctgtt gtcccgggag 20220 cactggggcc tgctggctga gtgccagaca tctgagaagg ccctgctccg gcgccaggcc 20280 tgtgcccgct ggtgtctggc ccgcagcctc cgcaagcact tccactccat cccgccagct 20340 gcaccgggtg aggccaagag cgtgcatgcc atgcccgggt tcatctggct catccggagc 20400 ctgtacgaga tgcaggagga gcggctggct cggaaggctg cacgtggcct gaatgttggg 20460 cacctcaagt tgacattttg cagtgtgggc cccactgagt gtgctgccct ggcctttgtg 20520 ctgcagcacc tccggcggcc cgtggccctg cagctggact acaactctgt gggtgacatt 20580 ggcgtggagc agctgctgcc ttgccttggt gtctgcaagg ctctgtagtg agtgttactg 20640 ggcattgctg ttcaggtatg ggggagcacc atcaaggcta agtgtgggag caccgagctg 20700 ggctctagaa gtctgggccc agcttcgcct ctgccaccct gctttgcaac actgcccaga 20760 tcccttccct tctgggcctt aatttcaata tgtgatgatg acagccacac tttattgact 20820 ggcctatgtg ctgggtctgg tgctatgctt tccggaatga cctcatctaa tctctacaac 20880 caccctggcg ggtaggcagg aatgttatta tctccattat ccttgacttg aggctcagag 20940 aagtgaagta acttgtccag gaaatggcag agctggggtt cacaaattgc atcattctga 21000 ttacaggttt tctgcctccc accagtctat ggatacactt cagaggctcc ctgaaaacct 21060 tgaggtcact tgcagaaagt tttgtgtagt atgtgtccgt atcaggaaca acaccaaatc 21120 agaggtgact tgtgccccat cagagacttt aacaccccaa ccagatggga atttcaggac 21180 ccaagaaata gaaagtggct gcagggttac aactactgtt ggattcctga ggtagcacag 21240 tgtccaaaca ggatttcagc actacccgta ttgcttagag ccccagccaa agatgtgagg 21300 ttttgccctt tggagaatct gtgcccctga actcgggggc ctctttccac atcttggggg 21360 caggcaaggg cagagggtgt gcctaggcct gcggatcagc atgcgacaga ttccccaaca 21420 tccttccagc ttgaaagggg attgccctgc ttctatttag aacctatagg aaagcagaag 21480 ttctagattg aagttaaaat tgattcccag cctccagggg ctttgggcta cacctggatg 21540 accttaattg accctaagca tgggacaaac cacttcctga gagtattagg atggtataca 21600 tcttctctgg gggcaaagca acaagattta tttttcatca tggaccaaac acatggatac 21660 ccactagaaa ctgtgtagtg aattttgtta accctgacat agggaccatg gtctttaggt 21720 taaagcataa taacaacata atacataaca tatatagcga atatatatat gtattatatg 21780 caatgaatgt aaatatgatt atacccatca tggtcttgga ggaaacagat gacacactta 21840 aaatgggtgt tttgaggaga gtttgaaaaa cagattgttt acaagccatg ggcaggagtt 21900 aggaagagtg agagggttgg tgcaggggcc tggggttagt aacagctggg ggagggtaga 21960 cttgaagggg gaaggggagg gagactaatt agctgggggg aaggtatgga gacggctgcc 22020 tgagcttctg caaagtggaa gaataccgct tggccctaac tcctcacccc aactcttgct 22080 cctggccagc gccttccacc agctggaccc atcagggagg ccgagtgggc tgtctgctgg 22140 agtagtcccc aggcatcagc ctcccaggag ccagggacgg gtagagaagg gggagagtgg 22200 atctggccag gcaaatggaa aacagccagc accaaactct atttccctag gagggaggat 22260 catgatactt tgagtgggaa tttggaaacc tgtctgttgg agcaatttcc ctgatagaaa 22320 taagaatgtg cattttcctg ggtagtagac tcagttttta ccccaagagg ccaggcatca 22380 ctggcctgtg tgatcctcat aggccagtcc atctctggaa ttcttgaatg gatcatccat 22440 ccttgattag ggatgtcccc gtgattacca gggtgtgcag aagggctctg ggaaacctgt 22500 gggtctgtct ctgtgttcag agaaaggtga gggtggcctg gttctagctc atggtgctca 22560 gactgtggtg tgtaaaggca ctcgtggcaa tgcagattcc tgggcctgcc tctagtgatt 22620 cccattcagt aggtttgggg tggggcccag gaaatctata tttttcacag acacccctgg 22680 tgattctgat acaagtggtc tcgccctggg agaactactg gtctgcagca accagcttgg 22740 ttttccatta gcaattactg tccttgagcg agttttactg ctcttcacct tacacacact 22800 aaaactgcca aggccgtagg ggaggggaag caaccatgag gttgctgtga gtgcactgtg 22860 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtatg agagagagag agagattgag 22920 aaagagagga agggaggaag ggggagggca caggctcctc tcccacagtg ccaacctgcc 22980 tctctcccac ttgaagtgtt tccatgccaa ctgaaatcct cagcctctag gaaaccctat 23040 atacacagtg cccctatata ggtttcttta gactctggct ctctcagact ctagagtgat 23100 ggctttaaaa gttttttgaa tgttacccac aatgattgag aaagagagag agagagcacg 23160 caccaccatg taaacatgga acctaagttt cacaaaatga cttcgcttta tgaactctga 23220 gacactctgc tctcttctgt tctgttctat ttccatttta gaaatgctgc tcaggacctt 23280 caaaatgatt tgcatgacct gcaacctgca gtctgaaaaa tcactgcact acagaagtgg 23340 ccataagagg ccctgaggga gaagctgcac aatgtcatgg ttaagagtgg ggtttggagc 23400 caagccgcct aggctcaaag cctttatgtg ccgtacaacc ttggcaaagt cacttcgctt 23460 gtctgtgcct cagtttcttt ctcacgaatg ctcataataa tggttcccat ttcactggct 23520 tgttgtgagg atgaaatagt gttattattg agaagtggta agggtagtga tcagtgctag 23580 cgatcatgat tctaggtgac ttttactgtg taccgggtgc tcacaaggct ttatgtgcac 23640 agcctggtga ggctgataat actattgttc cctctttttt tttttttgag acggagtctc 23700 gttctgttgc ccaggcaggg ggtacagtgg cacaatctcg gctcatgcaa gctctgcctc 23760 ccgggttcac gccattctcc tgcctcagcc tcccaagtag ctgggactac aggcgcctgc 23820 caccacgccc ggctaatttt tttgtatttt tggtagcgac agggtttcac tgtgttaacc 23880 aggatggtct cgatctcctg acctcgtgat ccgcccgcct cggcctccca aagtgctggg 23940 attacaggcg tgagccaccg tgcccggcct gttccctctt ttatagatga agagaccagc 24000 aaataactag taagtcgctg atcaggatca caatatccag ctgaggcact ccagagcctg 24060 agctgttaac cattcagtca gggcctccca agtttgccta aagataaaga atcatgtgca 24120 cagttgttaa aatatacaga ttcctgggcc ccaccccgca gatacttgat tgccagctcc 24180 agggtatggg cctgagaatc tgtcttttag ggaagctttc agatgatgtt gtgatcaggt 24240 gagttttggg aatggtgccc caagaggagt ggcagacagg gcttgctcgg cagggactag 24300 cctgttggag tggtgccatt ggggttaagg actgggcagc agggcctcac taaccacagc 24360 ctatatgcct gtttctgaag ttttggccac tctcatccag ctggtctact gtctgctgac 24420 ctagatgatg gtaaattgtc cccaggggta gcctgtctag ttcaggctgc acctttcgca 24480 tatatcagct cctttccacc atcatcccct ttgtgaggct gctgtgatta tcatgttcct 24540 tttgcagaga tggaaacatt gcctcaaatt agctctgtca tttcctaagg attccagggt 24600 tctttagtag ggggtctgga tcctacgtcc tgggccatcc ccatcatagt gcaccacgtc 24660 acctccctgg ccagggaccg tggggtctcc acttttttgg ggtgctccat ctatgcaggg 24720 tttcctggaa gcacagatgc tggcacttca gggatgaatg aaagtctttt tgggggattt 24780 gtagattttt ttcttgtctt actagctcca ttttcaaatg tatttatttt gtctctttag 24840 tttgcgcgat aacaatatct cagaccgagg catctgcaag ctcattgaat gtgctcttca 24900 ctgcgagcaa ttgcagaagt tagcgtaagt cagcctgggc tgtggacaat gggctccaag 24960 tgccctggtc tcaccccagg tcgtgcagcc tgggaagctg tgagtgatgg gctggggcag 25020 gggctgtttg catgatgggg ggtgcaggtg attcctgccc agaggggaag ggcaaccctg 25080 ggatttggtg ctcactgtcc aatgtgcttt gcttctgtgt ctcctctctt ctggaactga 25140 acagtctatt caacaacaaa ttgactgacg gctgtgcaca ctccatggct aagctccttg 25200 catgcaggca gaacttcttg gcattgaggt gagcccaggt tttccttatt ccctggaaac 25260 tattttttgc cccattcctg agtcagtctg atctggtctt ggcctggcac tgcccacact 25320 ggctcctgac ctcctgattg aatgcaggga cagtgtctca ttttaagcag gggttctcta 25380 atgctgtgat ctccccagta aactctggac tagctctgct gaggacttcc tgtcttttga 25440 cctttagccc gtagggcaag aaagcttttc taggcccctt tccttttctg tgtctaagag 25500 tgtcacagct ttctgggttt actgagttcc acgatgcatg ttgagctcgt cctggtgggg 25560 gaggcataca cagttacttg ccaccccagc tgtggcagcg agttgctgca acactcccag 25620 gaggtccttt caccactcag agcatgcaag gtttgcagtc catctggttc tgcatttctg 25680 ctactccagt gtctcccagt ttcaacagga gtctctctct ctcctacctg atgcctttaa 25740 attgcccctc tagctggccg ctgggttggc ctggcttctc tctccttctc tctctctcag 25800 atattcttgc ctcctgtgat ttgtgaggca gtaaaaaaag acaaagtaaa gaattgcttc 25860 catctattct tttacctctt gggctgggtt tgtggatggg agccgccatt ttaaaatggc 25920 gggccacata gctcagtctc ggcaagggct actgagatca gaaccacagg tgccaatttg 25980 tacaaaggac tcagtcctgc taccactgcc tgatccctca gactcacaag cctggaatag 26040 gctgtggcca gacctggctg gcccatccct gagaagggtg ctagtttcag aaatggaggc 26100 tgagtttgtg gccaacacag tagtcctccg gtatgtgcag gagagatgtt ctaagacccc 26160 agtggatgcc tgaaaccatg gagagtatca agccctacac ataccatgct tttcccaata 26220 cctacacacc tgcaataaag tgtagtttat aaattaggca cagtaagaga gtaatagcaa 26280 ctcataataa aatagaacaa ttataacaat caatgtacta taataacact atgtgaatgt 26340 ggactctctc catctccctc aaaatatctt cttgtactgt actcaccctt cttcttggga 26400 agatgtgtgg tggtaaaatg cctgtgtgat gggaggaagt gaggtggatg acgcatgcag 26460 cactgtgctc tagcgctggg ctgctgttga cctgaccaca cttcaggagg agaatcatct 26520 gctcccagag atccctaatc tttgagcaac aatgaggtcg gcagctggat gtcaggagca 26580 gacgatcttg atgattacca aatgggagcg tatagagcgt ggatgcgctg gacggggggc 26640 tgattcacgt cctgggtggg atggagctgg atggcacgtg atcagaatag catgcaattt 26700 aaaatgtatg aattgtttat ctctagaatt ttccatttaa tatttttgga ctgcagttga 26760 tttcagataa ctgaaaccat agaaggcgaa gctgcggata agcagggggc agggattacc 26820 gtatatcatt gtaatagaga gcacaggctc tggagccaga ctgcccgagg tttgaaccct 26880 cattagctgc gtgacctcag gtcagcccaa tgtctgtgtg cctccgtttc cccttctgta 26940 gaatggaggt aataaccctg gctacctcac aggctgtagt gatgagcaag caagttaatc 27000 cacatgaagg gctgcaccgt ctggcagggg ctttatatag taagcgagtg gctgaaagat 27060 gatgggtaaa tcacacaagc actcagcttg tttctcctta tgtgagtccg gtcctccaag 27120 cagggattca atgtgccacc catttattgg ggaaaagtcc taaaagggga agtggggaag 27180 ggagctgggg gaggctggga ggtgtgtccc tgagtgaagg agagagggaa ggaaggaagg 27240 ttgagactgg gcaccttgga cttcagtgca gtcctaagac atcttggcaa ggctgatgag 27300 gagttcttga accaaattca ccaggcaggg gagcctgatg tctcaggcag gggctggcaa 27360 gtgcagatgc gaggatgtta gattttggag cacagcagct ggggcccttg gctacctcca 27420 aggagctgag gctggagacc tgaaaggcga gttctcctag ctgccacacc ccttctccaa 27480 ggatacaata atatctgcct tataggattg ttgtgagctg agtggcttga cgttccttga 27540 aagaatgaaa gcgtatagtt atcccaggaa gcctagggtt gcaggtgaga gctctggggc 27600 ttctccgaag ctctccgagg tgtctggatt cagttgcagc aggagccttc cttgctggga 27660 tcttccccca cccctagcct tggccctccc tctctccttc ctttctggaa ggctcagtgg 27720 gccccacccc tccctccagc cacctggacc tgcccagcgc tcttgtgcaa caggtaaagc 27780 ctacctgtag caacaacaga tctgggaagg ctgcagaggg cacgatgggg tctggatcga 27840 gggcggctga gaccagaggg aaaggtgtga ccctgagtca ccctcgctgt cccggggaaa 27900 ccacctccca ggacagctgc ctactgtggc tcctgcctgg aattgtcaca ctgctgtgca 27960 aacagcgtcc cgctgcccct ttccctttgc tgggggaaaa tgaagttgtg ggagccgctg 28020 agtaaactag acctagcagc gagggcacct gatgtggctg ctgcctcccg ggcaggtctt 28080 caatgctttc ttcctgtgtt tccctggcca gggcacagac ggccctcctt ttctgcctgc 28140 cgctgtgttc tctcagcctc ctctgtcttc ccttccaggc tggggaataa ctacatcact 28200 gccgcgggag cccaagtgct ggccgagggg ctccgaggca acacctcctt gcagttcctg 28260 gggtaggttg gattccagga agagggacct gcatggaggg gcttgggact tttgaggatt 28320 taggggcagg tgaaactctt cagccaggag gccccagagg cagcccagct ccagtgggga 28380 ggacaagcca gggagagagt gggcggccct tgactgccac cttcatactt ggtctatgcc 28440 tgacaaacag gaagtttggg atgttggggc taggggagga cagtgcccac gagctggtga 28500 caggaagccc tctgatcctc agggggcgct agggctgtac tttagctgca tattaaaacc 28560 acctggaagc ttctaaacac tattgccagg cctcccaccc cagactgatg aaatgcaaat 28620 atctaggtgc aaggcccagg tatcaggagt tttaaaaagc ttcccagggg atgtacagcc 28680 aggggtgagg acccctgacc taagaaagag aaggaaatgg ggaaggatag gaaggcaccc 28740 aggataagag gggctgtgct aggtccctcg gagctcttgc tccctgtagg accatgctag 28800 ggcctgccag ggaggggagt accccaacct gcagccccag ggtgggcttc ctctgtttgc 28860 taggcaccca ggcttgcacc tgtgctgttt ccagcagcct ctctcctatc ctgtcatgcc 28920 ctagtgtgaa ctggagtcca tttgacaaga actgggagtt ttagaacctg ggactgtagg 28980 aagagagaat aaccttaggg cctaggtgtt ccagcccatt tcacagggag gcaagttgcc 29040 cccaagctca gttttttgtt ttgttttgtt ttgtttgaga tgtagtctca ctctgttgcc 29100 caggctagag tgcagtggca cgatcttggc tcactgcaac ctccgcctcc ttggttcaag 29160 cgattcacct gcctcagctt ctcaagtagc tgggattata ggcacccacc accacgccca 29220 gctaattttt gtatttttag tagagacagg gtttcaccat gttggcccgg ctggtcttga 29280 actcctgatc tcagatgatc cgcccgcctc ggcctcccaa agtgctggga ttacaggtgt 29340 gagccaccgc acccggcccc caagctcagt ttgagccaca aatgggacta tgttgctcta 29400 gaaatcaaca tcttttccac actgcattag tagcaacaga gtctagaaca aaggaggcca 29460 cagccccact gaactctctt ctgcttgagg tcacatctgc cacatcaggg gtatttacct 29520 ctttcaacac atatttatta gggcacctgt ctgggccagg cgttgtgcta aaacccccaa 29580 acgctgtcat atgatacaaa gtgttctgta acttgcttgg tttttttttt tgtttgtttg 29640 tttgttttgt tttgtttttg ttgttgtttt tttttgcttc gccatatatt ataggaattt 29700 ttttaggtca ttatgacctc tttatttact taattatcta tttatttatt ttactaatat 29760 ttacagaaag ggtctcactc tgtcacccag gctggagtgc agtggttgca atcatagctc 29820 attgtagcct tgaactcctg agctcaagtg atcttcctac ctcggcctcc tgagtagctg 29880 ggactacagg cacaagccac catgcctggc cgatattttt atgttttgta gagacggggt 29940 ctcactatgt tgcccaggct ggtctcaaac tcctgggctc aggtgatcct ccctcctttg 30000 cctcccaaag tattgggatt acacaagtga gccaccttgc tcagcctgac ctcatttttc 30060 aaagagctgc agagtgttac ataatgtatt taactggtca ctttttgatg actattaagt 30120 tgttttcagg ttttttgtta ttacagtgtc atatccctgg ggcacagagc agtgctggca 30180 catagccaga gctcaatcga tacataccta atgaatgaaa gtacagtgga catcctaatt 30240 cagccattct ttgctaactt gtgtacatac ctgtccaggg taggtcccta gaatacagtc 30300 aataagtcag aaggtgtgag ttgggatcta ccttttggaa agggatgttt tcaaactaca 30360 gtgagtcaga ggaggatggc ccagaagctg ggggagttga agctgatggc gtgaaggaat 30420 taggggtgtt aggaagaagc aggagataaa gagctagctt gcagaagaag tgttagactt 30480 gttatgggca ggtactggag ggtagctaag gacttgtggg tggcagttac caggaagcgt 30540 atctgaacta agtgtcagaa aaagtgtcac aactgtaaat tactcttgtc agtgagttcc 