KR20220159110A - Composition for regulating neuronal differentiation and biomarker for detecting neuronal differentiation containing HAX1 - Google Patents
Composition for regulating neuronal differentiation and biomarker for detecting neuronal differentiation containing HAX1 Download PDFInfo
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- KR20220159110A KR20220159110A KR1020210067050A KR20210067050A KR20220159110A KR 20220159110 A KR20220159110 A KR 20220159110A KR 1020210067050 A KR1020210067050 A KR 1020210067050A KR 20210067050 A KR20210067050 A KR 20210067050A KR 20220159110 A KR20220159110 A KR 20220159110A
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- protein
- hax1
- cpne1
- neuronal differentiation
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Abstract
Description
본 발명은 HAX1을 포함하는 신경세포 분화 조절용 조성물 및 신경세포 분화 탐지용 바이오마커에 관한 것이다.The present invention relates to a composition for regulating neuronal differentiation comprising HAX1 and a biomarker for detecting neuronal differentiation.
CPNE1은 코파인(Copine) 단백질 중 하나로, 암세포 발달 및 신경세포 분화에 관여한다고 알려져 있다. CPNE1은 N 말단에 두 가지 C2 도메인(C2A, C2B)과 C 말단에 A 도메인을 포함하고 있는데, C2 도메인은 칼슘 의존성 인지질 결합 활성에 관여하는 반면, A 도메인은 결합 단백질을 통해 CPNE1의 기능을 조절한다. 최근, CPNE1의 C2A 도메인은 14-3-3γ 및 Jab1(Jun activation domain-binding protein 1) 단백질과 결합하여 CPNE1-매개 신경세포 분화를 유도한다고 보고된 바 있다(J.C. Yoo 등, 2017. Exp. Cell Res. 356:85-92 및 J.C. Yoo 등, 2018. Biochem. Biophys. Res. Commun. 497:424-429). CPNE1 is one of the Copine proteins and is known to be involved in cancer cell development and neuronal differentiation. CPNE1 contains two C2 domains (C2A, C2B) at the N-terminus and an A domain at the C-terminus. The C2 domain is involved in calcium-dependent phospholipid binding activity, while the A domain regulates the function of CPNE1 through binding proteins. do. Recently, it has been reported that the C2A domain of CPNE1 binds to 14-3-3γ and Jab1 (Jun activation domain-binding protein 1) proteins to induce CPNE1-mediated neuronal differentiation (J.C. Yoo et al., 2017. Exp. Cell Res. 356:85-92 and J. C. Yoo et al., 2018. Biochem. Biophys. Res. Commun.
한편, HAX1(Hematopoietic cell-specific protein-associated protein X-1)은 세포 내 편재하여 발현하는 단백질로, 결핍되면 호중구의 성숙이 저해되어 선천성 호중구 감소증(congenital neutropenia)이 유발된다고 알려져 있다. 또한, HAX1단백질은 건선 또는 암 환자의 세포에서 과발현되며, 세포의 증식, 이동 및 사멸에 관여한다고 보고된 바 있다. 그러나, 이러한 HAX1 단백질의 역할에 대한 분자생물학적인 메커니즘이나 신경세포 분화에 대한 역할은 알려져 있지 않다. On the other hand, HAX1 (Hematopoietic cell-specific protein-associated protein X-1) is a protein expressed ubiquitously in cells, and it is known that when it is deficient, maturation of neutrophils is inhibited and congenital neutropenia is induced. In addition, HAX1 protein has been reported to be overexpressed in cells of psoriasis or cancer patients, and to be involved in cell proliferation, migration, and death. However, the molecular biological mechanism of the role of the HAX1 protein or its role in neuronal differentiation is not known.
한편, 한국등록특허 제1508936호에는 '메탈로프로테이즈 ADAM10에 의한 도파민성 신경세포로의 분화 조절 조성물'이 개시되어 있고, 한국등록특허 제1268561호에는 '백스 저해제-1을 도입한 배아줄기세포로부터 초기신경세포의 분화 촉진 방법 및 그 조성물'이 개시되어 있으나, 본 발명의 'HAX1을 포함하는 신경세포 분화 조절용 조성물 및 신경세포 분화 탐지용 바이오마커'에 대해서는 기재된 바가 없다.On the other hand, Korean Patent No. 1508936 discloses 'a composition for regulating differentiation into dopaminergic neurons by metalloproteinase ADAM10', and Korean Patent No. 1268561 discloses 'embryonic stem cells introduced with Bax inhibitor-1'. Although a method for promoting differentiation of early neurons and a composition thereof' are disclosed, there is no description of 'a composition for regulating neural cell differentiation containing HAX1 and a biomarker for detecting neural cell differentiation' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 HAX1 단백질의 N 말단이 분화 유도 단백질인 CPNE1 단백질의 C2A 도메인과 특이적으로 결합하는 것을 확인하였고, HiB5 세포(해마 전구체 세포)에서 CPNE1/HAX1 동시 과발현시켰을 때, CPNE1 단독 과발현 및 CPNE1/N 말단이 결손된 HAX1 동시 과발현시켰을 때보다 신경돌기의 분화가 억제되는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present inventors confirmed that the N-terminus of HAX1 protein specifically binds to the C2A domain of CPNE1 protein, a differentiation-inducing protein, and CPNE1 in HiB5 cells (hippocampal precursor cells). The present invention was completed by confirming that the simultaneous overexpression of /HAX1 suppresses neurite differentiation compared to the simultaneous overexpression of CPNE1 alone and the simultaneous overexpression of CPNE1/N-terminal-defective HAX1.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1의 HAX1(Hematopoietic cell-specific protein-associated protein X-1) 단백질 또는 서열번호 1의 아미노산 서열에서 1 내지 30번째 아미노산 잔기로 이루어진 폴리펩티드를 유효성분으로 포함하는 신경세포 분화 조절용 조성물을 제공한다.In order to solve the above problems, the present invention is HAX1 (Hematopoietic cell-specific protein-associated protein X-1) protein of SEQ ID NO: 1 or a polypeptide consisting of the 1st to 30th amino acid residues in the amino acid sequence of SEQ ID NO: 1 as an active ingredient. It provides a composition for regulating neuronal differentiation comprising the.
또한, 본 발명은 서열번호 1의 HAX1 단백질 코딩 서열 또는 서열번호 1의 아미노산 서열에서 1 내지 30번째 아미노산 잔기로 이루어진 폴리펩티드 코딩 서열을 포함하는 재조합 벡터를 신경세포에 도입하여 HAX1 단백질 또는 폴리펩티드의 발현을 조절하는 단계를 포함하는, 신경세포 분화 조절 방법을 제공한다.In addition, the present invention introduces a recombinant vector comprising the HAX1 protein coding sequence of SEQ ID NO: 1 or the polypeptide coding sequence consisting of the 1st to 30th amino acid residues in the amino acid sequence of SEQ ID NO: 1 into neurons to express the HAX1 protein or polypeptide. It provides a method for regulating neural cell differentiation, comprising the step of regulating.
또한, 본 발명은 CPNE1 단백질을 과발현하는 신경세포에서 HAX1 단백질의 발현 수준을 측정하는 제제를 포함하는 신경세포 분화 탐지용 조성물을 제공한다.In addition, the present invention provides a composition for detecting neuronal differentiation comprising an agent for measuring the expression level of HAX1 protein in neurons overexpressing CPNE1 protein.
본 발명에 따른 HAX1 단백질의 발현을 조절하면 신경세포의 분화를 억제하거나 촉진할 수 있으므로, 중추 신경계 질환의 예방 또는 치료에 사용할 수 있으며, HAX1 단백질을 신경세포 분화 초기의 마커로 이용하여 신경계 질환의 진단 및 치료에 유용하게 사용할 수 있을 것으로 기대된다. Regulating the expression of the HAX1 protein according to the present invention can inhibit or promote the differentiation of nerve cells, so it can be used for the prevention or treatment of central nervous system diseases, and using the HAX1 protein as a marker in the early stage of nerve cell differentiation can prevent nervous system diseases. It is expected to be useful for diagnosis and treatment.
