KR20210123861A - Composition for diagnosing and treating anti-cancer drug resistance - Google Patents

Composition for diagnosing and treating anti-cancer drug resistance Download PDF

Info

Publication number
KR20210123861A
KR20210123861A KR1020200041598A KR20200041598A KR20210123861A KR 20210123861 A KR20210123861 A KR 20210123861A KR 1020200041598 A KR1020200041598 A KR 1020200041598A KR 20200041598 A KR20200041598 A KR 20200041598A KR 20210123861 A KR20210123861 A KR 20210123861A
Authority
KR
South Korea
Prior art keywords
cancer
drug resistance
anticancer drug
sdha
kit
Prior art date
Application number
KR1020200041598A
Other languages
Korean (ko)
Other versions
KR102393682B1 (en
Inventor
정재호
김정민
Original Assignee
연세대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 연세대학교 산학협력단 filed Critical 연세대학교 산학협력단
Priority to KR1020200041598A priority Critical patent/KR102393682B1/en
Priority to PCT/KR2021/004253 priority patent/WO2021206406A1/en
Publication of KR20210123861A publication Critical patent/KR20210123861A/en
Application granted granted Critical
Publication of KR102393682B1 publication Critical patent/KR102393682B1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Wood Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Endocrinology (AREA)

Abstract

The present invention relates to a composition for diagnosing or treating anticancer drug resistance diagnosing and overcoming the anticancer drug resistance using a novel biomarker composition for diagnosing the anticancer drug resistance. It is expected that patient's cost burden can be reduced by predicting the suitability of anticancer drugs for each patient prior to administration and providing accurate basic information, and a survival rate can be improved by treating anticancer drug resistance.

Description

항암제 내성 진단 또는 치료용 조성물{Composition for diagnosing and treating anti-cancer drug resistance}Composition for diagnosing and treating anti-cancer drug resistance

본 발명은 항암제 내성 진단용 조성물 및 항암제 내성 치료용 조성물에 관한 것이다.The present invention relates to a composition for diagnosing anticancer drug resistance and a composition for treating anticancer drug resistance.

암은 인류가 해결해야 할 난치병 중의 하나로, 전 세계적으로 이를 치유하기 위한 개발에 막대한 자본이 투자되고 있는 실정이며, 우리나라의 경우, 질병 사망 원인 중 제 1위의 질병으로서 연간 약 10만 명 이상이 진단되고, 약 6만 명 이상이 사망하고 있다. 특히 위암은 2018년 전 세계에서 5번째로 가장 빈번하게 진단되었으며 지난 10년간 암 진단과 치료에 있어 다양한 항암 요법이 비약적으로 발전하고 있지만, 암 발병으로 인한 치사율은 여전히 높다. 또한 다양한 항암제 및 여러 항암 요법을 시도할 때 수반되는 부작용도 여전히 존재한다. 이러한 부작용을 줄이기 위한 연구가 활발히 진행되고 있는 실정이다.Cancer is one of the incurable diseases that mankind needs to solve, and huge capital is being invested in development to cure it worldwide. Diagnosed and more than 60,000 people have died. In particular, gastric cancer was diagnosed as the 5th most frequently diagnosed worldwide in 2018, and although various anticancer therapies have been developed rapidly in cancer diagnosis and treatment over the past decade, the fatality rate due to cancer is still high. In addition, side effects that accompany various anticancer drugs and various anticancer therapies still exist. Research to reduce these side effects is being actively conducted.

약물에 대한 저항성(drug resistance)은 성공적인 항암 치료 효과를 반감시키는 주된 요소로 다양한 암종의 예후를 나쁘게 한다. 암 세포에서의 약물 저항성(chemo-resistance)은 기존에 내재되어 있던 약물 저항성(pre-existing of resistance-mediating factor)과 약물 투여로 인하여 새롭게 획득된 약물 저항성(acquired drug resistance)으로 나뉜다(Cancer drug resistance: an evolving paradigm. Nat Rev Cancer. 2013 Oct;13(10):714-26.). 후천적으로 획득된 약물 저항성을 일으키는 요인으로는 약물 유출 증가, 약물 표적의 돌연변이 발생, DNA 손상 복구, 대체 신호 경로의 활성화 또는 약물 저항성에 의해 유도된 세포 사멸의 회피 등을 예로 들 수 있다. 이렇듯 약물 내성을 지닌 환자에게 해당 약물로 치료한다 할지라도 치료의 효과를 보장할 수 없어 임상 의사 및 환자들의 시간 및 비용이 불필요하게 소모되는 상황이 발생할 가능성이 존재하게 된다. 따라서 항암 치료를 시작할 시에는 치료에 따른 환자의 개개인별 특성을 고려하여 결정하여야 하며, 무작정 치료를 감행하기보다는 개개인에게 맞는 맞춤형 항암제를 선택적으로 사용할 수 있도록 특정 바이오 마커를 통해 미리 치료 효율을 가늠할 수 있는 척도가 현실적으로 필요한 상황이다. 이렇듯 약물에 대한 내성과 관련된 연구는 거의 없는 실정이기에 본 발명자들은 항암제 내성이 있는 환자를 미리 선별할 수 있는 신규한 마커를 발굴함으로써 본 발명을 완성하기에 이르렀다.Drug resistance is a major factor that counteracts the effectiveness of successful chemotherapy, and worsens the prognosis of various carcinomas. Chemo-resistance in cancer cells is divided into pre-existing of resistance-mediating factor and newly acquired drug resistance due to drug administration (Cancer drug resistance). : an evolving paradigm. Nat Rev Cancer. 2013 Oct;13(10):714-26.). Factors causing acquired drug resistance include increased drug efflux, mutagenesis of drug targets, DNA damage repair, activation of alternative signaling pathways, or avoidance of cell death induced by drug resistance. Even if a patient with drug resistance is treated with the drug, the effect of the treatment cannot be guaranteed, so there is a possibility that the time and money of the clinician and the patient are wasted unnecessarily. Therefore, when starting anticancer treatment, it is necessary to make a decision in consideration of the individual characteristics of the patient according to the treatment, and the treatment efficiency can be estimated in advance through a specific biomarker so that a customized anticancer drug can be selectively used for each individual rather than blindly conducting treatment. It is a situation in which there is a realistic need for a measure. As such, since there are few studies related to drug resistance, the present inventors have completed the present invention by discovering a novel marker capable of pre-selecting patients with anticancer drug resistance.

본 발명의 일 목적은 항암 약제에 대한 내성을 진단하기 위한 항암제 내성 진단용 조성물을 제공하고자 한다.An object of the present invention is to provide a composition for diagnosing anticancer drug resistance for diagnosing resistance to anticancer drugs.

본 발명의 다른 목적은 항암 약제 치료 시 내성 발생 유무를 사전에 예측할 수 있는 항암제 내성 진단용 키트를 제공하고자 한다.Another object of the present invention is to provide a kit for diagnosing anticancer drug resistance that can predict in advance whether or not resistance develops during anticancer drug treatment.

본 발명의 또 다른 목적은 항암 약제 치료 시에 나타날 내성 발생 유무를 진단하기 위한 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하고자 한다.Another object of the present invention is to provide a method for providing information for diagnosing anticancer drug resistance for diagnosing whether or not resistance occurs during anticancer drug treatment.

본 발명의 또 다른 목적은 항암 약물 내성을 극복하고 더 나아가 그에 대한 감수성을 증진시켜 암을 치료할 수 있는 항암제 내성 치료용 조성물을 제공하고자 한다.Another object of the present invention is to provide a composition for anticancer drug resistance treatment capable of treating cancer by overcoming anticancer drug resistance and further enhancing its sensitivity.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

이하, 본원에 기재된 다양한 구체예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세 사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구체예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구체예" 또는 "구체예"에 대한 본 명세서 전체를 통한 참조는 구체예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구체예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구체예에서" 또는 "구체예"의 상황은 반드시 본 발명의 동일한 구체예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구체예에서 어떠한 적합한 방법으로 조합될 수 있다.Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

명세서 내에 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다. Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

본 발명의 일 구현예에 따르면, SDHA(succinate dehydrogenase complex flavoprotein subunit A) 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 제제를 포함하는 항암제 내성 진단용 바이오 마커 조성물을 제공한다.According to one embodiment of the present invention, there is provided a biomarker composition for diagnosing anticancer drug resistance, comprising an agent for measuring the expression level of a succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein or a gene encoding the same.

본 발명의 일 구체예에서, 상기 "SDHA(succinate dehydrogenase complex flavoprotein subunit A)"란 미토콘드리아 호흡 사슬의 복합체인 숙신산-유비퀴논 산화환원효소(succinate-ubiquinone oxidoreductase)의 주요 촉매 서브 유닛을 암호화한다. 복합체는 4개의 핵으로 인코딩된 서브 유닛으로 구성되며 미토콘드리아 내막에 위치한다. 상기 유전자는 FAD 결합 부위를 포함하며 이 부위에서 숙신산이 푸마르산으로 전환되고 탈양성자화되는 것으로 알려져 있다. 상기 유전자 돌연변이는 리 증후군(Leigh Syndrome)으로 알려진 미토콘드리아 호흡 사슬 결핍으로 에너지 생산과정의 이상이 생겨 발생하는 신경변성질환과 관련이 있다. 상기 SDHA 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며, 본 발명에서의 SDHA의 아미노산 서열, 및 이를 코딩하는 핵산 염기 서열을 각각 서열번호 1과 서열번호 2에 나타내었다.In one embodiment of the present invention, the term "succinate dehydrogenase complex flavoprotein subunit A (SDHA)" encodes a major catalytic subunit of succinate-ubiquinone oxidoreductase, a complex of the mitochondrial respiratory chain. The complex consists of four nuclear-encoded subunits located in the inner mitochondrial membrane. The gene contains a FAD binding site, where it is known that succinic acid is converted to fumaric acid and deprotonated. The gene mutation is related to a neurodegenerative disease caused by an abnormality in the energy production process due to a deficiency of the mitochondrial respiratory chain known as Leigh Syndrome. The SDHA protein and genetic information is registered with the National Center for Biotechnology Information (NCBI), and the amino acid sequence of SDHA in the present invention and the nucleic acid base sequence encoding it are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

본 발명에서 상기 "SDHA 단백질 또는 이를 코딩하는 유전자"는 서열번호 1로 표시되는 아미노산 서열 또는 서열번호 2로 표시되는 염기서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다. 비제한적인 예에서 상기 SDHA의 서열과 99% 이상 내지 100% 미만, 95% 이상 내지 99% 미만, 90% 이상 내지 95% 미만, 85% 이상 내지 90% 미만, 또는 80% 이상 내지 85% 미만의 상동성을 가지는 경우일 수 있으며, 당해 분야의 통상의 기술자에게 본 발명의 목적하는 효과를 발휘한다는 것이 자명한 범위 내에서 이에 제한 없이 모두 포함할 수 있다.In the present invention, the "SDHA protein or a gene encoding the same" may consist of an amino acid sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto. 99% to less than 100%, 95% to less than 99%, 90% to less than 95%, 85% to less than 90%, or 80% to less than 85% of the sequence of SDHA in a non-limiting example It may be a case of having the homology of, and it may be included without limitation within the range that is obvious to those skilled in the art to exert the desired effect of the present invention.

본 발명에서 “약제" 또는 "항암 치료제”와 “항암제”는 혼용하여 사용될 수 있으며, 상기 약제는 암 줄기세포뿐만 아니라 암 세포를 사멸시켜 항암 효과가 있는 약물에 해당하며, 보다 바람직하게는 위암 치료 효과가 있는 약물을 말한다. In the present invention, "drug" or "anticancer agent" and "anticancer agent" may be used interchangeably, and the agent corresponds to a drug having an anticancer effect by killing cancer cells as well as cancer stem cells, and more preferably gastric cancer treatment drugs that are effective.

본 발명의 일 구체예에서, 상기 "항암제"란 암 세포를 사멸시키는 기전을 가진 약물로서 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투모맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 데시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르모퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈크리스틴, 빈블라스틴, 테니포시드, 독소루비신, 이다루비신, 에피루비신, 미톡산트론, 미토마이신, 블레로마이신, 다우노루비신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테모졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 나벨빈, 파드라졸, 타목시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상을 포함하는 것 일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the "anti-cancer agent" is a drug having a mechanism to kill cancer cells, such as nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib , nirotinib, semasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab Mab, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, Uracil, cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmopher, raltitrexed, docetaxel, paclitaxel, irinotecan, velotecan , topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin , pyrarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melparan, altretmine, dacarbazine, thiotepa, nimustine, Chlorambucil, Mitolactol, Leucovorin, Tretonin, Exmestane, Aminoglutethimide, Anagrelide, Nabelbine, Padrazole, Tamoxifen, Torremifene, Testolactone, Anastrozole, Letrozole , vorozol, bicalutamide, lomustine and carmustine may be one or more selected from the group consisting of, but is not limited thereto.

본 발명의 일 구체예에서, 상기 "항암제"는 OXPHOS 억제제에 해당할 수 있으며, 상기 약제는 미토콘드리아의 산화적 인산화를 억제하여 암 세포를 사멸시키는 새로운 기전을 갖는 항암제에 해당한다. 다양한 유형의 면역 세포는 세포 생존, 발달 및 기능을 유지하기 위해 특정한 세포 대사 과정을 이용하므로 상기 약제는 ATP 생성을 억제함으로써 암 치료 효과를 높이는 데에 사용될 수 있다. 상기 항암제는 OXPHOS 억제제의 일 유형에 해당한다면 이에 제한 없이 모두 포함할 수 있다.In one embodiment of the present invention, the "anticancer agent" may correspond to an OXPHOS inhibitor, and the agent corresponds to an anticancer agent having a new mechanism to kill cancer cells by inhibiting oxidative phosphorylation of mitochondria. Since various types of immune cells utilize specific cellular metabolic processes to maintain cell survival, development and function, the drug can be used to enhance the effectiveness of cancer treatment by inhibiting ATP production. The anticancer agent may include all without limitation, if it corresponds to one type of OXPHOS inhibitor.

본 발명의 일 구체예에서, 상기 "OXPHOS 억제제"란 유비퀴논 환원 활성 억제제(ubiquinone reduction activity inhibitor), 미토콘드리아 전자 수송 사슬 Ⅰ 억제제(mitochondrial electron transport chain I inhibitor) 또는 철 킬레이터(iron chelator)로 이루어진 군에서 선택된 1종 이상을 포함할 수 있으며, 미토콘드리아의 산화적 인산화를 억제하여 암 세포를 사멸시키는 기전을 가진 약물이라면 이에 제한되지 않고 모두 포함될 수 있다.In one embodiment of the present invention, the "OXPHOS inhibitor" refers to a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator. It may include one or more selected from the group, and any drug having a mechanism to kill cancer cells by inhibiting oxidative phosphorylation of mitochondria may be included without being limited thereto.

본 발명에서 상기 유비퀴논 환원 활성 억제제는 IACS-010759일 수 있으나, 미토콘드리아 내막에 존재하는 지용성 전자운반체인 유비퀴논의 활성에 영향을 미치는 약물에 해당한다면 이에 제한되는 것은 아니다. In the present invention, the ubiquinone reducing activity inhibitor may be IACS-010759, but is not limited thereto as long as it corresponds to a drug that affects the activity of ubiquinone, a fat-soluble electron transporter present in the inner mitochondrial membrane.

본 발명에서 상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156, 메트포민(Metformin), 펜포르민(Phenformin) 및 로테논(Rotenone)으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 보다 바람직하게는 IM156 또는 로테논일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the mitochondrial electron transport chain I inhibitor may be at least one selected from the group consisting of IM156, metformin, phenformin, and rotenone, more preferably IM156 or rotenone. However, the present invention is not limited thereto.

본 발명에서 상기 철 킬레이터는 VLX 600일 수 있으나, 미토콘드리아 호흡을 억제하는 기능을 가진 화합물이라면 이에 제한되는 것은 아니다.In the present invention, the iron chelator may be VLX 600, but is not limited thereto as long as it is a compound having a function of inhibiting mitochondrial respiration.

본 발명에서 상기 "IM156 약물"은 이전에 HL156A로 알려진 새로운 비구아니드 유도체(biguanide derivative) 화합물의 일종으로 다른 비구아니드 약물과 유사하게 미토콘드리아 복합체 I(mitochondrial complex I)을 차단하는 작용을 하는 약물에 해당한다. 비구아니드에서 추출한 소분자 경구 약물로 강력한 산화성인산화(OXPHOS) 억제제로 알려져 있다. 최근 연구 결과에 따르면 IM156을 이용한 시험관내 배양된 쥐 복막 중피 세포 및 쥐 신장 근위관 세포의 치료 후, AMPK 활성은 메트포민과 같은 다른 AMPK (AMP-activated protein kinase)보다 효과가 더욱 좋다는 것이 밝혀져 있다(Am J Physiol Renal Physiol. 2016 Mar 1;310(5):F342-50.). 그러나 IM156 약물은 세포 유형에 따라 상이한 작용 방식으로 영향을 미치기 때문에 다양한 작용 기전에 관한 연구가 진행 중에 있다. In the present invention, the "IM156 drug" is a kind of a new biguanide derivative compound previously known as HL156A, and similarly to other biguanide drugs, a drug that blocks mitochondrial complex I corresponds to It is a small molecule oral drug derived from biguanide and is known as a potent oxidative phosphorylation (OXPHOS) inhibitor. According to the results of a recent study, after treatment of in vitro cultured rat peritoneal mesothelial cells and rat renal proximal duct cells using IM156, it was found that AMPK activity was more effective than other AMPK (AMP-activated protein kinase) such as metformin ( Am J Physiol Renal Physiol. 2016 Mar 1 ;310(5):F342-50.). However, since IM156 drugs affect different cell types in different ways of action, studies on various mechanisms of action are ongoing.

본 발명에서 상기 "로테논 약물"은 미토콘드리아 호흡 사슬 복합체 I(Mitochondrial Complex I)에 작용하여, 산화적 대사에 의존하는 종양 생장을 억제하는 기능을 하는 약물이다. 친유성을 띠며 콩과 식물인 론코카르푸스(Lonchocarpus) 및 데리스(Derris) 종의 뿌리와 줄기에서 자연적으로 발생하여 얻어지는 산물로 살충제로 널리 사용되었지만, 독성 효과가 있어 많은 국가에서 사용이 제한되기도 하였다. 또한, 로테논은 다양한 인간 암 세포주에서 세포 사멸을 일으켜 세포 증식을 억제하는 것으로 알려져 암 치료에 이용될 수 있도록 개발 중이다. 로테논 외 미토콘드리아 호흡 사슬 복합체 I에 작용하는 미토콘드리아 전자 수송 사슬 Ⅰ 억제제의 예로는 IM156, 메트포민(Metformin), 펜포르민(Phenformin) 등이 있다.In the present invention, the "rotenone drug" is a drug that functions to inhibit tumor growth dependent on oxidative metabolism by acting on mitochondrial respiratory chain complex I (Mitochondrial Complex I). It is a lipophilic, naturally occurring product obtained from the roots and stems of the legumes Lonchocarpus and Derris species. It has been widely used as an insecticide, but its toxic effect has limited its use in many countries. did. In addition, rotenone is known to inhibit cell proliferation by causing apoptosis in various human cancer cell lines, and is being developed for use in cancer treatment. Examples of mitochondrial electron transport chain I inhibitors that act on mitochondrial respiratory chain complex I other than rotenone include IM156, metformin, and phenformin.

본 발명에서 상기 "바이오 마커"란 체내 세포나 혈관, 단백질, DNA, RNA, 대사 물질 등을 이용하여 체내 변화를 알아낼 수 있는 생물학적 지표로, 미국 국립보건원(NIH)은 상기 바이오 마커를 정상적인 생물학적 과정, 질병 진행 상황, 치료 방법에 대한 약물의 반응성을 객관적으로 측정하고 평가할 수 있는 지표라고 정의하였다. 즉, 특정 질병이나 암의 경우 정상이나 병적인 상태를 구분할 수 있거나 치료 반응을 예측할 수 있고 이를 객관적으로 측정할 수 있는 표지자를 의미한다. 따라서 바이오 마커는 정상적인 생물학적 과정, 질병 진행 상황, 치료 방법에 대한 약물의 반응성을 객관적으로 측정하고 평가할 수 있는 역할을 하여야 한다. 활용도에 따라 약물 타깃의 존재를 확인하는 타깃 마커, 병의 유무를 진단하는 진단 마커, 특정 약물에 대한 반응군과 비반응군을 구별할 수 있는 예상 마커, 약물 치료 효과를 모니터링 할 수 있는 대리 표지자 마커, 질병의 예후를 알려주는 예후 바이오 마커 등이 존재한다. In the present invention, the "biomarker" is a biological indicator that can detect changes in the body using cells, blood vessels, proteins, DNA, RNA, metabolites, etc. in the body, and the National Institutes of Health (NIH) of the United States uses the biomarker as a normal biological process. , disease progression, and drug responsiveness to treatment methods were defined as indicators that can objectively measure and evaluate. That is, in the case of a specific disease or cancer, it refers to a marker that can distinguish normal or pathological conditions or predict treatment response and objectively measure it. Therefore, biomarkers should play a role that can objectively measure and evaluate a drug's responsiveness to normal biological processes, disease progression, and treatment methods. A target marker that confirms the presence of a drug target according to the utilization, a diagnostic marker that diagnoses the presence or absence of a disease, a predictive marker that can distinguish a responder from a non-responder to a specific drug, and a surrogate marker that can monitor the effect of a drug treatment There are markers, prognostic biomarkers indicating the prognosis of a disease, and the like.

본 발명에서 상기 "약제 내성"이란 약물을 정량 반복적으로 사용했을 때 해당 약물의 효과가 감소하는 것을 말하며, 약제 내성이 있는 환자에게 이전에 경험한 동일한 효과를 얻기 위해서는 그 사용량을 늘리거나 사용 빈도를 증가시켜야 하거나 혹은 이전과 같은 용량의 물질을 투여해도 전과 똑같은 효과를 얻지 못하는 상태를 말한다. 본 발명에서 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준의 변화를 대조군과 비교하여 증가하는 경우 약제 내성이 있는 것으로 진단하였다. In the present invention, the term "drug resistance" refers to a decrease in the effect of the drug when the drug is used repeatedly in a quantitative manner, and in order to obtain the same effect previously experienced in patients with drug resistance, increase the amount of the drug or increase the frequency of use It is a condition in which the same effect as before is not obtained even if the dose must be increased or the same dose of the substance is administered. In the present invention, when the change in the expression level of the SDHA protein or the gene encoding it is increased compared to the control group, drug resistance was diagnosed.