30600 tgtccttaag ggttagggct gggtagccct ctactattct ctaagtctgt aatgtaaagc 30660 cactgaaaac tcttgggtta agtttggcca tcccacccaa aagatggagg caggtccact 30720 ttgctgggac caggagcccc agtgaggcca ctctgggatt gagtggtcct gcccctctgg 30780 ctgggactgc agagggagga ggactgttag ttcatgtcta gaacacatat caggtactca 30840 ctgacactgt ctgttgactc ttttggcctt ttcagattct ggggcaacag agtgggtgac 30900 gagggggccc aggccctggc tgaagccttg ggtgatcacc agagcttgag gtggctcagg 30960 taagcttcag agtctatcct gcagttttct tggggagatc aggtgaagag ggaggagctg 31020 gggccagttc tgaaggtctt tgaactttat ttctacccca caatgttagg caatggagta 31080 aggaaaaaag accattggat ttcaagagag gacactcgag tctttctggg tgacttggaa 31140 atgtcccttg tcctctcagg gttttgatac agtatctgta aattgaagat attgggctgg 31200 atcaggtaca ttttatctta aggaccaatt ccaatccatt ggtagtgggt gcccagtgca 31260 ccacattaaa aagaattcta aggctgcacc tgggcttaaa gaagagcact ataatcaatt 31320 agtgatgtct aaaaaagcta aaaaaaaaaa aaaaagagca ctgcattcaa ttagtgatgt 31380 ctaaaaaggg tagaaaaaaa aaaaaaaaga aaaaagaaag agcaccgcaa tcaattagtg 31440 atgtctgaaa tggagcagac caggagagca ccacgaattt tgccctccat aggttagctc 31500 atctctgagg tctttccctg ctctgacata cttttgttcc atgattacct ccagcctggt 31560 ggggaacaac attggcagtg tgggtgccca agccttggca ctgatgctgg caaagaacgt 31620 catgctagaa gaactctggt gagtttgggg gattctctgc tctggggaag tggatcacaa 31680 tctctgttga tcccctggcc tcatccatag gagcggttgt gtggacagac aaaggtggat 31740 gattgagtga ttgactgatt gattgattgt gtttgtcttt atatgtactg agtggtatga 31800 agcttataga gcctggtatg tacatgctaa tttttttatt taataaaata tatgggtttg 31860 ctggtttggt gactgcctcc acatggcata agtgttaaga gcacagactc tgtaatcaag 31920 caggccgtga tcttaggcaa gttaaataac aatttcagaa tctcaagttt catgtctgta 31980 aaatgagggt aagaatactt ccaaccataa aggatttttg caagaattag ataaagtagt 32040 gcctgtgaag accttaatat agtgcctggc atatttgtaa gtgctccata aatgttaaat 32100 tagaataatg gcagggttac tactactatt actgctgctg ctgctgctgc tgctgctaca 32160 actactatag tactgtgact actactacta ataaagtttt gttattttaa agtgattttg 32220 agttcctagg agcactgggt attcaagtct taggtcattt tggaaggtgt aatggagttt 32280 tgatagttga aagaggaacc atgaatcatg cttatactgt tgacctgaag cagattctaa 32340 gtttctcatc ctttagatgc cactagtata gttttctgac atgttctggg cagcttcaga 32400 ttatgtcagg gagataaaat actgaatgtt tgattttccc gggaagcaga aaggcactgc 32460 aacatatggg cattgccata aacagatttt atggatggac cttggctgtt gcagggctta 32520 ctagctctac tcaagtatga ttgattctat cctgactgga ttttgccact tggaatttct 32580 tagtagagga gaaccttgtt atgagagcat cagttatgat tactgttaaa agaaaaactt 32640 taggcaaatt aaatttagca gaactggttt gaacatacag caatttatga attgggcagc 32700 attcagaact gggagtgctc cacccagcaa ggtaggcaag cagtatctat agacaggaaa 32760 aggaagtgat gtacaaaaca gcttgattgg ttgcagctgg gcatttgcct tatatgggca 32820 tggtgtgatg aggcattttc tttatatgga tatagactga tcagctggta gactgtgact 32880 gactgaagcc tggctgctgt gattggctaa gacttagctg tttgttataa ggatatgttg 32940 ttaggttgca gtttgctaca taggaactca aagtacagag gcagtctcag gccaaattta 33000 gtttaactat atgttaagct gcaggtgaca gaatacctcc atctatagag gtttaaacaa 33060 ggaaagggtt tattttttcc tgtataggca gctggatgta ggcagtgtag ggtttgtaca 33120 gtggctacaa gaggccagga ggggtctcag ctctgtctca ttctcttcct gttccatcat 33180 tcttagcctg tgacttcatt cacatggttg gttgtctcat gatcacagga tggctgctcc 33240 aggtgcagca ctacttctgt attcccggat tcgatctata tacccaggaa agccatctgg 33300 gttctctcct ttaaaaagca ttcctggaag ccccacctgt cgacttcccc ttatgtatca 33360 accatgtgta tgtcacttga ccaacccact tgtatgttgt ttgaccagcc ctggctgcaa 33420 tggagagtgg gaaatacagt tttttcacca agtgcatggc tgtccaaatg aaatgagact 33480 tccattaata aggaagaaag gaaagatgga gatcaggaag ctgggggatc agggaactta 33540 ttacattgag agcccttgga gtgaattctc ttgcaaatat gtccctggaa ttgagaatcc 33600 ccacaacgta ctttatctgt tctttcttta tccatgagtt tgggttttca gatgttggat 33660 ttcctatatg gggggcatgt gagttcatca tcttccataa tcaatgttgt atcaactgga 33720 ttttctctct tcttctcacc agcctggagg agaaccatct ccaggatgaa ggtgtatgtt 33780 ctctcgcaga aggactgaag aaaaattcaa gtttgaaaat cctgaagtaa ggaacccata 33840 agcaggaaac aggacaataa ttgctggcct ttggaagggg catttctgaa taagatctgg 33900 gccgctctcc gctgggctaa ctcatgtgag gtggcctggt agaacagctt gccttggtct 33960 aggtggacaa ggattccagt gcaagttgtt tatctgggag gtggtcccag taaatgctga 34020 taggagagtg gtgaagtgag atggggaagt gaaggtaacc aataaagggg agttatcaag 34080 ccagttatca atgagggaaa ttggagctca gtactctggg gcactcctgg agccagtgca 34140 gaacacacat ggtcacctac ccaaccaatg ggcaagaaag ccatggcatt tatccaccaa 34200 ccctctgtcc ttcctatgtt gatgtgcgct catggggcac tgattctcca gcacttccag 34260 ctcaccctca cccagctgaa catgcttctg gggtcaggag aatggcctca ggcagagagt 34320 ggaaggtctt ctctgcaagc agtggctggg gaggtgatgt gatggggagt actgtggcct 34380 cctccagtgg ctgactcagt ggcttgggac ttgtgccaca aagagatgga cagctcaggt 34440 gaacatgaac ccacctagtg accatcatgg gtttgtcagg gtgctctctg aggctgatgc 34500 caaaattctt atttcaagta gacctcagga accccatcag atggctcctt ttgctggagg 34560 aaagtggcat ctgcctaggc aaatgtggtc ctaggaaaac gcttgccttt agagacagac 34620 agacagacag ctgcctctgt gagtgccagc tttgctgcca ggctgctacc cactctggcg 34680 acactcattt gtgttgcttt cacaagctag gaagtttcca aatatttgga gaaaacactt 34740 ccactaatta tttgggtgga aatgggctgg gaagttgggg tgaagcccgg atgtgtctga 34800 gccagatgcc agctttgcac tgagggtcgg cctttgggaa taccaagccc attatcaacc 34860 aggtgtggat atggcaggtt tgtcttccct ccttgtcaca gccttactcc acttgactcc 34920 catggatgcc aggcaatgag gctggggttg gtcccatgcc accctgtcat cagccttatt 34980 tttcagcatc ctaaactata tcatccccca caaaaattga acttctgata tatcttttat 35040 aaaaaagaga aatgcctaca tctttctttt ccaggcttag tttctgccaa gagttggttg 35100 agagcccagg cttgctgggt gcagtggctc acacctgtaa tcccagcact ttgggaggct 35160 gaggcgggtg gatcacctga agtggggagt tccataccag cctgaccaac atggagaaac 35220 cccatctcta ctaaaaatac aaaattagcc gggcgtggtg gcatacacct gtaatcccat 35280 ctactcagga agctgaggca ggagaatcac ttgaacctgg gaggtggagg ttgccatgag 35340 ccaagatcac accattgcac cctagactgg acaagagaga aacttccatc tcaaaaaaaa 35400 aaaaaaaaag gatgagaaaa ataataattt aaaaaaaaga gtccaggctc tggaaccaga 35460 cagcctgggt cttacccctg ctccaccatt accagccagt tcttcttgga tgagtgcctc 35520 agttgcctca agtgtaaatg gagataatgg ctggaccttc attataggcc atgagcattc 35580 actgagagaa tgtagctaac aaaagtgagt tgtaggttgg agcaaaagta attgtggttt 35640 cagaccatga actttaaatt attataacta ggctaaaata catctttatt aatcaaaata 35700 ggaaccatta aaatcaacac atttttgcca ataagaaata agtttgttta ttcctgtagc 35760 ataaaaattc atgcttcggg attcaacaaa ctcttggaaa gcattttctg catcctcctg 35820 gttgtggaag catttttcct gcagaaagtt gtcaagattc ttgaagaaat ggtagtcagt 35880 tggctagagg tcaggtaaat atggcggatg aggcaaaact tcatagtcca attcattcaa 35940 cttttgaagc tttggttgtg tgacatgcag tcgggtgttg tcgtggagaa ttggaccctt 36000 tctgttgacg aatgccggct gcaggtgttg cagttttcag tgcatctcat tgacttgccg 36060 agcatacttc tcatatgtaa tggtttcgca gggattcaga aagctgtagg ggatcagact 36120 agcagcagac caccagtgac catgaccttt tttttttggt gcgaatttgc ctttgggaag 36180 tgctttggag cttcttctcg gtccaaccac tgagctagtc attgccagtt gtataaaatc 36240 cacttttcat cgcacgtcac aatcagatca agaaatggtt cgctgttgtt gtgtagaata 36300 agagaagatg acacttcaaa atgacgattt tcttggtttt cactcagctc atgaggcaca 36360 cacttatcga ggtttttcac ctttccaatt tgcttcaaat gctgaatgac catggaatgg 36420 tcgatgttga gttctcaagt agttgtaaga aaatcagctt tgatgattgc tctcaattgg 36480 tcattgtcag cttctgatgg cctgccagta cactcctcat cttcaaggct cttatctcct 36540 tcgcaaaact tcttgaacca ccactgcact atacgttagt tagcagttcc tgggccaaat 36600 gcattgctga tgttgtgagt tgtctccgct gctttacaac ccattttgaa ttcaaataag 36660 aaaattgctt gaatttgctt tttgtctaac atcattttca tagtctaaaa taaatataaa 36720 ataaacagaa agtattaagt cattagcaaa aaatcataaa gtgagaattg tgcattaaaa 36780 tgatgtatag cataaccaca tttatttaag aatgtattcc aatatcaaat ggcaaatttc 36840 aacaatgcaa aaactgcaat tacttttgca ccaatctaat agaagttcaa taaatactgg 36900 caattacaat tggcattgcc ttagggtcaa cttgtaagac attcctgaaa ttgtgggaaa 36960 gggggaggac ctggagtgga cattattgga aggcaaagct gtaaccaaaa gagcaacctg 37020 ggaaacacat gactcctctg ttgctgtccc tggccctatc ctgtctcccc tccctgttgt 37080 cagctacctc atatgttctc taatctctgt ctctgtgccc tcaaagaccc ccctgaaaat 37140 agaaatatta ctgctcattg gttattttct atcaattaag tactgtatta gtccgttttc 37200 atgctgatga taaagatata cccaagactg ggcactttat gaaagaaaga gttttattga 37260 acttacagtt ccacgtggct ggggaggtct cacaatcatg gctgaaggtg aaaggcacat 37320 ctcacatggc agcagacagg agaagagggc ttgttcaggg aaactcccct ttttaaaacc 37380 atcagatatc atgaaactta tttactgtaa tgagaacagg atgggattca attaccttcc 37440 actgggtcgc tcccacaaca cgtgggaatt caagagattt gggtggggac acagccaaac 37500 catatcaagt actgtgcaag tgttttaggc atgcagagag tggtgggtct tcccagcaag 37560 cagagtgtgg ggaggtaatg ggggactggt ggctgactta atggcccagg acccatgcca 37620 caaggagatg gatggtggat gtgaatagga gcctgcttac acccatcaca atttagattc 37680 ttatgctcga tggcacgggt actcttttag gcccatttta ccaatgagga gattgggact 37740 aatttgctcg agatcaaaaa agaagtggtg taggtgggat ttaaacccag gatgtctagc 37800 actaaaatgc aggtacttaa ccactatcct aagggagtgg ctacttaatt tgataaactc 37860 atctagtgaa tggaagagag acggttacat ttcactgatg gtactgagcc tttgttgatg 37920 agctcattgg gaatctcaga catgagcagg atgtgtctaa gggacaggtg ggcttcagta 37980 gactggctaa ctcctgcagt ctctttaact ggacagtttc aagaggaaaa ccaagaatcc 38040 ttgaagctca ccattgtatc ttcttttcca ggttgtccaa taactgcatc acctacctag 38100 gggcagaagc cctcctgcag gcccttgaaa ggaatgacac catcctggaa gtctggtaag 38160 gcccctgggc aggcctgttt tagctctccg aacctcagtt tttctatctg taaaatgggg 38220 tgacgggaga gaggaatggc agaattttga ggatcccttc tgattctgac attcagtgag 38280 aatgattctg catgtgaagg atctgattct ctgtctaaga aagaagtctt tacctcttta 38340 agtagggagc aatgatttca tttttaaacc ttgactattt attcagcaac ttctctgctc 38400 tatgagatag tgtaggaatg gggatgtggt tgaagaatga aaagaaaagt cagctcccgc 38460 cctcctagaa attgcatctg ccttcacagg tcaaggatat tggatcagac cttctgcggt 38520 tctgaatgga gattacacag gttaggagca ggttgcacag tgtttccaat tctctataat 38580 taaagccata gactttcatg tattgaaaaa agcaagaatt gcattcttga cagattcttt 38640 cattgcctta aaaagaatga ctagccttgg gagtctgggc agctgggtcc agtgttgtag 38700 actttctctc tgctgagcca cagcttcaaa gatttgtcct tcttgtttcc agggatctat 38760 ttctcagaca ataagtaaag gctttccctg gcctaatgtg ctgtaagtga atgctactat 38820 atatgttcca ggcactgggc tagagactaa tatttaaaag ccaggaaatt tcctatagaa 38880 aatctatatc tcagggtttt ctcaaaagag ctgggaactc tggatgccca ttcatgattc 38940 cagtagttaa ccagagtaca agaagggctg agtcttctca gatgggcaaa cccactctgg 39000 ctgactgcag atccaccaag cctattgtct tagaccagga ccctttggca actcattccc 39060 ataagcctgt gacccttgct ttaaatatgc aggccttgtc ttctctcaaa aagcacatca 39120 aggctgcagc gaatgcagat atcaaatgat gaagttaaaa acaaaagctt tgctgggcgt 39180 ggcagctcac acctgtaatc ctagcacttt gggaggctga ggcaggagga tcactttagg 39240 ccagaggttc aacaccagac cttgtctctc aaaaaataaa aaattcagct gggtgcggtg 39300 tagttcctag ccacttggga ggctgggatg gaaggatccc ttgaacccag gagttcaagg 39360 ctgcagtggg ccatgattgc atcactgcac aggcgacaga attagatccc atctcttaaa 39420 aaaataaaaa atttaaaagt gacttcaaaa atctatgctg tgatggagag atttttcctt 39480 ctgtatgatt gtgatagctc tgtggcctat gacgtcatca ggttctgggc aaagtgtagg 39540 ttttctgttt ctttgttttt gaaaccattg cacagtccta agaaacatca cattctgggt 39600 cctgggcacc agccaacatg aggttgatat tttaaggggt ctgatgttat ttgctgcctg 39660 ttgatggaac ttgggagggc accagggttt gctcattgca ttcttgacag attctcttat 39720 tgccttaaaa agaatcactg gccttgggga gtctgtggct ggctgggtgc agtgttgtgg 39780 actctctctg cagagtcatg gagccttgtt cagaatgctt cctgagctgc cctggttggc 39840 caagggtaaa aacagccctg acttccctgc aagaaacact gcagctgggc cagagagtca 39900 gcccatccca ggcatgggtt taaaaagtgg aggcttttgt ttgaaagccc tgctctaatt 39960 ttgtcctcac tcaaacctct gttcacttga tctgctttag gctccgaggg aacactttct 40020 ctctagagga ggttgacaag ctcggctgca gggacaccag actcttgctt tgaagtctcc 40080 gggaggatgt tcgtctcagt ttgtttgtga gcaggctgtg agtttgggcc ccagaggctg 40140 ggtgacatgt gttggcagcc tcttcaaaat gagccctgtc ctgcctaagg ctgaacttgt 40200 tttctgggaa caccataggt cacctttatt ctggcagagg agggagcatc agtgccctcc 40260 aggatagact tttcccaagc ctacttttgc cattgacttc ttcccaagat tcaatcccag 40320 gatgtacaag gacagcccct cctccatagt atgggactgg cctctgctga tcctcccagg 40380 cttccgtgtg ggtcagtggg gcccatggat gtgcttgtta actgagtgcc ttttggtgga 40440 gaggcccggc ctctcacaaa agacccctta ccactgctct gatgaagagg agtacacaga 40500 acacataatt caggaagcag ctttccccat gtctcgactc atccatccag gccattcccc 40560 gtctctggtt cctcccctcc tcctggactc ctgcacacgc tccttcctct gaggctgaaa 40620 ttcagaatat tagtgacctc agctttgata tttcacttac agcaccccca accctggcac 40680 ccagggtggg aagggctaca ccttagcctg ccctcctttc cggtgtttaa gacatttttg 40740 gaaggggaca cgtgacagcc gtttgttccc caagacattc taggtttgca agaaaaatat 40800 gaccacactc cagctgggat cacatgtgga cttttatttc cagtgaaatc agttactctt 40860 cagttaagcc tttggaaaca gctcgacttt aaaaagctcc aaatgcagct ttaaaaaatt 40920 aatctgggcc agaatttcaa acggcctcac taggcttctg gttgatgcct gtgaactgaa 40980 ctctgacaac agacttctga