도 1은 CPNE1과 HAX1 단백질의 결합을 확인한 결과로, A는 효모의 영양제한 조건에서 효모단백질잡종분석(Yeast two-hybrid assay) 결과이고, B는 MBP 풀다운 분석(MBP pull-down assay) 결과이고, C는 HEK293T 세포에서의 동시 면역침전(co-Immunoprecipitation) 분석 결과이고, D는 COS7 세포에서의 단백질 발현 위치를 확인한 결과이며, E는 분화 조건의 HiB5 세포 및 출생 후 1일째의 마우스(postnatal P1 mouse) 뇌 추출물에서의 웨스턴 분석 결과이다. Input: 5% 용해물, 흰색 화살표: HAX1과 CPNE1 단백질의 공존 위치, 스케일 바(scale bar)=20 μm
도 2는 HAX1과 CPNE1 단백질의 결합에 관여하는 CPNE1 단백질의 결합부위를 확인한 결과로, A는 CPNE1 단백질의 각 도메인만 발현하는 돌연변이체 제작을 위한 모식도이고, B는 HEK293T 세포에서의 동시 면역침전 분석 결과이고, C는 효모의 영양제한 조건에서 효모단백질잡종분석한 결과이다.
도 3은 HAX1과 CPNE1 단백질의 결합에 관여하는 HAX1 단백질의 결합부위를 확인한 결과로, A는 HAX1 단백질의 각 도메인만 발현하는 돌연변이체 제작을 위한 모식도이고, B는 HEK293T 세포에서의 동시 면역침전 분석 결과이고, C는 HAX1 단백질의 N1 부위에서 각 도메인만 발현하는 돌연변이체 제작을 위한 모식도이고, D는 HEK293T 세포에서의 동시 면역침전 분석 결과이다.
도 4는 CPNE1 단백질과 관련된 신경세포 분화 조절능에 대한 HAX1 단백질의 영향을 확인한 결과로, A는 HiB5 세포의 분화 조건에 대한 모식도이고, B는 HiB5 세포에 CPNE1과 HAX1△1-30(CPNE1과 결합하지 않는 부위 포함)를 동시 과발현시킨 후, 인산화-AKT 및 Tuj1 단백질 발현양을 분석한 결과이고, C는 HiB5 세포에 CPNE1과 HAX1(CPNE1과 결합하는 부위 포함)을 동시 과발현시킨 후, 인산화-AKT 및 Tuj1 단백질 발현양을 분석한 결과이며, D는 CPNE1 단독 과발현시킨 대조구대비 CPNE1과 HAX1을 동시 과발현시킨 실험구의 인산화-AKT 및 Tuj1 단백질 발현양이 통계적으로 유의미하게 감소한 것을 보여주는 결과로, ***은 p<0.001이며, E는 HiB5 세포에서 각 단백질의 과발현을 유도하는 아데노바이러스로 형질감염시킨 후, 신경세포의 분화적 형태 변화를 관찰한 결과이다. 스케일 바(scale bar)=20 μmFigure 1 is the result of confirming the binding of CPNE1 and HAX1 protein, A is the result of yeast protein hybrid analysis (Yeast two-hybrid assay) under nutrient-restricted conditions of yeast, B is the result of MBP pull-down assay (MBP pull-down assay) , C is the result of co-immunoprecipitation analysis in HEK293T cells, D is the result of confirming the location of protein expression in COS7 cells, E is HiB5 cells under differentiated conditions and mice at
Figure 2 is a result of confirming the binding site of CPNE1 protein involved in the binding of HAX1 and CPNE1 protein. A is a schematic diagram for constructing a mutant expressing only each domain of CPNE1 protein, and B is a co-immunoprecipitation analysis in HEK293T cells. result, and C is the result of yeast protein hybridization analysis under nutrient-restricted conditions of yeast.
Figure 3 is a result of confirming the binding site of the HAX1 protein involved in the binding of the HAX1 and CPNE1 proteins. A is a schematic diagram for constructing a mutant expressing only each domain of the HAX1 protein, and B is a co-immunoprecipitation analysis in HEK293T cells. Results, C is a schematic diagram for constructing a mutant expressing only each domain in the N1 region of the HAX1 protein, and D is the result of co-immunoprecipitation analysis in HEK293T cells.
Figure 4 is a result of confirming the effect of HAX1 protein on the neuronal differentiation control ability related to CPNE1 protein. A is a schematic diagram of the differentiation conditions of HiB5 cells, and B is a schematic diagram of CPNE1 and HAX1Δ1-30 (combined with CPNE1) in HiB5 cells. C is the result of analyzing the amount of phosphorylated-AKT and Tuj1 protein expression after simultaneous overexpression of CPNE1 and HAX1 (including the site that binds to CPNE1) in HiB5 cells, followed by phosphorylation-AKT and Tuj1 protein expression, and D is a result showing that the phospho-AKT and Tuj1 protein expression levels in the experimental group in which CPNE1 and HAX1 were simultaneously overexpressed were statistically significantly decreased compared to the control group in which CPNE1 was overexpressed alone. *** is p<0.001, and E is the result of observing differentiated morphological changes of neurons after transfection with adenovirus inducing overexpression of each protein in HiB5 cells. scale bar=20 μm
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1의 HAX1(Hematopoietic cell-specific protein-associated protein X-1) 단백질 또는 서열번호 1의 아미노산 서열에서 1 내지 30번째 아미노산 잔기로 이루어진 폴리펩티드를 포함하는 신경세포 분화 조절용 조성물을 제공한다.In order to achieve the object of the present invention, the present invention includes the HAX1 (Hematopoietic cell-specific protein-associated protein X-1) protein of SEQ ID NO: 1 or a polypeptide consisting of the 1st to 30th amino acid residues in the amino acid sequence of SEQ ID NO: 1. It provides a composition for regulating neuronal differentiation.
본 발명의 신경세포 분화 조절용 조성물에 있어서, 상기 HAX1 단백질은 바람직하게는 CPNE1(Copine 1) 단백질과 특이적으로 결합할 수 있으며, 더욱 바람직하게는 CPNE1 단백질의 C2A 도메인 또는 A 도메인에 특이적으로 결합할 수 있으나, 이에 제한되지 않는다.In the composition for regulating neural cell differentiation of the present invention, the HAX1 protein may preferably bind specifically to CPNE1 (Copine 1) protein, and more preferably bind specifically to the C2A domain or A domain of CPNE1 protein. It can, but is not limited thereto.
또한, 본 발명의 조성물에 있어서, 상기 서열번호 1의 아미노산 서열 N 말단의 1 내지 30번째 아미노산 잔기로 이루어진 폴리펩티드는 CPNE1 단백질과 특이적으로 결하는 영역이다. In addition, in the composition of the present invention, the polypeptide consisting of the 1st to 30th amino acid residues of the N-terminus of the amino acid sequence of SEQ ID NO: 1 is a region that specifically binds to the CPNE1 protein.
본 발명에 따른 HAX1 단백질의 범위는 서열번호 1로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. 본 발명에 있어서, 용어 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. "실질적으로 동질의 생리활성"이란 신경세포 분화를 조절하는 활성을 의미한다.The scope of the HAX1 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents of the protein. In the present invention, the term "functional equivalent" is at least 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. More preferably, it refers to a protein having a sequence homology of 95% or more, and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially homogeneous physiological activity" means an activity that regulates neuronal differentiation.
본 발명은 또한, 본 발명의 HAX1 단백질을 코딩하는 유전자를 포함하며, 상기 유전자의 범위는 HAX1 단백질을 코딩하는 게놈 DNA, cDNA 및 합성 DNA를 모두 포함한다. 바람직하게는, 본 발명의 HAX1 단백질을 코딩하는 유전자는 서열번호 2로 표시되는 염기서열을 포함할 수 있다. 또한, 상기 염기서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 2의 염기 서열과 각각 70% 이상, 더 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The present invention also includes a gene encoding the HAX1 protein of the present invention, and the scope of the gene includes both genomic DNA, cDNA and synthetic DNA encoding the HAX1 protein. Preferably, the gene encoding the HAX1 protein of the present invention may include the nucleotide sequence represented by SEQ ID NO: 2. In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 2. can do. The "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence (not containing additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to
또한, 본 발명의 조성물에 있어서, 상기 조성물은 배아 줄기세포, 성체 줄기세포, 유도만능 줄기세포, 신경 줄기세포 및 신경 전구세포로 이루어진 군으로부터 선택되는 하나 이상의 세포 또는 이들 세포의 배양물과 접촉함으로써 신경세포로 분화를 조절할 수 있으며, 바람직하게는 신경 전구세포일 수 있으며, 더욱 바람직하게는 CPNE1 단백질을 과발현하는 신경 전구세포일 수 있으나, 이에 제한되지 않는다.In addition, in the composition of the present invention, the composition is contacted with one or more cells selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells, neural stem cells and neural progenitor cells, or a culture of these cells. Differentiation into neurons can be controlled, preferably neural progenitor cells, more preferably neural progenitor cells overexpressing the CPNE1 protein, but not limited thereto.