본 발명에서 상기 "진단"은 약제에 대한 감수성(susceptibility)을 판정하는 것, 발병한 질환이 약제 내성을 현재 가지고 있는지 여부를 판정하는 것, 또는 약제 내성 암의 예후(예컨대, 해당 약물에 대한 암의 반응성 결정)를 판정하는 것, 또는 상기 진단에 대한 정보를 제공하는 것을 포함하는 넓은 개념으로 정의한다.In the present invention, the "diagnosis" refers to determining susceptibility to a drug, determining whether an onset disease currently has drug resistance, or prognosis of drug-resistant cancer (eg, cancer to the drug) It is defined as a broad concept that includes determining the reactivity of

본 발명의 “예후”란, 질병의 경과 및 사망 또는 생존의 결과를 미리 예측하는 행위를 말한다. 상기 예후 또는 예후 진단이란 질환의 경과가 환자의 생리적 또는 환경적 상태에 따라 달라질 수 있으며, 이러한 환자의 상태를 종합적으로 고려하여 치료 전/후 질병의 경과를 예측하는 모든 행위를 의미하는 것으로 해석될 수 있다. 본 발명의 목적상 상기 예후는 항암제 치료, 바람직하게는 OXPHOS 억제제 치료 후 치료 반응성을 미리 예상하는 행위 또는 이를 토대로 OXPHOS 억제제 사용 여부를 적절하게 선택하는 행위로 해석될 수 있다. The "prognosis" of the present invention refers to an act of predicting in advance the course of a disease and the outcome of death or survival. The prognosis or prognostic diagnosis may be interpreted to mean any act of predicting the course of a disease before/after treatment by comprehensively considering the patient's condition, as the course of the disease may vary depending on the physiological or environmental condition of the patient. can For the purposes of the present invention, the prognosis may be interpreted as an act of predicting in advance the therapeutic reactivity after an anticancer drug treatment, preferably an OXPHOS inhibitor treatment, or an act of appropriately selecting whether to use an OXPHOS inhibitor based on this.

본 발명에서 “종양” 또는 “암”은 세포 주기가 조절되지 않아 세포 분열을 계속하는 질병으로서, 발생 부위에 따라 암종(Carcinoma)과 육종(Sarcoma)으로 나뉜다. 암종(Carcinoma)은 점막, 피부 같은 상피성 세포에서 발생한 악성 종양을 뜻하고, 육종(Sarcoma)은 근육, 결합 조직, 뼈, 연골, 혈관 등의 비상피성 세포에서 발생한 악성 종양을 뜻한다. In the present invention, “tumor” or “cancer” refers to a disease in which cell division is continued due to uncontrolled cell cycle, and is divided into Carcinoma and Sarcoma depending on the site of occurrence. Carcinoma refers to a malignant tumor that arises from epithelial cells such as mucous membranes and skin, and sarcoma refers to a malignant tumor that arises from non-epithelial cells such as muscle, connective tissue, bone, cartilage, and blood vessels.

본 발명에서 상기 암 환자의 항암 치료는 OXPHOS 억제제를 이용한 것일 수 있으며, 상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제, 미토콘드리아 전자 수송 사슬 Ⅰ 억제제, 또는 철 킬레이터로 이루어진 군으로부터 선택된 1종 이상일 수 있으며, 보다 바람직하게는 미토콘드리아 전자 수송 사슬 Ⅰ 억제제를 이용할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the anticancer treatment of the cancer patient may be one using an OXPHOS inhibitor, and the OXPHOS inhibitor may be at least one selected from the group consisting of an ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator, More preferably, a mitochondrial electron transport chain I inhibitor may be used, but is not limited thereto.

본 발명에서 상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156, 메트포민(Metformin), 펜포르민(Phenformin) 및 로테논(Rotenone)으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 보다 바람직하게는 IM156일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the mitochondrial electron transport chain I inhibitor may be at least one selected from the group consisting of IM156, metformin, phenformin, and rotenone, more preferably IM156, However, the present invention is not limited thereto.

본 발명에서 상기 예후는 암 환자의 상기 항암제 치료 후 치료 반응성일 수 있으며, 상기 항암제에 내성이 발생하는지 여부를 예측하는 것일 수 있다.In the present invention, the prognosis may be therapeutic responsiveness after the anticancer drug treatment of a cancer patient, and may be predicting whether resistance to the anticancer drug occurs.

본 발명에서 상기 치료의 대상이 되는 질환으로 상기 "암"은 포유류에서 전형적으로 조절되지 않는 세포 성장으로 특징 지어진 생리적 상태를 나타내거나 가리킨다. 상기 암은 갑상선암, 부갑상선암, 위암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있으나, 종양의 분화 및/또는 증식 등 암의 진행이 본 발명에서 기술하는 암 세포 및/또는 암 줄기세포에 의존적인 암의 종류라면 이에 제한되지 않는다. In the present invention, the "cancer" as a disease to be treated refers to or refers to a physiological condition characterized by uncontrolled cell growth typically in mammals. The cancer is thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervoussystem tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma If the cancer progression, such as differentiation and/or proliferation, is a cancer cell and/or cancer stem cell-dependent type of cancer described in the present invention, the present invention is not limited thereto.

본 발명의 일 구현 예에 따르면, SDHA(succinate dehydrogenase complex flavoprotein subunit A) 단백질, 또는 이를 코딩하는 유전자를 포함하는, 항암제 내성 진단용 바이오 마커 조성물에 관한 것이다.According to one embodiment of the present invention, it relates to a biomarker composition for diagnosing anticancer drug resistance, comprising a succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein or a gene encoding the same.

본 발명에서 상기 SDHA 단백질은 서열번호 1로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the SDHA protein may consist of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

본 발명에서 상기 SDHA 유전자는 서열번호 2로 표시되는 염기서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the SDHA gene may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

본 발명에서 상기 서열은 해당 서열과 80% 이상의 상동성을 가지는 서열을 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the sequence may include a sequence having 80% or more homology with the corresponding sequence, but is not limited thereto.

본 발명의 바람직한 일 예시에서 상기 바이오 마커는 SDHA 단백질 또는 이를 코딩하는 유전자를 포함할 수 있으나, 이에 제한되는 것은 아니다. In a preferred embodiment of the present invention, the biomarker may include an SDHA protein or a gene encoding the same, but is not limited thereto.

본 발명에서 상기 바이오 마커는 개체로부터 얻어진 시료 발현이 측정되는 것일 수 있다.In the present invention, the biomarker may be one in which expression of a sample obtained from an individual is measured.

본 발명에서 바람직한 일 예시로 상기 항암제는 OXPHOS 억제제로서 미토콘드리아 전자 수송 사슬 Ⅰ 억제제일 수 있으며, 보다 바람직하게는 IM156일 수 있으나, 이에 제한되는 것은 아니다.As a preferred example in the present invention, the anticancer agent may be a mitochondrial electron transport chain I inhibitor as an OXPHOS inhibitor, more preferably IM156, but is not limited thereto.

본 발명의 다른 구현 예에 따르면, SDHA(succinate dehydrogenase complex flavoprotein subunit A) 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 제제를 포함하는, 암 환자의 항암제 내성 진단용 바이오 마커 조성물에 관한 것이다.According to another embodiment of the present invention, it relates to a biomarker composition for diagnosing anticancer drug resistance in cancer patients, comprising an agent for measuring the expression level of a succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein, or a gene encoding the same.

본 발명의 항암제 내성 진단용 조성물은 상기 SDHA 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 제제를 포함할 수 있으나, 이에 제한되는 것은 아니다.The composition for diagnosing anticancer drug resistance of the present invention may include an agent for measuring the expression level of the SDHA protein or a gene encoding the same, but is not limited thereto.

본 발명의 상기 항암제 내성 진단용 조성물에서 상기 SDHA 단백질의 발현 수준울 측정하는 제제는 SDHA 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함할 수 있으나, 이에 제한되는 것은 아니다. The agent for measuring the expression level of the SDHA protein in the anticancer drug resistance diagnostic composition of the present invention is an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and an aptamer that specifically binds to SDHA protein. It may include one or more selected from, but is not limited thereto.

본 발명에서 상기 "항체"는 항원과 특이적으로 결합하여 항원-항체 반응을 일으키는 물질을 가리킨다. 본 발명의 목적상, 항체는 상기 SDHA 단백질에 대해 특이적으로 결합하는 항체를 의미한다. 본 발명의 항체는 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다. 상기 항체는 당 업계에 널리 공지된 기술을 이용하여 용이하게 제조될 수 있다. 예를 들어, 다클론 항체는 상기 단백질의 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 과정을 포함하는 당 업계에 널리 공지된 방법에 의해 생산될 수 있다. 이러한 다클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소, 개 등의 임의의 동물로부터 제조될 수 있다. 또한, 단클론 항체는 당 업계에 널리 공지된 하이브리도마 방법(hybridoma method; Kohler 및 Milstein (1976) European Journal of Immunology 6:511-519 참조), 또는 파지 항체 라이브러리 기술(Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991 참조)을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제될 수 있다. 또한, 본 발명의 항체는 2개의 전장의 경쇄 및 2개의 전장의 중쇄를 갖는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등이 있다.In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the SDHA protein. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibody can be easily prepared using techniques well known in the art. For example, the polyclonal antibody can be produced by a method well known in the art, including the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like. In addition, monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or the phage antibody library technology (Clackson et al, Nature, 352). :624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.

본 발명에서 상기 "올리고펩타이드"는 펩타이드로 2 내지 20 개의 아미노산으로 구성되며 디 펩티드, 트리 펩티드, 테트라 펩티드 및 펜타 펩티드를 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the "oligopeptide" is a peptide consisting of 2 to 20 amino acids and may include a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide, but is not limited thereto.

본 발명에 상기 "PNA(Peptide Nucleic Acid)"는 인공적으로 합성된, DNA 또는 RNA와 비슷한 중합체를 가리키며, 1991년 덴마크 코펜하겐 대학교의 Nielsen, Egholm, Berg와 Buchardt 교수에 의해 처음으로 소개되었다. DNA는 인산-리보스당 골격을 갖는데 반해, PNA는 펩타이드 결합에 의해 연결된 반복된 N-(2-아미노에틸)-글리신 골격을 가지며, 이로 인해 DNA 또는 RNA에 대한 결합력과 안정성이 크게 증가되어 분자 생물학, 진단 분석 및 안티센스 치료법에 사용되고 있다. PNA는 문헌[Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500]에 상세하게 개시되어 있다.In the present invention, "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized, DNA or RNA-like polymer, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark in 1991. Whereas DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy. PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.

본 발명에서 상기 "앱타머"는 올리고핵산 또는 펩타이드 분자이며, 앱타머의 일반적인 내용은 문헌[Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727(1998)]에 상세하게 개시되어 있다.In the present invention, the "aptamer" is an oligonucleic acid or a peptide molecule, and the general description of the aptamer is described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727 (1998).

본 발명에서 상기 SDHA 단백질을 코딩하는 유전자의 발현 수준을 측정하는 제제는 상기 SDHA 단백질을 코딩하는 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the agent for measuring the expression level of the gene encoding the SDHA protein may include one or more selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding the SDHA protein. , but is not limited thereto.

본 발명에서 상기 "프라이머"는 표적 유전자 서열을 인지하는 단편으로서, 정방향 및 역방향의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다. 프라이머의 핵산 서열이 시료 내 존재하는 비-표적 서열과 불일치하는 서열이어서, 상보적인 프라이머 결합 부위를 함유하는 표적 유전자 서열만 증폭하고 비특이적 증폭을 유발하지 않는 프라이머일 때, 높은 특이성이 부여될 수 있다.In the present invention, the "primer" is a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but preferably, a primer pair that provides analysis results with specificity and sensitivity. High specificity can be conferred when the primer's nucleic acid sequence is a sequence that is inconsistent with the non-target sequence present in the sample, so that only the target gene sequence containing the complementary primer binding site is amplified and the primer does not cause non-specific amplification. .

본 발명에서 상기 "프로브"란 시료 내의 검출하고자 하는 표적 물질과 특이적으로 결합할 수 있는 물질을 의미하며, 상기 결합을 통하여 특이적으로 시료 내의 표적 물질의 존재를 확인할 수 있는 물질을 의미한다. 프로브의 종류는 당 업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA(peptide nucleic acid), LNA(locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA일 수 있으며, 가장 바람직하게는 PNA이다. 보다 구체적으로, 상기 프로브는 바이오 물질로서 생물에서 유래되거나 이와 유사한 것 또는 생체 외에서 제조된 것을 포함하는 것으로, 예를 들어, 효소, 단백질, 항체, 미생물, 동식물 세포 및 기관, 신경세포, DNA, 및 RNA일 수 있으며, DNA는 cDNA, 게놈 DNA, 올리고뉴클레오타이드를 포함하며, RNA는 게놈 RNA, mRNA, 올리고뉴클레오타이드를 포함하며, 단백질의 예로는 항체, 항원, 효소, 펩타이드 등을 포함할 수 있다.In the present invention, the "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA. More specifically, the probe includes a biomaterial derived from or similar thereto or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

본 발명에서 상기 "LNA(Locked nucleic acids)"란, 2'-O, 4'-C 메틸렌 브릿지를 포함하는 핵산 아날로그를 의미한다 [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA 뉴클레오사이드는 DNA와 RNA의 일반적 핵산 염기를 포함하며, Watson-Crick 염기 쌍 규칙에 따라 염기 쌍을 형성할 수 있다. 하지만, 메틸렌 브릿지로 인한 분자의 'locking'으로 인해, LNA는 Watson-Crick 결합에서 이상적 형상을 형성하지 못하게 된다. LNA가 DNA 또는 RNA 올리고뉴클레오티드에 포함되면, LNA는 보다 빠르게 상보적 뉴클레오티드 사슬과 쌍을 이루어 이중 나선의 안정성을 높일 수 있다. In the present invention, the "LNA (Locked nucleic acids)" means a nucleic acid analog including a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502) ]. LNA nucleosides include common nucleic acid bases in DNA and RNA, and can form base pairs according to Watson-Crick base pairing rules. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA does not form an ideal shape in the Watson-Crick bond. When LNAs are incorporated into DNA or RNA oligonucleotides, LNAs can pair with complementary nucleotide chains more rapidly, increasing the stability of the double helix.

본 발명에서 상기 "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어, 표적서열 내에서 전형적으로 mRNA와 RNA: 올리고머 헤테로이중체의 형성을 허용하는, 뉴클레오티드 염기의 서열 및 서브유닛간 백본을 갖는 올리고머를 의미한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 근사 상보성을 가질 수 있다.In the present invention, the "antisense" means that the antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, and typically mRNA and RNA in the target sequence: A sequence of nucleotide bases allowing the formation of an oligomeric heteroduplex. and oligomers having an inter-subunit backbone. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.

본 발명에 따른 SDHA 단백질이나, 이들을 코딩하는 유전자의 정보는 알려져 있으므로, 당 업자라면 이를 바탕으로 상기 단백질을 코딩하는 유전자에 특이적으로 결합하는 프라이머, 프로브 또는 안티센스 뉴클레오티드를 용이하게 디자인할 수 있을 것이다. Since information on the SDHA protein according to the present invention or genes encoding them is known, those skilled in the art can easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein based on this information. .

본 발명에서 상기 예후는 암 환자의 항암제에 대한 내성 발생 유무일 수 있다.In the present invention, the prognosis may be whether a cancer patient develops resistance to an anticancer agent.

본 발명에서 상기 암은 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병 및 이들의 조합으로 구성된 군으로부터 선택된 암일 수 있고, 바람직하게는 위암일 수 있다.In the present invention, the cancer is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer , thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, and a combination thereof may be a cancer selected from the group consisting of, preferably stomach cancer.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 암 환자의 항암제 내성 진단용 바이오 마커 조성물을 포함하는, 항암제 내성 진단용 키트에 관한 것이다.According to another embodiment of the present invention, it relates to a kit for diagnosing anticancer drug resistance, comprising the biomarker composition for diagnosing anticancer drug resistance of a cancer patient of the present invention.

본 발명에서 "키트"는 바이오 마커 성분에 특이적으로 결합하는 프로브 또는 항체를 검출 가능한 표지로 표지하여 바이오 마커의 발현 수준을 평가할 수 있는 도구를 말한다. 프로브 또는 항체 관련하여 검출 가능한 물질을 기질과의 반응에 의해서 직접적으로 표지하는 것뿐만 아니라, 직접적으로 표지된 다른 시약과의 반응성에 의한 발색하는 표지체가 접합된 간접적 표지도 포함한다. 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 기타 다른 용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하여 제작될 수 있다. 본 발명에서 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있으며, 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소, 역전사효소, DNase, RNase 억제제, 멸균수 등을 포함할 수 있다. 또한, 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 항암제 내성 진단용 유전자를 검출하기 위한 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조군 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다. 본 발명의 키트는 당 업계에 공지되어 있는 것이라면, 이에 제한되지 않는다. In the present invention, "kit" refers to a tool capable of evaluating the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. In addition to direct labeling of a detectable substance with respect to a probe or antibody by reaction with a substrate, it includes indirect labeling in which a color-generating label is conjugated by reactivity with another directly labeled reagent. It may include a chromogenic substrate solution, a washing solution, and other solutions to undergo a color reaction with the label, and may be prepared including reagent components used. In the present invention, the kit may be a kit including essential elements necessary for performing RT-PCR, and in addition to each primer pair specific for a marker gene, a test tube, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerization enzymes, reverse transcriptase, DNase, RNase inhibitors, sterile water, and the like. In addition, the kit may be a kit for detecting a gene for anticancer drug resistance diagnosis including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

본 발명에서 상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트일 수 있다. In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

본 발명의 상기 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치를 더 포함할 수 있다. 예를 들면, 본 발명에서 상기 키트는 역전사 중합효소반응을 수행하기 위해 필요한 필수 요소를 더 포함할 수 있다. 역전사 중합효소반응 키트는 마커 단백질을 코딩하는 유전자에 대해 특이적인 프라이머 쌍을 포함한다. 프라이머는 상기 유전자의 핵산 서열에 특이적인 서열을 가지는 뉴클레오티드로써, 약 7 bp 내지 50 bp의 길이, 보다 바람직하게는 약 10 bp 내지 30 bp의 길이를 가질 수 있다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그 외 역전사 중합효소반응 키트는 테스트 튜브 또는 다른 적절한 용기, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNase 억제제 DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다.The kit of the present invention may further include one or more other component compositions, solutions or devices suitable for the assay method. For example, in the present invention, the kit may further include essential elements necessary for performing the reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a pair of primers specific for a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably, about 10 bp to 30 bp. It may also include primers specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

또한, 본 발명의 항암제 내성 진단용 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함할 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드(oligonucleotide)가 부착되어 있는 기판, 및 형광표지 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드를 포함할 수 있다.In addition, the anticancer drug resistance diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently-labeled probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

또한, 본 발명의 항암제 내성 진단용 키트는 ELISA를 수행하기 위해 필요한 필수 요소를 포함할 수 있다. ELISA 키트는 상기 단백질에 대해 특이적인 항체를 포함한다. 항체는 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromophores), 효소(예: 항체와 컨주게이트됨) 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다.In addition, the anticancer drug resistance diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit contains an antibody specific for this protein. Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibody, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and substrates thereof or capable of binding the antibody. other materials and the like.

본 발명의 항암제 내성 진단용 키트에서 항원-항체 결합반응을 위한 고정체로는 니트로셀룰로오즈 막, PVDF 막, 폴리비닐(polyvinyl) 수지 또는 폴리스티렌(polystyrene) 수지로 합성된 웰 플레이트(Well plate), 유리로 된 슬라이드 글래스 등이 사용될 수 있으나, 이에 제한되는 것은 아니다.In the anticancer drug resistance diagnostic kit of the present invention, as a fixture for antigen-antibody binding reaction, a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, a well plate synthesized from a glass, A slide glass or the like may be used, but is not limited thereto.

또한, 본 발명의 항암제 내성 진단용 키트에서 2차 항체의 표지체는 발색 반응을 하는 통상의 발색제가 바람직하며, HRP(horseradish peroxidase), 염기성 탈인산화효소(alkaline phosphatase), 콜로이드 골드(coloid gold), FITC(폴리 L-라이신-플루오르세인 아이소티오시아네이트), RITC(로다민-B-아이소티오시아네이트) 등의 형광물질(fluorescein) 및 색소(dye) 등의 표지체가 사용될 수 있으나, 이에 제한되는 것은 아니다.In addition, in the anticancer drug resistance diagnostic kit of the present invention, the label of the secondary antibody is preferably a color developing agent that exhibits a color reaction, HRP (horseradish peroxidase), basic dephosphorylation enzyme (alkaline phosphatase), colloid gold (colloid gold), Labels such as fluorescein and dye such as FITC (poly L-lysine-fluorescein isothiocyanate) and RITC (rhodamine-B-isothiocyanate) may be used, but are limited thereto it is not

또한, 본 발명의 항암제 내성 진단용 키트에서 발색을 유도하기 위한 발색 기질은 발색 반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB(3,3',5,5'-테트라메틸 베지딘), ABTS[2,2'-아지노-비스(3-에틸벤조티아졸린-6-설폰산)], OPD(o-페닐렌다이아민) 등을 사용할 수 있다. 이때, 발색 기질은 완충 용액(0.1 M NaAc, pH 5.5)에 용해된 상태로 제공되는 것이 더욱 바람직하다. TMB와 같은 발색기질은 이차 항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 발색 침적체를 생성하고, 이 발색 침적체의 침적 정도를 육안으로 확인함으로써 상기 마커 단백질들의 존재 유무를 검출한다.In addition, the chromogenic substrate for inducing color development in the anticancer drug resistance diagnostic kit of the present invention is preferably used according to a colorimetric marker, TMB (3,3',5,5'-tetramethyl bezidine), ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD(o-phenylenediamine), etc. can be used. In this case, the chromogenic substrate is more preferably provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a label of the secondary antibody conjugate to generate chromogenic deposits, and the presence or absence of the marker proteins is detected by visually confirming the degree of deposition of the chromogenic deposits.