aatagaccca caagaggcag ttccatttca tttgtgccag 41040 aatgctttag gatgtacagt tatggattga aagtttacag gaaaaaaaat taggccgttc 41100 cttcaaagca aatgtcttcc tggattattc aaaatgatgt atgttgaagc ctttgtaaat 41160 tgtcagatgc tgtgcaaatg ttattatttt aaacattatg atgtgtgaaa actggttaat 41220 atttataggt cactttgttt tactgtctta agtttatact cttatagaca acatggccgt 41280 gaactttatg ctgtaaataa tcagagggga ataaactgtt gagtcaaaac agccatcttc 41340 cttgtgacca aacatttaaa aatattctgg ctgggcacag tggctcacgc ctgtaatccc 41400 agcactttgg gaggccgagg tgggcagatc acctgaggtt gggggtttga gaccaacctg 41460 accaacatgg agaaaccctg tctctactaa aaatagtgga ggttgcagtg agccaagatc 41520 acaccgttgt cctccagcct gggcaacaag agtggaactc catctcaaat aaataaagaa 41580 aataaaaaat aaaaatatta ctaaaacttg tcaaatttta aagcacaatt ttctacttga 41640 agcggaggaa aatgatgttt tcaacccgat aatgtatagc ttaaggcaaa agcaattctt 41700 agaaaatagc caggcaatca aaacactttt ggcttcagtt tagaaaagtt taaccccgtg 41760 aagttttcag ctgtgcagga gcctccccgt acccagccta aaaggctgca acacatgtga 41820 aaaggcccct tggcccccct cgattccctc aaagtggctc caggagtctt taacagaagg 41880 atggagaagg tttacttatg gctggctctc cacagcgagc agcctctcat gcaggccttg 41940 tgttaaaata gtgcttagct gcgtttaata agcaagggat caggtggtca cccagcaagg 42000 taaaatccta cttccttgaa aggttttgcg tctggctgac tcggtggtgt ggcatctccc 42060 tggaccctgc atgggagtgt gtctatgggt tttctttgtg attggtgccc tgttctgccc 42120 agaccattgt ttctgataca gcagatcagg aggaaggtgg tgttgagtct cctttcgctg 42180 aggagtacac acagcagccc atttgcatag cactgtattg ggttgtcagt ttgtcatctt 42240 taaattcatt cagcaatgag gccaggcatg gtggctcatg cctgtaatct cagcactttg 42300 ggaggctgag gcagctggat cacttgaggt cagaagttca agaccagcct ggccaacatg 42360 gcaaaaccct gtctctaaaa atagaagaag aaaaaaaaaa gttagctggg tgtggcagcg 42420 cacgcctgta atcccagcta cttgggaagc agaggcagga gaatcgcttg aatcttggag 42480 gaggaggttg cagtgagctg agatcatgcc actgcactcc agcctgggcg acacagcgag 42540 actgtcataa ataaataaat aaataaattc agcaatgatt tattaaccag ttatattagg 42600 atgaccagtg gattgacaaa acagacatgg tcctcaaggt gcttaggtga gggtggaggg 42660 agaggcaggc agaaaacaag tcagttgaaa aagacatgaa aaagtcttca tccaggcact 42720 gtggctcatg cctgtaatct cagcactttg ggaggccaag gtgggcagat cacttgcggt 42780 cgggagttcg agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa 42840 aattagccgg gcatggtagc aggtgcctgt aatcccagct acttgggagg ttgaagcagg 42900 agaactgctt gaacccggga ggcagaggtt gcagtgagct gagatcacac tactgcactc 42960 tagcctgggc aacagagcaa gactccgtat caaaaaaaaa aaaaaaaaag tcttcaggct 43020 gttaaggggc tggtccgaga caggttgtct gaggaggtgc caggtaagct gtgtccccaa 43080 gaatgagaaa gatatggcca agcaaagagt gagagggaag aagggagcat tctagtaatg 43140 ggggacaaga ggtacaaaga ccctggtgag ggaaggagcg tggtatgcat ggtaggctca 43200 atagtcactc ccgaacatat gtagatccta atccccggga cctgggaatg ttgccttacc 43260 tggcaaaagg gactttggag gtgggattca gttaagggtc ttgagatggg aagatcatct 43320 tggattacct gggtgggtct taaatgcaat cgcagtttcc ttataagaga aatttaaagg 43380 acaatgtaaa gcaaagggaa atatgacact ggagaagaaa gaaatgtgat caccaagtaa 43440 gatgctacac tgctggctct gaaggtggag gaaggcgcca tgagccaagg attgcagctc 43500 tagaagccaa agaagaccaa gaaatagatt ctcccctaga acctctgcaa gcagcatagc 43560 cctgcctgcc ttggctccag cccattttgg acttctggct tccacgacta taaggggata 43620 atttgtgtca tttcctgcag cagccatagg aaactaatac actatgttgg aggcaagcta 43680 agggaaaggt gtcactagat gggtttgcag aggtaggtag gaccagttca ggaaaggtct 43740 cactggccac agtcaagagt ttggtcttga tcttggaata atgtcaaggc cttggaaagt 43800 tctaagcagg ggagatgtga accaatttac agttaaagaa gatcttgttt gcttctgggt 43860 ggagatagga caggaatatg taggggtggg gatgggaggc agaagaagtg gggaggctgc 43920 ctcagagcta ttgcaggtga gaggcggttg gcagtggagg tggagagaag aggatggatt 43980 ccagagacat tttggaggta gattcccaag agcctacgaa tgggttggat gggagggatg 44040 agggaatggc aaacatcaag gatgactcat agatttctga cttgagaaac tgggggcctg 44100 ggaaagttgg gtaggaaacc agtttggaag tgggagggag ttctgttttg aattgtcaag 44160 tttgagacac cgaaatgggg aagttaagtg gagacgtcaa aagatggtgt gaatttggag 44220 ctcgtgggaa aggtcacagc cagagataat agcgtgggtg tcctcagtgt caaactgaga 44280 atggggccct cgagtctgga tgacatgagc tagaaagaga ttatggagaa agaagaggtc 44340 aagagggtct aggtaagatg gtactgagat cttgatatgt gattcctggt agagttcaga 44400 gtaggagaaa ttccctaatt ttcaaaagtc cagcggatac caccaaaagc ctcctgggcc 44460 tgtgtaggga actagggaag taggattctt gattgtaagc ttctctgata gggaagttac 44520 ctgaaaggta catgctcatg gccagaagtt tatccatgag cagacggatt ccagttttgc 44580 agctaagaat acataattct tatgcacaga aaaacaacgt attagagcag aattttaaaa 44640 acagcctcag actttcataa tattcatcat catcctggaa gaaaaaggat gccagtctga 44700 gggcccttcc ctccccatag caccaaacaa aaagctgaaa tgaatacacc ccacagaaat 44760 gtgagagttg gagtcatatt ccaagtggct gagctgagac tagcgcttca ttccattgtt 44820 gtaccaggac agcttcaagc ctccttattc actccggtga gtggtccagc ccagtgtgtg 44880 aagtgcccag tactcagggc aatgcctgga agtgcccggg cacgagagga tgtgctgcct 44940 cgggccctag gtggagggtt ccaggtgagc tgtaaggtca taaaggacat ttcccctgga 45000 gaatcgcagg cggagatcgc agcagggaag actggcaatc caggctcttt gattggcttc 45060 ccttggatcg tttttttccc tgtaaattta atcccaacct attattattt cctgaacaat 45120 ccacagatcc agtcccctaa gcatcaccac tcccctgatc actcaccctg aaaatgcttc 45180 ttttcaggag tgctacatct ctttctgctt tgtaacaact tctcgatatc ttcagttcca 45240 catccccctg cacccctccc tctcacctgc tctgcctcca cccccaggaa acaagggggt 45300 ccctttggga tccatccttg cctgggttac tgcatcagtc tccctgttac tgactttgac 45360 ctgaggcatg aacctattaa aacataagtc agatcagcta ctgctctgat gtaaaccctc 45420 caggacttct tgttggtgca gggtaaaagg taaggtctca tctgacttgg accccctgta 45480 tcctctctgg cctccccttt gccactcaca cctgttctct gggctccaac ctcactggct 45540 tctttgctgt tccctgagca taccaggcac attgctgagg acaccaggca cattgctgct 45600 tccagacctc tgcactcgcc tttcccctcc tatgggaaca ctgttccctg agacagtggt 45660 gtggttggct tcttcatttc cttccagtct tagttcaaat gccacctaca tgaggtagtc 45720 cttccctgaa caatctacct aaatagcagt ttccagccac tgcctgcctt cctttgtatt 45780 gtcttctctg catgaactac ctgatgcatg gtcatttttc ccctccccta gaatgtcagc 45840 ttcttgagaa caggaaccac gctcactgca gtgtcctcag agtctacaaa ggtgcctagc 45900 acatagtagg tgctcaatac atatttgttg aatgaaggaa ggatccactt tctttattaa 45960 agggggcaga aaatagaagt tggggcagct tataagaatg agtcaccctt tttctgacat 46020 catttgtgtc tccttggtaa aggaggaaga ttccctggga catggaacaa tccctaatgc 46080 atcaactgcc caaaatggga tccagatgta gcagaaagtt ccctttaata aagagctttc 46140 tgtgtgtgtt ttaaaaatca aacatagaaa ttatttgtca ttgataccag caaatagctc 46200 aaatgggaga gcatggattt atagttcggc tggattagtt taaagatgat taaagtaatc 46260 ccctttatct tatcaaaaag agcttttagg tggtcagagt aaaagtcctc agacagaatg 46320 ttcagttgca cttggaattt cccttcccta agagataagc acatctcaga gatggaagac 46380 ctcagcccgt tatccatgga tcaaaacact gtgctgggct actgtggaga aagcgcctgt 46440 gacgtatcct tgcttgtgcg gatacctggt ccagttagcc acttggcctg gtgttcattc 46500 tgaatgccca tattcacagg attagtggga aaatggctgg aaccatggga ggttggatgg 46560 acatctggga gttgccattt actaagatgg ggagtgcaga gggattgcag ggggtggtgg 46620 tggtgaatat caggagttcg cttttggctg tgttgagtgg agatgctgag aaggcagtag 46680 gatataaaag agacaccaaa tttggagaga ctgcaagtag 46720 <210> 2 <211> 1040 <212> PRT <213> Homo sapiens <400> 2 Met Gly Glu Glu Gly Gly Ser Ala Ser His Asp Glu Glu Glu Arg Ala 1 5 10 15 Ser Val Leu Leu Gly His Ser Pro Gly Cys Glu Met Cys Ser Gln Glu 20 25 30 Ala Phe Gln Ala Gln Arg Ser Gln Leu Val Glu Leu Leu Val Ser Gly 35 40 45 Ser Leu Glu Gly Phe Glu Ser Val Leu Asp Trp Leu Leu Ser Trp Glu 50 55 60 Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe His Leu Leu Gly Gln Pro 65 70 75 80 Leu Ser His Leu Ala Arg Arg Leu Leu Asp Thr Val Trp Asn Lys Gly 85 90 95 Thr Trp Ala Cys Gln Lys Leu Ile Ala Ala Ala Gln Glu Ala Gln Ala 100 105 110 Asp Ser Gln Ser Pro Lys Leu His Gly Cys Trp Asp Pro His Ser Leu 115 120 125 His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala Ile Val Arg Arg 130 135 140 Leu His Ser His Val Glu Asn Met Leu Asp Leu Ala Trp Glu Arg Gly 145 150 155 160 Phe Val Ser Gln Tyr Glu Cys Asp Glu Ile Arg Leu Pro Ile Phe Thr 165 170 175 Pro Ser Gln Arg Ala Arg Arg Leu Leu Asp Leu Ala Thr Val Lys Ala 180 185 190 Asn Gly Leu Ala Ala Phe Leu Leu Gln His Val Gln Glu Leu Pro Val 195 200 205 Pro Leu Ala Leu Pro Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met Ala 210 215 220 Lys Leu Arg Thr Thr Val Ser Ala Gln Ser Arg Phe Leu Ser Thr Tyr 225 230 235 240 Asp Gly Ala Glu Thr Leu Cys Leu Glu Asp Ile Tyr Thr Glu Asn Val 245 250 255 Leu Glu Val Trp Ala Asp Val Gly Met Ala Gly Pro Pro Gln Lys Ser 260 265 270 Pro Ala Thr Leu Gly Leu Glu Glu Leu Phe Ser Thr Pro Gly His Leu 275 280 285 Asn Asp Asp Ala Asp Thr Val Leu Val Val Gly Glu Ala Gly Ser Gly 290 295 300 Lys Ser Thr Leu Leu Gln Arg Leu His Leu Leu Trp Ala Ala Gly Gln 305 310 315 320 Asp Phe Gln Glu Phe Leu Phe Val Phe Pro Phe Ser Cys Arg Gln Leu 325 330 335 Gln Cys Met Ala Lys Pro Leu Ser Val Arg Thr Leu Leu Phe Glu His 340 345 350 Cys Cys Trp Pro Asp Val Gly Gln Glu Asp Ile Phe Gln Leu Leu Leu 355 360 365 Asp His Pro Asp Arg Val Leu Leu Thr Phe Asp Gly Phe Asp Glu Phe 370 375 380 Lys Phe Arg Phe Thr Asp Arg Glu Arg His Cys Ser Pro Thr Asp Pro 385 390 395 400 Thr Ser Val Gln Thr Leu Leu Phe Asn Leu Leu Gln Gly Asn Leu Leu 405 410 415 Lys Asn Ala Arg Lys Val Val Thr Ser Arg Pro Ala Ala Val Ser Ala 420 425 430 Phe Leu Arg Lys Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly Phe Ser 435 440 445 Glu Gln Gly Ile Glu Leu Tyr Leu Arg Lys Arg His His Glu Pro Gly 450 455 460 Val Ala Asp Arg Leu Ile Arg Leu Leu Gln Glu Thr Ser Ala Leu His 465 470 475 480 Gly Leu Cys His Leu Pro Val Phe Ser Trp Met Val Ser Lys Cys His 485 490 495 Gln Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys Thr Thr Thr Asp 500 505 510 Met Tyr Leu Leu Ile Leu Gln His Phe Leu Leu His Ala Thr Pro Pro 515 520 525 Asp Ser Ala Ser Gln Gly Leu Gly Pro Ser Leu Leu Arg Gly Arg Leu 530 535 540 Pro Thr Leu Leu His Leu Gly Arg Leu Ala Leu Trp Gly Leu Gly Met 545 550 555 560 Cys Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val Ser 565 570 575 Pro Asp Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys Gly Val Val 580 585 590 Pro Gly Ser Thr Ala Pro Leu Glu Phe Leu His Ile Thr Phe Gln Cys 595 600 605 Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala Asp Val Pro Pro Ala 610 615 620 Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly Asn Ser Pro Met 625 630 635 640 Ala Arg Leu Leu Pro Thr Met Cys Ile Gln Ala Ser Glu Gly Lys Asp 645 650 655 Ser Ser Val Ala Ala Leu Leu Gln Lys Ala Glu Pro His Asn Leu Gln 660 665 670 Ile Thr Ala Ala Phe Leu Ala Gly Leu Leu Ser Arg Glu His Trp Gly 675 680 685 Leu Leu Ala Glu Cys Gln Thr Ser Glu Lys Ala Leu Leu Arg Arg Gln 690 695 700 Ala Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys His Phe His 705 710 715 720 Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys Ser Val His Ala Met 725 730 735 Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met Gln Glu Glu 740 745 750 Arg Leu Ala Arg Lys Ala Ala Arg Gly Leu Asn Val Gly His Leu Lys 755 760 765 Leu Thr Phe Cys Ser Val Gly Pro Thr Glu Cys Ala Ala Leu Ala Phe 770 775 780 Val Leu Gln His Leu Arg Arg Pro Val Ala Leu Gln Leu Asp Tyr Asn 785 790 795 800 Ser Val Gly Asp Ile Gly Val Glu Gln Leu Leu Pro Cys Leu Gly Val 805 810 815 Cys Lys Ala Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp Arg Gly Ile 820 825 830 Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu Gln Leu Gln Lys Leu 835 840 845 Ala Leu Phe Asn Asn Lys Leu Thr Asp Gly Cys Ala His Ser Met Ala 850 855 860 Lys Leu Leu Ala Cys Arg Gln Asn Phe Leu Ala Leu Arg Leu Gly Asn 865 870 875 880 Asn Tyr Ile Thr Ala Ala Gly Ala Gln Val Leu Ala Glu Gly Leu Arg 885 890 895 Gly Asn Thr Ser Leu Gln Phe Leu Gly Phe Trp Gly Asn Arg Val Gly 900 905 910 Asp Glu Gly Ala Gln Ala Leu Ala Glu Ala Leu Gly Asp His Gln Ser 915 920 925 Leu Arg Trp Leu Ser Leu Val Gly Asn Asn Ile Gly Ser Val Gly Ala 930 935 940 Gln Ala Leu Ala Leu Met Leu Ala Lys Asn Val Met Leu Glu Glu Leu 945 950 955 960 Cys Leu Glu Glu Asn His Leu Gln Asp Glu Gly Val Cys Ser Leu Ala 965 970 975 Glu Gly Leu Lys Lys Asn Ser Ser Leu Lys Ile Leu Lys Leu Ser Asn 980 985 990 Asn Cys Ile Thr Tyr Leu Gly Ala Glu Ala Leu Leu Gln Ala Leu Glu 995 1000 1005 Arg Asn Asp Thr Ile Leu Glu Val Trp Leu Arg Gly Asn Thr Phe Ser 1010 1015 1020 Leu Glu Glu Val Asp Lys Leu Gly Cys Arg Asp Thr Arg Leu Leu Leu 1025 1030 1035 1040 <110> University of Ulsan Foundation for Industry Cooperation <120> Compositions for treatment of bone disease comprising inhibitor against NOD2 <130> NP13-1188 <150> KR 12/078699 <151> 2012-07-19 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 46720 <212> DNA <213> Homo sapiens <400> 1 catctctggt gccagaggaa agggggcatc tcgaggctgg ggaggcccct ggcaggagtg 60 agaagtcctg aggagcccgg gcttcaggtc tcagaggggg ccactgtttt ttgagtacct 