본 발명은 또한, 서열번호 1의 HAX1 단백질 코딩 서열 또는 서열번호 1의 아미노산 서열에서 1 내지 30번째 아미노산 잔기로 이루어진 폴리펩티드 코딩 서열을 포함하는 재조합 벡터를 신경세포에 도입하여 HAX1 단백질 또는 폴리펩티드의 발현을 조절하는 단계를 포함하는, 신경세포 분화 조절 방법을 제공한다. The present invention also relates to the expression of HAX1 protein or polypeptide by introducing a recombinant vector comprising the HAX1 protein coding sequence of SEQ ID NO: 1 or the polypeptide coding sequence consisting of the 1st to 30th amino acid residues in the amino acid sequence of SEQ ID NO: 1 into neurons. It provides a method for regulating neural cell differentiation, comprising the step of regulating.
본 발명의 방법에 있어서, 상기 HAX1 단백질 및 폴리펩티드는 전술한 것과 같다. In the method of the present invention, the HAX1 protein and polypeptide are as described above.
또한, 본 발명의 방법은 바람직하게는 CPNE1 단백질을 과발현하는 것을 특징으로 하는 신경세포에서 HAX1 단백질 또는 폴리펩티드를 과발현시켜 신경세포로의 분화를 억제하는 것일 수 있으나, 이에 제한되지 않는다. In addition, the method of the present invention may preferably inhibit differentiation into neurons by overexpressing the HAX1 protein or polypeptide in neurons characterized by overexpressing the CPNE1 protein, but is not limited thereto.
본 명세서에서, 용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포 내 재도입된 것이다.As used herein, the term "recombinant" refers to a cell that replicates, expresses a heterologous nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. A recombinant cell may also express a gene found in the cell in its natural state, but the gene has been reintroduced into the cell by artificial means as a modified one.
본 명세서에서, 용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다.In this specification, the term "vector" is used to refer to DNA fragment(s) or nucleic acid molecules that are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells. The term “delivery vehicle” is often used interchangeably with “vector”.
본 발명의 벡터는 전형적으로 발현 또는 클로닝을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵세포 또는 진핵세포를 숙주로 하여 구축될 수 있다. 예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예를 들면, pLλ 프로모터, trp 프로모터, lac 프로모터, T7 프로모터, tac 프로모터 등), 해독의 개시를 위한 리보솜 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주세포로서 대장균(Escherichia coli)이 이용되는 경우, E. coli 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위, 그리고 파아지 λ의 좌향 프로모터(pLλ 프로모터)가 조절 부위로서 이용될 수 있다.Vectors of the invention may typically be constructed as vectors for expression or cloning. In addition, the vector of the present invention can be constructed using a prokaryotic or eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (eg, pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) ), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When Escherichia coli is used as a host cell, promoter and operator sites of the E. coli tryptophan biosynthetic pathway and leftward promoter (pLλ promoter) of phage λ may be used as control sites.
본 발명의 재조합 벡터에서, 상기 프로모터는 형질전환에 적합한 프로모터들로서, 바람직하게는 T7 프로모터, CaMV 35S 프로모터, 액틴 프로모터, 유비퀴틴 프로모터, pEMU 프로모터, MAS 프로모터 또는 히스톤 프로모터일 수 있으며, 바람직하게는 T7 프로모터일 수 있으나, 이에 제한되지 않는다.In the recombinant vector of the present invention, the promoter may be promoters suitable for transformation, preferably T7 promoter, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably T7 promoter. It may be, but is not limited thereto.
본 명세서에서, 용어 "프로모터"란 용어는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다. As used herein, the term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.
본 발명의 재조합 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관 내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 벡터는 번역 개시 부위로서 리보솜 결합 부위 및 전사 터미네이터를 포함할 수 있다.The recombinant vector of the present invention can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. In addition, the vector may include a ribosome binding site and a transcription terminator as a translation initiation site.
본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주세포는 당업계에 공지된 어떠한 숙주세포도 이용할 수 있으며, 원핵세포의 예로는, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스(Bacillus subtilis), 바실러스 츄린겐시스(Bacillus thuringiensis)와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움(Salmonella typhimurium), 세라티아 마르세슨스(Serratia marcescens) 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다.Any host cell known in the art can be used as a host cell capable of stably and continuously cloning and expressing the vector of the present invention, and examples of prokaryotic cells include E. coli JM109, E. coli BL21, and E. coli. Bacillus genus strains such as RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis , Bacillus thuringiensis , and Salmonella typhimurium Enterobacteriaceae and strains such as Salmonella typhimurium , Serratia marcescens and various Pseudomonas species.
본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주세포로서, 효모(Saccharomyce cerevisiae), 곤충세포, 사람세포(예컨대, CHO 세포주(Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용될 수 있다. 숙주세포는 바람직하게는 사람세포이다.When the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2 , 3T3, RIN and MDCK cell lines) and plant cells, etc. may be used. The host cell is preferably a human cell.
본 발명의 벡터를 숙주세포 내로 운반하는 방법은, 숙주 세포가 원핵 세포인 경우, CaCl2 방법, 하나한 방법(Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) 및 전기천공 방법 등에 의해 실시될 수 있다. 또한, 숙주세포가 진핵세포인 경우에는, 바이러스-매개 형질감염법, 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있다.Methods for delivering the vectors of the present invention into host cells, when the host cells are prokaryotic cells, include the CaCl 2 method, the Hanhan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580), and electroporation. It can be implemented by methods and the like. In addition, when the host cell is a eukaryotic cell, virus-mediated transfection, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, etc. Vectors can be injected into host cells.
본 발명은 또한, CPNE1 단백질을 과발현하는 신경세포에서 HAX1 단백질의 발현 수준을 측정하는 제제를 포함하는 신경세포 분화 탐지용 조성물을 제공한다. The present invention also provides a composition for detecting neuronal differentiation comprising an agent for measuring the expression level of HAX1 protein in neurons overexpressing the CPNE1 protein.
본 발명의 신경세포 분화 탐지용 조성물에 있어서, 상기 HAX1 단백질의 범위는 전술한 것과 같다. In the composition for detecting neuronal differentiation of the present invention, the range of the HAX1 protein is the same as described above.
또한, 신경세포 분화 탐지용 조성물에 있어서, 바람직하게는 CPNE1 단백질을 과발현하는 것을 특징으로 하는 신경세포에서 HAX1 단백질의 발현 수준이 대조군 시료보다 과발현된 경우에는 신경세포 분화가 억제된 것일 수 있다. In addition, in the composition for detecting neuronal differentiation, preferably, when the HAX1 protein expression level is overexpressed in a control sample in neurons characterized by overexpressing the CPNE1 protein, neuronal differentiation may be suppressed.
또한, 본 발명에서 '단백질의 발현 수준 측정'은, 신경세포 분화 탐지를 위해 생물학적 시료에서의 신경세포 분화 마커 유전자(HAX1)에서 발현되는 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 통상적으로 단백질의 양을 확인함으로써 수행될 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏(Western blot), ELISA(enzyme linked immunosorbent assay), 방사선면역분석(Radioimmunoassay, RIA), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), FACS(Fluorescence Activated Cell Sorting) 및 단백질 칩(protein chip) 등이 있으나, 이에 제한되지 않는다.In addition, in the present invention, 'measurement of protein expression level' is a process of confirming the presence and expression level of a protein expressed in the neuronal differentiation marker gene (HAX1) in a biological sample in order to detect neuronal differentiation. This can be done by checking the amount of protein. Analysis methods for this include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket ) immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence Activated Cell Sorting) and protein chip, but are not limited thereto.