본 발명의 항암제 내성 진단용 키트에서 세척액은 인산염 완충 용액, NaCl 및 트윈 20(Tween 20)을 포함하는 것이 바람직하며, 0.02 M 인산염 완충용액, 0.13 M NaCl, 및 0.05% 트윈 20으로 구성된 완충 용액(PBST)이 더욱 바람직하다. 세척액은 항원-항체 결합 반응 후 항원-항체 결합체에 2차 항체를 반응시킨 다음 적당량을 고정체에 첨가하여 3 내지 6회 세척한다. 반응 정지 용액은 황산 용액(H2SO4)이 바람직하게 사용될 수 있다.In the anticancer drug resistance diagnostic kit of the present invention, the washing solution preferably includes a phosphate buffer solution, NaCl and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20. ) is more preferable. After the antigen-antibody binding reaction, the secondary antibody is reacted with the antigen-antibody conjugate, and an appropriate amount is added to the immobilizer and washed 3 to 6 times. As the reaction stop solution, a sulfuric acid solution (H 2 SO 4 ) may be preferably used.

본 발명의 또 다른 구현 예에 따르면, 목적하는 개체로부터 분리된 생물학적 시료에서 SDHA 단백질 또는 상기 단백질을 코딩하는 유전자의 발현 수준을 측정하는 단계를 포함하는 암 환자의 항암제 내성 진단을 위한 정보 제공 방법에 관한 것이다.According to another embodiment of the present invention, in a method for providing information for diagnosing anticancer drug resistance in a cancer patient, comprising measuring the expression level of the SDHA protein or a gene encoding the protein in a biological sample isolated from a target individual it's about

본 발명의 상기 항암제 내성 진단을 위한 정보 제공 방법은 상기 목적하는 개체로부터 분리된 생물학적 시료에서 상기 SDHA 단백질 또는 상기 단백질을 코딩하는 유전자의 발현 수준을 측정하는 단계를 포함할 수 있다.The method for providing information for diagnosing anticancer drug resistance of the present invention may include measuring the expression level of the SDHA protein or a gene encoding the protein in a biological sample isolated from the target subject.

본 발명에서 상기 생물학적 시료는 개체로부터 얻어지거나 개체로부터 유래된 임의의 물질, 생물학적 체액, 조직 또는 세포를 의미하는 것으로, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 혈장(plasma), 혈청(serum), 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 복수(ascites), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 및 뇌척수액(cerebrospinal fluid) 등으로 이루어진 군에서 선택된 1종 이상일 수 있다.In the present invention, the biological sample refers to any material, biological fluid, tissue or cell obtained from or derived from an individual, whole blood, leukocytes, peripheral blood mononuclear cells ), buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, respiration (breath), urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid , glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate (joint aspirate), organ secretions (organ secretions), cells (cell), cell extract (cell extract), and may be at least one selected from the group consisting of cerebrospinal fluid (cerebrospinal fluid).

본 발명에서 상기 SDHA 단백질의 발현 수준을 측정하는 제제는 상기 SDHA 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함할 수 있다.In the present invention, the agent for measuring the expression level of the SDHA protein is at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to the SDHA protein. may include.

본 발명에서 상기 SDHA 단백질의 발현 수준의 측정은 단백질 칩 분석, 면역 측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, SELDI-TOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, 방사선 면역 분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏팅 또는 ELISA(enzyme linked immunosorbentassay)에 의해 수행될 수 있다. In the present invention, the measurement of the expression level of the SDHA protein is a protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption) /Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, octeroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography-mass spectrometry (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay) may be performed.

또한, 본 발명에서 상기 SDHA 단백질의 발현 수준의 측정은 다중 반응 모니터링 (multiple reaction monitoring; MRM) 방법에 의할 수 있다. In addition, in the present invention, the measurement of the expression level of the SDHA protein may be performed by a multiple reaction monitoring (MRM) method.

본 발명에서 상기 다중 반응 모니터링 방법 시 내부 표준 물질은 타깃 펩타이드를 구성하는 특정 아미노산을 동위원소로 치환한 합성 펩타이드 또는 대장균 베타 갈락토시다아제를 사용할 수 있다. In the present invention, in the method for monitoring multiple reactions, a synthetic peptide in which a specific amino acid constituting a target peptide is substituted with an isotope or E. coli beta galactosidase may be used as the internal standard material.

본 발명에서 상기 SDHA의 타깃 펩티드는 서열번호 1로 표시되는 아미노산 서열로 이루어질 수 있다.In the present invention, the target peptide of SDHA may consist of the amino acid sequence represented by SEQ ID NO: 1.

본 발명에서 상기 SDHA 단백질을 코딩하는 유전자의 발현 수준을 측정하는 제제는 상기 SDHA 단백질을 코딩하는 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함할 수 있다. In the present invention, the agent for measuring the expression level of the gene encoding the SDHA protein may include one or more selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding the SDHA protein. .

본 발명에서 상기 SDHA 단백질을 코딩하는 유전자의 발현 수준의 측정은 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting) 또는 DNA 칩에 의할 수 있다.In the present invention, the measurement of the expression level of the gene encoding the SDHA protein is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR) ), RNase protection assay (RPA), Northern blotting or DNA chip.

본 발명의 정보 제공 방법에서 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid), 앱타머(aptamer) 등에 관한 기재와 프라이머, 프로브 등에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다. In the information providing method of the present invention, the description of the antibody, oligopeptide, ligand, PNA (peptide nucleic acid), aptamer, etc. and the description of primers, probes, etc. are overlapped with those described above. The detailed description is omitted.

본 발명에서 상기 목적하는 개체의 생물학적 시료에 대하여 측정된 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군에 비하여 증가하는 경우, 상기 목적하는 개체의 항암 치료제 사용 후의 예후가 나쁠 것으로 예측할 수 있다.In the present invention, when the expression level of the SDHA protein or a gene encoding it measured with respect to the biological sample of the target individual increases compared to the control group, the prognosis after the use of the anticancer therapeutic agent of the target individual can be predicted to be poor.

본 발명에서 상기 목적하는 개체의 항암 치료제는 OXPHOS 억제제를 이용한 것일 수 있으며, 상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제, 미토콘드리아 전자 수송 사슬 Ⅰ 억제제, 또는 철 킬레이터로 이루어진 군으로부터 선택된 1종 이상일 수 있으며, 보다 바람직하게는 미토콘드리아 전자 수송 사슬 Ⅰ 억제제를 이용할 수 있다. 상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156, 메트포민(Metformin), 펜포르민(Phenformin) 및 로테논(Rotenone)으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 보다 바람직하게는 IM156일 수 있으나, 이에 제한되는 것은 아니다. 상기 약물들에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the anticancer therapeutic agent of the subject may be one using an OXPHOS inhibitor, and the OXPHOS inhibitor may be at least one selected from the group consisting of an ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator. , more preferably a mitochondrial electron transport chain I inhibitor. The mitochondrial electron transport chain I inhibitor may be at least one selected from the group consisting of IM156, metformin, phenformin, and rotenone, and more preferably IM156, but is limited thereto. it is not The description of the drugs overlaps with those described above, and detailed description thereof will be omitted below to avoid excessive complexity of the specification.

본 발명에서 상기 정보 제공 방법은 상기 목적하는 개체의 상기 항암제 내성 발생 여부 또는 발생 가능성을 진단하는 것일 수 있다.In the present invention, the information providing method may be to diagnose whether or not the anticancer drug resistance of the target individual or the possibility of occurrence.

본 발명에서 상기 목적하는 개체의 생물학적 시료에 대하여 측정된 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군에 비하여 증가하는 경우, 상기 항암 치료제 사용 후의 항암제 내성이 발생하거나 발생할 가능성이 높은 것으로 예측할 수 있다.In the present invention, when the expression level of the SDHA protein or the gene encoding it measured with respect to the biological sample of the subject of interest increases compared to the control group, it can be predicted that anticancer drug resistance occurs or is highly likely to occur after the use of the anticancer drug. .

본 발명에서 바람직한 일 예시로 상기 목적하는 개체의 생물학적 시료 중 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군에 비하여 증가한 경우, 상기 항암 치료제에 대한 치료 반응성이 낮은 것으로 예측할 수 있다.As a preferred example in the present invention, when the expression level of the SDHA protein or a gene encoding the same in the biological sample of the target individual is increased compared to the control group, it can be predicted that the therapeutic responsiveness to the anticancer agent is low.

본 발명에서 "대조군"이란 항암제 내성이 발생하지 아니한 정상 대조군이거나, 항암제 감수성 세포에서의 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준의 평균 내지 중간 값일 수 있다. 대조군에서의 마커 단백질 또는 이를 코딩하는 핵산분자의 발현량과 분석 대상이 되는 암 환자 유래의 생물학적 시료에서의 마커 단백질 또는 이를 코딩하는 핵산분자의 발현량을 비교할 수 있으며, 상기 발현량의 유의한 변화 여부를 판단하여 항암제 내성 여부를 진단할 수 있다. 상기 정상 대조군 시료의 범위로는 해당 약제에 대하여 내성을 획득하지 않은 것으로 확인된 암 환자 유래의 세포, 이의 배양액, 혈액, 혈청, 혈장, 및 조직도 포함된다.In the present invention, "control group" may be a normal control in which anticancer drug resistance does not occur, or an average to median value of the expression level of SDHA protein or a gene encoding the same in anticancer drug sensitive cells. The expression level of the marker protein or nucleic acid molecule encoding it in the control group can be compared with the expression level of the marker protein or the nucleic acid molecule encoding the same in a biological sample derived from a cancer patient to be analyzed, and whether there is a significant change in the expression level can be used to diagnose anticancer drug resistance. The range of the normal control sample also includes cells derived from a cancer patient confirmed not to have acquired resistance to the drug, a culture solution thereof, blood, serum, plasma, and tissue.

본 발명에서 상기 목적하는 개체의 생물학적 시료에 대하여 측정된 SDHA 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군에 비하여 증가한 경우, 상기 암 면역 치료 후의 예후가 나쁠 것으로 예측할 수 있다. In the present invention, when the expression level of the SDHA protein or a gene encoding it measured with respect to the biological sample of the target individual is increased compared to the control group, it can be predicted that the prognosis after the cancer immunotherapy is poor.

본 발명의 일 구현예에서, SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제를 포함하는 항암제 내성 치료용 조성물을 제공한다. In one embodiment of the present invention, there is provided a composition for treating anticancer drug resistance comprising an agent that reduces the expression level of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA), or a gene encoding the same.

본 발명에서 상기 SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제를 포함하는 조성물은 단독으로 투여하여 사용될 수 있으며, 통상적으로 사용되는 항암제와 병용 투여할 수도 있으며, 병용 투여를 통하여 항암제 내성 치료 효과로 인한 암 치료 효과를 현저하게 향상시킬 수 있다. In the present invention, the composition comprising an agent that reduces the expression level of the succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein, or a gene encoding it, may be administered alone, and may be administered in combination with a commonly used anticancer agent. Also, through combination administration, it is possible to significantly improve the cancer treatment effect due to the anticancer drug resistance treatment effect.

본 발명에서 상기 항암제에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the description of the anticancer agent is omitted below in order to avoid excessive complexity of the specification because it overlaps with that described above.

본 발명에서 상기 SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제은 SDHA의 FAD 결합 부위를 타깃으로 하여 작용하는 제제이거나 SDHB 또는 SDHC/D의 유비퀴논 결합 부위를 타깃으로 하여 작용하는 제제일 수 있으나, 세포 호흡 저해 기능을 수행하는 제제에 해당한다면 이에 제한되는 것은 아니다.In the present invention, the agent for reducing the expression level of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA) or a gene encoding it is an agent that targets the FAD binding site of SDHA, or a ubiquinone binding agent of SDHB or SDHC/D It may be an agent that targets and acts on a site, but is not limited thereto if it corresponds to an agent that performs a cellular respiration inhibitory function.

본 발명에서 상기 SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제로 3-니트로 프로피온산(3-nitropropionic acid; 3NPA) 또는 아트페닌 A5(atpenin A5)일 수 있다. 그러나, 세포 호흡 저해 기능을 가진 것이라면 이에 제한되지 아니하고 모두 포함될 수 있다.In the present invention, 3-nitropropionic acid (3NPA) or atpenin A5 (atpenin A5) is an agent for reducing the expression level of the SDHA (succinate dehydrogenase complex flavoprotein subunit A) protein, or a gene encoding it. can However, as long as it has a function of inhibiting cellular respiration, the present invention is not limited thereto, and all of them may be included.

본 발명의 일 구체예에서, 상기 3-니트로 프로피온산(3-nitropropionic acid; 3NPA)은 5개의 수소 원자, 3개의 탄소 원자, 1개의 질소 원자 그리고 4개의 산소 원자로 구성되어 총 13개의 원자로 구성된 화합물로서 미토콘드리아 전자 수송 사슬 복합체 Ⅱ에 작용하는 숙신산 탈수소화효소 억제제(inhibitor of succinate dehydrogenase)로 알려져 있다.In one embodiment of the present invention, the 3-nitropropionic acid (3NPA) is a compound consisting of 5 hydrogen atoms, 3 carbon atoms, 1 nitrogen atom and 4 oxygen atoms, and a total of 13 atoms. It is known as an inhibitor of succinate dehydrogenase that acts on mitochondrial electron transport chain complex II.

본 발명의 일 구체예에서, 상기 아트페닌 A5(atpenin A5)는 유비퀴논 결합 부위인 미토콘드리아 복합체 Ⅱ의 억제제로 미토콘드리아의 ATP 민감성 칼륨 채널(ATP-sensitive potassium channel; KATP channel)을 자극하며, 호흡 저해제로도 기능을 한다. 생체 내에서 항허혈 작용 및 심장 보호 효과 기능 또한 수행하는 것으로 알려져 있는 물질 중 하나이다. In one embodiment of the present invention, the atpenin A5 (atpenin A5) is an inhibitor of mitochondrial complex II, which is a ubiquinone binding site, and stimulates mitochondrial ATP-sensitive potassium channel (KATP channel), and is a respiratory inhibitor. also functions as It is one of the substances known to also perform anti-ischemic and cardioprotective functions in vivo.

본 발명의 일 구체예에서, 상기 SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제로 SDHA 활성 또는 발현 억제제를 포함할 수 있다. 상기 SDHA 활성 억제제는 SDHA에 특이적으로 결합하는 항체, 또는 그 항원 결합 단편을 포함하는 것일 수 있으며, SDHA 발현 억제제는 SDHA 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 마이크로RNA로 구성된 군으로부터 선택된 것일 수 있으나, 표적 단백질 또는 유전자인 SDHA에 직간접적으로 작용하여 그의 활성 또는 발현이 저해되는 효과를 도출하는 수단에 해당하는 것으로 당 업계에서 통상적으로 사용되는 방법으로 공지된 기술에 의하여 용이하게 도출 가능한 것이라면 이에 제한되지 아니하고 모두 포함할 수 있다.In one embodiment of the present invention, an agent for reducing the expression level of the succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein, or a gene encoding it, may include an SDHA activity or expression inhibitor. The SDHA activity inhibitor may include an antibody that specifically binds to SDHA, or an antigen-binding fragment thereof, and the SDHA expression inhibitor is selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and microRNA specific for SDHA gene. However, it corresponds to a means for deriving an effect that inhibits its activity or expression by acting directly or indirectly on SDHA, which is a target protein or gene, and can be easily derived by a known technique in a method commonly used in the art. It is not limited thereto, and may include all of them.

본 발명에 있어서, SDHA 발현 억제제는 인간 SDHA를 녹다운시킨 siRNA로서, 상기 siRNA는 서열번호 3과 서열번호 4로 표시되는 뉴클레오티드 서열이다. 상기 siRNA의 용도는 SDHA의 단백질 또는 이를 코딩하는 유전자의 발현 수준을 감소시켜 암 치료 효과를 향상시키는데 그 목적이 있는 것으로 서열번호 3은 정방향 서열(sense)이며, 서열번호 4는 역방향 서열(antisense)이다.In the present invention, the SDHA expression inhibitor is an siRNA that knocked down human SDHA, and the siRNA is the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4. The purpose of the siRNA is to improve the cancer treatment effect by reducing the expression level of the SDHA protein or a gene encoding it. SEQ ID NO: 3 is a forward sequence (sense), and SEQ ID NO: 4 is a reverse sequence (antisense) am.

본 발명의 일 구체예에서, SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 제제를 포함하는 항암제 내성 진단용 키트를 제공하고, 상기 SDHA 단백질은 서열번호 1과 80% 이상의 상동성을 가지는 서열로 표시되는 것인 항암제 내성 진단용 키트를 제공하며, 상기 SDHA 단백질은 서열번호 1로 표시되는 것인 항암제 내성 진단용 키트를 제공하며, 상기 SDHA 유전자는 서열번호 2와 80% 이상의 상동성을 가지는 서열로 표시되는 것인 항암제 내성 진단용 키트를 제공하며, 상기 SDHA 유전자는 서열번호 2로 표시되는 것인 항암제 내성 진단용 키트를 제공하며, 상기 항암제는 OXPHOS 억제제인 항암제 내성 진단용 키트를 제공하며, 상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제(ubiquinone reduction activity inhibitor), 미토콘드리아 전자 수송 사슬 Ⅰ 억제제(mitochondrial electron transport chain I inhibitor) 또는 철 킬레이터(iron chelator)로 이루어진 군에서 선택된 1종 이상을 포함하는 것인 항암제 내성 진단용 키트를 제공하며, 상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156인 항암제 내성 진단용 키트를 제공하며, 상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함하는 항암제 내성 진단용 키트를 제공하며, 상기 유전자의 발현 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함하는 항암제 내성 진단용 키트를 제공하며, 상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트인 항암제 내성 진단용 키트를 제공하며, 상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인 항암제 내성 진단용 키트를 제공한다.In one embodiment of the present invention, there is provided a kit for diagnosing anticancer drug resistance comprising an agent for measuring the expression level of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA) or a gene encoding the same, wherein the SDHA protein is SEQ ID NO: 1 It provides a kit for diagnosing anticancer drug resistance, which is represented by a sequence having at least 80% homology with It provides a kit for diagnosing anticancer drug resistance, which is represented by a sequence having at least 80% homology, wherein the SDHA gene provides a kit for diagnosing anticancer drug resistance that is represented by SEQ ID NO: 2, wherein the anticancer agent is an OXPHOS inhibitor for diagnosing anticancer drug resistance Provided is a kit, wherein the OXPHOS inhibitor is one selected from the group consisting of a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator. It provides a kit for diagnosing anticancer drug resistance, comprising the above, wherein the mitochondrial electron transport chain I inhibitor is IM156. It provides a kit for diagnosing anticancer drug resistance comprising at least one selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer, wherein the agent for measuring the expression level of the gene is the gene It provides a kit for diagnosing anticancer drug resistance comprising at least one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to, wherein the kit includes an RT-PCR kit, a DNA chip kit, an ELISA kit, It provides a kit for diagnosing anticancer drug resistance, which is a white matter chip kit, a rapid kit or a multiple reaction monitoring (MRM) kit, wherein the anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer , cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma or any of these Provided is a kit for diagnosing anticancer drug resistance for the treatment of cancer selected from the group consisting of combinations.

본 발명의 다른 구체예에서, 목적하는 개체로부터 분리된 생물학적 시료에서 SDHA의 단백질 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 단계를 포함하는 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하고, 상기 SDHA의 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군 시료에 비하여 증가된 것으로 나타난 경우, 상기 항암제 내성으로 인하여 치료 예후가 좋지 않을 것으로 예측하는 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 SDHA 단백질은 서열번호 1과 80% 이상의 상동성을 가지는 서열로 표시되는 것인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 SDHA 유전자는 서열번호 2와 80% 이상의 상동성을 가지는 서열로 표시되는 것인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 항암제는 OXPHOS 억제제인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제(ubiquinone reduction activity inhibitor), 미토콘드리아 전자 수송 사슬 Ⅰ 억제제(mitochondrial electron transport chain I inhibitor) 또는 철 킬레이터(iron chelator)로 이루어진 군에서 선택된 1종 이상을 포함하는 것인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공하며, 상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인 항암제 내성 진단을 위한 정보를 제공하는 방법을 제공한다.In another embodiment of the present invention, there is provided a method for providing information for diagnosing anticancer drug resistance, comprising measuring the expression level of a protein of SDHA or a gene encoding the same in a biological sample isolated from a subject of interest, When the expression level of the SDHA protein or the gene encoding it is shown to be increased compared to the control sample, it provides a method of providing information for diagnosing anticancer drug resistance, which predicts that the treatment prognosis will be poor due to the anticancer drug resistance, SDHA protein provides a method for providing information for anticancer drug resistance diagnosis, which is represented by a sequence having at least 80% homology with SEQ ID NO: 1, wherein the SDHA gene is a sequence having at least 80% homology with SEQ ID NO: 2 It provides a method of providing information for diagnosing anticancer drug resistance, which is represented by , wherein the anticancer agent provides a method of providing information for diagnosing anticancer drug resistance, which is an OXPHOS inhibitor, wherein the OXPHOS inhibitor is a ubiquinone reducing activity inhibitor (ubiquinone). reduction activity inhibitor), mitochondrial electron transport chain I inhibitor (mitochondrial electron transport chain I inhibitor) or iron chelator (iron chelator) that includes at least one selected from the group consisting of a method of providing information for diagnosing resistance to anticancer drugs It provides a method for providing information for diagnosing anticancer drug resistance, wherein the mitochondrial electron transport chain I inhibitor is IM156, and the anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, Ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma or Provided is a method for providing information for diagnosing resistance to anticancer drugs for treatment of a cancer selected from the group consisting of combinations thereof.

본 발명의 또 다른 구체예에서, SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질의 활성, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제를 포함하는 항암제 내성 치료용 조성물을 제공하고, 상기 제제는 3-니트로 프로피온산(3-nitropropionic acid; 3NPA) 또는 아트페닌 A5(atpenin A5)인 항암제 내성 치료용 조성물을 제공하며, 상기 제제는 SDHA 활성 억제제 또는 SDHA 발현 억제제인 항암제 내성 치료용 조성물을 제공하며, 상기 활성 억제제는 SDHA에 특이적으로 결합하는 항체인 항암제 내성 치료용 조성물을 제공하며, 상기 발현 억제제은 -서열의 siRNA를 포함하는 항암제 내성 치료용 조성물을 제공하며, 상기 항암제는 IM156인 항암제 내성 치료용 조성물을 제공하며, 상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인 항암제 내성 치료용 조성물을 제공한다.In another embodiment of the present invention, there is provided a composition for treating anticancer drug resistance comprising an agent that reduces the activity of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA), or the expression level of a gene encoding the same, the agent comprising: It provides a composition for treating anticancer drug resistance which is 3-nitropropionic acid (3NPA) or atpenin A5 (atpenin A5), wherein the agent provides a composition for treating anticancer drug resistance which is an SDHA activity inhibitor or an SDHA expression inhibitor, The activity inhibitor provides a composition for treating anticancer drug resistance which is an antibody that specifically binds to SDHA, the expression inhibitor provides a composition for treating anticancer drug resistance comprising an siRNA of -sequence, and the anticancer agent is IM156 for anticancer drug resistance treatment Provides a composition, wherein the anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, It provides a composition for the treatment of anticancer drug resistance for the treatment of cancer selected from the group consisting of head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, or a combination thereof.