120 tcctctctgt gggactcagt ttccccatct tcaccatgcc agtgagtggg tgggtgtaag 180 tgagcgagtc tgcctggagg atcctactgc tgctctctgc acctcttagg ggaatctcct 240 gcctcctcct tctcccttgg tgggcgtgag atcaaagttc aaatgtgtac ctcagtttct 300 gcatctataa gttagaggca ataatagtca ataccctaga ggtggaagta actgagttac 360 tgtctgtaaa gactagcaca tggtaaacac tttctagatg ccagttattt tcgttatttt 420 taaaaattga cttcacattt tttaaaacaa ccaaatatga gtttcaaaag gaacatttac 480 attaacatgt taaatggaaa accggtgtca tttgctccgg agaacagaag aatgtaaaat 540 aaaatatatt taatggaaac aaaaacaatg ctattaaagc taattagata gtttcagagc 600 caagtaatct aggcagtgct agaaaggtac tcaaacccta ttagtactaa actgagaaat 660 aaactccatt caacataaac ggaaaaactg aaaggaaaat gaaaaaaaag aaccttaaat 720 gaccagtcca ctcttaccta acatgtaacc tgtcacactc tggaaaagtt ttcctagttt 780 tggctgtgtg tgcgtgtgtg tgtgtgtgtg tgttgggaag tgaggcgtgt gttgggaagc 840 gatgtgtgtg ttgggaagcg atgtgtgtgt gttgggaagt ggggtgtgtg tatattggga 900 agcatgtttg tgtgtgttgg gaagcggtgt gtgtgttggg aagtggtgtg tgtggggtgt 960 ggtgtgtgtg tgttgggaag tggtgtgtgt gtggggtgtg tgtatgtgca cgtgtgtgtt 1020 gggaagcagt gtgtgtgggg tgtgtgtgtg ttgggaaagc agggagtgta gtgtgtgttt 1080 gtgttggaaa gtggtgtgtg tgtggggtgt gtgtgtgtgt gcacgtgtgt gttgggaagc 1140 ggtgtgtgtg gggtgtatgt gttgggaaag cagggtgtgt gtgtgttggg aagtggtgtg 1200 tgtgtgagtg ttgggaagca gggtgtgtgt gttggaagta gggggtgtat gtgtatcggg 1260 aagcatgttt gtggtggggg gaagcagggc gtgtgtgtgt gtgtatgtga gtgttgggaa 1320 gcaaggtatg tgtgttgggg gaagcgaggg ctgtgtgtgt attgggaagc atgtttgtgt 1380 gtggggggaa gcgagggctg tgtgtgtgta ttgggaaaca tgtttgcgtg tgggggggaa 1440 gcagggcgtg tgtgtgtgtt gcgaagcggt gtgtgtgtgg tgtgtgtgt tgcgcgtgtg 1500 tgctgggaag cggtgtgtgt ggtgtgtggt gtgtgtgtgt gtgtatgtgt atgtgttggg 1560 aagcgggttg ttccgaggac tccccgattc cctcctggtt gaaacgtggc ccagaaagag 1620 aggcacactg gctagggcac acagccaggg ccgtccagcc ccgcaggaag ccccggtcct 1680 gccctgtccc aggtaagcct ggccagccct ggagccggcg ctgcggggct gcggggctgc 1740 ggggctgcgg ggccacggga caggagcggg tgctgcggtc ccggcagagc aggggcgggg 1800 tccgggcggg ggcggagccg agcgcccgcg cacccggagc cgcagcccga gccggagccg 1860 gagccgggag tcgtggcccg gagtgggcct tggagtcggc gcgcaggcgg ctcgcggtga 1920 gtgctgtgcc cagcgcctgg gggtgggctg gggggagact gtgggcgccg agggtcccac 1980 ccccgggact tgaacacttg gcttctgagc cgtcctggcc gcagggcttg ggcaagggtt 2040 gcctcggttt ctcctggggg agtcccagaa tgtagatggg aagtgagacg gaaagggggc 2100 atctcgaggt tggggaggcc cctgggccac tcttttgatt tccttcctct ctgtgggact 2160 cagtttcccc agcttcacca tgccagtgag tgggtgggta taagtgagag agtctgcctg 2220 gaggatcctg ctgctgctct ctgtacctct tatgggaatc tcctgcctcc tccttctccc 2280 ttgttgggtg tgagatctaa gttcaaatcc atcccttccc agacagccca caccaggacc 2340 tgcgaggcca catcccccag ctgtttgctc tcctatgctt ctcaggttga tggagcagcc 2400 atctggtcct gctttcattc caacaaactg aaaagccctc caccccaatc cccctgggga 2460 ggacctcctg aagctgcatg agggctgggg gctggacccc tgcttctgag tttgctcttt 2520 ctgagcctct cctgccatgg ccttggtttc cccatgggct gtgacctagc ctcctccagg 2580 gtccttgttc tacagagggg aaacacctgc cctgtcaccc tcccctgacc ccttagtgaa 2640 aaagaccaga gtgacatccc caccacctgt acttgcctgt cccgatcact cagccatggg 2700 gctgctgggg cccctgagtt tgttggctgc tcaaggcccc tttgcctcgc tgcaccctgg 2760 gatctgaggc tggaagcatc ctcatccagc ccacctccct cctacccagt gcagagcccc 2820 tctgtgacac atgcccagtg gtggggagct tattactgat gaggtggctt tttagtgcct 2880 tgcagctctt gtttccttta gctaaaatct gtggcttaca gattctacac cttggttcca 2940 tgtctgtctc ccggagccat gcagagcaag tcagttcttt cattctttcc agcagtcatc 3000 attcatgcac tggatgagac acgagacaga gcagtaggaa aatcagacaa aaccccctgc 3060 cctcatggag tgcacactct agaaaggaat ggacagcaat gaaataagta aatacatggc 3120 gtagcaggtg gcgggaagta ctacgagtaa aacaaagctg ggaagcagga tggggtgctg 3180 gggctgggta ttgcattgta tatagcacaa ccagggaagg cttccccaag accacgatag 3240 ctgggcaaag tcataaggaa ttgaggggtg agccacatga gtatctgggg ggctgggagg 3300 ctttggtttt cactctgagc atgatgggag cacgggtttg gaacagagga gtcacctgct 3360 ttgacttggg gtctggcagg cccatctagc tgtggggtgg agaatggatc atgggggggg 3420 tggggtaagg gcagaaggag agcccgttga gcggatcatt gctgtcattc tcgggagaga 3480 ccatggtgtc ttgcccatgg cccagaggtg gttaaaaaaa agaaaaccag acattcagat 3540 ttgggatata tttcaaaggc cgtgtcgata gggtttgggg tgcaagagaa aggtagagtc 3600 aaagatgatt ctgagtgttt ggctcgagct tctggaggtt tgaagctgcc atgaactgaa 3660 atgggagttg cttagcggag agatcaagag tttggttttg tgcttgtgga gtttgagctg 3720 cccattaggc atcaatgatg agtaagcagt tgggaaaagt taagggcgga ggttggagtt 3780 gaaaataaaa ctttgggcat catcaccata taactggtat ttaaagccac aagagcaggt 3840 gggctcatct agggatggag tgatatggag aagagaaggg gtctaaccat tgagtgctgg 3900 ggcccccagt gttaggaacc agccaagaag acagaaagag tgaaaatcag agagttgggg 3960 tgtcctggag gaaatgaaga aaatgcccca aagaggaagg agggaacaaa tatgaccaat 4020 gcccctggca gagcaagcag gctgagggct gaggattgag caatgggagg tcactggtga 4080 cagtttcact ggagctggat ggggaactag agggaatggg aggggatggg aggacttggg 4140 gacagcagta caggcaacag acaagggggc ctgctgtaaa gggagcagat aaatgggatt 4200 ggagccaaat gaagaagggg agtgtcaaga gagtgcttta cttttacaat ggagaattag 4260 agtgcattgt gcactggtgg ggggatttga tctcttaggg agagaacagt gttagggagg 4320 gagaatgcag gatagctggg ggagggtggg gggcttggcc ccagcagaga ctcaggacac 4380 ttgggaagtt gagcttccct gggcttcccc tcctctcctg tctgcaaggg gtcagtgggc 4440 tgagatttca gcacttaagc aaagcatttg ctcttggccc cagagaaacc gggctggctg 4500 tggtctcagg aaggaaggag gtgtccaggc tcaggcctgg gcctgggttt cagggagggc 4560 ccacgtgggt cgccccttga ccctctcttt cagcaaggaa gtgatccttt ctctacatgg 4620 gcctcacctt ggggaggaca atggtgtctt tgaagttgta gtaactgaag tagagatcaa 4680 aaggcaatgc agatagactg acagatttcg cctgaagagg ggaagcccga ccaggtaata 4740 aaggagtaag aggaaggatg ttaaggacaa ttttaggaaa cagataatga gtgaatattt 4800 tttctctctc tttcccaatt taaactgaag caggagaaac tgaagctaga cataatgatt 4860 aacttcccaa gctggtgagc ttcctgagct ggttagtgag aacagcacta aggccaggtt 4920 ctcctcccca gatgtttaag atgagacagg acaatgcctg ctcagagaca gggcctggct 4980 gaattggccc tcaggattct ctctgctctg aggtttctgg aagaaggcca gggcagaggt 5040 gtggtgatgt agctgctggg aggacagagc tccgagtcac gtggcttggg cgggcctccc 5100 cttcctggtg tccacagaag cccaacgtca ctagctgggg tgtgtatggc tcacacgtag 5160 gccaggctgc cctaggcttg gtgtgcaagg gaggggcccc tacttacttg tggcctgtcc 5220 cctcgtgaat gtgtctcatg tccccagtgg ggtttttcag tgagggtcat ggtctccagg 5280 atgcacaagg ctttgtgcca gaattgcttg gaattgccta gttctggaag gctggttggc 5340 caactctggc ctccggcttt tcctttggga atttcccttg aaggtggggt tggtagacag 5400 atccaggctc accagtcctg tgccactggg cttttggcgt tctgcacaag gcctacccgc 5460 agatgccatg cctgctcccc cagcctaatg ggctttgatg ggggaagagg gtggttcagc 5520 ctctcacgat gaggaggaaa gagcaagtgt cctcctcgga cattctccgg gtaagaggag 5580 caggcattgt cccgtcccag cttgatcctc agccttcttt catccttggc cgcgacatgc 5640 tcccaggcct ggggtcagat ggggagtgct gactctgttt ctgggctgtt ttctggggag 5700 aatgggtcgg cgggtttttt tccccaggac ctgggcaggg tcaatggtgg gggccgctgt 5760 cgcatccttg gctggtgttt ccacagctga gaaccactcc agggccaagc ccagagctta 5820 ttctaccctt ttttgtcctc tcttcccctg tcctcggcca ccccaccctc ttggctcctc 5880 tgcttagatg tgggcacaag gaggagaact ccttggcctg agagaactac cttagatcct 5940 ggcttccagt ggcctctgca ggggggtaca ccctctctcc caagcagcca gacacacaag 6000 taacctcatt gcctcagttt ccccatctga ccagcacagg gccccctgtg ccccagcagc 6060 gttctgagag attggagctt tctccttttg cttaccttgg ctaccgtatg aggacggata 6120 cagagtgttc cccccacccc cagcccaggg gatatttgat tcatgaacat tccctcagtg 6180 tctttgtggg ggacaatgct gtgccaggct cagggatgcc aggacgagta agacccaggc 6240 tcccacgtgg cccaggcagg gagagagaca cataaacaac catcaggaaa gaggtaaaat 6300 ccccaggcca cttggcatct gctcccttga gtgtctggga atgtccctga tttataaaaa 6360 gaagctgacg gccctctttg ttgtccatgc ctacaccctt tcactttcgt ttcttcgggg 6420 cactgcagca gcccttgtcc acagacccca tgacaatcgc agaactgacc atgctgagag 6480 attttcttgg ctgctcaggg accctgccag ggcttgaagc tcctggaggg tcacttgccc 6540 tcaaattccc agaacgcaca gcaggtcact gatgatagca gtggcagcag tctgtgcacg 6600 gtggtttcga gggcgtggga gggaggtgag ggccctaggg caagtgtgtg tgggaagtgt 6660 tgatggggga caaggcacca gaacgctcgg aaacaactta gtttgcaccg taatttttca 6720 cttcgcctag gacaggacct ttagagcaat attctgagtc taccccttgg agtagcagtg 6780 tgcaaaacac acagcacggg cttggggccc ccgtggggaa cccaaatgta agagttagag 6840 acatgcattc cggagtcata catggctcgt gttgaaatcc tgactctgcc tgtctagctg 6900 tgacacatcg tacaaatcac ttagcttctt ggtgcctcag tgtcttcctc tgtagaatgg 6960 gtagatcata ggcactactt cagagtggct gggagggttc agtgaattcc tgcaggagag 7020 cacttagaat ggcacttggt gtgtagttta tgcttaatta atattagccg ttactgaaac 7080 tgctgtagcc tgaatccagc cagcatgaaa gagcccctct caccctgctt cgaagagaat 7140 gaattccctg attgtttgga agatctctct ctctctctct gtcttttttt tttttttttg 7200 agaaacggtc ttgctctctt gcccaggctg gagcgcaatg gtgccatctt ggctcactgc 7260 aacctctgcc tcccgggttc aagtgattct cctgtctcag cctcctgagt agctgggatt 7320 acaggcgctc gccaccacgc ctggctaatt tttgtatttt tagtagagac agcgtttcac 7380 cgtgttggcc gggctggtct agcgctcctg atctcaagtg accttgggag atctcttgct 7440 cctaatatta cctcaagcct ttttaaacgt tttaagccgg agaccaagca tggatatggg 7500 agttaggggt cttgatttaa ttcttggttg cttcaaactc tgtggaacct tgaggtgttt 7560 cttgccttct ctgggtctca attttcacat ctatatggtg gggagcttgg attgggtaat 7620 gtctgaggct agaaccatgg ccaactcggg ttctgctggg gctgacttgc cctggccttc 7680 cctgaccacc ctgcatctgg cttctggaga agtcccgcac tgaccttgtt ctcctcccca 7740 ggttgtgaaa tgtgctcgca ggaggctttt caggcacaga ggagccagct ggtcgagctg 7800 ctggtctcag ggtccctgga aggcttcgag agtgtcctgg actggctgct gtcctgggag 7860 gtcctctcct gggaggacta cgagggcttc cacctcctgg gccagcctct ctcccacttg 7920 gccaggcgcc ttctggacac cgtctggaat aagggtactt gggcctgtca gaagctcatc 7980 gcggctgccc aagaagccca ggccgacagc cagtccccca agctgcatgg ctgctgggac 8040 ccccactcgc tccacccagc ccgagacctg cagagtcacc ggccagccat tgtcaggagg 8100 ctccacagcc atgtggagaa catgctggac ctggcatggg agcggggttt cgtcagccag 8160 tatgaatgtg atgaaatcag gttgccgatc ttcacaccgt cccagagggt gaggcactcc 8220 tggtgtgcat cacagagttc tcaggaaagg ggtgcttagt caccaagact gatttgtcct 8280 catgaagtca gcctgtgggg taacttggtc catgggattt cccctaaaaa ggtagccagg 8340 caggtaaaat ttgctcttga ctcttggcag gaaacgtaca actctttctt tcttcttttc 8400 ttttcttttt ctcactctgt taccctggct agaatgcagt ggcacaatca tagctcactg 8460 tagccttgaa ttcctgcgct caagtgatct tctggcctta gagtagctgg gactacggct 8520 gctgtaccac catgaacagc taattttttt tttttctttt agagatgggg tgttgctatg 8580 ttgcccaggc tggtctccag ctcctggctt taagcaatcc tcccgccttg gcctcccaaa 8640 ctgttgggat tgcaggcatg agccactttg cctggccaac agaacacttc tgccgagagg 8700 aagtgtgtgg tggccaggaa ctcagattct ggagccagaa tggtgcaggc tcaaggtcaa 8760 ccctgtgtga tctcaggctt ccctatggag cctctccagc ctcagtctcc cttgtttcag 8820 tttcctcatc tacaaaacaa tgttaatagt caaatggtgc ctatcctata aggctcttgg 8880 gaggattcag tgagttaatt tgagtaatgc ttaggatagt gtctattacc actggctgct 8940 atttattatt tctgttatga gtgatactct gtacttgtac acttttattt ctgtctgttt 9000 taaattaaca gcacaacaga ccataacact gcagtatatt gaatttattt tataattaac 9060 atagcatatt ataaactaat atagcttaaa tgtttatgta ggatttctga catgaaattg 9120 cattagatca tagatgttca gagttggtat ataacagccc ctgagaatgt agtaactcag 9180 cagagaccag aaggtcagag aaatgaccac tgagtatttt tgaaactctt ttgttttctt 9240 ccaaatagtg attcttaggg ctcctgagag gcagatggaa caatcattaa cattccactt 9300 tataaatcgg gaagttgaga ccaaggaaag tagtttgaat aagctcacag tagttaatga 9360 gggggccagt gctggaccaa ttggccagca ctggtcattg acttattcat ccatcattca 9420 tttattcagc cagaatctat taggtgcttc atacatattt gcttaaagtt tgttgtgttc 9480 atagagcttt gcacacggta ggtactccat aaacatttgt tgatgaaata agtgagttac 9540 tgaatgaatg attgaattag aatgacactg cagtgttaaa atgggctggg ttggggaaca 9600 ttttagtttt tgtttttgtc tgttttccaa aaatgtatgt gttgttcaca tgagtctgga 9660 taaccctaga ttgagattga tgacataaat aaatttgtct tcaaggctgc actaaagctg 9720 gctcacatgg ctaggtattt acagagcaga agtggtgcag tcctctctga ttagttgcac 9780 gtacagaaga catattcgtt attggactga ccttagtttc tcttataatt tgttagggga 9840 attgaatcag cccatctgag aagttacaag attgtgtctt gtcatcttta aaagttcagc 9900 aatgtgatgt ggtacagatg gtctgagggg tttggagaag gtagcctaga tccctagggc 9960 ccagagaaga caggatgtga acagaggaag tacatggatt ggtgaagaaa agaaatggga 10020 taactcatgg gtcaaagaag aaatcatgat ggaaatcaga aaatattcag aaccatacaa 10080 taatgagaat attatttatc aaaatctatt ggatgcagct aaagcaggac atagggggaa 10140 atttacaacc ttaggtgcct agattaggaa agaaggaagg catttgttta tttatttgtt 10200 tatttattta tttgagatgg gggtctcact gtgtcaccca ggctgctgga gtgcagtagc 10260 acgatcataa atcactgaag tctcgaactt ctgggctgaa gtgatcctcc cgcctcagcc 10320 ttccaagtag gtgggacaca ggctagcacc accataccag gctaattttt tttttgtaga 10380 cacagggtct tgctatgttg aggtctcaaa ctcctgggct caagtaatcc tcctccctcg 10440 gcttcccaaa gtgctgggat tacaggcatg agccactgcg cccatctaag gctgaatttt 10500 aatgagctaa gaattcatct taagaaaggg ctaaatagac agcaaaagca aacattgaag 10560 gttgggactg agctgagtgg gtagcaggga tgggagacaa cagatctgag gagagcagga 10620 gattttgaaa ggattgcact gcctgaggtt taagccttta gaatccagct