본 발명에서 단백질의 발현 수준을 측정하는 제제는 이에 한정되지는 않으나, 바람직하게는 HAX1 단백질에 대하여 특이적으로 결합하는 항체 또는 압타머일 수 있다.In the present invention, the agent for measuring the expression level of the protein may be, but is not limited to, an antibody or an aptamer that specifically binds to the HAX1 protein.
본 발명에서, 용어 "항체"는 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커인 HAX1 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는, HAX1 유전자의 전장 혹은 일부를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 HAX1 유전자의 전장 혹은 일부에 의해 코딩되는 단백질을 얻고, 얻어진 단백질을 면역원(항원)으로 사용하여, 통상적인 방법에 의해 제조될 수 있다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 기능적인 단편도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다. 상기 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab')2 및 ScFv 등이 있다.In the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the HAX1 protein, which is the marker of the present invention, and such an antibody is obtained by cloning the full length or part of the HAX1 gene into an expression vector according to a conventional method, and the HAX1 gene. It can be produced by a conventional method by obtaining a protein encoded by the full length or part of a gene and using the obtained protein as an immunogen (antigen). The form of the antibody of the present invention is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or functional fragments having antigen-binding properties are also included in the antibodies of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Functional fragments of the antibody molecule refer to fragments having at least an antigen-binding function, and include Fab, F(ab'), F(ab')2, and ScFv.
본 발명에서, 용어 "압타머(aptamer)"는 안정된 삼차구조를 유지하면서 특정 분자에 특이적으로 강하게 결합할 수 있는 핵산을 의미한다. 특이적 결합을 하는 기능 때문에 항체와 비교되며 항체의 대체 기술로 평가되고 있다.In the present invention, the term "aptamer" refers to a nucleic acid that can specifically and strongly bind to a specific molecule while maintaining a stable tertiary structure. Because of its specific binding function, it is compared with antibodies and is being evaluated as an alternative technology for antibodies.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명은 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
1. 재조합 벡터 구성 1. Recombinant Vector Construction
CPNE1 cDNA(GenBank No.BC001142) 및 HAX1 cDNA(GenBank No.NM006118)를 한국생명공학연구원(KRIBB)으로부터 얻어 실험에 사용하였다. cDNA를 주형으로 사용하는 PCR 기반의 게놈 클로닝 방법을 통해 전장 클론을 생성하였다. CPNE1의 C2A, C2B, ΔC2A 및 A 도메인 돌연변이와 HAX1의 N 말단 및 C 말단 도메인 돌연변이는 각 영역에 특이적인 프라이머를 이용한 directed PCR을 통해 얻었다. 생성된 PCR 산물을 게이트웨이 클로닝 시스템(gateway cloning system, Invitrogen)을 사용하여 목적 벡터 pDEST-AD-GFP/mcherry에 클로닝하였다.CPNE1 cDNA (GenBank No.BC001142) and HAX1 cDNA (GenBank No.NM006118) were obtained from Korea Research Institute of Bioscience and Biotechnology (KRIBB) and used in the experiments. Full-length clones were generated through a PCR-based genome cloning method using cDNA as a template. Mutations in the C2A, C2B, ΔC2A and A domains of CPNE1 and the N-terminal and C-terminal domains of HAX1 were obtained by directed PCR using primers specific for each region. The resulting PCR product was cloned into the target vector pDEST-AD-GFP/mcherry using the gateway cloning system (Invitrogen).
2. 세포 배양 및 분화 유도2. Cell culture and induction of differentiation
HEK293A, HEK293T 및 COS7 세포를 1% 페니실린-스트렙토마이신(PS) 및 10% 소태아혈청(FBS)이 첨가된 DMEM(Dulbecco Modified Eagle Medium) 배지에서 37℃, 5% CO2 조건으로 배양하였다. HiB5 세포(해마 전구체 세포)는 1% PS 및 10% FBS가 첨가된 DMEM 배지에서 33℃, 5% CO2 조건으로 배양하여 증식시켰으며, 30 ng/㎖의 PDGF(Platelet-derived growth factor, Millipore)가 포함된 화학적으로 한정된(chemically defined) N2 배지에서 39℃, 5% CO2 조건으로 배양하여 신경세포의 분화를 유도하였다. HEK293A, HEK293T, and COS7 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 conditions. HiB5 cells (hippocampal progenitor cells) were grown by culturing in DMEM medium supplemented with 1% PS and 10% FBS at 33°C and 5% CO 2 conditions, and 30 ng/ml PDGF (Platelet-derived growth factor, Millipore ) was cultured in a chemically defined N2 medium containing 39° C. under 5% CO 2 conditions to induce differentiation of neurons.
3. 형질감염(Transfection)3. Transfection
아데노바이러스 발현 시스템(ViraPower, Invitrogen)을 사용하여 HEK293A 세포에서 아데노바이러스를 증식시킨 후, 2×105 세포/㎖의 HiB5 세포에 감염다중도(MOI)가 100인 아데노바이러스를 감염시키고 33℃, 5% CO2 조건으로 48시간 동안 배양하여 형질감염하였다. After propagating adenovirus in HEK293A cells using an adenovirus expression system (ViraPower, Invitrogen), 2×10 5 cells/ml of HiB5 cells were infected with an adenovirus having a multiplicity of infection (MOI) of 100 and maintained at 33° C. Transfection was performed by culturing for 48 hours under 5% CO 2 conditions.
4. 효모단백질잡종분석(Yeast two-hybrid assay, Y2B)4. Yeast two-hybrid assay (Y2B)
효모단백질잡종분석은 J.C. Yoo 등(2017. Exp. Cell Res. 356:85-92)에 기술된 방법에 따라 수행하였다. 결합 도메인(binding domain, BD)을 암호화하는 pGBKT7-/CPNE1/C2A/A 도메인과 활성화 도메인(activation domain, AD)을 암호화하는 pGADT7-HAX1을 효모 AH109 균주로 형질전환시켰다. 형질전환된 효모를 류신(leucine), 트립토판(trptophan) 및 히스티딘(histidine)이 결핍된(LTH-) SD(synthetic dropout) 배지에서 배양하여, 살아남은 콜로니를 선발하였다. Yeast protein hybridization was performed by J.C. It was performed according to the method described by Yoo et al. (2017. Exp. Cell Res. 356:85-92). The pGBKT7-/CPNE1/C2A/A domain encoding the binding domain (BD) and pGADT7-HAX1 encoding the activation domain (AD) were transformed into yeast strain AH109. The transformed yeast was cultured in a synthetic dropout (SD) medium lacking leucine, tryptophan, and histidine (LTH-), and surviving colonies were selected.
5. MBP 풀다운 분석(MBP pull-down assay)5. MBP pull-down assay
MBP 풀다운 분석은 J.C. Yoo 등(2018. Biochem. Biophys. Res. Commun. 497:424-429)에 기술된 방법에 따라 수행하였다. MBP-CPNE1 단백질을 정제하고, GFP 태그된 HAX1는 HEK293T 세포로 형질감염시키고 24시간 후 용균시켰다. 세포 용해물을 정제된 MBP-CPNE1 단백질 1 mg과 함께 MBP 결합 아가로오스 수지(Elpis Biotech Inc. Korea)에 넣고 4℃에서 1시간 동안 반응시켰다. 이후, 용해 완충액으로 3회 세척하고, 결합된 단백질을 SDS 샘플 완충액을 넣고 가열하여 용리시키고 웨스턴 블랏 분석을 하였다.The MBP pull-down assay was performed by J.C. It was performed according to the method described by Yoo et al. (2018. Biochem. Biophys. Res. Commun. 497:424-429). MBP-CPNE1 protein was purified and GFP tagged HAX1 was transfected into HEK293T cells and lysed after 24 hours. The cell lysate was added to MBP-binding agarose resin (Elpis Biotech Inc. Korea) together with 1 mg of purified MBP-CPNE1 protein and reacted at 4°C for 1 hour. Thereafter, the cells were washed three times with a lysis buffer, and the bound proteins were eluted by heating in SDS sample buffer and subjected to Western blot analysis.
6. 면역세포화학염색법(Immunocytochemistry)6. Immunocytochemistry
GFP-HAX1 및 mcherry-CPNE1 유전자를 형질감염한 COS7 세포를 공초점 현미경(Olympus FluoView FV1000)을 사용하여 관찰하고 촬영하였다. COS7 cells transfected with GFP-HAX1 and mcherry-CPNE1 genes were observed and photographed using a confocal microscope (Olympus FluoView FV1000).