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

본 발명은 항암제에 대한 내성 진단용 바이오 마커 조성물 및 항암제 내성을 치료하기 위한 조성물에 대한 것으로, 향후 치료 계획에 있어 암 환자별 항암제 적합성에 대한 정확한 기초 정보를 제공한다. 더 나아가 항암제 내성 치료용 조성물을 항암제와 병용 투여할 경우 치료 효과가 향상되므로 궁극적으로 생존율을 높일 수 있을 것으로 기대된다.The present invention relates to a biomarker composition for diagnosing resistance to anticancer drugs and a composition for treating anticancer drug resistance, and provides accurate basic information on the suitability of anticancer drugs for each cancer patient in future treatment plans. Furthermore, when a composition for anticancer drug resistance treatment is administered in combination with an anticancer agent, the therapeutic effect is improved, so it is expected to ultimately increase the survival rate.

도 1a 및 도 1b는 본 발명의 일 실시예에 따른, 다양한 세포주에서 IM156 약물 투여 후 세포 생존력(cell viability)을 측정한 결과를 나타낸 도이다.
도 2a는 본 발명의 일 실시예에 따른, 다양한 세포주에서의 SDHA 유전자의 발현을 웨스턴 블롯 분석을 통해 발현 정도를 확인한 도이다.
도 2b는 본 발명의 일 실시예에 따른, 유방암 세포주(MDA-MB-231 또는 MCF7)와 위암 세포주(SK4, MKN45 또는 AGS)를 P 세포와 S 세포로 분류하여 SDHA의 발현 정도를 웨스턴 블롯을 통해 비교 확인한 도이다.
도 3a 및 도 3b는 본 발명의 일 실시예에 따른, IM156 내성 세포주(Hs746T)와 IM156 감수성 세포주(SNU638)에서의 로테논(Rotenone) 또는 IM156 약물 투여에 따른 SDHA 발현 변화의 정도를 비교 확인한 도이다.
도 4a 내지 4c는 본 발명의 일 실시예에 따른, IM156 내성 세포주(Hs746T)에서의 로테논(Rotenone) 또는 IM156 약물 투여 후 세포 호흡 과정에서의 산소 소모율(Oxygen Consumption Rate; OCR)을 측정한 결과를 확인한 도이다.
도 5a 내지 5c는 본 발명의 일 실시예에 따른, IM156 감수성 세포주(SNU638)에서의 로테논(Rotenone) 또는 IM156 약물 투여 후 세포 호흡 과정에서의 산소 소모율(Oxygen Consumption Rate; OCR)을 측정한 결과를 확인한 도이다.
도 6a 및 도 6b는 본 발명의 일 실시예에 따른, 3-니트로 프로피온산(3-nitropropionic acid; 3NPA) 또는 아트페닌 A5(atpenin A5) 약물 투여시 산소 소모율(OCR) 측정을 통하여 항암제 감수성 변화를 비교 확인한 도이다.
도 7a 및 도 7b는 본 발명의 일 실시예에 따른, SDHA 억제제 약물 투여 또는 SDHA 유전자의 넉다운시 산소 소모율(OCR) 측정을 통하여 항암제 감수성 변화를 비교 확인한 도이다.1A and 1B are diagrams showing the results of measuring cell viability after administration of IM156 drug in various cell lines, according to an embodiment of the present invention.
2A is a diagram illustrating the expression level of SDHA gene in various cell lines through Western blot analysis according to an embodiment of the present invention.
2b is a Western blot showing the expression level of SDHA by classifying a breast cancer cell line (MDA-MB-231 or MCF7) and a gastric cancer cell line (SK4, MKN45 or AGS) into P cells and S cells according to an embodiment of the present invention. It is a diagram confirmed by comparison.
Figures 3a and 3b, according to an embodiment of the present invention, IM156 resistant cell line (Hs746T) and IM156 sensitive cell line (SNU638) Rotenone (Rotenone) or a diagram confirming the degree of SDHA expression change according to the administration of the IM156 drug am.
4a to 4c are results of measuring the oxygen consumption rate (OCR) in the cellular respiration process after administration of rotenone or IM156 drug in the IM156 resistant cell line (Hs746T) according to an embodiment of the present invention; is a confirmation of
5a to 5c are results of measuring Oxygen Consumption Rate (OCR) in the cellular respiration process after administration of rotenone or IM156 drug in IM156 sensitive cell line (SNU638) according to an embodiment of the present invention; is a confirmation of
Figures 6a and 6b, according to an embodiment of the present invention, 3-nitropropionic acid (3-nitropropionic acid; 3NPA) or atpenin A5 (atpenin A5) when the drug administration oxygen consumption rate (OCR) through the measurement of the anticancer drug sensitivity change This is a comparatively verified diagram.
7A and 7B are diagrams comparing and confirming changes in anticancer drug sensitivity through oxygen consumption rate (OCR) measurement during SDHA inhibitor drug administration or SDHA gene knockdown, according to an embodiment of the present invention.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예 1: 항암제 내성 세포의 준비Example 1: Preparation of anticancer drug-resistant cells

1.1 세포의 배양1.1 Cell culture

본 발명의 발명자들은 연세대학교 의과대학 평가위원회(Institutional Review Board, IRB)의 승인을 얻어 모든 실험을 수행하였다. 실험 진행을 위하여 미국 세포주 은행(American Type Culture Collection; ATCC)으로부터 인간 위암 세포주인 Hs 746T, MKN-45, YCC-11 및 YCC-16을 수득하였으며, 한국 세포주 은행(Korean Cell Line Bank)으로부터 인간 위암 세포주인 SK4, MKN-1, NCC-59, SNU-1750, SNU-1967, SNU-484, SNU-601, SNU-638, SNU-668 및 SNU-719를 수득하여, 미국 또는 한국 세포주 은행의 가이드에 따라 10% FBS가 포함된 RPMI 1640 배지에 1% 페니실린-스트렙토마이신(Gibco)을 추가하여 37℃, 5% CO2 incubator(HERAcell 150i, Thermo Scientific, Waltham, MA, USA)에서 배양하여 실험에 이용하였다.The inventors of the present invention performed all experiments with the approval of the Yonsei University School of Medicine Evaluation Committee (Institutional Review Board, IRB). Human gastric cancer cell lines, Hs 746T, MKN-45, YCC-11, and YCC-16, were obtained from the American Type Culture Collection (ATCC) for the experiment, and human gastric cancer from the Korean Cell Line Bank. Cell lines SK4, MKN-1, NCC-59, SNU-1750, SNU-1967, SNU-484, SNU-601, SNU-638, SNU-668 and SNU-719 were obtained, and the guide of the US or Korean cell line bank 1% penicillin in RPMI 1640 medium containing 10% FBS according to the - by adding streptomycin (Gibco) in the experiment and incubated at 37 ℃, 5% CO 2 incubator (HERAcell 150i, Thermo Scientific, Waltham, MA, USA) was used.

1.2 IM156 약물 처리 후 내성 세포주 및 감수성 세포주의 선별1.2 Selection of resistant and sensitive cell lines after IM156 drug treatment

IM156 약물의 민감도 확인을 위하여 실시예 1.1에서 수득하여 배양한 다양한 위암 세포주 각각에 농도별로 OXPHOS 억제제의 일종인 IM156 약물을 처리하였다. 상기 약물의 농도 별로 MTT assay를 통하여 세포 생존 능력(cell viability)을 측정한 값을 도 1의 그래프에 나타내었다. 그 결과 6.33uM 내지 63.29uM 농도 범위 내에서 세포 생존력(cell viability)이 급격히 변화하는 모습을 확인할 수 있었다(도 1a 참조). IM156 약물의 농도가 약 20uM 범위인 때 세포주간 민감도를 살펴본 결과 내성 세포주로서 가장 높은 세포 생존율을 가지는 Hs746T와 감수성 세포주로서 가장 낮은 세포 생존율을 가지는 SNU638을 선별하였다(도 1b 참조). In order to confirm the sensitivity of the IM156 drug, each of the various gastric cancer cell lines obtained and cultured in Example 1.1 was treated with the IM156 drug, a type of OXPHOS inhibitor, at each concentration. The values measured for cell viability through the MTT assay for each concentration of the drug are shown in the graph of FIG. 1 . As a result, it was confirmed that the cell viability rapidly changed within the concentration range of 6.33uM to 63.29uM (see FIG. 1a ). As a result of examining the cell line sensitivity when the concentration of the IM156 drug was in the range of about 20 uM, Hs746T having the highest cell viability as a resistant cell line and SNU638 having the lowest cell viability as a sensitive cell line were selected (see FIG. 1b).

실시예 2: 줄기세포적 특성과 SDHA 발현량과의 상관 관계 확인Example 2: Confirmation of correlation between stem cell characteristics and SDHA expression level

상기에서 수득한 위암 세포주 외에 다른 암종의 세포주(유방암 세포주인 MDA-MB-231 및 MCF7, 대장암 세포주인 HCT116 및 SW480, 폐암 세포주인 A549, 위암 세포주인 Hs746T, YCC9 및 SK4)를 별도로 수득하여 실시예 1.1과 같은 방법으로 배양하였다. 이들로부터 SDHA 바이오 마커의 발현 수준을 측정하고자 웨스턴 블롯을 수행하였다(도 2a 참조). 일반적으로 암 세포에서 SDHA의 발현이 관찰되는 것을 확인할 수 있었다. 이들 중에서 유방암 세포주와 위암 세포주만을 선택하여 추가적으로 모세포(parent cell; P cell) 및 모세포를 연속 배양하여 30일 이후에 생존한 세포(selected cell; S cell)로 나누어 SDHA 단백질의 발현 정도를 확인하기 위하여 웨스턴 블롯을 수행하였다. 그 결과 줄기세포성/난치암 아형에서의 SDHA의 발현이 높게 나타났으며 특히 위암 세포주에서 차이가 현저한 것으로 확인되었다. 다양한 위암 세포주 모두에서 줄기세포적 특성을 갖는 S 세포인 경우 상대적으로 상기 단백질의 발현량이 높은 것으로 나타났다(도 2b 참조). 이는 결국 SDHA의 발현량이 높은 것은 상대적으로 치료율이 낮을 수 있음을 시사한다.In addition to the gastric cancer cell lines obtained above, cell lines of other carcinomas (breast cancer cell lines MDA-MB-231 and MCF7, colon cancer cell lines HCT116 and SW480, lung cancer cell lines A549, gastric cancer cell lines Hs746T, YCC9 and SK4) were separately obtained and carried out. It was cultured in the same manner as in Example 1.1. Western blot was performed to measure the expression level of the SDHA biomarker from them (see FIG. 2a ). In general, it was confirmed that SDHA expression was observed in cancer cells. Among them, only breast cancer cell lines and gastric cancer cell lines were selected and additionally, parent cells (P cells) and parent cells were continuously cultured and divided into cells that survived after 30 days (selected cells; S cells) to check the expression level of SDHA protein. Western blot was performed. As a result, the expression of SDHA was high in stem cell/refractory cancer subtypes, and it was confirmed that the difference was particularly significant in gastric cancer cell lines. In all of the various gastric cancer cell lines, the expression level of the protein was relatively high in the case of S cells having stem cell characteristics (see FIG. 2b ). This suggests that the high expression level of SDHA may result in a relatively low therapeutic rate.

실시예 3: IM156 약물 특이적인 SDHA 마커 발현량의 변화 확인Example 3: Confirmation of change in expression level of IM156 drug-specific SDHA marker

3.1. SDHA 마커의 발현량 변화 확인3.1. Confirmation of change in expression level of SDHA marker

IM156 약물에 대한 SDHA 마커 특이성이 인정되는지 확인을 위해 감수성 세포주와 내성 세포주에 같은 OXPHOS 억제제 중의 하나인 로테논 약물을 추가로 이용하여 비교 실험을 수행하였다. IM156 약물과 로테논 약물 투여 전 후의 SDHA 발현량 변화를 측정하기 위해 웨스턴 블롯을 수행하였으며 그에 따른 결과를 도 3a 및 도 3b에 나타내었다. In order to confirm whether SDHA marker specificity for the IM156 drug is recognized, a comparative experiment was performed using rotenone, one of the OXPHOS inhibitors, in addition to the sensitive cell line and the resistant cell line. Western blot was performed to measure the change in SDHA expression levels before and after administration of IM156 drug and rotenone drug, and the results are shown in FIGS. 3A and 3B.

실시예 1에서 선별한 약물 내성 세포주인 Hs746T와 감수성 세포주인 SNU638을 배양하여 로테논과 IM156을 각각 투여하였다. 그 결과 로테논을 투여한 군에서 내성 세포주의 SDHA 발현량에 변화가 나타나지 않은 반면, IM156을 투여한 군에서 SDHA 발현량이 약물 투여 전에 비하여 증가한 모습을 관찰할 수 있었다(도 3a 참조). 이를 명확히 비교하기 위하여 약물 감수성 세포주에 각 약물을 투여하고 관찰한 결과 로테논을 투여한 군에는 약물 투여 전 후에 SDHA 발현량에 변화가 없었으나, IM156을 투여한 군에서 SDHA 발현량이 감소하는 결과를 확인할 수 있었다(도 3b 참조). 이러한 결과를 종합하면 약물 투여 전 후의 SDHA 발현량 변화를 통하여 IM156 약물에 대한 내성을 확인하기 위한 마커로서 SDHA가 사용될 수 있음을 시사한다. Hs746T, a drug-resistant cell line selected in Example 1, and SNU638, a sensitive cell line, were cultured and administered with rotenone and IM156, respectively. As a result, the SDHA expression level of the resistant cell line did not show change in the group administered with rotenone, whereas it was observed that the SDHA expression level increased in the group administered with IM156 compared to before drug administration (see FIG. 3a ). In order to clearly compare this, each drug was administered to a drug-sensitive cell line and observed. As a result, there was no change in SDHA expression level in the group administered with rotenone before and after drug administration. was confirmed (see FIG. 3b ). Taken together, these results suggest that SDHA can be used as a marker to confirm resistance to the IM156 drug through the change in SDHA expression level before and after drug administration.

3.2 각 약물 투여시 미토콘드리아 호흡률 비교3.2 Comparison of mitochondrial respiration rates upon administration of each drug

상기 약물에 대한 내성 발생 유무를 확인하고자 실시예 1에서 선별된 Hs746T와 SNU638 세포주를 비교하였다. 먼저, 내성 세포주인 Hs746T에 로테논과 IM156 약물을 각각 투여하여 세포 호흡 과정에서의 산소 소모율(Oxygen Consumption Rate; OCR)을 측정하여 분석한 결과를 도 4a 내지 4c에 나타내었으며, 감수성 세포주인 SNU638도 동일하게 측정하여 분석한 결과를 도 5a 내지 도 5c에 나타내었다. 이하 모든 실시예에 따른 도면 상의 수치는 평균±표준오차범위(standard error of the mean; SEM)로 나타내었으며, *는 통계적 유의성을 나타낸다(*P<0.05, **P<0.005). 산화적 인산화의 지표인 산소 소모량은 암 세포의 대사 과정으로서 암 세포의 분열이 얼마나 활발하게 일어나는지를 확인할 수 있는 척도가 될 수 있다. The Hs746T and SNU638 cell lines selected in Example 1 were compared to determine whether resistance to the drug occurred. First, rotenone and IM156 drugs were administered to the resistant cell line Hs746T, respectively, and the oxygen consumption rate (OCR) was measured and analyzed in the process of cellular respiration. The results of measurement and analysis are shown in FIGS. 5A to 5C. Numerical values on the drawings according to all Examples below are expressed as mean±standard error of the mean (SEM), and * indicates statistical significance (*P<0.05, **P<0.005). Oxygen consumption, which is an indicator of oxidative phosphorylation, is a metabolic process of cancer cells and can be a measure of how actively the division of cancer cells occurs.

센서 카트리지(Agilent Technologies, Palo Alto, CA, USA)에 XF Calibrant buffer(Agilent Technologies) 1mL을 넣고 37℃, non-CO2 incubator 조건에서 오버나이트하였다. 위암 세포주인 A549 세포를 XF24 세포배양 마이크로플레이트(Agilent Technologies)에 7×104 cells/well로 분주한 후, 37℃, 5% CO2 incubator에서 24시간 후 로테논 약물을 50nM 또는 IM156 약물을 15uM을 투여한 후 37℃, 5% CO2 incubator에서 오버나이트시켰다. 약물 투여한 마이크로플레이트에 XF base media(pH 7.4, Agilent Technologies)를 각각 500uL/well 넣은 후 37℃, non-CO2 incubator에서 1시간 동안 추가로 배양하였다. 센서 카트리지의 A port에 1.5uM 올리고마이신(Agilent Technologies), B port에 2.0uM FCCP(Agilent Technologies), C port 0.5uM 로테논/안티마이신 A(Agilent Technologies)을 넣고 추가로 XF24 마이크로플레이트도 함께 Agilent seahorse XF24 (Agilent Technologies)기기에 넣어 OCR을 측정하여 비교하였다. 이때, 측정된 값은 1분 당 picomole 단위(pM/min)를 기준으로 하여 퍼센티지 값을 비교하였다. 1mL of XF Calibrant buffer (Agilent Technologies) was added to the sensor cartridge (Agilent Technologies, Palo Alto, CA, USA), and overnight at 37°C, non-CO 2 incubator conditions. After dispensing A549 cells, a gastric cancer cell line, at 7×10 4 cells/well on an XF24 cell culture microplate (Agilent Technologies), after 24 hours at 37°C, 5% CO 2 incubator, 50 nM of rotenone drug or 15 uM of IM156 drug After administration, it was overnight at 37°C, 5% CO 2 in an incubator. After each 500uL/well of XF base media (pH 7.4, Agilent Technologies) was added to the drug-administered microplate, it was further cultured at 37°C, non-CO 2 in an incubator for 1 hour. 1.5uM oligomycin (Agilent Technologies) in port A of the sensor cartridge, 2.0uM FCCP (Agilent Technologies) in port B, and 0.5uM rotenone/antimycin A (Agilent Technologies) in port C of the sensor cartridge, and an Agilent XF24 microplate. OCR was measured and compared in a seahorse XF24 (Agilent Technologies) instrument. In this case, the measured values were compared with percentage values based on picomole units per minute (pM/min).

IM156 약물에 대한 내성 세포주와 감수성 세포주에서의 결과를 살펴보면, 로테논 약물을 투여한 경우 시간에 따른 OCR 측정 정도의 값이 동일한 양상(도 4a 및 도 5a 참조)을 보이는 것을 확인할 수 있는 반면, IM156 약물을 투여한 경우 OCR 측정 정도의 값은 내성 세포주와 감수성 세포주에서 반대의 양상(도 4b 및 도 5b 참조)을 보이는 것을 확인하였다. 이를 더욱 명확하게 비교하기 위하여 기초 호흡량(basal respiration), 호흡용량(spare respiratory capacity) 및 ATP 생산량(ATP production)을 음성 대조군 기준으로 하여 환산한 그래프로 나타내었다. 도 4c 및 도 5c를 참조하면, 내성 세포주와 감수성 세포주에서 현저한 차이를 보이는 것은 IM156 약물에 해당하는 것을 확인할 수 있다. 따라서 상기 결과를 종합할 때, 동일한 OXPHOS 약물 중에서 IM156 약물 특이적인 내성 마커로서 SDHA가 활용될 수 있다는 것을 확인하였다.Looking at the results in the IM156 drug-resistant cell line and the sensitive cell line, it can be seen that when the rotenone drug is administered, the OCR measurement degree over time shows the same pattern (see FIGS. 4a and 5a), whereas IM156 When the drug was administered, it was confirmed that the OCR measurement degree showed the opposite pattern (see FIGS. 4b and 5b ) in the resistant cell line and the sensitive cell line. In order to more clearly compare this, basal respiration, spare respiratory capacity, and ATP production are shown as a converted graph based on the negative control standard. Referring to Figures 4c and 5c, it can be confirmed that the significant difference between the resistant cell line and the sensitive cell line corresponds to the IM156 drug. Therefore, combining the above results, it was confirmed that SDHA can be utilized as an IM156 drug-specific resistance marker among the same OXPHOS drugs.

실시예 4: SDHA 발현 조절을 통한 항암제 내성 치료 가능성 확인Example 4: Confirmation of anticancer drug resistance treatment potential through SDHA expression regulation

4.1 줄기세포성/난치암의 치료 효과4.1 Therapeutic effect of stem cell/refractory cancer

위암 세포주인 SK4 세포주로부터 P 세포(PSK4)와 S 세포(SSK4)를 준비하였으며, P 세포군과 S 세포군에 SDHA의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제인 3-니트로 프로피온산(3-nitropropionic acid; 3NPA) 또는 아트페닌 A5(atpenin A5)을 투여한 후 OCR을 측정한 결과를 도 6a 및 도 6b에 나타내었다. 줄기세포적 특성을 띠는 SSK4 세포에 상기 두 가지 제제가 작용하여 암 치료 효과가 있는 것을 확인하였으며, 아트페닌 A5에 비하여 3-니트로 프로피온산이 더욱 효과적으로 암 세포와 함께 암 줄기세포도 치료할 수 있음을 확인하였다. P cells (PSK4) and S cells (SSK4) were prepared from the SK4 cell line, a gastric cancer cell line, and 3-nitropropionic acid (3 After administration of -nitropropionic acid; 3NPA) or atpenin A5 (atpenin A5), the results of measuring OCR are shown in FIGS. 6a and 6b. It was confirmed that the above two agents act on SSK4 cells with stem cell characteristics to have a cancer treatment effect. Confirmed.