ctctctgagc 10680 tccctttgag ctctgacatt ctgtgactct gatttggtgg ccttccctta gtggccttac 10740 tgatttcatt tggatggtgc ttgtggtata tccaaccaac atgtcttccc aaatggcctt 10800 ttaatttcct ataaagaagt agttgtcatt gattgcaggt tagggacaga aaatgctgtg 10860 gaatgaaaca aaatgcaagt taaagaacta aattccaaaa atacccattg ctactattga 10920 ctgagtgaat tcctactgtg tgccagacac tgtacccagt ccattccctg tattgtttta 10980 tttaagcctc acaagggtat agtgtgacta cactgtttct taacaatgaa gaaactgccc 11040 aaatcgccca tctgggaagc ggcccagcta gaatttgaat ccaggcctgt tttcctccag 11100 agcttgtgct attctctgtc tgtcataaaa tgtgggggct ttgtgtggta aacttgctca 11160 gttgggcata gcagttgtta ggaaacctga ggctggtaac accagctgta ataccagctg 11220 tccgtctgac tcatgcaact gttaaagttg atagggctga ggtgtcagac tgagctctga 11280 attgcctgat tcctataaca atattaactt aaacattttt taaattggga aatgcaccat 11340 gcatacagaa gagtgtgtat atttcatatg tatagtgtaa actgttccca tcacccaggt 11400 taaaaaacag gatgttgcca gtacctgggg ccttctttaa ctgcaactgc tagaggtaaa 11460 cactggcttg acttttgtgt aaatcatctc tttgcctttc tttaatgttt tagcatcttt 11520 taaaataaat ccccaaataa tgtattgttc tattttgaaa aactgagtag caagccaaaa 11580 atagctgtgt aaagaaaggt cacttaaatt aggctgggtg cagtggctca agcctttaat 11640 cccagtactt tgggaggctg aggcaggtgg atcacaaggt caggagatcg agaccatcct 11700 ggccaacatg gagaaacccc gtctctacta aaaatacaaa aaattagcca agaatagtgg 11760 catgtgcctg tagtcccagc tactcgggag gctgaggcag gagaatcgct tgaacccggg 11820 aggcagatgt tgcagtgagc cgagatcgca ctgcttgaac ccgggaggca gaggttgcag 11880 tgagccaaga tcgcaccact gcactctagc ctgggtcaca gagcaagact ctgtctcaaa 11940 aaaaaaaaaa aaaaaaagaa aggttactat tgccttttct tagatgaagg ttcccaaggc 12000 agggaaagct aagtggagtc tcagggactt ggtctggctt ttccttccct gggaatttat 12060 aaggacctct tctgggaagt cagtcggcaa tgccatgaat gagtctgggg aaatattggg 12120 ctcattgcaa ctggagggtc tggtaggact gatgtgaatt aggtgctgtg tccggaggaa 12180 aatggccaga ggaagtgggc tgctttgtac agtcagtggt aaagttgcca aaggctatta 12240 tagctcacag gaatgggcca aggctaaaca ctcctgtgga gtgaaatgaa tgtcctcagc 12300 tgactgaggc agcgggagtt gaaaagaaac gatattagtt catggtgaag acaagtcaaa 12360 tatagataaa ggttagggtc aggcttgcct ggacatctag gagataactg ccctcaactt 12420 gtttgaatct tgagtcactg ctccattttg tttgaactgg tggccatcta cttatagtat 12480 acagccatca acctgagatt tccctacatg gtcttcctgc cttggtctcc tgtatcctga 12540 atcctatggc ctcttcttcc ctggtttact acattttgct agaccgtatc ctccagtcaa 12600 ttccttagaa tgaatgtatg aaagttaaaa tttctgaggt ctcacatgtc ttaaagttcc 12660 ctcatactgg attgatagtt tggctgggta taaaattctg ggctggccat cattttcctt 12720 cagaattttg attgcattat tccattatcc tctcttttca atattgcttc taagaattcc 12780 aaaacctttt tttttttttc tttttgagac agtgtctcac tctgtcaccc aggctggaat 12840 gcagtagtgt gatctcagct cactgcaacc tccacctcct gggtttaagt gattcttctt 12900 cctcagcctc ctgagcagct gggattacag gcacccacca ccacaccctt tagtagagat 12960 ggggttttgc tatgttggcc aggctggtct tgaacttctg actttaggtg atctgcctac 13020 ttcggcctcc caaagtgctg ggattaaagg cgtgagccac cacacccagc ctccaaaacc 13080 attttaaaac tctttctgga agcttttaaa attttctttt agtccccaga attttaaaat 13140 ttcaattatg tgccttggtg ttcttccatt atgttagtca cccaagaggt actttcaatc 13200 tggaaacttc tctatgtttt gggaaatgtt cttgattagt ttacaggtga tttcttcctc 13260 tccattttat ctcttctctt ttcatgaaac tactattaat tcaatgttag aattccttga 13320 ctgatcattt aattttcttc tattttccat ctctgtgtct ttttgctcta cttttctatg 13380 atagtcacag ctctatcttt aaactcttga gtttttcatt tttgatgtca tgattttaat 13440 ttgcaagggg taggtttgac tgattctttt ttgtagtatc ttactcttgt tttatggatg 13500 caacatcttc tttgacttaa ggatcataag ataggtgggt tctttgtttg tttgtttgac 13560 tgtttttcac cctatgtaaa ctttttctac aagtttcttt ccccttcccc cctttttggc 13620 ttctatctcc cacattagat gctttctctg ggctcatgat actctttggt tttctttctc 13680 aagattgaca ggtaggactt taaaacttgt tgagcatgcg ggtgaaactt gtctaccatg 13740 aatttcactg tagatatttt ggagattgac agtgtttata tctttagatc tcgcctcctg 13800 ggttgatcaa gttatctgag tacaccacag accttttgcc tggggataaa ccagaaatct 13860 gtttcagaaa ccactttgat tcagtcttcc ttgttttagt catttccttc agttccggag 13920 gtccctcatg ctgatcattc cagagccctt tacagatcct agggtacaca ctgcatggtt 13980 ttcaactttc ttgttttggg gttaagattt ggctttcagg agtctcctca gtccgttact 14040 attcattcaa tcagcaagtc cttgagcacc tgatttgtgc cagacattct tctaggtgtt 14100 agggatacct cagtgaacaa aacagacaaa aatctttgtc ttggaaatac acacactcca 14160 gtcaggggag agggacaata agccaaagga aggaaattac agcgtgtgct agaaggtgat 14220 aagtgctgta gaaagtaagt aaagtgggtt tgggagttga gagtttggga aggggataaa 14280 tgatggcaat tgtaaataga gtagtcagag ttctcactta gaaggtgaaa ttcaagtaaa 14340 gacttgaagg aggacaggga attagccaca tggatggcta ggggaaggct tccaagctga 14400 gaggacagcc agagccaagg cccagaggca ggagcatacc tggtagtttt aggaaacagg 14460 aggccaggat gctgagtgga gtaagagggg gcatgaaagg agaaacttgg gtccacgtgg 14520 ttctagacag gtatttttgt ctgttttggg ccctgaaggt tactattgga cttggactct 14580 tactctgagg aaatagggac gctattggga cgtttgtaca ggagcaatgt gacctgagtt 14640 ttgtttgtaa aggattagac tctggctgtg gcattaaggc taggctgtgg gggcaggaac 14700 agaagcaggg ggaccagttt tgcagcctgt gcagctttcc agataagcag ggattgtggc 14760 ttggaggagg atggtataga ggaggtgaca agaaatgact ctatgtctgg tatgtagata 14820 ttggccacag atggcatttg agcactagag acctggctgg tccacatgga gtttccataa 14880 gcacataata cacatcagat ttcaaagact taatatgaaa gaaagaatga aacgtacctt 14940 gctatttctt tctttttttt ttttttttga gacccagtct tgctctgtca cccaggctgg 15000 agtgcagtgg tgtgatctcg gctcactgca acctccgcct cccaggttca agtgattctc 15060 ctgcctcagc ctcctgagta cctgggacta caggcacctg ccaccacgcc tggctaattt 15120 tttgtatttt tagtagtgat ggggtttcac catgttgtcc aggctggtct ggaactccgg 15180 accttagggg atctacccgc cttggcctcc caaattgctg ggattacagg catgagccac 15240 catgctcagc catatcttgc tattttctac atggattaca tgttgaaatg gtaatgtttt 15300 ggctattgtg gattaaatag aatatatgat taaagttgat ttcatctatt tcttttaact 15360 ttaaaaaata tgtctgttag aggatttgaa attccacatg cggcttgcat ttgtgacctg 15420 catttcattt ctgtggaaca gtgccctttt tgggacatgc tttgaaggtg gagtcaacag 15480 gatttggcag attacagacg agaggcttca agggtgactc caagacttcg gggcagagca 15540 cctggaagaa aggggttaat attagccaag atgaggaagg ctgtcggttt ggcaggtgca 15600 tgggcaggtt aggagtttag ttttgaatat gttggaggtg tttatgaaac ttttaagtgg 15660 agatggaaaa taggcagttg gatgtgcaag tccagggttc agggagacag ttcaggctgg 15720 agatgaagat gtgggagtct gaggagagat tgtattcaaa tattcaatcc atgagacttg 15780 atgaaatcac ttctcttcca aatgatttac agcctgcaga atcattttcc ctatctttgt 15840 aggtttatgt cttcattttg tttcatttat ttttcagtta ttcactgttt tagtgagttt 15900 tgagtaggag ccagattgga tgcatgcgtt caattcacca tccaacactg tattaactac 15960 ttgaaactca tgtggttgtt cggttgtttt tttgaccttt tattctggat ggaagagaga 16020 tgcttatgaa gttgcagtaa tcagtaagcc ttcccacatt gctccatcag ccttcctgga 16080 agaataatgt cttctgcctt tcctgtaggc aagaaggctg cttgatcttg ccacggtgaa 16140 agcgaatgga ttggctgcct tccttctaca acatgttcag gaattaccag tcccattggc 16200 cctgcctttg gaaggtaggt gtatgttctc agttaatcag aaagggaagg gcagtcagtg 16260 cagatccatg gttaagagca gaacacacct cggttaacat cccatatgct ggcagtatag 16320 cctccctatg actcaatttc cttgttttaa ggctagcacc accccgtctc attgggattt 16380 tgggagcatt aaaaggacaa aagcgtgtaa tgttagctat tagctttcat tatctcccac 16440 acagtatact gacaattggg ctaccatata ttgagggcta actaaaggtg ttacttacca 16500 tccaaactct cattatctgt accgaaaaga tatggacaca tgttttgagt tagggctggt 16560 atctcttgat ctctgaaatt tagcagctca caatgggaaa ctcaagaacc aagtggatct 16620 agagactctg gtatccctca gtgcccaggg tcaccaccca aactcaggaa caggaggggc 16680 ttggaccgca ccacttgaac ataccaggca tcctgccagg tgctttatgg acaatgtcta 16740 ccctttgcaa caaccctgag aagtaggtgg tgtttttttc caccttatag atgtggaaac 16800 tgggcaggga ggttaagtga cgagggaggg gaagatgggt ctgattgtaa attgtcccca 16860 cctacacttt ctcttttctt gggagaagaa atgtcagttg taaagagaga gtgcaagcct 16920 ggcactcttt agggcttgtt cctacaccac tgtagggaaa gctcattggc actgaagccc 16980 cctgagctgt gtgtggtgct ggcagatggg tctatcaccc tgggctgtgt cctctgggca 17040 gcaagcaagc ctgtgggcgg ggtggctgga agtctgtgcc tggcactcgc gagtgcaccg 17100 tctcattgaa gaacaggatc taaacatcag tgcgccacag cagggtgcgc ggcacggagt 17160 gcaggccctg gtttggccct tggttgaggt ttgctgttga catcatcaag cacagctagt 17220 cactgtaaga ccaggccagg gtgcaagatt ccccacactt ctaaaggtga caattggtgt 17280 gtttatttct ctataaaatg acattttttt tttctggaga attttagtat cattggtgat 17340 gactggaaaa cctgcatcag aaatcaggtc ggaagaggaa gatatatatc tgatatgtac 17400 tggagaggaa gatatctatc ttatggtcta agttcaggga tcctggtata ttcagagggc 17460 agaaagctca gcaataatca tcaactctgg gaacagaggt gacataaaca cagggcgtcc 17520 cctttgtgtg actgcagata gtcatcagtg agctcagagc tctatgaaaa ttacttgcta 17580 gtttttgggt tgaaaatagt gggccagtgt ttggttgggg gcagtgaggc tgtgatggcg 17640 ggggaccatg ccaagctcct accagcctgg gacgctaaac cagcacttcc ccatttcctg 17700 aaaggggaac taaactctga cacaggaaat ggtttgcttg cattactttc aggatgagaa 17760 aggaagagca ctggccttcc aaacacaccc cgtgcatgaa aactctccct gcatggggtg 17820 catggggagg atggggaagt ggaggcagga tcacagactc ttgttcgagt gctcagctgg 17880 ggcaccccgg tgaccccgag gccttccctt gctaggtcca cccagatcaa tcaggatcat 17940 ctccccatct cgaagtttaa ctttatcaca tctcagagtt ccttttgcca cgtaaggtaa 18000 catattcaca ggttctgaga atccggacat ggacatcttt gagggtctat tgttgtgcct 18060 actatatcca tgaataataa tgataataag caccattttt tgagagtttg ccatgtcaga 18120 tattctttta aactgtattt tatctcgctg cctcctgaaa aaatccttcc aggtgtatat 18180 tgtccccatt tttacagatg agagaactga ggcccagaaa ggctaaatgg cttgcccaag 18240 tgtatggtgg acccaggttt tcaaactcag gtgtgtctgg cttcagagac tgggctcctg 18300 agcccttaag ccctttgttc ccctttagaa aaagtcacct gaggctgagt ggtgaaggga 18360 tttatccaaa gccacccggc cactatggca ggacagatat cagaatacag gtcttccgat 18420 cccagcccag agccccttcc cgtcatctag aactcctcct ggtgtcagta atgataacgg 18480 cagtcactga tgtcttttga gcacttactt tgtgttgagc acttacactg tgctaagcac 18540 ttgacatagg tcatcttagt tgatccgtgt aaaactctgt gaggtagtga ccaacatttc 18600 tcccacctta cagaggtgga aactgagggt taggaagttt ccttgactgt cctcaaagtg 18660 cacagcttgt gaatggagga gccaggatgg gcgcccgctg gctctcctat cccttcagtt 18720 atgtcagcgt cccccgcagc agcccattgt ctggttaggt cccgtcttca ccatggtgcc 18780 accttcatct gcctcttctt ctgccttcca gctgccacat gcaagaagta tatggccaag 18840 ctgaggacca cggtgtctgc tcagtctcgc ttcctcagta cctatgatgg agcagagacg 18900 ctctgcctgg aggacatata cacagagaat gtcctggagg tctgggcaga tgtgggcatg 18960 gctggacccc cgcagaagag cccagccacc ctgggcctgg aggagctctt cagcacccct 19020 ggccacctca atgacgatgc ggacactgtg ctggtggtgg gtgaggcggg cagtggcaag 19080 agcacgctcc tgcagcggct gcacttgctg tgggctgcag ggcaagactt ccaggaattt 19140 ctctttgtct tcccattcag ctgccggcag ctgcagtgca tggccaaacc actctctgtg 19200 cggactctac tctttgagca ctgctgttgg cctgatgttg gtcaagaaga catcttccag 19260 ttactccttg accaccctga ccgtgtcctg ttaacctttg atggctttga cgagttcaag 19320 ttcaggttca cggatcgtga acgccactgc tccccgaccg accccacctc tgtccagacc 19380 ctgctcttca accttctgca gggcaacctg ctgaagaatg cccgcaaggt ggtgaccagc 19440 cgtccggccg ctgtgtcggc gttcctcagg aagtacatcc gcaccgagtt caacctcaag 19500 ggcttctctg aacagggcat cgagctgtac ctgaggaagc gccatcatga gcccggggtg 19560 gcggaccgcc tcatccgcct gctccaagag acctcagccc tgcacggttt gtgccacctg 19620 cctgtcttct catggatggt gtccaaatgc caccaggaac tgttgctgca ggaggggggg 19680 tccccaaaga ccactacaga tatgtacctg ctgattctgc agcattttct gctgcatgcc 19740 acccccccag actcagcttc ccaaggtctg ggacccagtc ttcttcgggg ccgcctcccc 19800 accctcctgc acctgggcag actggctctg tggggcctgg gcatgtgctg ctacgtgttc 19860 tcagcccagc agctccaggc agcacaggtc agccctgatg acatttctct tggcttcctg 19920 gtgcgtgcca aaggtgtcgt gccagggagt acggcgcccc tggaattcct tcacatcact 19980 ttccagtgct tctttgccgc gttctacctg gcactcagtg ctgatgtgcc accagctttg 20040 ctcagacacc tcttcaattg tggcaggcca ggcaactcac caatggccag gctcctgccc 20100 acgatgtgca tccaggcctc ggagggaaag gacagcagcg tggcagcttt gctgcagaag 20160 gccgagccgc acaaccttca gatcacagca gccttcctgg cagggctgtt gtcccgggag 20220 cactggggcc tgctggctga gtgccagaca tctgagaagg ccctgctccg gcgccaggcc 20280 tgtgcccgct ggtgtctggc ccgcagcctc cgcaagcact tccactccat cccgccagct 20340 gcaccgggtg aggccaagag cgtgcatgcc atgcccgggt tcatctggct catccggagc 20400 ctgtacgaga tgcaggagga gcggctggct cggaaggctg cacgtggcct gaatgttggg 20460 cacctcaagt tgacattttg cagtgtgggc cccactgagt gtgctgccct ggcctttgtg 20520 ctgcagcacc tccggcggcc cgtggccctg cagctggact acaactctgt gggtgacatt 20580 ggcgtggagc agctgctgcc ttgccttggt gtctgcaagg ctctgtagtg agtgttactg 20640 ggcattgctg ttcaggtatg ggggagcacc atcaaggcta agtgtgggag caccgagctg 20700 ggctctagaa gtctgggccc agcttcgcct ctgccaccct gctttgcaac actgcccaga 20760 tcccttccct tctgggcctt aatttcaata tgtgatgatg acagccacac tttattgact 20820 ggcctatgtg ctgggtctgg tgctatgctt tccggaatga cctcatctaa tctctacaac 20880 caccctggcg ggtaggcagg aatgttatta tctccattat ccttgacttg aggctcagag 20940 aagtgaagta acttgtccag gaaatggcag agctggggtt cacaaattgc atcattctga 21000 ttacaggttt tctgcctccc accagtctat ggatacactt cagaggctcc ctgaaaacct 21060 tgaggtcact tgcagaaagt