7. 면역침전(Immunoprecipitation, IP)7. Immunoprecipitation (IP)
면역침전 분석은 J.C. Yoo 등(2018. Biochem. Biophys. Res. Commun. 497:424-429)에 기술된 방법에 따라 수행하였다. P1(postnatal 1) 마우스 뇌 추출물, HEK293T 및 HiB5 세포를 프로테아제-인히비터 칵테일(protease-inhibitor cocktail)을 포함하는 RIPA 완충액(50 mM Tris-HCl, pH 7.4, 150 mM EDTA, 1 mM PMSF 및 1% NP-40)으로 용해시켰다. 전체 세포 용해물을 얼음에서 30분 동안 반응시킨 다음, 4℃에서 20분 간 20,000g로 원심분리하여 상등액을 분리하였다. 얻어진 상등액을 지시(indicated) 항체와 함께 4℃에서 3시간 배양하였고, 단백질 A/G+아가로스(Santa Cruz Biotechnology)를 첨가한 후 3시간 동안 추가 배양하였다. 아가로스 겔에 포획된 단백질을 RIPA 완충액으로 3회 세척하고, SDS 샘플 완충액을 넣고 가열하여 용리시키고 웨스턴 블랏 분석을 하였다.The immunoprecipitation assay was performed according to J.C. It was performed according to the method described by Yoo et al. (2018. Biochem. Biophys. Res. Commun. 497:424-429). P1 (postnatal 1) mouse brain extracts, HEK293T and HiB5 cells were cultured in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM EDTA, 1 mM PMSF and 1% PMSF) containing a protease-inhibitor cocktail. NP-40) was dissolved. The whole cell lysate was incubated on ice for 30 min, and then the supernatant was separated by centrifugation at 20,000 g for 20 min at 4°C. The obtained supernatant was incubated with the indicated antibody at 4° C. for 3 hours, and after adding protein A/G+agarose (Santa Cruz Biotechnology), it was additionally incubated for 3 hours. Proteins captured on the agarose gel were washed three times with RIPA buffer, eluted with SDS sample buffer and heated, and subjected to Western blot analysis.
8. 웨스턴 블랏 분석 8. Western blot analysis
단백질 샘플을 10% 겔을 사용하여 SDS-PAGE로 분리하고 PVDF 멤브레인에 블로팅하였다. 1차 항체로 사용한 anti-FLAG, anti-MBP, anti-Tuj1 항체는 Sigma에서 구입하였고, anti-HA 항체는 Roche에서 구입하였고, anti-GFP, anti-HAX1, anti-CPNE1 항체는 Santa Cruz Biotechnology에서 구입하였으며, anti-AKT1/2/3, anti-phospho-AKT1/2/3(S473), anti-GAPDH 항체는 Cell signaling에서 구입하여 사용하였다. HRP(horseradish peroxidase) 결합 2차 항체와 함께 항온 배양한 후, ECL(enhanced chemiluminescence, Amersham)을 이용하여 단백질 밴드를 검출하였다.Protein samples were separated by SDS-PAGE using a 10% gel and blotted onto PVDF membranes. Anti-FLAG, anti-MBP, and anti-Tuj1 antibodies used as primary antibodies were purchased from Sigma, anti-HA antibodies were purchased from Roche, and anti-GFP, anti-HAX1, and anti-CPNE1 antibodies were purchased from Santa Cruz Biotechnology. Anti-AKT1/2/3, anti-phospho-AKT1/2/3 (S473), and anti-GAPDH antibodies were purchased from Cell Signaling and used. After incubation with a horseradish peroxidase (HRP)-coupled secondary antibody, protein bands were detected using ECL (enhanced chemiluminescence, Amersham).
9. 통계분석9. Statistical analysis
통계분석은 unpaired Student's t-test로 평가하였다. 통계적 유의성은 *** P<0.001로 설정하였다. 데이터는 세 번의 독립적인 실험의 평균±표준 편차로 기재하였다.Statistical analysis was evaluated by unpaired Student's t-test. Statistical significance was set at ***P<0.001. Data are presented as the mean±standard deviation of three independent experiments.
실시예 1. CPNE1과 HAX1 단백질의 결합 확인Example 1. Confirmation of CPNE1 and HAX1 protein binding
CPNE1 단백질에 결합하는 새로운 단백질을 규명하기 위해, CPNE1 단백질을 미끼로 효모단백질잡종분석(Y2H)을 수행하여, HAX1 단백질을 분리하였다. 이후, CPNE1과 HAX1 단백질의 결합을 in vitro 및 in vivo에서 재확인하였다.In order to identify a new protein that binds to the CPNE1 protein, yeast protein hybridization (Y2H) was performed using the CPNE1 protein as a bait, and the HAX1 protein was isolated. Then, the binding between CPNE1 and HAX1 protein was reconfirmed in vitro and in vivo .
효모의 영양제한 조건에서의 효모단백질잡종분석을 수행한 결과, BD-CPNE1과 AD-HAX1 단백질이 결합하는 것을 확인하였고(도 1A), MBP 풀다운 분석 결과, MBP-CPNE1과 GFP-HAX1 단백질이 결합하는 것을 확인하였다(도 1B). 또한, HEK293T 세포에서의 동시 면역침전(co-IP) 분석 결과, GFP-CPNE1과 FLAG-HAX1 단백질이 결합하는 것을 확인하였고(도 1C), GFP-HAX1 및 mcherry-CPNE1으로 형질감염된 COS7 세포를 공초점 현미경(confocal microscopy)으로 관찰하여 각 단백질의 발현 위치를 확인한 결과, HAX1 단백질은 핵과 세포질 내에 발현하는 반면, CPNE1 단백질은 세포질 내 전반적으로 퍼져 발현하며, CPNE1과 HAX1 단백질은 세포질 및 세포막 부위에서 공존하는 것을 확인하였다(도 1D). 또한, 분화 조건의 HiB5 세포(in vitro)와 출생 후 1일째의 마우스(postnatal P1 mouse) 뇌 추출물(in vivo)에서의 동시 면역침전 분석 결과, HAX1과 CPNE1 단백질은 서로 결합되어 발현하는 것을 확인하였다(도 1E).As a result of yeast protein hybridization analysis under nutrient-limited yeast conditions, it was confirmed that BD-CPNE1 and AD-HAX1 proteins bind (Fig. 1A), and as a result of MBP pull-down analysis, MBP-CPNE1 and GFP-HAX1 proteins bind It was confirmed that (Fig. 1B). In addition, as a result of co-immunoprecipitation (co-IP) analysis in HEK293T cells, it was confirmed that GFP-CPNE1 and FLAG-HAX1 proteins bind (Fig. 1C), and COS7 cells transfected with GFP-HAX1 and mcherry-CPNE1 were co-administered. As a result of confirming the expression location of each protein by observing it under a confocal microscopy, HAX1 protein is expressed in the nucleus and cytoplasm, whereas CPNE1 protein is expressed throughout the cytoplasm, and CPNE1 and HAX1 proteins coexist in the cytoplasm and membrane regions. It was confirmed that (Fig. 1D). In addition, as a result of co-immunoprecipitation analysis in differentiated HiB5 cells ( in vitro ) and
실시예 2. HAX1-CPNE1 결합에 관여하는 CPNE1 단백질의 결합부위 확인Example 2. Identification of CPNE1 protein binding site involved in HAX1-CPNE1 binding
HAX1과 CPNE1 단백질의 결합에 관여하는 CPNE1 단백질의 결합부위를 확인하기 위해, CPNE1의 C2A, C2B, A 및 ΔC2A 도메인을 각각 분리하였고, 각 도메인의 N 말단을 GFP로 표지하였다(도 2A). 이렇게 만들어진 각각의 CPNE1 돌연변이체와 FLAG-HAX1을 HEK293T 세포 내로 동시 형질감염(co-transfection)시키고 FLAG에 대한 항체를 이용하여 동시 면역침전 분석을 수행한 결과, HAX1이 CPNE1의 C2B 도메인에 결합하지 않고, C2A 및 A 도메인에 결합하는 것을 확인하였다(도 2B). 또한, HAX1과 CPNE1의 C2A 및 A 도메인으로 효모단백질잡종분석을 수행한 결과, HAX1이 CPNE1의 C2A 및 A 도메인에 결합하는 것을 재확인하였다(도 2C).In order to identify the CPNE1 protein binding site involved in the binding between HAX1 and CPNE1 protein, the CPNE1 C2A, C2B, A, and ΔC2A domains were isolated, respectively, and the N terminus of each domain was labeled with GFP (FIG. 2A). Each CPNE1 mutant thus prepared and FLAG-HAX1 were co-transfected into HEK293T cells, and co-immunoprecipitation analysis was performed using an antibody against FLAG. As a result, HAX1 did not bind to the C2B domain of CPNE1 and , it was confirmed that it binds to the C2A and A domains (FIG. 2B). In addition, as a result of yeast protein hybridization analysis with HAX1 and the C2A and A domains of CPNE1, it was confirmed that HAX1 binds to the C2A and A domains of CPNE1 (FIG. 2C).