4.2 IM156 약물 내성 치료 용도의 확인4.2 Identification of IM156 Drug Resistance Therapeutic Uses

내성 세포주인 Hs746T에도 같은 암 치료 효과가 나타나는지 확인하기 위하여 로테논 또는 IM156 약물 단독 투여한 경우를 100%로 환산하여 3-니트로 프로피온산과 병용 투여 후의 ATP 생산량을 각각 측정하였다. 그 결과 줄기세포성 특성을 갖는 세포주에 효과가 있었던 것과 마찬가지로 두 약물과 병용 투여시 모두에서 암 치료 효과가 나타난 것을 확인하였다(도 7a 및 도 7b 참조). 또한, SDHA 발현 억제제의 효과를 확인하기 위하여 추가 실험을 수행하였다. 6 웰 플레이트에 세포를 60% 컨플루언시(confluency)로 분주하고, 37℃, 5% CO2 인큐베이터에서 오버나이트 시킨 후 2% FBS(without antibiotics) RPMI-1650 배지로 교체하여 배양하였다. siRNA 50nM(바이오니아 제품)과 트렌스펙션 시약(transfection reagent, mirus bio 제품)을 섞은 뒤 총 부피를 200uL로 맞추어 20분 동안 상온에서 인큐베이션시켰다. 6 웰 플레이트에 siRNA와 트렌스펙션 시약 혼합물을 천천히 넣어주어 37℃, 5% CO2 인큐베이터에서 오버나이트 후 10% FBS(with antibiotics) RPMI-1650 배지로 교체하여 72시간 인큐베이션시켜 SDHA 발현 억제제를 제조하였다. 상기와 같은 실험을 반복 수행하였다. 하기 표 1을 참조하면, 로테논과 IM156 모두에서 효과가 나타났으며, 특히 IM156 약물에 내성을 갖는 세포주에서 IM156 약물의 내성이 극복되어 암 치료 효과가 나타난 것을 확인할 수 있었다(도 7b 하단 참조).In order to check whether the same cancer treatment effect is also shown in the resistant cell line Hs746T, the ATP production after administration with 3-nitropropionic acid and 3-nitropropionic acid was measured in terms of 100% when rotenone or IM156 was administered alone. As a result, it was confirmed that the cancer treatment effect was shown in both drugs when administered in combination with the two drugs similarly to the effect on the cell line having stem cell characteristics (see FIGS. 7a and 7b ). In addition, an additional experiment was performed to confirm the effect of the SDHA expression inhibitor. Cells were aliquoted to 60% confluency in a 6-well plate, and incubated at 37° C., 5% CO 2 overnight in an incubator, followed by replacement with 2% FBS (without antibiotics) RPMI-1650 medium. After mixing siRNA 50nM (Bioneer product) and transfection reagent (Mirus bio product), the total volume was adjusted to 200uL and incubated at room temperature for 20 minutes. After slowly putting the mixture of siRNA and transfection reagent in a 6-well plate at 37° C., 5% CO 2 overnight in an incubator, it was replaced with 10% FBS (with antibiotics) RPMI-1650 medium and incubated for 72 hours to prepare an SDHA expression inhibitor. . The above experiment was repeated. Referring to Table 1 below, effects were shown in both rotenone and IM156, and in particular, it was confirmed that the resistance of the IM156 drug was overcome in a cell line resistant to the IM156 drug, thereby showing a cancer treatment effect (see the bottom of FIG. 7b ).

ATP production(%)ATP production (%) 로테논rotenone IM156IM156 단독Exclusive 100100 100100 병용(3NPA)Combination (3NPA) 50.6450.64 47.0047.00 병용(siSDHA)Combination (siSDHA) 37.6637.66 12.3012.30

상기 결과를 종합하면, SDHA발현 억제와 직간접적으로 관련이 있는 3-니트로 프로피온산 약물 또는 아트페닌 A5 약물을 처리하거나 SDHA의 발현을 siRNA를 이용해 넉다운 시킴으로써 항암제 내성이 치료되는 것을 확인할 수 있다. Summarizing the above results, it can be confirmed that the anticancer drug resistance is treated by treating the 3-nitropropionic acid drug or the atfenin A5 drug directly or indirectly related to the inhibition of SDHA expression, or by knocking down the SDHA expression using siRNA.