tttgtgtagt atgtgtccgt atcaggaaca acaccaaatc 21120 agaggtgact tgtgccccat cagagacttt aacaccccaa ccagatggga atttcaggac 21180 ccaagaaata gaaagtggct gcagggttac aactactgtt ggattcctga ggtagcacag 21240 tgtccaaaca ggatttcagc actacccgta ttgcttagag ccccagccaa agatgtgagg 21300 ttttgccctt tggagaatct gtgcccctga actcgggggc ctctttccac atcttggggg 21360 caggcaaggg cagagggtgt gcctaggcct gcggatcagc atgcgacaga ttccccaaca 21420 tccttccagc ttgaaagggg attgccctgc ttctatttag aacctatagg aaagcagaag 21480 ttctagattg aagttaaaat tgattcccag cctccagggg ctttgggcta cacctggatg 21540 accttaattg accctaagca tgggacaaac cacttcctga gagtattagg atggtataca 21600 tcttctctgg gggcaaagca acaagattta tttttcatca tggaccaaac acatggatac 21660 ccactagaaa ctgtgtagtg aattttgtta accctgacat agggaccatg gtctttaggt 21720 taaagcataa taacaacata atacataaca tatatagcga atatatatat gtattatatg 21780 caatgaatgt aaatatgatt atacccatca tggtcttgga ggaaacagat gacacactta 21840 aaatgggtgt tttgaggaga gtttgaaaaa cagattgttt acaagccatg ggcaggagtt 21900 aggaagagtg agagggttgg tgcaggggcc tggggttagt aacagctggg ggagggtaga 21960 cttgaagggg gaaggggagg gagactaatt agctgggggg aaggtatgga gacggctgcc 22020 tgagcttctg caaagtggaa gaataccgct tggccctaac tcctcacccc aactcttgct 22080 cctggccagc gccttccacc agctggaccc atcagggagg ccgagtgggc tgtctgctgg 22140 agtagtcccc aggcatcagc ctcccaggag ccagggacgg gtagagaagg gggagagtgg 22200 atctggccag gcaaatggaa aacagccagc accaaactct atttccctag gagggaggat 22260 catgatactt tgagtgggaa tttggaaacc tgtctgttgg agcaatttcc ctgatagaaa 22320 taagaatgtg cattttcctg ggtagtagac tcagttttta ccccaagagg ccaggcatca 22380 ctggcctgtg tgatcctcat aggccagtcc atctctggaa ttcttgaatg gatcatccat 22440 ccttgattag ggatgtcccc gtgattacca gggtgtgcag aagggctctg ggaaacctgt 22500 gggtctgtct ctgtgttcag agaaaggtga gggtggcctg gttctagctc atggtgctca 22560 gactgtggtg tgtaaaggca ctcgtggcaa tgcagattcc tgggcctgcc tctagtgatt 22620 cccattcagt aggtttgggg tggggcccag gaaatctata tttttcacag acacccctgg 22680 tgattctgat acaagtggtc tcgccctggg agaactactg gtctgcagca accagcttgg 22740 ttttccatta gcaattactg tccttgagcg agttttactg ctcttcacct tacacacact 22800 aaaactgcca aggccgtagg ggaggggaag caaccatgag gttgctgtga gtgcactgtg 22860 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtatg agagagagag agagattgag 22920 aaagagagga agggaggaag ggggagggca caggctcctc tcccacagtg ccaacctgcc 22980 tctctcccac ttgaagtgtt tccatgccaa ctgaaatcct cagcctctag gaaaccctat 23040 atacacagtg cccctatata ggtttcttta gactctggct ctctcagact ctagagtgat 23100 ggctttaaaa gttttttgaa tgttacccac aatgattgag aaagagagag agagagcacg 23160 caccaccatg taaacatgga acctaagttt cacaaaatga cttcgcttta tgaactctga 23220 gacactctgc tctcttctgt tctgttctat ttccatttta gaaatgctgc tcaggacctt 23280 caaaatgatt tgcatgacct gcaacctgca gtctgaaaaa tcactgcact acagaagtgg 23340 ccataagagg ccctgaggga gaagctgcac aatgtcatgg ttaagagtgg ggtttggagc 23400 caagccgcct aggctcaaag cctttatgtg ccgtacaacc ttggcaaagt cacttcgctt 23460 gtctgtgcct cagtttcttt ctcacgaatg ctcataataa tggttcccat ttcactggct 23520 tgttgtgagg atgaaatagt gttattattg agaagtggta agggtagtga tcagtgctag 23580 cgatcatgat tctaggtgac ttttactgtg taccgggtgc tcacaaggct ttatgtgcac 23640 agcctggtga ggctgataat actattgttc cctctttttt tttttttgag acggagtctc 23700 gttctgttgc ccaggcaggg ggtacagtgg cacaatctcg gctcatgcaa gctctgcctc 23760 ccgggttcac gccattctcc tgcctcagcc tcccaagtag ctgggactac aggcgcctgc 23820 caccacgccc ggctaatttt tttgtatttt tggtagcgac agggtttcac tgtgttaacc 23880 aggatggtct cgatctcctg acctcgtgat ccgcccgcct cggcctccca aagtgctggg 23940 attacaggcg tgagccaccg tgcccggcct gttccctctt ttatagatga agagaccagc 24000 aaataactag taagtcgctg atcaggatca caatatccag ctgaggcact ccagagcctg 24060 agctgttaac cattcagtca gggcctccca agtttgccta aagataaaga atcatgtgca 24120 cagttgttaa aatatacaga ttcctgggcc ccaccccgca gatacttgat tgccagctcc 24180 agggtatggg cctgagaatc tgtcttttag ggaagctttc agatgatgtt gtgatcaggt 24240 gagttttggg aatggtgccc caagaggagt ggcagacagg gcttgctcgg cagggactag 24300 cctgttggag tggtgccatt ggggttaagg actgggcagc agggcctcac taaccacagc 24360 ctatatgcct gtttctgaag ttttggccac tctcatccag ctggtctact gtctgctgac 24420 ctagatgatg gtaaattgtc cccaggggta gcctgtctag ttcaggctgc acctttcgca 24480 tatatcagct cctttccacc atcatcccct ttgtgaggct gctgtgatta tcatgttcct 24540 tttgcagaga tggaaacatt gcctcaaatt agctctgtca tttcctaagg attccagggt 24600 tctttagtag ggggtctgga tcctacgtcc tgggccatcc ccatcatagt gcaccacgtc 24660 acctccctgg ccagggaccg tggggtctcc acttttttgg ggtgctccat ctatgcaggg 24720 tttcctggaa gcacagatgc tggcacttca gggatgaatg aaagtctttt tgggggattt 24780 gtagattttt ttcttgtctt actagctcca ttttcaaatg tatttatttt gtctctttag 24840 tttgcgcgat aacaatatct cagaccgagg catctgcaag ctcattgaat gtgctcttca 24900 ctgcgagcaa ttgcagaagt tagcgtaagt cagcctgggc tgtggacaat gggctccaag 24960 tgccctggtc tcaccccagg tcgtgcagcc tgggaagctg tgagtgatgg gctggggcag 25020 gggctgtttg catgatgggg ggtgcaggtg attcctgccc agaggggaag ggcaaccctg 25080 ggatttggtg ctcactgtcc aatgtgcttt gcttctgtgt ctcctctctt ctggaactga 25140 acagtctatt caacaacaaa ttgactgacg gctgtgcaca ctccatggct aagctccttg 25200 catgcaggca gaacttcttg gcattgaggt gagcccaggt tttccttatt ccctggaaac 25260 tattttttgc cccattcctg agtcagtctg atctggtctt ggcctggcac tgcccacact 25320 ggctcctgac ctcctgattg aatgcaggga cagtgtctca ttttaagcag gggttctcta 25380 atgctgtgat ctccccagta aactctggac tagctctgct gaggacttcc tgtcttttga 25440 cctttagccc gtagggcaag aaagcttttc taggcccctt tccttttctg tgtctaagag 25500 tgtcacagct ttctgggttt actgagttcc acgatgcatg ttgagctcgt cctggtgggg 25560 gaggcataca cagttacttg ccaccccagc tgtggcagcg agttgctgca acactcccag 25620 gaggtccttt caccactcag agcatgcaag gtttgcagtc catctggttc tgcatttctg 25680 ctactccagt gtctcccagt ttcaacagga gtctctctct ctcctacctg atgcctttaa 25740 attgcccctc tagctggccg ctgggttggc ctggcttctc tctccttctc tctctctcag 25800 atattcttgc ctcctgtgat ttgtgaggca gtaaaaaaag acaaagtaaa gaattgcttc 25860 catctattct tttacctctt gggctgggtt tgtggatggg agccgccatt ttaaaatggc 25920 gggccacata gctcagtctc ggcaagggct actgagatca gaaccacagg tgccaatttg 25980 tacaaaggac tcagtcctgc taccactgcc tgatccctca gactcacaag cctggaatag 26040 gctgtggcca gacctggctg gcccatccct gagaagggtg ctagtttcag aaatggaggc 26100 tgagtttgtg gccaacacag tagtcctccg gtatgtgcag gagagatgtt ctaagacccc 26160 agtggatgcc tgaaaccatg gagagtatca agccctacac ataccatgct tttcccaata 26220 cctacacacc tgcaataaag tgtagtttat aaattaggca cagtaagaga gtaatagcaa 26280 ctcataataa aatagaacaa ttataacaat caatgtacta taataacact atgtgaatgt 26340 ggactctctc catctccctc aaaatatctt cttgtactgt actcaccctt cttcttggga 26400 agatgtgtgg tggtaaaatg cctgtgtgat gggaggaagt gaggtggatg acgcatgcag 26460 cactgtgctc tagcgctggg ctgctgttga cctgaccaca cttcaggagg agaatcatct 26520 gctcccagag atccctaatc tttgagcaac aatgaggtcg gcagctggat gtcaggagca 26580 gacgatcttg atgattacca aatgggagcg tatagagcgt ggatgcgctg gacggggggc 26640 tgattcacgt cctgggtggg atggagctgg atggcacgtg atcagaatag catgcaattt 26700 aaaatgtatg aattgtttat ctctagaatt ttccatttaa tatttttgga ctgcagttga 26760 tttcagataa ctgaaaccat agaaggcgaa gctgcggata agcagggggc agggattacc 26820 gtatatcatt gtaatagaga gcacaggctc tggagccaga ctgcccgagg tttgaaccct 26880 cattagctgc gtgacctcag gtcagcccaa tgtctgtgtg cctccgtttc cccttctgta 26940 gaatggaggt aataaccctg gctacctcac aggctgtagt gatgagcaag caagttaatc 27000 cacatgaagg gctgcaccgt ctggcagggg ctttatatag taagcgagtg gctgaaagat 27060 gatgggtaaa tcacacaagc actcagcttg tttctcctta tgtgagtccg gtcctccaag 27120 cagggattca atgtgccacc catttattgg ggaaaagtcc taaaagggga agtggggaag 27180 ggagctgggg gaggctggga ggtgtgtccc tgagtgaagg agagagggaa ggaaggaagg 27240 ttgagactgg gcaccttgga cttcagtgca gtcctaagac atcttggcaa ggctgatgag 27300 gagttcttga accaaattca ccaggcaggg gagcctgatg tctcaggcag gggctggcaa 27360 gtgcagatgc gaggatgtta gattttggag cacagcagct ggggcccttg gctacctcca 27420 aggagctgag gctggagacc tgaaaggcga gttctcctag ctgccacacc ccttctccaa 27480 ggatacaata atatctgcct tataggattg ttgtgagctg agtggcttga cgttccttga 27540 aagaatgaaa gcgtatagtt atcccaggaa gcctagggtt gcaggtgaga gctctggggc 27600 ttctccgaag ctctccgagg tgtctggatt cagttgcagc aggagccttc cttgctggga 27660 tcttccccca cccctagcct tggccctccc tctctccttc ctttctggaa ggctcagtgg 27720 gccccacccc tccctccagc cacctggacc tgcccagcgc tcttgtgcaa caggtaaagc 27780 ctacctgtag caacaacaga tctgggaagg ctgcagaggg cacgatgggg tctggatcga 27840 gggcggctga gaccagaggg aaaggtgtga ccctgagtca ccctcgctgt cccggggaaa 27900 ccacctccca ggacagctgc ctactgtggc tcctgcctgg aattgtcaca ctgctgtgca 27960 aacagcgtcc cgctgcccct ttccctttgc tgggggaaaa tgaagttgtg ggagccgctg 28020 agtaaactag acctagcagc gagggcacct gatgtggctg ctgcctcccg ggcaggtctt 28080 caatgctttc ttcctgtgtt tccctggcca gggcacagac ggccctcctt ttctgcctgc 28140 cgctgtgttc tctcagcctc ctctgtcttc ccttccaggc tggggaataa ctacatcact 28200 gccgcgggag cccaagtgct ggccgagggg ctccgaggca acacctcctt gcagttcctg 28260 gggtaggttg gattccagga agagggacct gcatggaggg gcttgggact tttgaggatt 28320 taggggcagg tgaaactctt cagccaggag gccccagagg cagcccagct ccagtgggga 28380 ggacaagcca gggagagagt gggcggccct tgactgccac cttcatactt ggtctatgcc 28440 tgacaaacag gaagtttggg atgttggggc taggggagga cagtgcccac gagctggtga 28500 caggaagccc tctgatcctc agggggcgct agggctgtac tttagctgca tattaaaacc 28560 acctggaagc ttctaaacac tattgccagg cctcccaccc cagactgatg aaatgcaaat 28620 atctaggtgc aaggcccagg tatcaggagt tttaaaaagc ttcccagggg atgtacagcc 28680 aggggtgagg acccctgacc taagaaagag aaggaaatgg ggaaggatag gaaggcaccc 28740 aggataagag gggctgtgct aggtccctcg gagctcttgc tccctgtagg accatgctag 28800 ggcctgccag ggaggggagt accccaacct gcagccccag ggtgggcttc ctctgtttgc 28860 taggcaccca ggcttgcacc tgtgctgttt ccagcagcct ctctcctatc ctgtcatgcc 28920 ctagtgtgaa ctggagtcca tttgacaaga actgggagtt ttagaacctg ggactgtagg 28980 aagagagaat aaccttaggg cctaggtgtt ccagcccatt tcacagggag gcaagttgcc 29040 cccaagctca gttttttgtt ttgttttgtt ttgtttgaga tgtagtctca ctctgttgcc 29100 caggctagag tgcagtggca cgatcttggc tcactgcaac ctccgcctcc ttggttcaag 29160 cgattcacct gcctcagctt ctcaagtagc tgggattata ggcacccacc accacgccca 29220 gctaattttt gtatttttag tagagacagg gtttcaccat gttggcccgg ctggtcttga 29280 actcctgatc tcagatgatc cgcccgcctc ggcctcccaa agtgctggga ttacaggtgt 29340 gagccaccgc acccggcccc caagctcagt ttgagccaca aatgggacta tgttgctcta 29400 gaaatcaaca tcttttccac actgcattag tagcaacaga gtctagaaca aaggaggcca 29460 cagccccact gaactctctt ctgcttgagg tcacatctgc cacatcaggg gtatttacct 29520 ctttcaacac atatttatta gggcacctgt ctgggccagg cgttgtgcta aaacccccaa 29580 acgctgtcat atgatacaaa gtgttctgta acttgcttgg tttttttttt tgtttgtttg 29640 tttgttttgt tttgtttttg ttgttgtttt tttttgcttc gccatatatt ataggaattt 29700 ttttaggtca ttatgacctc tttatttact taattatcta tttatttatt ttactaatat 29760 ttacagaaag ggtctcactc tgtcacccag gctggagtgc agtggttgca atcatagctc 29820 attgtagcct tgaactcctg agctcaagtg atcttcctac ctcggcctcc tgagtagctg 29880 ggactacagg cacaagccac catgcctggc cgatattttt atgttttgta gagacggggt 29940 ctcactatgt tgcccaggct ggtctcaaac tcctgggctc aggtgatcct ccctcctttg 30000 cctcccaaag tattgggatt acacaagtga gccaccttgc tcagcctgac ctcatttttc 30060 aaagagctgc agagtgttac ataatgtatt taactggtca ctttttgatg actattaagt 30120 tgttttcagg ttttttgtta ttacagtgtc atatccctgg ggcacagagc agtgctggca 30180 catagccaga gctcaatcga tacataccta atgaatgaaa gtacagtgga catcctaatt 30240 cagccattct ttgctaactt gtgtacatac ctgtccaggg taggtcccta gaatacagtc 30300 aataagtcag aaggtgtgag ttgggatcta ccttttggaa agggatgttt tcaaactaca 30360 gtgagtcaga ggaggatggc ccagaagctg ggggagttga agctgatggc gtgaaggaat 30420 taggggtgtt aggaagaagc aggagataaa gagctagctt gcagaagaag tgttagactt 30 480 gttatgggca ggtactggag ggtagctaag gacttgtggg tggcagttac caggaagcgt 30540 atctgaacta agtgtcagaa aaagtgtcac aactgtaaat tactcttgtc agtgagttcc 30600 tgtccttaag ggttagggct gggtagccct ctactattct ctaagtctgt aatgtaaagc 30660 cactgaaaac tcttgggtta agtttggcca tcccacccaa aagatggagg caggtccact 30720 ttgctgggac caggagcccc agtgaggcca ctctgggatt gagtggtcct gcccctctgg 30780 ctgggactgc agagggagga ggactgttag ttcatgtcta gaacacatat caggtactca 30840 ctgacactgt ctgttgactc ttttggcctt ttcagattct ggggcaacag agtgggtgac 30900 gagggggccc aggccctggc tgaagccttg ggtgatcacc agagcttgag gtggctcagg 30960 taagcttcag agtctatcct gcagttttct tggggagatc aggtgaagag ggaggagctg 31020 gggccagttc tgaaggtctt tgaactttat ttctacccca caatgttagg caatggagta 31080 aggaaaaaag accattggat ttcaagagag gacactcgag tctttctggg tgacttggaa 31140 atgtcccttg tcctctcagg gttttgatac agtatctgta aattgaagat attgggctgg 31200 atcaggtaca ttttatctta aggaccaatt ccaatccatt ggtagtgggt gcccagtgca 31260 ccacattaaa aagaattcta aggctgcacc tgggcttaaa gaagagcact ataatcaatt 31320 agtgatgtct aaaaaagcta aaaaaaaaaa aaaaagagca ctgcattcaa ttagtgatgt 31380 ctaaaaaggg tagaaaaaaa aaaaaaaaga aaaaagaaag agcaccgcaa tcaattagtg 31440 atgtctgaaa tggagcagac caggagagca