실시예 3. HAX1-CPNE1 결합에 관여하는 HAX1 단백질의 결합부위 확인Example 3. Identification of binding site of HAX1 protein involved in HAX1-CPNE1 binding
HAX1과 CPNE1 단백질의 결합에 관여하는 HAX1 단백질의 결합부위를 확인하기 위해, HAX1의 N 말단과 C 말단을 분리하였고, 분리된 각 단편들의 N 말단을 GFP로 표지하였다(도 3A). 이렇게 만들어진 각각의 HAX1 돌연변이체와 FLAG-CPNE1을 HEK293T 세포 내로 동시 형질감염시키고 FLAG 또는 GFP에 대한 항체를 이용하여 동시 면역침전 분석을 수행한 결과, CPNE1이 HAX1의 N 말단과 결합하는 것을 확인하였다(도 3B 왼쪽 패널). In order to confirm the binding site of the HAX1 protein involved in the binding of HAX1 and CPNE1 proteins, the N-terminus and C-terminus of HAX1 were separated, and the N-terminus of each of the separated fragments was labeled with GFP (FIG. 3A). Each of the HAX1 mutants thus prepared and FLAG-CPNE1 were co-transfected into HEK293T cells, and co-immunoprecipitation analysis was performed using antibodies against FLAG or GFP. As a result, it was confirmed that CPNE1 binds to the N-terminus of HAX1 ( Fig. 3B left panel).
HAX1 단백질의 결합 부위를 더 구체적으로 규명하기 위해, HAX1 단백질의 N 말단 부위에서 BH 도메인을 포함하는 N 말단(N1) 및 PEST 서열을 포함하는 N 말단(N2)을 분리하였고, GFP로 표지하였다(도 3A). 이렇게 만들어진 각각의 HAX1 돌연변이체와 HA-CPNE1을 HEK293T 세포 내로 공동 형질감염시키고 GFP 또는 HA에 대한 항체를 이용하여 동시 면역침전 분석을 수행한 결과, CPNE1이 HAX1의 N1 부위에 결합하는 것을 확인하였다(도 3B 오른쪽 패널). In order to more specifically identify the binding site of the HAX1 protein, the N-terminal (N1) containing the BH domain and the N-terminal (N2) containing the PEST sequence were isolated from the N-terminal region of the HAX1 protein and labeled with GFP ( Figure 3A). Each of the HAX1 mutants and HA-CPNE1 thus prepared were co-transfected into HEK293T cells, and co-immunoprecipitation analysis was performed using an antibody against GFP or HA. As a result, it was confirmed that CPNE1 binds to the N1 site of HAX1 ( Fig. 3B right panel).
HAX1의 결합 부위를 보다 더 구체적으로 규명하기 위해, HAX1 단백질의 BH 도메인을 포함하는 N 말단(N1) 부위에서 N1△0, N1△acid box, N1△BH1 및 N1△BH2를 분리하였고, GFP로 표지하였다(도 3C). 이렇게 만들어진 각각의 HAX1 돌연변이체와 FLAG-CPNE1을 HEK293T 세포 내로 공동 형질감염시키고 FLAG 또는 GFP에 대한 항체를 이용하여 동시 면역침전 분석을 수행한 결과, CPNE1이 HAX1의 N1△0 부위에 결합하지 않고 N1△acid box, N1△BH1 및 N1△BH2 부위에 결합하는 것을 확인하였다(도 3D). In order to more specifically identify the binding site of HAX1, N1Δ0, N1Δacid box, N1ΔBH1 and N1ΔBH2 were isolated from the N-terminal (N1) region containing the BH domain of the HAX1 protein, and GFP labeled (Fig. 3C). Each of these HAX1 mutants and FLAG-CPNE1 were co-transfected into HEK293T cells, and co-immunoprecipitation analysis was performed using antibodies against FLAG or GFP. As a result, CPNE1 did not bind to the N1Δ0 site of HAX1 and Binding to the Δacid box, N1ΔBH1 and N1ΔBH2 sites was confirmed (Fig. 3D).
실시예 4. HiB5 세포에서 HAX1에 의한 분화 조절능 분석Example 4. Analysis of differentiation control ability by HAX1 in HiB5 cells
CPNE1 단백질과 관련된 신경세포 분화 조절능에 대한 HAX1 단백질의 영향을 규명하기 위해, 도 4A에 나타낸 바와 같이 HiB5 세포에 각각의 아데노바이러스를 공동 형질감염시킨 후, 분화 관련 마커로 알려진 인산화-AKT 및 Tuj1 단백질의 발현 변화를 분석하였다. In order to investigate the effect of HAX1 protein on neuronal differentiation control ability related to CPNE1 protein, as shown in Figure 4A, after co-transfection of each adenovirus into HiB5 cells, phosphorylated-AKT and Tuj1 protein known as differentiation-related markers The expression change of was analyzed.
그 결과, HiB5 세포에서 CPNE1과 HAX1△1-30(CPNE1과 결합하지 않는 부위 포함)를 동시 과발현시킨 실험구의 인산화-AKT 및 Tuj1 단백질 발현양이 CPNE1 단독 과발현시킨 대조구와 유사한 것을 확인하였다(도 4B). 반면, HiB5 세포에서 CPNE1과 HAX1(CPNE1과 결합하는 부위 포함)을 동시 과발현시킨 실험구의 인산화-AKT 및 Tuj1 단백질 발현양이 CPNE1 단독 과발현시킨 대조구보다 현저하게 감소한 것을 확인하였다(도 4C, D). As a result, it was confirmed that the expression levels of phospho-AKT and Tuj1 protein in the experimental group in which CPNE1 and HAX1Δ1-30 (including the non-CPNE1 binding site) were simultaneously overexpressed in HiB5 cells were similar to those in the control group in which CPNE1 was overexpressed alone (FIG. 4B ). On the other hand, it was confirmed that the expression levels of phosphorylated-AKT and Tuj1 protein in the experimental group in which CPNE1 and HAX1 (including the CPNE1 binding site) were simultaneously overexpressed in HiB5 cells were significantly decreased compared to the control group in which CPNE1 was overexpressed alone (Fig. 4C, D).
또한, HiB5 세포에 각각의 아데노바이러스를 공동 형질감염시킨 후, 72시간 동안 분화를 유도한 결과, CPNE1/HAX1 동시 과발현시킨 실험구의 신경돌기 성장이 CPNE1 단독 과발현 또는 CPNE1/HAX1△1-30 동시 과발현시킨 실험구보다 감소한 것을 확인하였다(도 4E). In addition, as a result of inducing differentiation for 72 hours after co-transfection of each adenovirus into HiB5 cells, neurite outgrowth of experimental groups in which CPNE1/HAX1 co-overexpression was observed by CPNE1 alone overexpression or CPNE1/HAX1Δ1-30 co-overexpression It was confirmed that it decreased compared to the experimental group (Fig. 4E).