상기 실시예 1 내지 4를 종합적으로 고려하면, 약물의 종류에 따른 항암제 내성 진단 방법은 약물 종류에 따라 달라질 수 있음을 시사하며, 상기 SDHA발현 억제제를 투여할 경우 항암제 내성 보다 구체적으로 IM156약물 내성을 극복하여 암 치료 효과의 향상을 유도할 수 있을 것으로 기대된다.Comprehensively considering Examples 1 to 4, it is suggested that the method of diagnosing anticancer drug resistance according to the type of drug may vary depending on the type of drug, and when the SDHA expression inhibitor is administered, more specifically, IM156 drug resistance It is expected to lead to an improvement in the cancer treatment effect.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for diagnosing and treating anti-cancer drug resistance <130> PDPB194289 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 664 <212> PRT <213> Homo sapiens <400> 1 Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu 1 5 10 15 Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly 20 25 30 Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser 35 40 45 Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala 50 55 60 Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu 65 70 75 80 Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr 85 90 95 Arg Ser His Thr Val Ala Ala Gln Gly Gly Ile Asn Ala Ala Leu Gly 100 105 110 Asn Met Glu Glu Asp Asn Trp Arg Trp His Phe Tyr Asp Thr Val Lys 115 120 125 Gly Ser Asp Trp Leu Gly Asp Gln Asp Ala Ile His Tyr Met Thr Glu 130 135 140 Gln Ala Pro Ala Ala Val Val Glu Leu Glu Asn Tyr Gly Met Pro Phe 145 150 155 160 Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln 165 170 175 Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val 180 185 190 Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser 195 200 205 Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu 210 215 220 Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu 225 230 235 240 Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr 245 250 255 Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser 260 265 270 Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln 275 280 285 Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly 290 295 300 Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn 305 310 315 320 Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp 325 330 335 Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg 340 345 350 Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu 355 360 365 His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser 370 375 380 Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile 385 390 395 400 Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn 405 410 415 Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val 420 425 430 Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly 435 440 445 Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly 450 455 460 Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys 465 470 475 480 Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu 485 490 495 Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg 500 505 510 Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val 515 520 525 Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly 530 535 540 Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr 545 550 555 560 Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu 565 570 575 Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His 580 585 590 Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys 595 600 605 Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys 610 615 620 His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu 625 630 635 640 Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr 645 650 655 Val Pro Pro Ala Ile Arg Ser Tyr 660 <210> 2 <211> 46593 <212> DNA <213> Homo sapiens <400> 2 actgcagccc cgctcgactc cggcgtggtg cgcaggcgcg gtatcccccc tcccccgcca 60 gctcgacccc ggtgtggtgc gcaggcgcag tctgcgcagg gactggcggg actgcgcggc 120 ggcaacagca gacatgtcgg gggtccgggg cctgtcgcgg ctgctgagcg ctcggcgcct 180 ggcgctggcc aaggcggtga gtccgtgccg cggaccgggg cggggcaggc gggggccgag 240 gcggcggtag gagcgggacg gtccccagcg ggtccgagcg gagcgggcgc cgggtccctg 300 cgccctctgt cccgggatcg ggaaggggct gagagagccc tgggccggtg cgaggggaag 360 ccgcggggcg gactcgggga cccggggagc tcggtcctta gtagatagtc cgcgtccggg 420 tgaaggtcac aaccccgcgg gcttgctggg cgtcccctcc gccgccttgg tccgggcctg 480 gggtcctggg accccgcggg ctgaggtagc ccctcgcctc agtgcctggc aggtggactc 540 ggggaggagt cgtgtctgcc caaggtcacc cgggcggata gcggccggtg gccgccctgg 600 ctgggctggg cctctgccgc cctctgtgcg ggttgtcctg aggagcagcc cgcagcccgt 660 gggtggggcc ggcggggcgg gtgagaccgc ccgggtgggt gcgaggagtg gccgggctcg 720 gcccggtggg cgtccggtgg gaagcgtggc gcgcccgagc ttaggcttgc agttcgcctt 780 tccagaaagc gcaaatctgt ccatgtccac ttcgagacct tgtaagttaa gggcttctac 840 tttgggtcgt gtttggtggt ccttatgcca ccaaaaatgt gccagtgttt aaaagcagct 900 gtgccagttt ttaaaactca ggcggagagc tcagcgcact gaccgggcga ggacgccggg 960 acctgtgctt gcctgtgcgc tgagtgcctc tagggccggg ctcggcttag tccaggatgg 1020 tggtcagggt tatacttccc tgagcccttg ctctctgagt gtctgaatgt gccctctacg 1080 attgcatctt aagaatcggc cttctagggt tttatttaat caagctaaat tggataggtt 1140 tagttgtttg gttcttttaa atgaacttac ccacccacct ccttaattac aaagtaattt 1200 taaattgcag aataaaaatc tcaataggaa ccaaggcatt cagcaatatt gatttgaatt 1260 atgcctgtaa ttgtgcaatt ttctcctttt tgaaatactt attaaaaatc tcgttgagtt 1320 aaatgtgagg attagtcata cagccatcct gccaacatca gaaagcgtgt aaaccgttct 1380 agtgtgttgc tgtggttggt actgactgag cagagacccc tgccgcatct tgggctttta 1440 agagctgcta ggagggcgtc cacaagcagg aaggaaagcc catggtcagt ggggcttttt 1500 aggggaaatg gtagcttgtg attggagaaa agctagagct agtgcctttg tcctcaaccc 1560 agatggtgcc cggtgttcct tctgcagact aagaccctgc cagcggcgga ggcacctcag 1620 acgggatggc aagatagcaa aattgaacca aaatttagtc ttgggttttg taaaagtctt 1680 tttatcttga tgaagttagc ttttcctaca gaaattctgc ctgcaagcag catatttatc 1740 ggcatttcag ataggctttt tttcctgaag aattggagtt tacgaaggaa cagcataatc 1800 aaaaaattag cagaaaaaag aaagaaattc gtgatagcca actgagaaat gtgtttggaa 1860 aatagatgct gaggataatt tcagtaaagt ccagagagaa ttttttaacc tcattttggc 1920 cagttctgtg gtgttttgat cattctgtat ttacaactgc tttcaagtga cgaacttatc 1980 agacttattt tgcttttggt ttctttttat ggcccagtgc actgacacag cttgcaaact 2040 gttcgaaagc agtttaatca gaggaaaaag aaaaccaaga aacttcaata gttactggtt 2100 aactgcttac ttcaggtgaa gtttttcaaa tcccttaaca tgaacatcca atatattgtt 2160 ctaggacaaa ttgatttctt ttctttgaat ggattgttct gaatatgttt aatggtgttc 2220 tagaaataca ttatttaggc cattatagta gtaaaccttt gctttgcttc ttgggataat 2280 tgtaacaaca atggcattcc attctgcatt cctcatgatg tttttcctga tttatttgaa 2340 aaattgtgac ttgttttttg atatgaaacc tggatattaa tattagttgg gagtctcaga 2400 gatttttccc aagggtcttt agataggggg gcaagtccag gatccagagc caggccggcc 2460 gacttgaatc ctctctgagc tccgtctttc cgtggggtta gtgaggattc tgtgggtaat 2520 gtttgaaacc tttggtgatg ctcttgagaa ccagggctca cagcctatgc acagtaacca 2580 tgtgtgaact gaattttttt ttttttttga gacagagtct cactctgtca cccaggctgg 2640 agtgcagtgg cgcaatctcg gctcactgca agctctgcct cccgagttca cgccattctc 2700 ctgcctcagc ctcctgagta gctgggacta caggcacctg ccatcatgcc cggctaattt 2760 ttttgtgttt ttagtagaga cggggtttca ccatgttagc cagaatggtc tccatctcct 2820 gacctcgtga tccgcccgcc tcggtctccc aaagggctgg gattacaggt gtgagccacc 2880 acgcccggcc gaactgtatt ttaagtatcc ctgtttttaa aataataaac actaaaattc 2940 tagtgagaat aattacacgc agttgtaaaa gaatcaatga gtataccctc cctcacccag 3000 ttcattttcc atgaaagcaa ttcctgtttc tgatactgac tcctggcatt tgtaatacct 3060 ctgtttcttt agcggatatg caggtgcatt tttatcagtt ttagacttct tgcctagaga 3120 ggggatttag ttcacttaaa ctaccctgct tccgcttctt cctgatacag tcgtgcgttt 3180 ttaggttctt cgttggctgc ttttgtgatt gtgtaggttg tacatgcaga ttagtttctt 3240 gctcgtgatt tataggtgcg ttgtattaga tgaggacccc ttactttgct agatttcgga 3300 tatgaatgtc tctgcacttc ttacttttcc cctccacctc ctaattcagt catctgaaat 3360 tctgtattgt taagcaaggt ctaagtattc cttttagtta tatgttcccc attttttttc 3420 ttgaggaaat gtttgatagt ttctcctaaa aaaattaaca attggcacaa aagactagtt 3480 ttgtgtcaaa actagttttg agttttatct aaagactgaa attggcttaa agttgggctt 3540 tccaaattca aaaatctgcc ccagattaga tttagattga gagggttagt gtccttttcg 3600 ccagggggat ggcgtgataa tttgttcaag attgtgttat agtagctgcc ccttttaagc 3660 cagctctgtg tgtgtgtggt ggggggtggg cagtgggtct tccacatcaa catcctagaa 3720 agaacgaata aacattgagt gatctcactg tttctactta catttggtat aatatactgt 3780 tcttactggt gctattacta tgttaatagg gcactttaca acattttcaa gaacatttgt 3840 attaaaatta tttcaaagac ttctttctta aaatatgatt ttaccatgca aaaattttac 3900 taaggtagaa gaatatttgt tctttctcat tcattttctg aaaaaagaaa aaagctgaat 3960 tagcttatac caataaaaac agagactaga aattggagaa atgaagaata attttttatc 4020 ccacataata agtaatttct gaattgcaag tatttctaaa tacttgaaga cattcctcac 4080 atcccctctt ctgattgcat ttatttccta aagctttttt ttcttttctt tttttttttt 4140 ttggagacgt tgtctctctc tgttgccgag gccggagtgc agtggcacag tctaggctca 4200 ctgcagcctc tgccttctgg gttcaagcga ttctcctgcc tcagcctccc aagtagctgg 4260 gattacaggt gcccgccacg acgcccagct aattgttagt agagatgggg ttttgccatg 4320 ttggccagac tggtcttgaa ctcctgactt caggtgatcc acccaccttg gcctccccaa 4380 atgctgggat tacaggcatg agccaccgtg cccagcccct aaaactattc ttgatgatat 4440 ttctgagact attcagtggt cttctcaaat gtagccagca gaatggaaaa cgtatcccct 4500 aaatggctgg ccaaccttag catatggaca gtgtcacctc tctcacacag agccactaaa 4560 aactaaacac caaaaccagt tttcttgggt aaagttttct aagatggaaa atttaagcag 4620 caagatgtgt caagttgtag atgttggcca ggaaaaagcc agcagcagcc aggcagggga 4680 gagtgtgcat ccgacatcct cctgtgtgat gaagggatga cacctcgtcc ctctaggctg 4740 tcagctttta ctgttccagg atacagatct cctgattcaa tgttgagtgc cttttgaact 4800 gaccacaagc cctcctggac gattggaact gtaatgtgga aagggctatg atggagccag 4860 ttaaaatgct tcattacttg caaaatacca catacagtaa tacaatatac acgtctttcc 4920 ccttctccac taagtagcat acattaagac ttcacagagg aagtgtgcct ttttcatttc 4980 gtatctgagt cagtgagtat cctgtttgga aacaagcttc atcctggttt tctagagtgc 5040 caagtcaggg tggaaacgag gacctggggg ctcagtcctt ccttgcccct tgggctgcct 5100 tcagggttaa gtagagggtc ccagctgagc tctctggatg cacaggagca cctgggtaca 5160 taagaaggtg aacagtttgc aaggggaaat tactatcccc cacagcattt gttccttcag 5220 gacactaacc ctctggatct gtgtcttctg tgtctccagt ggccaacagt gttgcaaaca 5280 ggaacccgag gttttcactt cactgttgat gggaacaaga gggcatctgc taaagtttca 5340 gattccgtaa gttcatgctt tttgttccat tataaatgat ttttttggct tagggggtaa 5400 ggatctatac cagtttgttt tcatatgagt catagacata agggaaaaat ttctcatagg 5460 tatccaatgc atgctgaaat tattttcagt gtaataatac ttaattgcaa gtacaaatat 5520 aaacatagat gtttacattt ttacttgtat ctgttatgta tctataaact agatttaaat 5580 ttagatcaag taaagcaata aattaaaata aaatgaacag tgtctgctgt gatagatgat 5640 aaaattccta ctgaaaatag gacggtgggg ccagtgaaca gtatctgctg tgatagatga 5700 taaaatccta ctgaaaatag gatggtgggg cccctcgggt gtgggttgct ttgtatttag 5760 tgagcctatc tctgttgaaa aactgaaact tgctgagaag ttgttttctt ataagcccac 5820 aatagcgact gaatactcct tggcaccttc tcaggcataa gcataggcac ggccctgaag 5880 tagagttgtg gtcctcagtc tgaccccatg ggatagaccc tctaccattc gtagaatctg 5940 attgtcagtc ccttctccag gtgcgaatgt gcccacctct ccccgaactg ttcagggctc 6000 agccccagga caggatggag gccctgtgtg cccagcagtt gctcctttta tctttgtcaa 6060 gctctttcac tgtcacaaga ttcttcatgt ttggcatagt ggaacatgtg attgacaggt 6120 gaatttttct tttccagatt tctgctcagt atccagtagt ggatcatgaa tttgatgcag 6180 tggtggtagg cgctggaggg gcaggcttgc gagctgcatt tggcctttct gaggcagggt 6240 ttaatacagc atgtgttacc aagctgtttc ctaccaggtc acacactgtt gcagcacagg 6300 taagagaaag gtgccccact gtgctcccac tccgtgcagg tcccgcgcag cctcgcactt 6360 tctacctggg cagccccctg cctcctcccc ctgctccagc cacatggcct cttgctgtgc 6420 cttactcagc tcaccctctc aggggtctct ccctggagcc tcttccctgg ggactttgaa 6480 gggcgggaga cttgttgtca ctcctaattc agactccagt cacacttggg ttttctctga 6540 ccatcttgcc tactaccttc cccacctacc cccgccaccc caacacctta agaaaaggag 6600 atcacctaaa gaggaggaat cagaatttag gttggggaag aaaagggcaa gggtttcatt 6660 tgtccctgtg cttctgtctt ctgggactct ctgaggggta agacagtggt gggcacacac 6720 agccaaagga agctggggta caggggagtg cgactctgag tgtggagttt attacttggc 6780 aggaagcact tctagtcttt aacacatgcc cgtaaatgcc attgggaaga tttgttaata 6840 aaattatctg gaaagatttg ttgagtacct tttctgtgcc agataggtta ggttataagc 6900 atattacagg tagcctttca ctcactgctc cagtcagccc ttcctggagt tccctgtgtc 6960 tccaccacgc agatgaggag actgaggcta aggatggaat cactgggcga gtcggggagg 7020 ggttgtgatc tggaatctgt cgggtctcgc tgctcctctg ctgaggtcag ccctcactgg 7080 gagtcactgt gtgagtagtt ggctttctct gaatcccccc agcgggtgga tttgggcctg 7140 gaagacaaag ttggcgctcc tgtttgtggc ttgtaaggag tggttggtgt ttccagggag 7200 gaatcaatgc tgctctgggg aacatggagg aggacaactg gaggtggcat ttctacgaca 7260 ccgtgaaggg ctccgactgg ctgggggacc aggatgccat ccactacatg acggagcagg 7320 cccccgccgc cgtggtcgag gtgatgggcg ggaggctctg ggtgttctcg tggtctgttt 7380 ctagtacaaa agaatcctgg aaaaaaatgt aagcaattga ggcggatgtg gcagccaaaa 7440 gaatggtgat gagcaaagtt cacaagaaga gtctttttcc gttatgaact atgcattata 7500 tgtaacaaga aaacttctct ttgatgaagt gttgacattt tcataaaata ggttactttg 7560 ggtttgcaga tttgtgttaa acttgtttag tgtagattag ctgcgaatat cttgactcct 7620 ttaaggtgtt aaggtttttg tttgttttta tctttcacag ctagaaaatt atggcatgcc 7680 gtttagcaga actgaagatg ggaagattta tcagcgtgca tttggtggac agagcctcaa 7740 gtttggaaag ggcgggcagg cccatcggtg ctgctgtgtg gctgatcgga ctggccactc 7800 gctattgcac accttatatg gaagggtaag gccgcccccg tccacctgag acaggacacg 7860 tagtgctggg gcttatggtg acagtgggga atgggttagc gtgcccagtg agtcagccag 7920 agattacgta aaaagcaaca gagaacagca gcgtggggcg catgcagcga ctgtggatgt 7980 gacaggacca gacgtgtgcc ttgaggagct gtccctaagg cagtgtgtga cttcttgcat 8040 ctatgtcaga aagttgaatc gataatctta cataccaggt tttaacttgg gatatgtgac 8100 actcaacata caagagcaga gcaagcaggc caggcacagt ggctcaggtc tgtaatccca 8160 gcactttagg aggccaaggc aggaggatca cttgagacca gaagtttgag accagcctgg 8220 acaacatagc aagaccctgt ctctacaaaa agtttaaaaa ttagctgggc atggtggtac 8280 atgcttgtaa tcccagttac tcaggaggct gaggctcctg gatcacttga gaccaggagg 8340 ttgaggctac agtgagccat gttcgtacca ctgcactcca gcctgggcaa caggagaccc 8400 tgtctcaaaa aaagagaaag aacaggctgg gtgcggtggc tcacgcctgt aatcccagca 8460 ctttgtgagg cctaggcggg cagatcacaa ggtcaggagt ttgaggccat cttgcctaac 8520 actgtgaaac cccgtctcta ctaaaaatac aaaaaaatta gccaggcatg gtggcaggca 8580 cctgtagtcc cagctgcttg ggaggctgag gcaggagaat ggcgtgaacc caggaggcgg 8640 aacttgcagt gagccgagat cgcacccctg cactccagcc tgggcaacag aatgagactc 8700 catctcaaaa aaaaaaaaaa aaaaagaaca agtattttaa gtctctttta ccacctctga 8760 gttcctgaat ggattggttt tgtttgtttt gttttgtttt gtttttgaga cggagtctca 8820 ctctgtcacc caggctggag tgcagtagca caatctcagc tcactgctac ttccacctcc 8880 tggattcaag tgattctcct gcctcagcct cccgagtagc tgggactaca ggcgcacgcc 8940 accatgcctg gctaattttt tgtattttag tagagacagg gtttcccatg ttgcccaggc 9000 tggtcccgaa ctcctgagct caggcagtcc acctgccttg gcctccgaaa gtgctgggat 9060 tgcaggcgtg agccaccgca cccggccatg gattgtttta atattaactg ttaacactga 9120 agaaagactt gaggtgacaa tagttactgg gtaatcagag tcaactttgg catggccaaa 9180 taatattctg aacggtattg attcagagta atcaatattc tgaactttgt tgttttctga 9240 tgcatgggga cggatcgtta atttgcaggt tgttagaaca ccagtgactt ctctgtggct 9300 gagtgcatcg acaagtgtgt ggtgggagga gaccgcggct ccatctggag caggagctgt 9360 catgtgggga gctggcccag gcttacaaga gcgacttgtg ctggctgagg gaacggcagg 9420 tccaggcggg cagcgctgtc cggcgcctac ctttctgcgg tgccggaatc tgctcgtctg 9480 caaccgtccg ctttggtagc tgccagccac atggggctgt tgctaatgtg gcaggtgtag 9540 ctgaagagct gaatgttttg acttatttta attaattaag tggttatgtg ttgccagtag 9600 ctcccatctg ggctgtgact gcatggtctg cggacctcac tctggcacca gactccgagt 9660 ggagctgcat gcggccaccg gacagtgtgg agtgcctctt cgggttgtgt agaagtagga 9720 aatgtgtcac caaaatagga gctgttgctg ctgcgttctc tagcacacct gccttgtctg 9780 tactgctcgg cgtggaatgc ctctcgggct ctgacagtgt cactgacact gttgctgatc 9840 tccttggatt tacctggtcc atttggatca agttctttca cctattcaca tgagcagata 9900 ccaccttaaa accttaaagt ttggcttaac acttcttgcc cttttttttt cctttctttt 9960 agtctctgcg atatgatacc agctattttg tggagtattt tgccttggat ctcctgatgg 10020 agaatgggga gtgccgtggt gtcatcgcac tgtgcataga ggacgggtcc atccatcgca 10080 taagagcaaa gaacactgtt gttgccacag ggtaggaatc tcatttctac tttattttgt 10140 ttataaaaat gaataaattt catttagagt ttctttattt taatgaaaat agaggcattg 10200 tagaataacg gtttagacac aggccttgat ataaccatgt gagggtgatg gcctttccca 10260 gccgtggttc ctcacctgta aagggtgagg acaggagcac ctgcctcagg gtgagaaagc 10320 atggtcctca gtagatggta gtggttgccc tcaggttcac agcttacctc tcccaattta 10380 gatgagggaa ctgtggccca aagagtcaac atggggtttt ctggcaaaat ctctcttgtt 10440 ttagtgggta ctgtattgat actgattcct cgggtgggta agtccttctt ctccacatga 10500 tgaaaataag taactttaat tttatacact gtcagttact ttaaccattt taaaagttaa 10560 aaagtgtcag tacagccaaa caaaaaatca gcaaaactac aggttgggaa gaaatatttt 10620 ccaaaccata tgtgtaataa tatcttacta tctaaaatag caaaaaaaaa accctactaa 10680 aaacaaccta ctaaacccta ctaaaaaaca accctactaa aaatgggcaa aggacttgaa 10740 tagatatttt tctagagaag acatacaaat ggccaattga tgtatgaaaa aatgctcaac 10800 gtcactaagc accagagaaa tgcaaattaa aaccccaatg agatatcatc tcatctcgct 10860 ccagttagaa tgactgttac caagaggaca aaagagcaag tgttggtgag gatgtggaga 10920 aaagggaacc ctgtgtgctg ttggtgggaa tgtaaattag tacaactatt acggaaaact 10980 ctggaggttc ctcaaaattc ataggactac tatgtgctcc agcaacccca tttctgggtg 11040 tatatccaaa gggcatgaaa tcagaagctc aaagagacac ctggacccta tgttcattgc 11100 agcattattc acaatacccg agatatggaa acaacctaaa tatgtgttgg tgtttaatga 11160 caatgtggtg tgtatacaca actgaatatt attcggctat gaaaatgaag gaaatcctgt 11220 catgtgtgac aacgtggatg aacccaaagt cattatgtta agtgaaacga gccaggcaca 11280 gaaagacaga tactgcatgt cactcatgtg gaatctaaaa cagtcacaac tcacagaaac 11340 agagtaggac agtggttgcc aggggctggg ggaatgcaga ctgtggcgat gctgattaaa 11400 ggtgtaactt cccgttacaa ggtgaagttc tgaaggtcta atatacaaca tggtggctat 11460 agttaacatt atacagcatg gtggccagag ttaacattat acagcatggt ggctatagtt 11520 aacattatac agcatggtgg ttagagttaa cattatacag catggtggct agagttaaca 11580 ttatacagca tggtggctag agttaacatt atacagcatg ctggctatcg ttaacattat 11640 acagcatggt ggccagagtt aacattatac agcatggtgg ccagagttaa cattatacag 11700 cacggtggct agagttaata tacagcatgg tggctatcgt taatattata cagcatggtg 11760 gctagagtta acattataca gcatggtggc tagagttaat attatacagc atggtggcta 11820 tcgttaatat tataacttga aatttgcgaa gatagtagac gttaggcgtc tgtatctcca 11880 aaaaaaggat aattgtatga ggtgatagat gtgtattaac ttgattgtgt catcatttca 11940 caaaataaat catcacgctg tacaccataa atatatagtt ttttttttaa attcatcaat 12000 catacctcag taaatctggg ggagaaaaaa atccataaaa attttaaaat tttcattata 12060 aaagtagtat atgcttattg gggaaacctt ttgaacagca caaagctaaa atacaaatag 12120 ggccaggtgt gcagtggctc acgcctgtaa tcccggcact ttgggaggcc aaagtgggag 12180 gatcacttga ggtcaggagt tcgagaccag cctggccaac atggtgaaac cccatctcta 12240 ctaaaaatac aaaaattaac tgggcgtggt ggtgcacact gtaatcccag ctactcggga 12300 ggctgaggcg gaagaatcac ttgaacctgg gagccggagg ttgctgtgag ccgagatcgc 12360 accactctac tccagcctgg tcaacagagt tagatcctat ctcaaaataa ataaataaaa 12420 tacaaatgga agtcctttct ttgatcccag gcttctatcc tacctgtgca gggtagcagc 12480 accagtggcg cggcccctag tgatatgtgt ggggtgtgcg tgagtagggg gttgtgtgtg 12540 cacagcactg agaagacggt gctgggggct gccgtgtcca ttctgtgatc tcaccagata 12600 ggaggtccag atgtgggccg ctgtgtgcag tcactgctct ctattgtttc cagaggctac 12660 gggcgcacct acttcagctg cacgtctgcc cacaccagca ctggcgacgg cacggccatg 12720 atcaccaggg caggccttcc ttgccaggac ctagagtttg ttcagttcca ccctacaggt 12780 agggcaggac gccttgcccg gcaggtgttt ggcttgtgtg tgtcttgtaa gcatgtgatg 12840 cctactcatt gctcttccat agttttatgt aataacatgg ttttgaagat cagcttcctc 12900 agctctcagg tcctaacttc aacgtcattt acctaaggag acttttccca cactcccctt 12960 cccctaaggc agtttgggcc accgtcttat gtatttctca agagcgctaa accctgcctt 13020 ggtgatactt aagccagcag taaagcaggg attgaggccg agcacagtgg ctcactcctg 13080 tagtcccagc actttaggag gccaaggcgg gcggatcacc tgaggtcagg agttcgagac 13140 cagcctgacc aacatggtga cacctcgtct ctactaaaaa tacaaaaatc agctgggcgt 13200 gatggcgtgt acctgtaatc ccaggtactt gggaggctga ggtgggagga atgcttgaac 13260 ctgggaggca gaggttgcag tgagccgaga tcgcgccact gcactccagc ctgggcaaca 13320 gagtaagact ccgtctcaaa aaaaaaaaaa aaaaaagtaa agcagggatt gttcagtgtc 13380 cctcttcgca acgaggtcgt cagctcctcg ggcaggcagg tcttctcgtt agctgtggtg 13440 ctccatgccc agcacatggt agggtctcca ccggggttta ctgagtgaac attctgagag 13500 ctgggagaat gccatggaac cgagaagcag cacaggcaga cttcagcttt gtaggagaac 