ccacgaattt tgccctccat aggttagctc 31500 atctctgagg tctttccctg ctctgacata cttttgttcc atgattacct ccagcctggt 31560 ggggaacaac attggcagtg tgggtgccca agccttggca ctgatgctgg caaagaacgt 31620 catgctagaa gaactctggt gagtttgggg gattctctgc tctggggaag tggatcacaa 31680 tctctgttga tcccctggcc tcatccatag gagcggttgt gtggacagac aaaggtggat 31740 gattgagtga ttgactgatt gattgattgt gtttgtcttt atatgtactg agtggtatga 31800 agcttataga gcctggtatg tacatgctaa tttttttatt taataaaata tatgggtttg 31860 ctggtttggt gactgcctcc acatggcata agtgttaaga gcacagactc tgtaatcaag 31920 caggccgtga tcttaggcaa gttaaataac aatttcagaa tctcaagttt catgtctgta 31980 aaatgagggt aagaatactt ccaaccataa aggatttttg caagaattag ataaagtagt 32040 gcctgtgaag accttaatat agtgcctggc atatttgtaa gtgctccata aatgttaaat 32100 tagaataatg gcagggttac tactactatt actgctgctg ctgctgctgc tgctgctaca 32160 actactatag tactgtgact actactacta ataaagtttt gttattttaa agtgattttg 32220 agttcctagg agcactgggt attcaagtct taggtcattt tggaaggtgt aatggagttt 32280 tgatagttga aagaggaacc atgaatcatg cttatactgt tgacctgaag cagattctaa 32340 gtttctcatc ctttagatgc cactagtata gttttctgac atgttctggg cagcttcaga 32400 ttatgtcagg gagataaaat actgaatgtt tgattttccc gggaagcaga aaggcactgc 32460 aacatatggg cattgccata aacagatttt atggatggac cttggctgtt gcagggctta 32520 ctagctctac tcaagtatga ttgattctat cctgactgga ttttgccact tggaatttct 32580 tagtagagga gaaccttgtt atgagagcat cagttatgat tactgttaaa agaaaaactt 32640 taggcaaatt aaatttagca gaactggttt gaacatacag caatttatga attgggcagc 32700 attcagaact gggagtgctc cacccagcaa ggtaggcaag cagtatctat agacaggaaa 32760 aggaagtgat gtacaaaaca gcttgattgg ttgcagctgg gcatttgcct tatatgggca 32820 tggtgtgatg aggcattttc tttatatgga tatagactga tcagctggta gactgtgact 32880 gactgaagcc tggctgctgt gattggctaa gacttagctg tttgttataa ggatatgttg 32940 ttaggttgca gtttgctaca taggaactca aagtacagag gcagtctcag gccaaattta 33000 gtttaactat atgttaagct gcaggtgaca gaatacctcc atctatagag gtttaaacaa 33060 ggaaagggtt tattttttcc tgtataggca gctggatgta ggcagtgtag ggtttgtaca 33120 gtggctacaa gaggccagga ggggtctcag ctctgtctca ttctcttcct gttccatcat 33180 tcttagcctg tgacttcatt cacatggttg gttgtctcat gatcacagga tggctgctcc 33240 aggtgcagca ctacttctgt attcccggat tcgatctata tacccaggaa agccatctgg 33300 gttctctcct ttaaaaagca ttcctggaag ccccacctgt cgacttcccc ttatgtatca 33360 accatgtgta tgtcacttga ccaacccact tgtatgttgt ttgaccagcc ctggctgcaa 33420 tggagagtgg gaaatacagt tttttcacca agtgcatggc tgtccaaatg aaatgagact 33480 tccattaata aggaagaaag gaaagatgga gatcaggaag ctgggggatc agggaactta 33540 ttacattgag agcccttgga gtgaattctc ttgcaaatat gtccctggaa ttgagaatcc 33600 ccacaacgta ctttatctgt tctttcttta tccatgagtt tgggttttca gatgttggat 33660 ttcctatatg gggggcatgt gagttcatca tcttccataa tcaatgttgt atcaactgga 33720 ttttctctct tcttctcacc agcctggagg agaaccatct ccaggatgaa ggtgtatgtt 33780 ctctcgcaga aggactgaag aaaaattcaa gtttgaaaat cctgaagtaa ggaacccata 33840 agcaggaaac aggacaataa ttgctggcct ttggaagggg catttctgaa taagatctgg 33900 gccgctctcc gctgggctaa ctcatgtgag gtggcctggt agaacagctt gccttggtct 33960 aggtggacaa ggattccagt gcaagttgtt tatctgggag gtggtcccag taaatgctga 34020 taggagagtg gtgaagtgag atggggaagt gaaggtaacc aataaagggg agttatcaag 34080 ccagttatca atgagggaaa ttggagctca gtactctggg gcactcctgg agccagtgca 34140 gaacacacat ggtcacctac ccaaccaatg ggcaagaaag ccatggcatt tatccaccaa 34200 ccctctgtcc ttcctatgtt gatgtgcgct catggggcac tgattctcca gcacttccag 34260 ctcaccctca cccagctgaa catgcttctg gggtcaggag aatggcctca ggcagagagt 34320 ggaaggtctt ctctgcaagc agtggctggg gaggtgatgt gatggggagt actgtggcct 34380 cctccagtgg ctgactcagt ggcttgggac ttgtgccaca aagagatgga cagctcaggt 34440 gaacatgaac ccacctagtg accatcatgg gtttgtcagg gtgctctctg aggctgatgc 34500 caaaattctt atttcaagta gacctcagga accccatcag atggctcctt ttgctggagg 34560 aaagtggcat ctgcctaggc aaatgtggtc ctaggaaaac gcttgccttt agagacagac 34620 agacagacag ctgcctctgt gagtgccagc tttgctgcca ggctgctacc cactctggcg 34680 acactcattt gtgttgcttt cacaagctag gaagtttcca aatatttgga gaaaacactt 34740 ccactaatta tttgggtgga aatgggctgg gaagttgggg tgaagcccgg atgtgtctga 34800 gccagatgcc agctttgcac tgagggtcgg cctttgggaa taccaagccc attatcaacc 34860 aggtgtggat atggcaggtt tgtcttccct ccttgtcaca gccttactcc acttgactcc 34920 catggatgcc aggcaatgag gctggggttg gtcccatgcc accctgtcat cagccttatt 34980 tttcagcatc ctaaactata tcatccccca caaaaattga acttctgata tatcttttat 35040 aaaaaagaga aatgcctaca tctttctttt ccaggcttag tttctgccaa gagttggttg 35100 agagcccagg cttgctgggt gcagtggctc acacctgtaa tcccagcact ttgggaggct 35160 gaggcgggtg gatcacctga agtggggagt tccataccag cctgaccaac atggagaaac 35220 cccatctcta ctaaaaatac aaaattagcc gggcgtggtg gcatacacct gtaatcccat 35280 ctactcagga agctgaggca ggagaatcac ttgaacctgg gaggtggagg ttgccatgag 35340 ccaagatcac accattgcac cctagactgg acaagagaga aacttccatc tcaaaaaaaa 35400 aaaaaaaaag gatgagaaaa ataataattt aaaaaaaaga gtccaggctc tggaaccaga 35460 cagcctgggt cttacccctg ctccaccatt accagccagt tcttcttgga tgagtgcctc 35520 agttgcctca agtgtaaatg gagataatgg ctggaccttc attataggcc atgagcattc 35580 actgagagaa tgtagctaac aaaagtgagt tgtaggttgg agcaaaagta attgtggttt 35640 cagaccatga actttaaatt attataacta ggctaaaata catctttatt aatcaaaata 35700 ggaaccatta aaatcaacac atttttgcca ataagaaata agtttgttta ttcctgtagc 35760 ataaaaattc atgcttcggg attcaacaaa ctcttggaaa gcattttctg catcctcctg 35820 gttgtggaag catttttcct gcagaaagtt gtcaagattc ttgaagaaat ggtagtcagt 35880 tggctagagg tcaggtaaat atggcggatg aggcaaaact tcatagtcca attcattcaa 35940 cttttgaagc tttggttgtg tgacatgcag tcgggtgttg tcgtggagaa ttggaccctt 36000 tctgttgacg aatgccggct gcaggtgttg cagttttcag tgcatctcat tgacttgccg 36060 agcatacttc tcatatgtaa tggtttcgca gggattcaga aagctgtagg ggatcagact 36120 agcagcagac caccagtgac catgaccttt tttttttggt gcgaatttgc ctttgggaag 36180 tgctttggag cttcttctcg gtccaaccac tgagctagtc attgccagtt gtataaaatc 36240 cacttttcat cgcacgtcac aatcagatca agaaatggtt cgctgttgtt gtgtagaata 36300 agagaagatg acacttcaaa atgacgattt tcttggtttt cactcagctc atgaggcaca 36360 cacttatcga ggtttttcac ctttccaatt tgcttcaaat gctgaatgac catggaatgg 36420 tcgatgttga gttctcaagt agttgtaaga aaatcagctt tgatgattgc tctcaattgg 36480 tcattgtcag cttctgatgg cctgccagta cactcctcat cttcaaggct cttatctcct 36540 tcgcaaaact tcttgaacca ccactgcact atacgttagt tagcagttcc tgggccaaat 36600 gcattgctga tgttgtgagt tgtctccgct gctttacaac ccattttgaa ttcaaataag 36660 aaaattgctt gaatttgctt tttgtctaac atcattttca tagtctaaaa taaatataaa 36720 ataaacagaa agtattaagt cattagcaaa aaatcataaa gtgagaattg tgcattaaaa 36780 tgatgtatag cataaccaca tttatttaag aatgtattcc aatatcaaat ggcaaatttc 36840 aacaatgcaa aaactgcaat tacttttgca ccaatctaat agaagttcaa taaatactgg 36900 caattacaat tggcattgcc ttagggtcaa cttgtaagac attcctgaaa ttgtgggaaa 36 960 gggggaggac ctggagtgga cattattgga aggcaaagct gtaaccaaaa gagcaacctg 37020 ggaaacacat gactcctctg ttgctgtccc tggccctatc ctgtctcccc tccctgttgt 37080 cagctacctc atatgttctc taatctctgt ctctgtgccc tcaaagaccc ccctgaaaat 37 140 agaaatatta ctgctcattg gttattttct atcaattaag tactgtatta gtccgttttc 37200 atgctgatga taaagatata cccaagactg ggcactttat gaaagaaaga gttttattga 37260 acttacagtt ccacgtggct ggggaggtct cacaatcatg gctgaaggtg aaaggcacat 37320 ctcacatggc agcagacagg agaagagggc ttgttcaggg aaactcccct ttttaaaacc 37380 atcagatatc atgaaactta tttactgtaa tgagaacagg atgggattca attaccttcc 37440 actgggtcgc tcccacaaca cgtgggaatt caagagattt gggtggggac acagccaaac 37500 catatcaagt actgtgcaag tgttttaggc atgcagagag tggtgggtct tcccagcaag 37560 cagagtgtgg ggaggtaatg ggggactggt ggctgactta atggcccagg acccatgcca 37620 caaggagatg gatggtggat gtgaatagga gcctgcttac acccatcaca atttagattc 37680 ttatgctcga tggcacgggt actcttttag gcccatttta ccaatgagga gattgggact 37740 aatttgctcg agatcaaaaa agaagtggtg taggtgggat ttaaacccag gatgtctagc 37800 actaaaatgc aggtacttaa ccactatcct aagggagtgg ctacttaatt tgataaactc 37860 atctagtgaa tggaagagag acggttacat ttcactgatg gtactgagcc tttgttgatg 37920 agctcattgg gaatctcaga catgagcagg atgtgtctaa gggacaggtg ggcttcagta 37980 gactggctaa ctcctgcagt ctctttaact ggacagtttc aagaggaaaa ccaagaatcc 38040 ttgaagctca ccattgtatc ttcttttcca ggttgtccaa taactgcatc acctacctag 38100 gggcagaagc cctcctgcag gcccttgaaa ggaatgacac catcctggaa gtctggtaag 38160 gcccctgggc aggcctgttt tagctctccg aacctcagtt tttctatctg taaaatgggg 38220 tgacgggaga gaggaatggc agaattttga ggatcccttc tgattctgac attcagtgag 38280 aatgattctg catgtgaagg atctgattct ctgtctaaga aagaagtctt tacctcttta 38340 agtagggagc aatgatttca tttttaaacc ttgactattt attcagcaac ttctctgctc 38400 tatgagatag tgtaggaatg gggatgtggt tgaagaatga aaagaaaagt cagctcccgc 38460 cctcctagaa attgcatctg ccttcacagg tcaaggatat tggatcagac cttctgcggt 38520 tctgaatgga gattacacag gttaggagca ggttgcacag tgtttccaat tctctataat 38580 taaagccata gactttcatg tattgaaaaa agcaagaatt gcattcttga cagattcttt 38640 cattgcctta aaaagaatga ctagccttgg gagtctgggc agctgggtcc agtgttgtag 38700 actttctctc tgctgagcca cagcttcaaa gatttgtcct tcttgtttcc agggatctat 38760 ttctcagaca ataagtaaag gctttccctg gcctaatgtg ctgtaagtga atgctactat 38820 atatgttcca ggcactgggc tagagactaa tatttaaaag ccaggaaatt tcctatagaa 38880 aatctatatc tcagggtttt ctcaaaagag ctgggaactc tggatgccca ttcatgattc 38940 cagtagttaa ccagagtaca agaagggctg agtcttctca gatgggcaaa cccactctgg 39000 ctgactgcag atccaccaag cctattgtct tagaccagga ccctttggca actcattccc 39060 ataagcctgt gacccttgct ttaaatatgc aggccttgtc ttctctcaaa aagcacatca 39120 aggctgcagc gaatgcagat atcaaatgat gaagttaaaa acaaaagctt tgctgggcgt 39180 ggcagctcac acctgtaatc ctagcacttt gggaggctga ggcaggagga tcactttagg 39240 ccagaggttc aacaccagac cttgtctctc aaaaaataaa aaattcagct gggtgcggtg 39300 tagttcctag ccacttggga ggctgggatg gaaggatccc ttgaacccag gagttcaagg 39360 ctgcagtggg ccatgattgc atcactgcac aggcgacaga attagatccc atctcttaaa 39420 aaaataaaaa atttaaaagt gacttcaaaa atctatgctg tgatggagag atttttcctt 39480 ctgtatgatt gtgatagctc tgtggcctat gacgtcatca ggttctgggc aaagtgtagg 39540 ttttctgttt ctttgttttt gaaaccattg cacagtccta agaaacatca cattctgggt 39600 cctgggcacc agccaacatg aggttgatat tttaaggggt ctgatgttat ttgctgcctg 39660 ttgatggaac ttgggagggc accagggttt gctcattgca ttcttgacag attctcttat 39720 tgccttaaaa agaatcactg gccttgggga gtctgtggct ggctgggtgc agtgttgtgg 39780 actctctctg cagagtcatg gagccttgtt cagaatgctt cctgagctgc cctggttggc 39840 caagggtaaa aacagccctg acttccctgc aagaaacact gcagctgggc cagagagtca 39900 gcccatccca ggcatgggtt taaaaagtgg aggcttttgt ttgaaagccc tgctctaatt 39960 ttgtcctcac tcaaacctct gttcacttga tctgctttag gctccgaggg aacactttct 40020 ctctagagga ggttgacaag ctcggctgca gggacaccag actcttgctt tgaagtctcc 40080 gggaggatgt tcgtctcagt ttgtttgtga gcaggctgtg agtttgggcc ccagaggctg 40140 ggtgacatgt gttggcagcc tcttcaaaat gagccctgtc ctgcctaagg ctgaacttgt 40200 tttctgggaa caccataggt cacctttatt ctggcagagg agggagcatc agtgccctcc 40 260 aggatagact tttcccaagc ctacttttgc cattgacttc ttcccaagat tcaatcccag 40320 gatgtacaag gacagcccct cctccatagt atgggactgg cctctgctga tcctcccagg 40380 cttccgtgtg ggtcagtggg gcccatggat gtgcttgtta actgagtgcc ttttggtgga 40440 gaggcccggc ctctcacaaa agacccctta ccactgctct gatgaagagg agtacacaga 40500 acacataatt caggaagcag ctttccccat gtctcgactc atccatccag gccattcccc 40560 gtctctggtt cctcccctcc tcctggactc ctgcacacgc tccttcctct gaggctgaaa 40620 ttcagaatat tagtgacctc agctttgata tttcacttac agcaccccca accctggcac 40680 ccagggtggg aagggctaca ccttagcctg ccctcctttc cggtgtttaa gacatttttg 40740 gaaggggaca cgtgacagcc gtttgttccc caagacattc taggtttgca agaaaaatat 40800 gaccacactc cagctgggat cacatgtgga cttttatttc cagtgaaatc agttactctt 40860 cagttaagcc tttggaaaca gctcgacttt aaaaagctcc aaatgcagct ttaaaaaatt 40920 aatctgggcc agaatttcaa acggcctcac taggcttctg gttgatgcct gtgaactgaa 40980 ctctgacaac agacttctga aatagaccca caagaggcag ttccatttca tttgtgccag 41040 aatgctttag gatgtacagt tatggattga aagtttacag gaaaaaaaat taggccgttc 41100 cttcaaagca aatgtcttcc tggattattc aaaatgatgt atgttgaagc ctttgtaaat 41160 tgtcagatgc tgtgcaaatg ttattatttt aaacattatg atgtgtgaaa actggttaat 41220 atttataggt cactttgttt tactgtctta agtttatact cttatagaca acatggccgt 41280 gaactttatg ctgtaaataa tcagagggga ataaactgtt gagtcaaaac agccatcttc 41340 cttgtgacca aacatttaaa aatattctgg ctgggcacag tggctcacgc ctgtaatccc 41400 agcactttgg gaggccgagg tgggcagatc acctgaggtt gggggtttga gaccaacctg 41460 accaacatgg agaaaccctg tctctactaa aaatagtgga ggttgcagtg agccaagatc 41520 acaccgttgt cctccagcct gggcaacaag agtggaactc catctcaaat aaataaagaa 41580 aataaaaaat aaaaatatta ctaaaacttg tcaaatttta aagcacaatt ttctacttga 41640 agcggaggaa aatgatgttt tcaacccgat aatgtatagc ttaaggcaaa agcaattctt 41700 agaaaatagc caggcaatca aaacactttt ggcttcagtt tagaaaagtt taaccccgtg 41760 aagttttcag ctgtgcagga gcctccccgt acccagccta aaaggctgca acacatgtga 41820 aaaggcccct tggcccccct cgattccctc aaagtggctc caggagtctt