상기 결과를 바탕으로, HAX1 단백질은 CPNE1 단백질과 결합함으로써 CPNE1에 의한 신경세포의 분화를 억제하는 것을 알 수 있었다. Based on the above results, it was found that the HAX1 protein inhibits the differentiation of neurons by CPNE1 by binding to the CPNE1 protein.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Composition for regulating neuronal differentiation and biomarker for detecting neuronal differentiation containing HAX1 <130> PN21122 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 279 <212> PRT <213> Homo sapiens <400> 1 Met Ser Leu Phe Asp Leu Phe Arg Gly Phe Phe Gly Phe Pro Gly Pro 1 5 10 15 Arg Ser His Arg Asp Pro Phe Phe Gly Gly Met Thr Arg Asp Glu Asp 20 25 30 Asp Asp Glu Glu Glu Glu Glu Glu Gly Gly Ser Trp Gly Arg Gly Asn 35 40 45 Pro Arg Phe His Ser Pro Gln His Pro Pro Glu Glu Phe Gly Phe Gly 50 55 60 Phe Ser Phe Ser Pro Gly Gly Gly Ile Arg Phe His Asp Asn Phe Gly 65 70 75 80 Phe Asp Asp Leu Val Arg Asp Phe Asn Ser Ile Phe Ser Asp Met Gly 85 90 95 Ala Trp Thr Leu Pro Ser His Pro Pro Glu Leu Pro Gly Pro Glu Ser 100 105 110 Glu Thr Pro Gly Glu Arg Leu Arg Glu Gly Gln Thr Leu Arg Asp Ser 115 120 125 Met Leu Lys Tyr Pro Asp Ser His Gln Pro Arg Ile Phe Gly Gly Val 130 135 140 Leu Glu Ser Asp Ala Arg Ser Glu Ser Pro Gln Pro Ala Pro Asp Trp 145 150 155 160 Gly Ser Gln Arg Pro Phe His Arg Phe Asp Asp Val Trp Pro Met Asp 165 170 175 Pro His Pro Arg Thr Arg Glu Asp Asn Asp Leu Asp Ser Gln Val Ser 180 185 190 Gln Glu Gly Leu Gly Pro Val Leu Gln Pro Gln Pro Lys Ser Tyr Phe 195 200 205 Lys Ser Ile Ser Val Thr Lys Ile Thr Lys Pro Asp Gly Ile Val Glu 210 215 220 Glu Arg Arg Thr Val Val Asp Ser Glu Gly Arg Thr Glu Thr Thr Val 225 230 235 240 Thr Arg His Glu Ala Asp Ser Ser Pro Arg Gly Asp Pro Glu Ser Pro 245 250 255 Arg Pro Pro Ala Leu Asp Asp Ala Phe Ser Ile Leu Asp Leu Phe Leu 260 265 270 Gly Arg Trp Phe Arg Ser Arg 275 <210> 2 <211> 3247 <212> DNA <213> Homo sapiens <400> 2 gacgcctcgc tcaatttctc acagggctgc gcaggtttcc cccgtctgcg aatggaccac 60 tggaggggtt caaaggttcg cgtcccagta cgggaatgag cctctttgat ctcttccggg 120 gctttttcgg ctttcctgga cctcggaggt gagagtaggt ccggctcgga caagggtggg 180 ggtcgtctga ggggagcttg acccctacgt cttatttttg gaaaaacatc ctctgtccca 240 ctccttcaac tcctgcagag aggacagaag tcgcaacatt gaacactcac cccgccacag 300 taaccaaggc ggtcattaag aggagaaaca gcaaactaag cctttctcca acctggggtg 360 atgcaacagg gacccgggcg gggaagaccg tgagggtctg gggaataaga cagtgagcaa 420 gtgagcagga ccttgggaga gagaggaggg actggggcac actgaagaaa aactggggga 480 aggtgtatga agggagctgc gagctgaggt ctgactttga ttaaaaaaaa aaaaaaaaaa 540 aaagaactgt cagccattgt attaatgttt tgatgtggca gccagtcctc cgaccctctc 600 cctagcttcc cagacccctt gctcttgtcc cactttgcca cccatgagtt gatttaatgg 660 cttaaatagt gctgaaatat tggtggccaa tctgcctcca ctctcagcca cagagatccc 720 ttttttggag ggatgactcg agatgaagat gatgatgagg aagaagaaga agaagggggc 780 tcatggggcc gtgggaaccc aaggttccat agtcctcagc acccccctga ggaatttggc 840 ttcggcttca gcttcagccc aggaggaggg atacgtttcc acgataactt cggctttgat 900 gacctagtac gagatttcaa tagcatcttc agcgatatgg gggcctggac cttgccttcc 960 catcctcctg gtgtgtggct ttccctaagg ggcaacctgt ggtttctggt gggttggtgg 1020 gtgaaataaa gagcctgcag ggagtagctg ggggatggga agtgtgagaa gactgatgat 1080 ttcagagaga ttaatagagc ccaagtcctt tcccatccca gcaaacacct gccacctttc 1140 tgcagaactt ccaggtcctg agtcagagac acctggtgag agactacggg agggacagac 1200 acttcgggac tcaatgctta agtatccaga tagtcaccag cccaggatct ttgggggggt 1260 cttggagagt gatgcaagaa gtgaatcccc ccaaccagca ccagactggg gctcccagag 1320 gccatttcat agggtgagta tcccatctgg tcctgaagtg agagcttgtg agagaccact 1380 aataaagtgc aaagactggc taggtgcggt ggctcacgcc tgtaatccca gcactttggg 1440 aggccgaggt gggcagatca ccagaggtca ggagttggag accagcctgg tcaacatggt 1500 gaaaccccgt gtctactaaa aatacaaaaa attagccggg catggtggca ggtgcctgta 1560 atgccagcta ctcaggaggc tgaggcagga gaattggttg aacccaggag gtggagactc 1620 catctcaaaa attgtttgag acccagcctt gctctgttgc ccaggctgta gtgccgtggc 1680 acaatctcag ctcactgcaa cctccaattc ccaggttcaa gtgattctcc tgcctcagcc 1740 tcctgagtag ctgggattac agtcgcctgc caaacgccca gctaattttt gtgtttttag 1800 tagagatggc gtttcaccgt gttggccagg ctgcttttga gctcctgacc tcaggtgatc 1860 cacccacctc ggcctcccaa agtgctggaa ttacaagtgt gagccatcat gcctggccaa 1920 agattgcaat tcttgtttga atctgatagc cttgggttgg aatcctaagc cctccattta 1980 gtgttttctg ttttctatat taagcttgtg ggattttctt tggggggaga tgggtgttct 2040 gtttctttta aaaaacaaaa acaaaaaaac accactggtc tagataattc ttggataagg 2100 caggattgtg cacagaggat aggaatatat cagttcagga agtctttctg gtagaaggat 2160 acataaaaca gccaggaagg agtgtgtaaa taaggctatt ctgaatggaa ttatctcttc 2220 tgtggaaggg ggttttggag ctcgggagta gtttgaggtc tatgaccttt caaatttcag 2280 attggaagga gtcttttcac ttactatggt ttcttctgca gtttgatgat gtatggccta 2340 tggaccccca tcctagaacc agagaggaca atggtaagtc tggaggaagg ggaagtttac 2400 cagccttttg ttattcttct gaagttctgt gtgttctccc tccctgaagt ttcttcctgt 2460 acacttgtct ccttttttcc tttggactct ttctctcctg cttcttcatc tctctgctct 2520 tccagatctt gattcccagg tttcccagga gggtcttggc ccggttctac agccccagcc 2580 caaatcctat ttcaagagca tctctgtgac caagatcact aaaccagatg gggtgagttg 2640 aaagaaagag gtaaaggaaa gtatggccag ggaacgaatg ccctgaaaca gggatctgtg 2700 taaagaaact gctgagcata acctttagta acattcggaa tatggtgggg acttctcttt 2760 gtagatagtg gaggagcgcc ggactgtggt ggacagtgag ggccggacag agactacagt 2820 aacccgacac gaagcagata gcagtcctag gggtggtaag ttaaaagaca aaggggttca 2880 tctcaagatt ccttggggaa gggaaatctt actcttctac ccttgtcctt tgcttctgcc 2940 tagtctcttt catttagcct atttacgtgt atgactttct tccttagatc cagaatcacc 3000 aagacctcca gccctggatg atgccttttc catcctggac ttattcctgg gacgttggtt 3060 ccggtcccgg tagccttgtt aaccctcaga ggccttcaag tcctttccac ctctcaccca 3120 ttgcccacca ttaataagct tagcttctct tgccacctca