13560 acggagcttc tgtgacaatg ggatgtaaag ttaggacgca gccgtgacag aaccccatgt 13620 gacattgggc gctgggctca gcccacgtga ccactgagag agcttgtcgt ggggaagatg 13680 agctcgtctt ggggacgtct gacggttgag gttacgaatg tgcaattcgg agacattaac 13740 tcagaaatga cagttgaagt cttgagtttg gaggaggttc ttcagaatga gtcagagaca 13800 gacacacacc tgcctcttgt ttgaggtgac ttgtcctata cttttctggg ttgcttttct 13860 acctgtggac gatggagacc tctgaagtgg tgccaaagaa ccagacctgt gtcttctctt 13920 tctctgtcag tgtcagcttt ctgatccctg gaagggatga aaataagaaa tgaatttgtt 13980 gtagtgtttt ttttttattt gtttcgagat ggggtcttgc tctgttgctc aggctggagt 14040 gcagtggagc aatcttgact cactgcagcc tttgtctccc aggctcaaac gatccttcca 14100 cctcagcctc ccaaacagct gggactacag gcatgtgtta ccatgcccag ctaattttcg 14160 agggtttttt cgtttttggt agagacggga tgtcaccgtt ttgcccaggc tggtctcaaa 14220 ctccagggct taggccatcc tcctgcctcg gcccctcagg gtcctgggat tatagccatg 14280 agccactgca cccggcctgt tgtatttttt attactgttt ttaatgaata aaatgtcact 14340 gagcccctga attccctctg taatttgctt agagctccac tgctcatgct ttgtcttcag 14400 tatgtgagaa gtaaccacag aaaaaagagc attgaaactt agaaaatcaa aagacagaca 14460 agaatcacat tcgtttttta aaagcaagat tgcccttttt gtatactaat aaaaattgta 14520 gctttcgaaa tacatttagt ttagggtttt tgatctcctt tgttaaaatt cgagagcttg 14580 gcatgccctg tttctcatcg cactgaggag tcacagagcc gctgtttgga gcacagaccg 14640 ccggactacc caggtttggg tttgagctct gtcctcagct gcatgactgg atgttaccaa 14700 gtgttaattt gcttgtcact gagaaagggg gtcgtactac ccagagttgt tgtaagactt 14760 aaacgagtta atatgtggaa agcagctaga actgcccata gcaagtgcag tgtaaatatt 14820 atctaaaata atcattatta ctgtccttgt cactgtttgg ataatttaga tatgaatact 14880 tcaagctagt attcttaact agtcaccaca gtatcatagt gcaaaagaat gttcaaaaat 14940 taaaacaaaa tttgaggcat ccaacgtaca ctgggctgta atcagagtat tggccagagg 15000 tctgtgggcc ggcctttctc ctctctgggg acaccgctct gccccagcct ctgctgcagc 15060 tgtcagcctt gtcagtgctt tttgttatcc agactttcta ctgtatctta actgtcttgc 15120 tgtgcagttt tgcacataat gccttctgct tatctttttc ctctttcccc caaaaaatgt 15180 cttgaaaaaa ataatgcatt tgaaatagag atctagcaat tgttaggtaa taaatatgtg 15240 tggttttttg caggcatata tggtgctggt tgtctcatta cggaaggatg tcgtggagag 15300 ggaggcattc tcattaacag tcaaggcgaa aggtttatgg agcgatacgc ccctgtcgcg 15360 aaggacctgg cgtctagaga tgtggtgtct cggtccatga ctctggagat ccgagaagga 15420 aggtgcgtgt gatttaccac cagcactgtc tgagcgggca cacgggccgg ggttgcttct 15480 gtgagtttca gcaccgctcg ccctcacctt cgtgtgcagg cgcatgtgca cagccacctc 15540 tcttagctgc tggcaggcgt ctgttagtct gcgatatttt cctaaagacc tacattttga 15600 aaattttagc cagtttcttt ctcaaatctg tggaacagag tttctcttag tgtgtgtgag 15660 tatgtgacgg agtatgggag agagagacac acacccaacc tgaagtcggc atgtgagcct 15720 tgggtgttgt gtctgatacc cacagatgtt ttttggcagc tttcaaagtg tgtgggttat 15780 ttggctttca gtaaaacagt ttgcagctct ttcattgcct gaccctgttc tttaatgtaa 15840 tgacatttgc taaatatctg ctggtatggc ctttagaggt tttacatttt tatattaaaa 15900 aaacagagaa gtcaggtggg gcgcagtggc tcacgcctgt aatcccagca ctttgggagg 15960 ctgaagcggg cagatcagga ggtcaggaga tcgagaccat cctggctaac acggtgaaac 16020 cccgtctcta ctaaaaatac aaaaaattag ccgggcgtga tggcaggtgc ctgtagtccc 16080 agctactcgg gaggctgagg caggagaatg gcgtgaacct gggaggcgga gcttgcagtg 16140 agctgagatc acaccactgc actccagcct gggcgacaga gcaacactct gtctcaaaaa 16200 aaaaaaaaaa aaaaaaaaaa acagagaagt caaatggttt tttggaatat ggtggccctc 16260 cgtacccatt ggttccacat gtgtggtttc agccaactat gtattgaaaa taaaattgca 16320 tccttacaaa tatgcagact ttttttcctt gtcattgttc ccttaacaat acagtgtaac 16380 agctatttac gtagcattta cattgtatta ggtactatga gtcatcctgg agttgctgta 16440 aaacttaaac gtaaaacttg aaatgaggat gatttaaagt atagaggagg atgtgcatag 16500 gttatatgca aatactctcc cattttatat tagggacttg agcatccacg gattttggta 16560 tccgtggggg tcctggaccc aacctgccac ggatacgcag ggacgactgt atttggcata 16620 gaggcctttc taatgcactt accaaggaca cctgcagcag gctgtgatcc ctgagacgag 16680 cgtgagttta gtgagggcag agtttttgtt ctggttctca gctgtgtccc agcacctggg 16740 attgtccctg gcacacagta gctgcttaga aaagatttga tgagagggtg accatacatg 16800 aggggaaatt ttcctcagta tcaaaacatg ttgaaactca cacacttcca agatgacgta 16860 ttctcaggtc tcctgccgtt gccgttctct gccgtatgtg atggtgttct gtcttaccag 16920 aggctgtggc cctgagaaag atcacgtcta cctgcagctg caccacctac ctccagagca 16980 gctggccacg cgcctgcctg gcatttcaga gacagccatg atcttcgctg gcgtggacgt 17040 cacgaaggag ccgatccctg tcctccccac cgtgcattat aacatgggcg gcattcccac 17100 caactacaag gggcaggtga tggtgctggc tcctccccca cagctggaaa gaaggctggg 17160 acgacggggc ccacctcgca gttgtctctt tagatcttac aggaaaagat agatgtttcc 17220 ttcaagaaag tactgtattg ttttctagat tgcactttaa atttctatta ccggaggatg 17280 gagggggctt aataatttat tcctccttag taaattgtca gagatacatc atttgcagct 17340 ttttccattt tgtaattact ttcctatatg atcttgtgtt atttctaatg atcttacaca 17400 tcaagggatc tttataattc attcctttga gtggtttgtg gttcacacag agcttgtcag 17460 tcacttaggc tccttgttgg gcgaggtggg tggaagctgt tgctctccct gcgtagacga 17520 agaggtgaac ggggtagaac agtctggaac atcagtctcc cctgctgatg ttcctccacc 17580 tgccgtgctc ctgggtctga gccggagcac aggtggtgag ggccccggga acatgggaca 17640 cgggggacag tcgcagatgc tgacattgga ggtcctctga cctgcttgta acagcaggtg 17700 ctcaggggca gaggggaaac tgggggatac cttcggaagc ttccctctga agaagagtag 17760 ctatggtcct tacttccctc ttagatacgg tctttacttc cctctctttt tttttcttgg 17820 agatgcagtc tcactctgtt gcttcggctg gagagcagtg gtgcgatctc agctcactgc 17880 aacctctgcc tcccaggttc aagtgatttt tccgcctcag cctccctagt agctgggatt 17940 acaggcaccc gccattatgc cccgctaatt tttgtatttt tagtagagat ggggtttcac 18000 cgtgttagcc agacaggtct tgaacccctg acctcaggtg atcacccacc tcagcctccc 18060 aaagtgctga gattacaggc gtgagccacc acgcctggcc tacttccctc tctctgacct 18120 gcagcacaga caccctgttg gggaaggtgg gctggtggag gcatgggcac cttgacattt 18180 cacctgaaat cttcctttcc acaggtcctg aggcacgtga atggccagga tcagattgtg 18240 cccggcctgt acgcctgtgg ggaggccgcc tgtgcctcgg tacatggtgc caaccgcctc 18300 ggggcaaact cgctcttgga cctggttgtc tttggtcggg catgtgccct gagcatcgaa 18360 gagtcatgca ggcctggtaa gtgttttctt caggagccag actatttgag aaggcgcagg 18420 acgttagaaa gtcttttttc ttttttttga gaaagggtca gcccaggctg gagtgcagtg 18480 gcacagtcat agcagcctca acctcccggg ctcaagcagt cttcaacacc tcaaccttca 18540 gagtcccaag tagctgggac tacagatgtg caccaccaca cctggctaat ttttaaaaaa 18600 atttattttg tagagacagg gtctcacaat attgcccagg ctggtcttga actcctagac 18660 tcaaacagtc ctgcctcagc ttcccaaagt atcgggatta caggcatgag ccactgcacc 18720 cagccaggtt acaaagcctt gatttcttac tggaaatttg cttagtgatc atatagaggt 18780 agtctgggtt tttcccccag aagtgattaa actgagaaat ccagaaatta tatggtggta 18840 atgttgagac tagatagagg ctggttgggg atcttaacag ttaaggtgac atttttgggg 18900 ttacattttt ttttaattat tttgcagtca ttatgttctg tttagaaaaa gcactattag 18960 aaagttgtta tttttagggg aatcattaca tattacttgc ctgataaaaa tcacttattt 19020 gcaatgaaat attttaagta gttggcatga atgaatatgt aacttcttgg tacttagaaa 19080 agtaatttag gccattctag aaacaaagta cagctagcct ctattagagg agaagggatg 19140 acttacagtg aacaggattc ccacccttta cggacagatt ggatttcact tgctggtttt 19200 cttttcatga tagcatcaaa taatgtgcag taaaggaaat accatgtgtg ggagtgtgag 19260 tcttacgtgc actaagaacg gggcagttag catctctccc acctccagac atcctcacgg 19320 tggttatcca gcctcgtgtg ctcagaacag tgtgaggtgg atgaggcact ggtggatgtt 19380 tgcgtggcaa ggatggtggg accccaggcc catgttcttc ccgttacctt tctctggtgt 19440 taactgttag catcatttct gctgttttta tacaacaggt gctttttgta tggattcaag 19500 tgaaataaaa actagcacgg ctgtaactta taaacgtgcc cccttttgta tctgtagtta 19560 gaaaggtgca gatagtatta aaagggtagc ttacttcaga cactctgtct ctggatctga 19620 ccaccgctcg ggaggccagc acacgcagag ctggcgtctc atccccagcc attggtgatc 19680 atcggcgaag gcggagttca ggtccatcgt tcctgacgcc gcaggtagtg cttgtctcac 19740 tccatagccc tgcactttgt cgcagtgagg actgatacca cttctctcag agcaatgtag 19800 aaattttgag ctgctctttc tttgaaaatg caaaaaagaa cattttgtaa gaatacccta 19860 tactttacat ctgagaaacc gcccacgcat gcagcatctc acgcagaatg ctgtggagtc 19920 agactcaaaa ggctgcatgc ctgtggttct gttgatacga cattctggaa aaggcacatc 19980 tagggaagac aagggattgg tggttgccag aggctgcttc ctgattgtgc tgagacttac 20040 agacacaact ctgtgtgtgt caaaatttga aaaaccctac actaaaaatg atgagtttat 20100 tttactgtat ctttatgctt taattttcaa aaatgaaaag gaaagaaaaa atgcttgtag 20160 catcactatt ctcccccaaa accccctgca aaaaaaaata catatatata cacatatata 20220 tgtatttttt ttttttaaga gatagtctcc ctctgtcgcc caggctgtag tgcagtggta 20280 cgatcaggtg cacaccacca cacctggcta atttttaaaa atgttttgta gggacagggt 20340 ctccctgtgt tgcccaggat gggcttgaac tcctggcctc aagtgttcct cctgtctcag 20400 cctcccgaag tggttacatg cgcctataca tgtgttaaaa ttggtagaac tgaggctggg 20460 tgcagtggct cacgcctata atcccaccgc tttgggaggc cgaggcaggc agattgcttg 20520 agctcaggag ttcgagacca gcttgggcaa cgtggtgaaa ccccgtctct accaaaaata 20580 caaaaattag ctgggcatgg tggctcacac ctatgtagtc ccagctactt gggaggctga 20640 ggttggagga ttgctggagc ctggaaggca gaggttgcag tgagccaaga tcacaccact 20700 gtactccagc ctgggcaaga gagggaggag acactgtctc aaaaaaaaga aaaagtaaat 20760 tgtagaactg tccacctaaa gaaaaaagtc aattttactg aatgatcaat ttttaaagca 20820 ttattatcaa aaggaagaga aacaccagcg agcctagaag catttgagca gaccctcaag 20880 agacccatag cctggtcctg tggagaggtg gtgggcgggg tggggcctgt ttgactcctg 20940 catttcatac atcctacctc ctgcatgtat tacctgttga agaaaatata tataacgtta 21000 taaaaaaaac ttaaaaactt ttttcaagac atcgtagaaa cacaagagtt gcaaatcttg 21060 gctgtgcgca gtggctcaca cgtgatccca gcactttggg aagccaaggc aggtggctca 21120 cctgaggtca ggagttctag actggccaac atggtgaaac ccatttctac tgaaaataca 21180 aaaaattagc caggtatggt ggcatattcc tgtagtccca gctactctgg aggctgaggc 21240 aggaggcttg cttgaacccg gagatggagg ttgcagtgag ccgagatggt gccactgact 21300 gcactccaac ctgggctaca gaacaaaatt ccatctcaaa aaaaaaaaaa ttgctaatct 21360 tgaagtatag ttgagagcac ataagagtcc aaatcaacag gtgactttca agcacacagc 21420 agccaccttc cccccgctga tgtgaagggt ggccggcccc ttgggaccac catctggaag 21480 gtgtctcttt tttcccttag tggagtgaca tttatataca cttaatatat ataaatttgt 21540 atacatttaa tttttttttt ttttaagaca gggtctcgct ctgttgccca ggctggagtg 21600 cagtggcgcg atctcggctc actgcaacct ccacctttag ggttcaagca gttctcatgc 21660 ctcagcctcc cgagtagctg ggattataga cgcgtgccac catacccaga taatttttgt 21720 gtttttagtg aaacgaggtt ttgccatgtt ggccaggctg ctcttgaatt cctgacctca 21780 agtgatccac ctaccttggc ctcgtaaagt gctgggatta caggcgtgag ccaccgcacc 21840 cggcctacat ttaatttttt aattttagag atgatttcta gtttattcac tctaagatca 21900 cttaatggat atctactgtg tgccaacaat tttgccttta tgttctttaa aattggcccc 21960 aactcaacag atggcctcag ctgtagggtg ggctggcagt gtgttagctc aggagactta 22020 cagagtttcc aagctccttg agtggctgtg ctacatgttt gtgtgtcatt ctaaatccat 22080 ttggtttttt aaaacggttt tcaaaagtta aattctagct tttttttgtt ttaggagata 22140 aagtccctcc aattaaacca aacgctgggg aagaatctgt catgaatctt gacaaattga 22200 gatttgctga tggaagcata agaacatcgg aactgcgact cagcatgcag aaggtaagag 22260 cctggactcg ctctggagtg agcaggaggg ctgcatacct ggccctgcac tggttttgtt 22320 ttttaaaaac tagatctagg gggatgcagg tgcagttttg tgtggatgta ctgggaggtg 22380 gtggagtctg ggcttttcat gtacccgtca cacaagtcgt gtgtgttgta cccagtaggt 22440 aattgttcaa ccccacccct cccgcttttt ggagctccca gtctctgcta ctccactcca 22500 tgtgtccatg tgtactcacc attcagcttc cacttccaag tgagaatgtg tgacacttga 22560 ccttctgagt cacttcactt aggatagtga cctcctgttc catccgtgtg gctgcagaag 22620 acatgattgc gttctttttt tatggccgag tagtatttca tggtatatat gtaccacatt 22680 ttcttcatct ggtcatccgt tgatgggcac ttaggttgat tccatgactt tgctgttgtg 22740 actagtgctg cgataaatac ataaggctgc accagtatct ggaggtaaac agcggtagga 22800 cgtactcctc accgtatcaa gaatatgaaa gagaccagga ggcctgaact atacagaagt 22860 gcacttcttt tccacataga aggtcggcag actagggcag aattagtgac tgcttggcat 22920 ccaggacagc cttctgtggt tcactcgtgt gtgcttgggt gtgacctccg tgacctgacc 22980 attgctggct gtcatggatg agtcacagcg tggaggagag ggaactgcag gaccgctgga 23040 gaagctcagt ccccagcagg gcagctttct cttagaggtt tcctggagtt caacacaaca 23100 ctggtgctta catctcaggc ccggatatcg atcatgtcat catgcctggc ttccagcagc 23160 tcggaaatgt ctttaagctg gacttgttgc tgcctctaaa tatactcgga agagagggag 23220 agtgggcggc aggtggaaac tgatctgtgg cctgtgctgc tgggagtcgg tccagtagga 23280 ccatctgtga tgatggaaat gttccgagtg tttgccatcc agtatggcag ccattagcca 23340 tggggccgtg gagaacctga tacatggtca gtctaagaaa ctgaattcct ctaataacaa 23400 ggattcttgt ccatgaatga gatctcttgc ctactatttg caagaatttc tgtgtatttt 23460 ctaaaaagtc cattgcttta gtcctattct gaaataggtg tctaacacct atgtggtgtt 23520 agacacagga gaacaggttc cctgctgaca ttttcagagg cctgtgccct tcagtcttca 23580 agtgaagctg ggcttcaggg aggctttgtg gaatggtgag aagaacagtg tgactaaggc 23640 acagaaggct gagtgatgcc ctgcagtact attgtagggt tggaggccag ctgggaaaga 23700 aagaaccatt gcattagaga atgggaacat gccttcggat atagaaatgg caaatccatg 23760 agatagttta aagtgagaat actagaagca ttcccaccaa acatggtgtg tcttggtgcc 23820 tgctgtatcc caggctccac gagatgctgg agtcagcact gaacaaacag agtttcctct 23880 gctcgtggaa atgcgttccg ttggggaagg gatttttctg ctgagtctgg ctattcatag 23940 taagaagcaa aaaaaagaaa tgaggtaaat ttttgggaac aggcccccaa atgtggccca 24000 aaactgacca taaacaaaat ctctgcagca ctgtgacatg ctcttgatgg ccatgacgcc 24060 cacgctagaa ggctgttggt ttaccagaat gagggcaagg gacacctggc ccacccaggg 24120 tggaaaaccg cttaaggcat tcttaaacca caaacaatag catgagcgat ctgtgcctta 24180 aggacatgtt cctgctgcag ataactagcc agagcccata cctttgtttc ccgtaaggaa 24240 tacttttagt aaatcttatg actggcttgc tctcaataaa tatgtgggta aatctctgtt 24300 caaggctctc aactctgaag gctgtgagac ccctgatttc ccactccaca cactatattt 24360 ctgtgtgtgt gtctttaatt cctctagcgc cgctgggtta gggtctccat gaccgagctg 24420 gtctcggcaa atggcgccca tatatggggg ctcgaatcta ggtcgaaggg tcaccggagc 24480 gatggagaac atggaactaa gctggaggac acctgagtac tcttacgcag tccctgtggt 24540 gagtaagacg ggtagctcag aagcatcagg gtaacaatgg gacacgtgtg ggctctggtt 24600 cgttccacct tggaaccttt tcatgctaat aataaggggg aaggagagta taacgaagta 24660 acagaagaaa ttacagagca ggtttgtttg ccaactaaag ctaaagcggc aaaggaggga 24720 gaggttcatc cctacccttc tgcaccccct ccttattttg aagaaaaaga gtggcctgac 24780 cctccagatc tttcttttcc agaggacagt gggcgaaaag tagttgctcc agtgactgtc 24840 cgagcagcac cttgagcgac ccctctcagt tctattcagg caggaattca gcaagctaga 24900 tgagagggtg atttagaggc ttggcagttc cctgttagaa tacaccctgc agatcaacag 24960 ggaaatatta cagctacatt tgagcctttt ccctttaaat tacttaagga gtttaaacac 25020 gctatcaatc agtatggacc aggttctcct tttgtaacag gactgttaaa gaatgttgtt 25080 gtttccagtc ggatgattcc tactgacttg gacgctctta cttgagcttg tctaactcct 25140 gctcagttct tacaatttaa aacttggtgg gcagttgtag cttccattca gcctacttgc 25200 agcgcccagg cccaacctca aattaatata actgcagacc agcttttggg ggtcggcagc 25260 tgggctggtt tagatgcaca agtggtcgtg caggatgatg ccgtagaaca gcttagagga 25320 gtgtgcatta gagcttggga aaaaatcact tcaggtagag aacaataccc ttcctttagt 25380 gcagtaaaac agggaccaaa agaaccgtac gctgatttta tagctcggtt acaggagtct 25440 cttaaaaagg tgatcgcaga ttcggctgct caggatatag tgctgcagtt atcagctttc 25500 gacaatgcta atcccgattg ccaggctgct ctgtgaccta tcagagggaa agcacactta 25560 gttgattata tcaaggcctg tgatggtatc agaggtactc tgcataaagc gactttgttg 25620 gcacaagcta tggcaggact gaaagtgagt aaaggaaata ctccgtttcc tggaggttgt 25680 tttaactgtg ggaagcatgg tcatactaaa aaagaatgta gaaaaaaatc agtgagtgag 25740 ggtgccagat gggggaagaa agaaaactgc taagcctgaa atatgtccaa aatgtaaaaa 25800 aggaaaactt tgggctaatc agtgtcactc taagtttgat aaagatggga accccatttt 25860 gggaaatgcc atgaggggcc cgtcctgggt cccgttccaa accggggcat ttccggctca 25920 ggccactccc tcacccctgt acaatgtctg ttccccgcca cagccggtag tgccacagta 25980 gatttatgct gcacagtagc tgtgagtctt ctgcttgggg aacccccacc aaaggtccca 26040 gcaggagtct gtggaccctt gcaagcaggg acgataggat tactcctagg caggtctagt 26100 ttaagtttaa aagggtacaa atacatagag gagtcattga ttcagattac aatgaggaaa 26160 ttcaaattgt tacatctact tctgttccct ggaaagcaga gccaggagag tgcatagcac 26220 agctcctgat tgtgccatat gtggaaatgg ggaaaagtga aattaaatga acaaaaggat 26280 atggaagcac agataaacaa ggcaaagcag cctattgggt gaatcaaatt actgataaac 26340 gtcctacctg tgaaataact attcagggaa agaaatttaa aggtttggta gatgcaggag 26400 cggacatttc aatcatttct ctacagcact ggccgtctgt gtggccaatt caacctgctc 26460 aatttaacat agttggagtt ggtaaagccc ctgaagtata tcaaagtagt tatatttcca 26520 ttgtgaaggg cccaatggac aacctgggac tattcaatga taacttctgt acctataaat 26580 ttatggggaa gagatttatt acaacgatgg ggagcacaag ttctaattcc agagcaatta 26640 tatagccctc aaagtcaaca tatgcatgaa atggggtatg tccctggtat gggactagga 26700 aaaaatttgc agggtttgaa agaagtgctt caagtggaaa gacaaaagtt cctgccaagg 26760 tttaggatat tatttttgat ggcggccatt gttaagcttc cagaacctat acctttaaaa 26820 tggttaacat ataagccaat ttggatagaa caatggccac tgagtaaaga gaaactggag 26880 gctttagagg acttagttaa tgaacaatta gaaaagggac acatagctcc aacattttcc 26940 ccttgtaatt ctttggtttt cttaattaag aaaaaatcag gtaaatggag aatgttaact 27000 gacttaagag ccattaattc agttatacaa cctacgggga cattacagcc aggattgcct 27060 tctcctgcta tgattccaaa aaattggcct ttaatagtca tagatttaaa agactgtttc 27120 tttactatcc ccttagctga gcaagactgt gaacggtttg catttacaat tcgtgcagta 27180 aacaacctgc agcctgctaa gcgttttcat tggaaagtgt tgcgacaagg catgttaaac 27240 agtccaacga tttgccagac ttgtgtaggg caagcaattg aacctactca taaaacattt 27300 tcacagtgtt acattattca ttatatggat gatatacttt gtgctgcccc tactcgagaa 27360 atattactcc aatgttatga tcatttgcaa aattcaattt ctggtgctgg tttaattata 27420 gctcctaaca aaatccagat tactactctt tactcctact tgggaacctt agtaaatgac 27480 attaccattg tgctacagaa agtagccata cgtagggatc aattgaaaac attaaatgag 27540 actttcaaaa attactgggg acattaactg gatacaccct gctctaggca ttcctaccta 27600 tgccatgagt aatctatttt ctatccttag aggagatcct agtctcacta gccctcggct 27660 tcctacctat gccatgagta atctattttc tatccttaga ggagatccta gtctcactag 27720 ccctcggcta tgccatgagt aatctatttt ctatccttag aggagatcct ggtctcacta 27780 gccctcagca attaacaaaa gaaactaagg gagtgcctca gctgatcgaa aagcaagtcc 27840 ataaagccca aataaataga acagatccag agaagactcg agctgcagct gatcgaaaag 27900 caaggccata aagcccaaat aaatggaaca gatccagaaa agactcaagc tgcagccgat 27960 cgaaaagcaa ggccataaag cccaaataaa tagaatcaat gcagagaaga ctcaagctgc 28020 agctgatcga aaagcaaggc cataaagccc aagtaaatag aatcgatcca gagaagactc 28080 gagctgcagc tgatcgaaaa gcaaggccat aaagcccaag taaatagaat cgatccagag 28140 aagacttgag ctgcagctga tcgaaacgca aggccgtaaa gcccaaatca atagaatcga 28200 tccagagaag actcgagctg cggctgatca aaaagcaagg ccgtaaagct caaatcaata 28260 gaatagatcc agagaagact tgagctgcag ctgatcgaaa agcaaggcca taaagcccaa 28320 gtaaatagaa tagttccaga gaaggctcta gatttgctaa tttttccaac tccacattca 28380 cctactggtg ttattgttca agagcaagat tttgtagaat ggctttttct tccacatact 28440 aattcatgga ctctagagaa ggctctagat ttgctaattt ttccaactcc acattcagct 28500 actggtgtta ttgttcaaga gcaagatctt gtagaatggc tttttcttcc acatactaat 28560 tcatggactc taactcctta tttggatcaa attgctacta tgataggaaa tgggagaact 28620 tggattgtta aattacatgg atatgatcct gaaaaaattg tccctctcac gaaggcacaa 28680 atacagcaag attttttaaa tagtcttact tggcaaaccc atttagctga ctttgtgggt 28740 attcttgata ttttcctaaa atgaaactgt ttcaattttt gaaattaact aattggattc 28800 tccctaaaat aactaaattt aaaccaaatg aagatgcaga gaatgttttt acagatgggt 28860 ctagtaatgg taaagcttct tactctggct cgaaaggtaa agttttctag acaccctatg 28920 cttcagctca aaaagtggag cttgtagctg taattgaggt attgactgct tttaatatgc 28980 ttattaatgt gatttctggt tcttcataca tggttcattc cacacaatta attgaaaatg 29040 ctgagttacg atttcataca gatgaacaac tgatgacttt atttatgcaa ttgcaaacag 29100 cagttaggag tagaatgcac cctttttaca tcactcacat tagggctcat acacctcttc 29160 caggaccttt gactgcaggg aatcaaatgg ctgatcgcct agttgctact gcaatatcta 29220 atgctagaca ctttcacaat ttaacccgtg ttaatgcctc tggtctcaaa cgcagataca 29280 gcagtacccg gaaagaagct aaagctatta tccagcgatg cccaacttgc caaatggtac 29340 attcctcatc ttttacagga ggagttaatc ctcgaagatt ggaacctaat tctctttggg 29400 aaatggatgt cacacatgtt ccctcgtttg ggagactagc ttatgtacat gcatgtgtgg 29460 acaccttttc actttgggct gcatgccaat caggagagtc ttctgcctac gttaaacgtc 29520 accttttgca gtgttttgtg gtaattggca ttctagcttc tattaaaaca gataacgccc 29580 caggctatac tagccaagct ctagctacat ttttctctat acggaatatt aaacacatta 29640 ctggtatccc ttataattct caaggacaag ccatagtgga aagaatgaat ctttccctga 29700 aacagcagct gcaaaagtaa aagggggaaa acagggacta cgggacaccc catatgcaat 29760 tgaatcgcat tatttaaatt ttttgagcct gcctaaaggc cagatcttat cagcagctga 29820 acagcatcta cagaaaccag ctgcaaagac agaagcagaa caactggttt ggtggagaga 29880 cctgataata aaaagttggg aaataggtaa aataataact tggggtagag gttatgctta 29940 tgtttctcca ggacggaacc atcaagacac ctgaaacctt atgagctgac actgaggaag 30000 agattctggg aggatcccga