taacagaagg 41880 atggagaagg tttacttatg gctggctctc cacagcgagc agcctctcat gcaggccttg 41940 tgttaaaata gtgcttagct gcgtttaata agcaagggat caggtggtca cccagcaagg 42000 taaaatccta cttccttgaa aggttttgcg tctggctgac tcggtggtgt ggcatctccc 42060 tggaccctgc atgggagtgt gtctatgggt tttctttgtg attggtgccc tgttctgccc 42120 agaccattgt ttctgataca gcagatcagg aggaaggtgg tgttgagtct cctttcgctg 42180 aggagtacac acagcagccc atttgcatag cactgtattg ggttgtcagt ttgtcatctt 42240 taaattcatt cagcaatgag gccaggcatg gtggctcatg cctgtaatct cagcactttg 42300 ggaggctgag gcagctggat cacttgaggt cagaagttca agaccagcct ggccaacatg 42360 gcaaaaccct gtctctaaaa atagaagaag aaaaaaaaaa gttagctggg tgtggcagcg 42420 cacgcctgta atcccagcta cttgggaagc agaggcagga gaatcgcttg aatcttggag 42480 gaggaggttg cagtgagctg agatcatgcc actgcactcc agcctgggcg acacagcgag 42540 actgtcataa ataaataaat aaataaattc agcaatgatt tattaaccag ttatattagg 42600 atgaccagtg gattgacaaa acagacatgg tcctcaaggt gcttaggtga gggtggaggg 42660 agaggcaggc agaaaacaag tcagttgaaa aagacatgaa aaagtcttca tccaggcact 42720 gtggctcatg cctgtaatct cagcactttg ggaggccaag gtgggcagat cacttgcggt 42780 cgggagttcg agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa 42840 aattagccgg gcatggtagc aggtgcctgt aatcccagct acttgggagg ttgaagcagg 42900 agaactgctt gaacccggga ggcagaggtt gcagtgagct gagatcacac tactgcactc 42960 tagcctgggc aacagagcaa gactccgtat caaaaaaaaa aaaaaaaaag tcttcaggct 43020 gttaaggggc tggtccgaga caggttgtct gaggaggtgc caggtaagct gtgtccccaa 43080 gaatgagaaa gatatggcca agcaaagagt gagagggaag aagggagcat tctagtaatg 43140 ggggacaaga ggtacaaaga ccctggtgag ggaaggagcg tggtatgcat ggtaggctca 43200 atagtcactc ccgaacatat gtagatccta atccccggga cctgggaatg ttgccttacc 43260 tggcaaaagg gactttggag gtgggattca gttaagggtc ttgagatggg aagatcatct 43320 tggattacct gggtgggtct taaatgcaat cgcagtttcc ttataagaga aatttaaagg 43380 acaatgtaaa gcaaagggaa atatgacact ggagaagaaa gaaatgtgat caccaagtaa 43440 gatgctacac tgctggctct gaaggtggag gaaggcgcca tgagccaagg attgcagctc 43500 tagaagccaa agaagaccaa gaaatagatt ctcccctaga acctctgcaa gcagcatagc 43560 cctgcctgcc ttggctccag cccattttgg acttctggct tccacgacta taaggggata 43620 atttgtgtca tttcctgcag cagccatagg aaactaatac actatgttgg aggcaagcta 43680 agggaaaggt gtcactagat gggtttgcag aggtaggtag gaccagttca ggaaaggtct 43740 cactggccac agtcaagagt ttggtcttga tcttggaata atgtcaaggc cttggaaagt 43800 tctaagcagg ggagatgtga accaatttac agttaaagaa gatcttgttt gcttctgggt 43860 ggagatagga caggaatatg taggggtggg gatgggaggc agaagaagtg gggaggctgc 43920 ctcagagcta ttgcaggtga gaggcggttg gcagtggagg tggagagaag aggatggatt 43980 ccagagacat tttggaggta gattcccaag agcctacgaa tgggttggat gggagggatg 44040 agggaatggc aaacatcaag gatgactcat agatttctga cttgagaaac tgggggcctg 44100 ggaaagttgg gtaggaaacc agtttggaag tgggagggag ttctgttttg aattgtcaag 44160 tttgagacac cgaaatgggg aagttaagtg gagacgtcaa aagatggtgt gaatttggag 44220 ctcgtgggaa aggtcacagc cagagataat agcgtgggtg tcctcagtgt caaactgaga 44280 atggggccct cgagtctgga tgacatgagc tagaaagaga ttatggagaa agaagaggtc 44340 aagagggtct aggtaagatg gtactgagat cttgatatgt gattcctggt agagttcaga 44400 gtaggagaaa ttccctaatt ttcaaaagtc cagcggatac caccaaaagc ctcctgggcc 44460 tgtgtaggga actagggaag taggattctt gattgtaagc ttctctgata gggaagttac 44520 ctgaaaggta catgctcatg gccagaagtt tatccatgag cagacggatt ccagttttgc 44580 agctaagaat acataattct tatgcacaga aaaacaacgt attagagcag aattttaaaa 44640 acagcctcag actttcataa tattcatcat catcctggaa gaaaaaggat gccagtctga 44700 gggcccttcc ctccccatag caccaaacaa aaagctgaaa tgaatacacc ccacagaaat 44760 gtgagagttg gagtcatatt ccaagtggct gagctgagac tagcgcttca ttccattgtt 44820 gtaccaggac agcttcaagc ctccttattc actccggtga gtggtccagc ccagtgtgtg 44880 aagtgcccag tactcagggc aatgcctgga agtgcccggg cacgagagga tgtgctgcct 44940 cgggccctag gtggagggtt ccaggtgagc tgtaaggtca taaaggacat ttcccctgga 45000 gaatcgcagg cggagatcgc agcagggaag actggcaatc caggctcttt gattggcttc 45060 ccttggatcg tttttttccc tgtaaattta atcccaacct attattattt cctgaacaat 45120 ccacagatcc agtcccctaa gcatcaccac tcccctgatc actcaccctg aaaatgcttc 45180 ttttcaggag tgctacatct ctttctgctt tgtaacaact tctcgatatc ttcagttcca 45240 catccccctg cacccctccc tctcacctgc tctgcctcca cccccaggaa acaagggggt 45300 ccctttggga tccatccttg cctgggttac tgcatcagtc tccctgttac tgactttgac 45360 ctgaggcatg aacctattaa aacataagtc agatcagcta ctgctctgat gtaaaccctc 45420 caggacttct tgttggtgca gggtaaaagg taaggtctca tctgacttgg accccctgta 45 480 tcctctctgg cctccccttt gccactcaca cctgttctct gggctccaac ctcactggct 45540 tctttgctgt tccctgagca taccaggcac attgctgagg acaccaggca cattgctgct 45600 tccagacctc tgcactcgcc tttcccctcc tatgggaaca ctgttccctg agacagtggt 45660 gtggttggct tcttcatttc cttccagtct tagttcaaat gccacctaca tgaggtagtc 45720 cttccctgaa caatctacct aaatagcagt ttccagccac tgcctgcctt cctttgtatt 45780 gtcttctctg catgaactac ctgatgcatg gtcatttttc ccctccccta gaatgtcagc 45840 ttcttgagaa caggaaccac gctcactgca gtgtcctcag agtctacaaa ggtgcctagc 45900 acatagtagg tgctcaatac atatttgttg aatgaaggaa ggatccactt tctttattaa 45960 agggggcaga aaatagaagt tggggcagct tataagaatg agtcaccctt tttctgacat 46020 catttgtgtc tccttggtaa aggaggaaga ttccctggga catggaacaa tccctaatgc 46080 atcaactgcc caaaatggga tccagatgta gcagaaagtt ccctttaata aagagctttc 46140 tgtgtgtgtt ttaaaaatca aacatagaaa ttatttgtca ttgataccag caaatagctc 46200 aaatgggaga gcatggattt atagttcggc tggattagtt taaagatgat taaagtaatc 46260 ccctttatct tatcaaaaag agcttttagg tggtcagagt aaaagtcctc agacagaatg 46320 ttcagttgca cttggaattt cccttcccta agagataagc acatctcaga gatggaagac 46380 ctcagcccgt tatccatgga tcaaaacact gtgctgggct actgtggaga aagcgcctgt 46440 gacgtatcct tgcttgtgcg gatacctggt ccagttagcc acttggcctg gtgttcattc 46500 tgaatgccca tattcacagg attagtggga aaatggctgg aaccatggga ggttggatgg 46560 acatctggga gttgccattt actaagatgg ggagtgcaga gggattgcag ggggtggtgg 46620 tggtgaatat caggagttcg cttttggctg tgttgagtgg agatgctgag aaggcagtag 46680 gatataaaag agacaccaaa tttggagaga ctgcaagtag 46720 <210> 2 <211> 1040 <212> PRT <213> Homo sapiens <400> 2 Met Gly Glu Glu Gly Gly Ser Ala Ser His Asp Glu Glu Glu Arg Ala 1 5 10 15 Ser Val Leu Leu Gly His Ser Pro Gly Cys Glu Met Cys Ser Gln Glu 20 25 30 Ala Phe Gln Ala Gln Arg Ser Gln Leu Val Glu Leu Leu Val Ser Gly 35 40 45 Ser Leu Glu Gly Phe Glu Ser Val Leu Asp Trp Leu Leu Ser Trp Glu 50 55 60 Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe His Leu Leu Gly Gln Pro 65 70 75 80 Leu Ser His Leu Ala Arg Arg Leu Leu Asp Thr Val Trp Asn Lys Gly 85 90 95 Thr Trp Ala Cys Gln Lys Leu Ile Ala Ala Ala Gln Glu Ala Gln Ala 100 105 110 Asp Ser Gln Ser Pro Lys Leu His Gly Cys Trp Asp Pro His Ser Leu 115 120 125 His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala Ile Val Arg Arg 130 135 140 Leu His Ser His Val Glu Asn Met Leu Asp Leu Ala Trp Glu Arg Gly 145 150 155 160 Phe Val Ser Gln Tyr Glu Cys Asp Glu Ile Arg Leu Pro Ile Phe Thr 165 170 175 Pro Ser Gln Arg Ala Arg Arg Leu Leu Asp Leu Ala Thr Val Lys Ala 180 185 190 Asn Gly Leu Ala Ala Phe Leu Leu Gln His Val Gln Glu Leu Pro Val 195 200 205 Pro Leu Ala Leu Pro Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met Ala 210 215 220 Lys Leu Arg Thr Thr Val Val Ala Gln Ser Arg Phe Leu Ser Thr Tyr 225 230 235 240 Asp Gly Ala Glu Thr Leu Cys Leu Glu Asp Ile Tyr Thr Glu Asn Val 245 250 255 Leu Glu Val Trp Ala Asp Val Gly Met Ala Gly Pro Pro Gln Lys Ser 260 265 270 Pro Ala Thr Leu Gly Leu Glu Glu Leu Phe Ser Thr Pro Gly His Leu 275 280 285 Asn Asp Asp Ala Asp Thr Val Leu Val Val Gly Glu Ala Gly Ser Gly 290 295 300 Lys Ser Thr Leu Leu Gln Arg Leu His Leu Leu Trp Ala Ala Gly Gln 305 310 315 320 Asp Phe Gln Glu Phe Leu Phe Val Phe Pro Phe Ser Cys Arg Gln Leu 325 330 335 Gln Cys Met Ala Lys Pro Leu Ser Val Arg Thr Leu Leu Phe Glu His 340 345 350 Cys Cys Trp Pro Asp Val Gly Gln Glu Asp Ile Phe Gln Leu Leu Leu 355 360 365 Asp His Pro Asp Arg Val Leu Leu Thr Phe Asp Gly Phe Asp Glu Phe 370 375 380 Lys Phe Arg Phe Thr Asp Arg Glu Arg His Cys Ser Pro Thr Asp Pro 385 390 395 400 Thr Ser Val Gln Thr Leu Leu Phe Asn Leu Leu Gln Gly Asn Leu Leu 405 410 415 Lys Asn Ala Arg Lys Val Val Thr Ser Arg Pro Ala Ala Val Ser Ala 420 425 430 Phe Leu Arg Lys Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly Phe Ser 435 440 445 Glu Gln Gly Ile Glu Leu Tyr Leu Arg Lys Arg His His Glu Pro Gly 450 455 460 Val Ala Asp Arg Leu Ile Arg Leu Leu Gln Glu Thr Ser Ala Leu His 465 470 475 480 Gly Leu Cys His Leu Pro Val Phe Ser Trp Met Val Ser Lys Cys His 485 490 495 Gln Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys Thr Thr Thr Asp 500 505 510 Met Tyr Leu Leu Ile Leu Gln His Phe Leu Leu His Ala Thr Pro Pro 515 520 525 Asp Ser Ala Ser Gln Gly Leu Gly Pro Ser Leu Leu Arg Gly Arg Leu 530 535 540 Pro Thr Leu Leu His Leu Gly Arg Leu Ala Leu Trp Gly Leu Gly Met 545 550 555 560 Cys Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val Ser 565 570 575 Pro Asp Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys Gly Val Val 580 585 590 Pro Gly Ser Thr Ala Pro Leu Glu Phe Leu His Ile Thr Phe Gln Cys 595 600 605 Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala Asp Val Pro Pro Ala 610 615 620 Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly Asn Ser Pro Met 625 630 635 640 Ala Arg Leu Leu Pro Thr Met Cys Ile Gln Ala Ser Glu Gly Lys Asp 645 650 655 Ser Ser Val Ala Ala Leu Leu Gln Lys Ala Glu Pro His Asn Leu Gln 660 665 670 Ile Thr Ala Ala Phe Leu Ala Gly Leu Leu Ser Arg Glu His Trp Gly 675 680 685 Leu Leu Ala Glu Cys Gln Thr Ser Glu Lys Ala Leu Leu Arg Arg Gln 690 695 700 Ala Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys His Phe His 705 710 715 720 Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys Ser Val His Ala Met 725 730 735 Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met Gln Glu Glu 740 745 750 Arg Leu Ala Arg Lys Ala Ala Arg Gly Leu Asn Val Gly His Leu Lys 755 760 765 Leu Thr Phe Cys Ser Val Gly Pro Thr Glu Cys Ala Ala Leu Ala Phe 770 775 780 Val Leu Gln His Leu Arg Arg Pro Val Ala Leu Gln Leu Asp Tyr Asn 785 790 795 800 Ser Val Gly Asp Ile Gly Val Glu Gln Leu Leu Pro Cys Leu Gly Val 805 810 815 Cys Lys Ala Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp Arg Gly Ile 820 825 830 Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu Gln Leu Gln Lys Leu 835 840 845 Ala Leu Phe Asn Asn Lys Leu Thr Asp Gly Cys Ala His Ser Met Ala 850 855 860 Lys Leu Leu Ala Cys Arg Gln Asn Phe Leu Ala Leu Arg Leu Gly Asn 865 870 875 880 Asn Tyr Ile Thr Ala Ala Gly Ala Gln Val Leu Ala Glu Gly Leu Arg 885 890 895 Gly Asn Thr Ser Leu Gln Phe Leu Gly Phe Trp Gly Asn Arg Val Gly 900 905 910 Asp Glu Gly Ala Gln Ala Leu Ala Glu Ala Leu Gly Asp His Gln Ser 915 920 925 Leu Arg Trp Leu Ser Leu Val Gly Asn Asn Ile Gly Ser Val Gly Ala 930 935 940 Gln Ala Leu Ala Leu Met Leu Ala Lys Asn Val Met Leu Glu Glu Leu 945 950 955 960 Cys Leu Glu Glu Asn His Leu Gln Asp Glu Gly Val Cys Ser Leu Ala 965 970 975 Glu Gly Leu Lys Lys Asn Ser Ser Leu Lys Ile Leu Lys Leu Ser Asn 980 985 990 Asn Cys Ile Thr Tyr Leu Gly Ala Glu Ala Leu Leu Gln Ala Leu Glu 995 1000 1005 Arg Asn Asp Thr Ile Leu Glu Val Trp Leu Arg Gly Asn Thr Phe Ser 1010 1015 1020 Leu Glu Glu Val Asp Lys Leu Gly Cys Arg Asp Thr Arg Leu Leu Leu 1025 1030 1035 1040

Claims

A pharmaceutical composition for preventing or treating bone diseases induced by estrogen deficiency, comprising an inhibitor of a nucleotide-binding oligomerization domain 2 (NOD2) gene as an active ingredient.

According to claim 1, wherein the NOD2 gene is a pharmaceutical composition, characterized in that consisting of the base sequence encoding the amino acid sequence of SEQ ID NO: 2.

According to claim 2, wherein the NOD2 gene is a pharmaceutical composition, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.

The composition of claim 1, wherein the inhibitor is a substance that inhibits transcription of NOD2 gene, a substance that inhibits translation of transcribed NOD2 mRNA, or a substance that inhibits the function of NOD2 protein.

5. The composition of claim 4, wherein the agent that inhibits transcription of the NOD2 gene is a protein, a compound that binds to a promoter, enhancer or transcription factor that binds to the promoter.

The composition of claim 4, wherein the substance that inhibits translation of the transcribed NOD2 mRNA is an antisense molecule that specifically binds to the mRNA.

The composition of claim 4, wherein the substance that inhibits the function of the NOD2 protein is an antibody that specifically binds to the protein.

The bone disease induced by estrogen deficiency is selected from the group consisting of osteoporosis, osteomalacia, osteoopenia, bone atrophy and osteoarthritis. Composition.