ggggcttgga tatgtggaat 3180 agtgaactgg ggccatgtca gtttgtcact cacccaaact gaccaataaa acctttattt 3240 atgctaa 3247 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Composition for regulating neuronal differentiation and biomarker for detecting neuronal differentiation containing HAX1 <130> PN21122 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 279 <212> PRT <213> Homo sapiens <400> 1 Met Ser Leu Phe Asp Leu Phe Arg Gly Phe Phe Gly Phe Pro Gly Pro 1 5 10 15 Arg Ser His Arg Asp Pro Phe Phe Gly Gly Met Thr Arg Asp Glu Asp 20 25 30 Asp Asp Glu Glu Glu Glu Glu Glu Gly Gly Ser Trp Gly Arg Gly Asn 35 40 45 Pro Arg Phe His Ser Pro Gln His Pro Pro Glu Glu Phe Gly Phe Gly 50 55 60 Phe Ser Phe Ser Pro Gly Gly Gly Ile Arg Phe His Asp Asn Phe Gly 65 70 75 80 Phe Asp Asp Leu Val Arg Asp Phe Asn Ser Ile Phe Ser Asp Met Gly 85 90 95 Ala Trp Thr Leu Pro Ser His Pro Pro Glu Leu Pro Gly Pro Glu Ser 100 105 110 Glu Thr Pro Gly Glu Arg Leu Arg Glu Gly Gln Thr Leu Arg Asp Ser 115 120 125 Met Leu Lys Tyr Pro Asp Ser His Gln Pro Arg Ile Phe Gly Gly Val 130 135 140 Leu Glu Ser Asp Ala Arg Ser Glu Ser Pro Gln Pro Ala Pro Asp Trp 145 150 155 160 Gly Ser Gln Arg Pro Phe His Arg Phe Asp Asp Val Trp Pro Met Asp 165 170 175 Pro His Pro Arg Thr Arg Glu Asp Asn Asp Leu Asp Ser Gln Val Ser 180 185 190 Gln Glu Gly Leu Gly Pro Val Leu Gln Pro Gln Pro Lys Ser Tyr Phe 195 200 205 Lys Ser Ile Ser Val Thr Lys Ile Thr Lys Pro Asp Gly Ile Val Glu 210 215 220 Glu Arg Arg Thr Val Val Val Asp Ser Glu Gly Arg Thr Glu Thr Thr Val 225 230 235 240 Thr Arg His Glu Ala Asp Ser Ser Pro Arg Gly Asp Pro Glu Ser Pro 245 250 255 Arg Pro Pro Ala Leu Asp Asp Ala Phe Ser Ile Leu Asp Leu Phe Leu 260 265 270 Gly Arg Trp Phe Arg Ser Arg 275 <210> 2 <211> 3247 <212> DNA <213> Homo sapiens <400> 2 gacgcctcgc tcaatttctc acagggctgc gcaggtttcc cccgtctgcg aatggaccac 60 tggaggggtt caaaggttcg cgtcccagta cgggaatgag cctctttgat ctcttccggg 120 gctttttcgg ctttcctgga cctcggaggt gagagtaggt ccggctcgga caagggtggg 180 ggtcgtctga ggggagcttg acccctacgt cttatttttg gaaaaacatc ctctgtccca 240 ctccttcaac tcctgcagag aggacagaag tcgcaacatt gaacactcac cccgccacag 300 taaccaaggc ggtcattaag aggagaaaca gcaaactaag cctttctcca acctggggtg 360 atgcaacagg gacccgggcg gggaagaccg tgagggtctg gggaataaga cagtgagcaa 420 gtgagcagga ccttgggaga gagaggaggg actggggcac actgaagaaa aactggggga 480 aggtgtatga aggggagctgc gagctgaggt ctgactttga ttaaaaaaaa aaaaaaaaaa 540 aaagaactgt cagccattgt attaatgttt tgatgtggca gccagtcctc cgaccctctc 600 cctagcttcc cagacccctt gctcttgtcc cactttgcca cccatgagtt gatttaatgg 660 cttaaatagt gctgaaatat tggtggccaa tctgcctcca ctctcagcca cagagatccc 720 ttttttggag ggatgactcg agatgaagat gatgatgagg aagaagaaga agaagggggc 780 tcatggggcc gtgggaaccc aaggttccat agtcctcagc acccccctga ggaatttggc 840 ttcggcttca gcttcagccc aggagggaggg atacgtttcc acgataactt cggctttgat 900 gacctagtac gagatttcaa tagcatcttc agcgatatgg gggcctggac cttgccttcc 960 catcctcctg gtgtgtggct ttccctaagg ggcaacctgt ggtttctggt gggttggtgg 1020 gtgaaataaa gagcctgcag ggagtagctg ggggatggga agtgtgagaa gactgatgat 1080 ttcagagaga ttaatagagc ccaagtcctt tcccatccca gcaaacacct gccacctttc 1140 tgcagaactt ccaggtcctg agtcagagac acctggtgag agactacggg aggggacagac 1200 acttcgggac tcaatgctta agtatccaga tagtcaccag cccaggatct ttgggggggt 1260 cttggagagt gatgcaagaa gtgaatcccc ccaaccagca ccagactggg gctcccagag 1320 gccatttcat agggtgagta tcccatctgg tcctgaagtg agagcttgtg agagaccact 1380 aataaagtgc aaagactggc taggtgcggt ggctcacgcc tgtaatccca gcactttggg 1440 aggccgaggt gggcagatca ccagaggtca ggagttggag accagcctgg tcaacatggt 1500 gaaaccccgt gtctactaaa aatacaaaaa attagccggg catggtggca ggtgcctgta 1560 atgccagcta ctcaggaggc tgaggcagga gaattggttg aacccaggag gtggagactc 1620 catctcaaaa attgtttgag acccagcctt gctctgttgc ccaggctgta gtgccgtggc 1680 acaatctcag ctcactgcaa ccctccaattc ccaggttcaa gtgattctcc tgcctcagcc 1740 tcctgagtag ctgggattac agtcgcctgc caaacgccca gctaattttt gtgtttttag 1800 tagagatggc gtttcaccgt gttggccagg ctgcttttga gctcctgacc tcaggtgatc 1860 cacccacctc ggcctcccaa agtgctggaa ttacaagtgt gagccatcat gcctggccaa 1920 agattgcaat tcttgtttga atctgatagc cttgggttgg aatcctaagc cctccattta 1980 gtgttttctg ttttctatat taagcttgtg ggattttctt tggggggaga tgggtgttct 2040 gtttctttta aaaaacaaaa acaaaaaaac accactggtc tagataattc ttggataagg 2100 2160 acataaaaca gccaggaagg agtgtgtaaa taaggctatt ctgaatggaa ttatctcttc 2220 tgtggaaggg ggttttggag ctcgggagta gtttgaggtc tatgaccttt caaatttcag 2280 attggaagga gtcttttcac ttactatggt ttcttctgca gtttgatgat gtatggccta 2340 tggaccccca tcctagaacc agagaggaca atggtaagtc tggaggaagg ggaagtttac 2400 cagccttttg ttattcttct gaagttctgt gtgttctccc tccctgaagt ttcttcctgt 2460 acacttgtct ccttttttcc tttggactct ttctctcctg cttcttcatc tctctgctct 2520 tccagatctt gattcccagg tttcccagga gggtcttggc ccggttctac agccccagcc 2580 caaatcctat ttcaagagca tctctgtgac caagatcact aaaccagatg gggtgagttg 2640 aaagaaagag gtaaaggaaa gtatggccag ggaacgaatg ccctgaaaca gggatctgtg 2700 taaagaaact gctgagcata acctttagta acattcggaa tatggtgggg acttctcttt 2760 gtagatagtg gaggagcgcc ggactgtggt ggacagtgag ggccggacag agactacagt 2820 aacccgacac gaagcagata gcagtcctag gggtggtaag ttaaaagaca aaggggttca 2880 tctcaagatt ccttgggggaa gggaaatctt actcttctac ccttgtcctt tgcttctgcc 2940 tagtctcttt catttagcct atttacgtgt atgactttct tccttagatc cagaatcacc 3000 aagacctcca gccctggatg atgccttttc catcctggac ttatcctgg gacgttggtt 3060 ccggtcccgg tagccttgtt aaccctcaga ggccttcaag tcctttccac ctctcaccca 3120 ttgccaccca ttaataagct tagcttctct tgccacctca ggggcttgga tatgtggaat 3180 agtgaactgg ggccatgtca gtttgtcact cacccaaact gaccaataaa accttattt 3240 atgctaa 3247
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