ggacccctcg gttgcagcca tgtcaagact gatgctgagg 30060 aggaccccaa ctgtcatgag caacacccgt cgaacacagc cacccacctg aggtcagatc 30120 aagaagctgt cacagatggc agaagaaaac ctgaggaaag cgggacaacc agtcacaatg 30180 agtaatttaa tggtagctgt gatagcagtg atcaccattg ccatgagtat tccttcaaca 30240 agggctgaca cagagaacaa ttatacttat tgggcatatt tatcaatctt ggctggcaag 30300 aatgcctgga tgtaatcact ctatgatgca gttacacatg ctttctgatc tcagtattta 30360 ccatgataaa tctgcttcta taattgaggc ataccgccct caaaaaccta tctgtaaaca 30420 ggattggacc cagtcagaga aaatgaacgt acatgtttgg gaagattgca ttgcagaaca 30480 ggcaggggtg ctggcaacga ttcctatgga atcattattg attgatctcc taaggggatg 30540 tttagcttga attgcacctc tctgtctgcg tgccacggcc acactatgtt cagctggtct 30600 gaacaaaatt gtcagatggt agaaatgata agaaatacgg caagggttcc tattatctgg 30660 aaccatggcg gtgtagtggc acctcaacct caaatggtat ggccctttgt aggagctaaa 30720 cataaggatt tgtggaaact gttaatagct cttaataaga tcaaaatttg ggaaagaata 30780 aaaaagcatc tagaaggaca ccgtacaaac ttgtctttgg atattgcaaa attgaaagaa 30840 cagatatttg aagcatccca gcacacctga ccttaatgcc aggaactgga gtgcttgaag 30900 gagctgcaga cagattagca gctggtaatc cattaaaatg gataaaaaca cttggaagct 30960 ctgtgatttc aatgaagatt gtgcttttaa tctgtgttat ttgtatagtc tgcagatgtg 31020 gatcctgact ctgcaagaag tagctcaccg taacaaagct gcctttgctt ttatcacttt 31080 gcaaaacaaa taaaggggac acgttgggaa caggcccccc aatgtggcca taaactgtcc 31140 ccaaaactgg ccataaacaa aatctctgca gcactctgac atgctcttga tggccatgat 31200 gcccacgctg gaaggttgtc ggtttactgg aatgagggca agggacacct ggcccaccca 31260 gggtggaaaa ccgtttaagg cattcttaaa ccacaaacaa tagcatgagc gatctgtgcc 31320 ttaaggacat tttcctgctg cagataacta gccagagccc atccttttgt ttcccgtaag 31380 gaatactttt agtaaatctt atcactggct tgctgtcaat aaatatgtgg gtaaatctct 31440 gttcaaggct ctcagctctg aaggctgtaa gacccctgat ttcccacttc acatgctgta 31500 tttctgtgtg tgtgtcttta attcctccag caccgctggg ttagggtctc cacaaccgag 31560 ctggtctcag cagtaaatat tgaaaaggaa tagatacaat tgtcattatt tacagataga 31620 atttccaaac aattcccagg gaataaactg aaaaactaac agaaacaaaa caagaatgta 31680 gtaaggtgtc tggataagag atttgtatct aaaaatcagt agctttacaa tgtgccagca 31740 gtagtctgct cagacatcag taaatatctc attcacattt caaaaaaaat tgaaaatgcc 31800 ctgaaataat gtaaccagaa atacgaaaaa aaggatatga aaacatgtgg ctgctaatgg 31860 acatgaaaga atgtaatact agattctgag atgcaatgtt tttcatttgt tcttcctgaa 31920 aaaccattag gttgatgtgc attacagtgt tacgattatg tatgagtcta aggaaaatca 31980 gatgaaatgt ccaaattaaa ccatgaaggt gcattggtag aggaagagaa aattagggtc 32040 agtggagcaa agcacagtta gagggagaag caaggaggag gagggatcac tgaggtggtg 32100 cctgtcccac aggaagcaaa agctgacgcc tagttcccag catacctaca gtaaacttca 32160 ggtccactgc atagcacgtc tctcatcaca gtaaaactat gaaggaactc agtgtacaag 32220 gagcttctac aagataggca gaagacagta gccagatggg ccaagggccc cagccaccca 32280 cgcccctccc tctccttgaa gacgtgcagc tccaacacca ccatcaccag ggctctgctc 32340 agctcctcct agtgtgtatc accacagggc tgctggcttg tgtcacgttc accaccagac 32400 cccgcgtcag gagtcccgcc aggggtgtgg agaggcagcg ctgcctggtt ggccgtggag 32460 ctgtatggaa catggtgcct cacaggcagt ctgcttggag tcctggaccc tggctgtatc 32520 ctgctggaaa ggatgtgtgt gggtctaaga tgtgtatgta atagaaacat ttatttattc 32580 agaagcttta gtcaaaactt catttttaag tttggagtaa taaactcata gtctgaattt 32640 cctaattttt ctgtttaatt tatgtaactt ttaagtgaaa tgcaaaacaa caggtctaaa 32700 agttaagcag ttcttggtat ggctgcttct atggattaaa agtttacaaa taatattttg 32760 tgccacagtc aatgcaaaat catgctgccg tgttccgtgt gggaagcgtg ttgcaagaag 32820 gttgtgggaa aatcagcaag ctctatggag acctaaagca cctgaagacg ttcgaccggg 32880 gtgagcagac agtgggctct gtgcacactg ttgggccctt ccttctgcag ggtgggctgg 32940 tgtctgtccc gtcagtgctg acttagttcc gtgcttgctg tctggatggg tcctggccca 33000 cagctataaa gccacaacca gtgactccat ggactagcag gcccaggctg acagctcgga 33060 gggcccatgt gactgggtcc cgcctgcccc tgatggaact ttttgtgtcc ccaggaatgg 33120 tctggaacac ggacctggtg gagaccctgg agctgcagaa cctgatgctg tgtgcgctgc 33180 agaccatcta cggagcagag gcacggaagg agtcacgggg cgcgcatgcc agggaagact 33240 acaaggtggg ccttctcacc acgcccacct gcacctgcct tttcctgcca cctggtggga 33300 ctcagcccca cccctgcatt ttctctgcat tttctttcgt tgccccaaaa gtaaatccaa 33360 aaaatgcctt tttcccccct ggtaactttg atccctgggt tctcgccatc ttctggatca 33420 ctgtgacctt ttccttgctt tgggtcggca tccactgatg ccagcagtgg catctccaag 33480 ccaatgtgct ttgctgttag aaggccaagg ttagaagtgc agctagagtg gcaagaccag 33540 gaaataaatg ccagtttatt aaataacgag taagccatca tttcaagcct gccctgtgga 33600 ggaaatgcca gtttattaaa taacgagtaa gccaccgttt caaacctgcc ctgtggagga 33660 aatgccagtt tattaaataa cgagtaagcc accgtttcaa acctgccctg tggaggaaat 33720 gccagtttat taaataacga gtaagccacc gtttcaagcc tgccctgtgg aggaaatgcc 33780 agtttattaa ataacgagta agccaccgtt tcagacctgc cctgtggtgg aaatgccagt 33840 ttattaaata acgagtaagc cactgtttca aacctgccct gtggaggaaa tgccagttta 33900 ttaacgagta agtcactgtt tcaaacctgt cctgtggagg aaatgccagt ttattaacga 33960 ataagccacc gtttcaaacc tgccctgtga aggaaatgcc agtttattaa ataaggagta 34020 agccaccgtt tcaagcctgc cctgtggagg aaatgcccgt ttattaaata acgagtaagc 34080 caccgtttca agcctgccct gtggaggaaa tgcccgttta ttaaataagg agtaagccac 34140 catttcaagc ctgccctgtg gaggaaatgc cagtttatta aataacgagt aagccaccgt 34200 ttcaagcctg ccctgtggag gaaatgccag tttattaaat aacgagtaag ccaccatttc 34260 aaacctgccc tgtggaggaa atgccagttt attaacgagt aagtcaccgt ttcaaacctg 34320 ccctgtggag gaaatgccag tttagtaacg agtaagtcac cgttttaagc ctgtcctgtg 34380 gaggaaatgc cagtttatta acgagtaagc caccgtttca aacctgccct gtggaggaag 34440 tgccagttta ttaaataatg agtaagtcac cgtttcaagc ctgccctgtg gaggaaatgc 34500 cagtttatta aataacgagt aagtcaccgt ttcatacctg ccctgtggag gaaatgccag 34560 tttattaacg agtaagccac cgtttcagac ctgccctgtg gaggaaaatg ccagtttatt 34620 aacaagtaag tcactgtttc agacctgccc tgtggatgaa atgccagttt attaatgagt 34680 aagtcaccat ttcagacctg cgctgtggag gaaatgccag tttattaaat aaggagtaag 34740 ccaccatttc agacctgtcc tgtggtttgg aaaaggtatt atagagcctg ccctgtggtc 34800 acttgttctt cagatgaact gatttttgtg cagagcgcac gtgttggatt ctgcctggta 34860 agagtttttc ccatatgata gcaaaaaacg acggaaaggg aagcttggga tgcaaatgca 34920 agttcaggat aaaccacatc agcaaaagga caaaggctcc acaaggcagg cgcacaggct 34980 ggttcaggac cgtgtgtagg cggctggtgg cagcctttcc agtcagctga acatggtgaa 35040 tgggaaaatc atttttattc accatgaaat tttactgatt taccctccac tagaatatgc 35100 tgatggctgt gatcactgct cagaacttgc tcttgtctcc ttgtatgtat taagagtttc 35160 ctgcaaagta tatgaatccg tgtttgccag aatacagaat aatagtaaat ttattatttt 35220 tatttttatt tttttgagaa ggaatctcac tgtcccccag gctggagtgc agtggcgtga 35280 tctcagctca ctgcaacctc tgcctcccgg gttcaagtga ttctcctgcc tcagcctccc 35340 aagcagctgg gattacagac acactgccac catgcctaat tttgtatttt cagtagagat 35400 ggggtttcac catattggcc gggctggtct cgaactcctg acctcaagtg aaccacccac 35460 ctcggcctcc caaagtgcta gggttacagg catgagccac tgtgcctggc cagtgcataa 35520 atttagttgg tcacagtcag ttttaattag aatagaagcc aggtgcagtg cctcacacct 35580 gtaatcccag catttgggag gctgaggcag gccaatgact tgagccccag agttgtagac 35640 cagcctgggc aacatggcga aacgtgtctc tacaaaaaca taaaaaatga gccaggtgtg 35700 gtggtagcac aggaggctga ggcaggagga ttgattgagc ctgggaagtc aaggctccgg 35760 caagctgtga tcacaccact gcactccagc ccaggtgaca tagccagacc ctgtctcaaa 35820 aaaaaaattt ttttttaata aaaacaggct gaaagaaaag attgaggtag tctcccagca 35880 catggagcaa aaagacaaag tatttgataa actcttaggt acataaagga tgcttaaggg 35940 aacatgcgga catggattac tgtggactca ctgctggctg cacaccccct ggccagccat 36000 gcggcctccg tgggttctga acatgttgat ggtgccaacc tcctgggctg aagtggaaat 36060 ggaatgagtt ctagggcatc tgtctcttag atcatgttaa tgtctgctgt gttttttctg 36120 tattgctctg ttagagtaat aagaaacgtg atggtgtttc tggcctcagg tgcggattga 36180 tgagtacgat tactccaagc ccatccaggg gcaacagaag aagccctttg aggagcactg 36240 gaggaagcac accctgtcct atgtggacgt tggcactggg aaggtcagtg tggagctcgt 36300 tctcaccaca gcccagcacc cacacggccc cgcccaggcc tgcgggctgg ccttgctgat 36360 ggtgaacggg gaagagcagg ccagatttaa atcaactccc gacagattcg aggcaccgct 36420 gaaaaaggca ctccgacagc agtcgggctt cgggctggaa acagaatcca gttcctgcag 36480 gtggttcaga ggagccttaa ggaagggttg ctccgtggtg tgcgccagat ggacgtcact 36540 gggcaggagc aagtgtccaa ggcctggtgg taggggagga gatgatgatt gtggacctag 36600 cgagaaagca tctgtggggt ggggacacac agccattacc agaaaccagt cccacgttaa 36660 gggagcccag aagagacacc tcccctctcc tcccacgggc tgggccaact ggaagcgtct 36720 gcaggggagc tgaggggatg tggtgcagcc tttagcatcc cctgggcact gagcaagcag 36780 agaagggcag aaatggaggt ggggttgggg tgagcagtgt cctgggaaca gccagccgag 36840 ggtgtggtag ggggtcgcag cctttttcca cacacggtga gcagcgtcct gggaacagcc 36900 agccgagggt gtggtagggg ggtcgcagcc ttgttccaca caagcacagt tcacctgtgt 36960 ggcatttccg ctgggcatta agattcagaa ataatgaaga tagaaagctt ttacccttaa 37020 acttttcata gcttgtaaga gtcgtataat cacttagctg tgtctgtgga agttaccttt 37080 ggactctcac tatcatctag tgtgtctgtg attcaggcag taggtcattt tcaggacttc 37140 tcatgaaggc catttcttga tagtgtatag aaatcacact tcactcgctt agcacaagtc 37200 tatttttaat gtttccgggt tcgggttttt tggttttttt ttgtttttct gagatagagt 37260 ctcatctctg ttgcccaggc tgatatgtgt tggcttgatc tcgtctcact gcagcctcaa 37320 cctcccccag gctcaggtga tcctcccacc tcagcttcct gggcacatgg caccatgcct 37380 ggctaatttt tatatgtttt gtagagatgg ggtttctcca cgttgcccag gctagtcttg 37440 aactcctgcg ctcaagtgat ccacctgtct tggcctccca acaggcatga accaacaccc 37500 ccagccagtt ttagtgattt ttcaagaaat acatactcat tttagaaagt acaaagagat 37560 tgaaataaga gctcactaac cagagacgac ccattatggt ttaaatttct ttgtatatgt 37620 gcctaccttt tcctgtgtgt gcatatttaa tacacagctt gagtattcct tctctgaaat 37680 ccttgggacc agaagcgttg tggatttcag gcttgttcag actttggaat atttgcatta 37740 tacttactgt ctgagcatcc ctaatcggaa gatctgactc catagcacat ttcttttgaa 37800 tgtcatgctg gtgctcagaa agtttcagat tttagagaat ttcagatttt tggattaggg 37860 atgttcaacc tatataaata tttactttga agtagaaaaa ctggaagtag atggtttaaa 37920 cataaagtgt cttggtatag acaagggttc tcccacttta catgatggga gcatttttgt 37980 aaagcactga gaatcttaaa gttcacatgc cataaatcta cttttatagt taaaattttt 38040 caaaaggaac acaagagatg gctttttgta catttttgtg cttaacttac cactgactct 38100 tcttttcaag gtcactctgg aatatagacc cgtgatcgac aaaactttga acgaggctga 38160 ctgtgccacc gtcccgccag ccattcgctc ctactgatga gacaagatgt ggtgatgaca 38220 gaatcagctt ttgtaattat gtataatagc tcatgcatgt gtccatgtca taactgtctt 38280 catacgcttc tgcactctgg ggaagaagga gtacattgaa gggagattgg cacctagtgg 38340 ctgggagctt gccaggaacc cagtggccag ggagcgtggc acttaccttt gtcccttgct 38400 tcattcttgt gagatgataa aactgggcac agctcttaaa taaaatataa atgaacaaac 38460 tttcttttat ttccaaatcc atttgaaata ttttactgtt gtgactttag tcatatttgt 38520 tgacctaaaa atcaaatgta atctttgtat tgtgttacat caaaatccag atattttgta 38580 tagtttcttt tttctttttc ttttcttttt tttttttgag acaggatcgg tgcagtagta 38640 caatcacagc tcactgcagc ctcaaactcc tgggcagctc aggtgatctt cctgactcag 38700 ccttctgagt agttggggct acaggtgtgc accaccatgc ccagctcatt tattttgtaa 38760 ttgtagggac agggtctcac tgtgttgcct aggctggtct caagtgatcc tccctccttg 38820 gcctcccaag gtgctggaat tataggtgtg aacaaaccac catgcctggc cttgtagttt 38880 atttctaagt tcaaattaat gttggtgcct gagaacgaat ggagaaaact aacatttcct 38940 cctttttctt ggcagacggt ttactaggtg agtgtgtcct tgattattca aatcaggggt 39000 ccccaatccc caggccacag attgttacca gtccatggcc tgttaggaac caggccgcac 39060 agtaggaggt gagcagtggg ccggtgagct actgtgtgag ctccaccccc tgccagagca 39120 ttactgtgaa ctccgccacc tgtcagagca ttactgtgtg agctccgccc cctgccagag 39180 cattactctg tgaacaccac ctgtcagagc attaccgtgt gagctccgcc tgctgccaga 39240 gcattactct gtgagctccg cctcctgtca gagcgttacc gtgagctccg ccccctgttg 39300 gagcattact gtgtgagctc cgcgccctgc cagagcatta ctgtgtgagc tccgcccctg 39360 ccagagcatt accttgtgag ctccaccccc tgccagagca ttactgggtg agctccaccc 39420 cctgccagag cattactggg tgagctccac cccctgccag agcattactg tgagctccgc 39480 ctcccgacag cattaccgtg tgagctccgc gccctgccag agcattactg tgtgagctcc 39540 gcccctgcca gagcattacc ttgtgagctc caccccctgc cagagcatta ctgggtgagc 39600 tccaccccct gccagagcat tactgtgtga gctccgcctc ctgccatagc attactgtga 39660 gctccgcctc ccgacagcat taccgtgtga gctccgcccc cgttagagca ttactgtgtg 39720 agctccaccc cctgccagag cattactggg tgagctccgc ccctgccaga gcattacctt 39780 gtgagctcca ccccctgcca gagcattact gggtgagctc caccccctgc cagagcatta 39840 ctgtgtgagc tccgcctcct gccatagcat tactgtgagc tccgcctccc gacagcatta 39900 ccgtgtgagc tccgcccccg ttagagcatt actgtgtgag ctccaccccc tgccagagca 39960 ttactgggtg agctccaccc cctgccagag cattaccgtg agctccgccc cctgtcagag 40020 cattactgtg agctccgccc catgttagag cattactatg tgagctccgc cccctgccag 40080 agcattactg tgagctccgc ccccctgtca gagcattagt gtgtgagctc ctcctcctgt 40140 cagagcctta ccgtgagctc cgccccctgc cagagcatta ccgtttgagc tccgcctcct 40200 gtcagagcat tactgtgagc tccatctctt gtcagagcat taccgtgtga gctccacccc 40260 ctgccagagc attaccgtgt gagctccgcc ccctgccaga gcattactgt gagctccttc 40320 tcctctcaga gccttgccgt gagctccgcc ccccgccaga gcattaccgt gtgagctccg 40380 cctcctgcca gagcattacc gtgtgagctc cgcctcccgc cagagcatta ccgtgtgagc 40440 tccgcctcct gtcacagcat taccgtgtga gctccgcctc ccgccagagc attaccgtgt 40500 gagctccgcc tcccgtccga gcattaccgt gtgagctccg ccccccgcca cagcattacc 40560 gtgtgagctc cgcctcccgt cacagcatta ccgtgtgagc tccgccccct gccagagcat 40620 tactgtgagc tccttctcct ctcagagcct tgccgtgagc tccgccctcc gccagagcat 40680 taccgtgtga gctccgcctc ccgtccgagc attaccgtgt gagctccgcc tcccgtccga 40740 gcattaccgt gtgagctccg cctcccgtca cagcattacc gtgtgagctc cgcctcccgc 40800 cagagcatta ccgtgtgagc tccgcctccc gccacagcat taccgtgtga gctccgcctc 40860 ccgtcagagc attaccgtgt gagctccgcc tcccgtccga gcattaccgt gtgagctccg 40920 cctcccgtca cagcattacc gtgtgagctc cgcctcccgt cagagcatta ccgtgtgagc 40980 tccgcctccc gtccgagcat taccgtgtga gctccgcctc ccgtcacagc attaccgtgt 41040 gagctccgcc tcccgccaga gcattaccgt gtgagctccg ccccccgcca gagcattacc 41100 gtgtgagctc cgccccccgc cagagcatta ccgtgtgagc tccgcccccc gccacagcat 41160 taccgtgtga gctccgcctc ccgtcacagc attaccgtgt gagctccgcc tcccgacaga 41220 gcattaccgt gtgagctccg cctcctgcca gagcattacc gtgtgagctc cgcctcctgt 41280 cacagcatta ccgtgtgagc tccgcctccc gccagagcat taccgtgtga gctccgcctc 41340 ccgtcacagc attaccgtgt gagctccgcc tcccgccaga gcattaccgt gtgagctccg 41400 cctcccgtcc gagcattacc gtgtgagctc cgcctcccgc cagagcatta ccgtgtgagc 41460 tccgcccccc gccacagcat taccgtgtga gctccgcctc ccgtccgagc attaccgtgt 41520 gagctccgcc tcccgtccga gcattaccgt gtgagctccg cctcccgcca gagcattacc 41580 gtgtgagctc cgcctcccgt ccgagcatta ccgtgtgagc tccgcctccc gtcacagcat 41640 taccgtgtga gctccgcctc ccgtcacagc attaccgtgt gagctccgcc tcccgtcaga 41700 gcattaccgt gtgagctccg cctcccgcca gagcattacc gtgtgagctc cgcctcccgc 41760 cagagcatta ccgtgtgagc tccgcctccc cacagagcat taccgtgtga gctccgcctc 41820 ccggcagagc attaccgtgt gagctccgcc tcccgtcaga gcattaccgt gtgagctccg 41880 cctcccgtca gagcattacc gtgtgagctc cgcctcccgt cagagcatta ccgtgtgagc 41940 tccgcctccc gtcagagcat taccgtgtga gctccgcctc cgtcagagca ttaccgtgtg 42000 agctccgcct cccgccagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 42060 tgtgagctcc gcctcccgcc agagcattac cgtgtgagct ccgcctcccg tcagagcatt 42120 accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg agctccgcct cccgacagag 42180 cattaccgtg tgagctccgc ctcccgccag agcattaccg tgtgagctcc gcctcccgtc 42240 acagcattac cgtgtgagct ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc 42300 cgtcacagca ttaccgtgtg agctccgcct cccgtcacag cattaccgtg tgagctccgc 42360 ctcccgtcac agcattaccg tgtgagctcc gcctcccgtc agagcattac cgtgtgagct 42420 ccgcctcccg tcagagcatt accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg 42480 agctccgcct cccgacagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 42540 tgtgagctcc gcctcccgac agagcattac cgtgtgagct ccgcctcctg ccagagcatt 42600 accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg agctccgcct cccgtccgag 42660 cattaccgtg tgagctccgc ctcccgccag agcattaccg tgtgagctcc gccccccgcc 42720 acagcattac cgtgtgagct ccgcctcccg tccgagcatt accgtgtgag ctccgcctcc 42780 cgtcagagca ttaccgtgtg agctccgcct cccgccagag cattaccgtg tgagctccgc 42840 ctcccgtccg agcattaccg tgtgagctcc gcctcccgtc acagcattac cgtgtgagct 42900 ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc cgtcacagca ttaccgtgtg 42960 agctccgcct cccgtcagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 43020 tgtgagctcc gcctcccgtc acagcattac cgtgtgagct ccgcctcccc gcagagcatt 43080 accgtgtgag ctccgcctcc cggcagagca ttaccgtgtg agctccgcct cccggcagag 43140 cattaccgtg tgagctccgc ctcccggcag agcattaccg tgtgagctcc gcctcccgtc 43200 agagcattac cgtgtgagct ccgcctcccg gcagagcatt accgtgtgag ctccgcctcc 43260 cgccagagca ttaccgtgtg agctccgcct cccgtcacag cattaccgtg tgagctccgc 43320 ctcccgtcag agcattaccg tgtgagctcc gcctcccgcc agagcattac cgtgtgagct 43380 ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc ccacagagca ttaccgtgtg 43440 agctccgcct cccgtcacag cattaccgtg tgagctccgc ctcccggcag agcattaccg 43500 tgtgagctcc gcctcccggc agagcattac cgtgtgagct ccgccccccg ccagagcatt 43560 accgtgtgag ctccgccccc cgacagagca ttaccgtgtg agctccgccc tcccgtcaga 43620 gcattaccgt gtgagctccg cctcccgtca gagcattacc gtgtgagctc cgcctcccgt 43680 cacagcatta ccgtgtgagc tccgcctccc gtcacagcat taccgtgtga gctccgcctc 43740 ccgtcacagc attaccgtgt gagctccgcc tcccgtcaca gcattaccgt gtgagctccg 43800 cctcccgtca cagcattacc gtgtgagctc cgcctcccgt cacagcatta ccgtgtgagc 43860 tccgcctccc gtcacagcat taccgtgtga gctccgcctc ccggcagagc attaccgtgt 43920 gagctccgcc tcccgtcaga gcattaccgt gtgagctccg cctcccgcca gagcattacc 43980 gtgtgagctc cgcctcccga cagagcatta ccgtgtgagc tccgcctccc gtcacagcat 44040 taccgtgtga gctccgcctc ccgacagagc attaccgtgt gagctccgcc tcccgtcaga 44100 gcattaccgt gtgagctccg cctcccggca gagcattacc gtgtgagctc cgcctcctgt 44160 ccaagcatta ctgtgtgaga tccgccccct gtcatatcat tactgggcga gctccgcctc 44220 ctgccagatc agtggtggca ttagattctc ataggagtgg aaccctgttg tgaactgcac 44280 atgcgaagga tctaggttat gcgccgctta tgaaaatcta atgctgatga tctgagatgg 44340 aaccgtttca tctccaaacc atccccacca tccccacact tgtccgtgga aaaagtgtct 44400 tccatgaaac cagtccctgg tgccaacaag gttagggacc accagtttaa ataaccaaat 44460 actaaaagaa ctggcataga agtaaatggg ctgctgcttt atttttaggc tgttcttttt 44520 agagaacaat gacaattatt tccaagtttg tcattagaaa gtaatattag gttggtgcaa 44580 aagtaattgc agtttttgcc attactttca atggtaaaag ccacgattac ttttgcacca 44640 acttaatatg ataaatttgt tccttaaagt gtatttttgg taagaaaaac cttttgtttt 44700 tccttatatt aattttttgt tttttttctt ggtagagaca gagtcttgcc atgctgccca 44760 ggctggagtg cagtgctgtg atctcggctc actgcaacct ccacctccgg ggctccagca 44820 atcctcccac ctcagcctcc caaggagctg agactacagg tgtgagccac catgcctggc 44880 taatttttat attttttaca gagacagggt ttcaccatct tgcccaggct ggtctcaaac 44940 tcctgggctc aagcagtcct cctgcctcag cctcccagag tattgggatt ataggtgtga 45000 gccactgcca gaaaaacttt tcctaagaca aggcaggttt tacattatat ttagattttt 45060 tttttaatga tgtctttttt ggcagtgcac agccagagaa caacacatca cacacaggaa 45120 acagttgtgc tcatgtgatg ggggcctcag cactaggaag gagcggacta ttggtgcacg 45180 cagcagcttg aataaatctg aaagtcacta tgctgtgtaa gagaagccaa attttaaaag 45240 tgcatactgt gtacagaggg tgtcgagaat gcctcctacg tgacggaaag cagatccgtg 45300 gttccctgca gactggcagg agcagattcc aaaggcacag gaagaagctt gtgggtagag 45360 tgtgttcatt atcttctgtg cattacacca taaaaaagct ggtcataaaa atgcgaacca 45420 aaaacaaagg tgaaactagg ataaaatttc tcacctgtgt gattggtaaa tgtgcaggtt 45480 tgacatcatg ctttgtttat gaagctgtgg gatacaagga ctctcatacg tcactgtgga 45540 atgcagaaca ttgcaacctc atggaagagg atttggcagc atctaacaaa agtatgtggc 45600 atttgccctt tgactcagca attctagact gcctcaaaaa aaactctggc aaaaaaatga 45660 aaggacttta ctcacagagt tcttttcacg cctaaatatg tttgcaacca agttcttcac 45720 tgtggcattt gtaaaactgg aaacaatcaa aatgtccatc agtaggggat taggaacatt 45780 aattcgtaca gtggggaact cagtaccaga aggagggatg aggaagacct attgataagg 45840 ggcagagtac atatcatata atgcaaatat atatttgctt tttcttaaaa cagtacaaag 45900 ataaaaaact aaagtggttg ctgtggagga caggggtcaa tggtggaagt gagactgaaa 45960 tagactctga agtaatatct ggactttgaa attataagtg ttttacatat taccaaacta 46020 agttttaaga tagtccctaa aattgaaaga atagtatctg aaatgaatga atctaaattc 46080 cttggattgt cttctgcagg tgccaaccct gagaccaaaa tttggaaggt ggccctgagc 46140 agcagctgaa gggaagcggg aggtaagaca ggaaagaggt gagggcacag ggtgtctggg 46200 agccggatcc cacgtgggca gctgggcccg tgctcattgt gggagctggt gcattccttc 46260 accagcccac gctccacagg gtcaggatgg tcaattccgg gcaccccggc ctaatccagg 46320 acatgctcct gccaccagag aaagccccta ggcagcatcc cgggtgctgg tggtgtcaga 46380 atcaagtttg agtctgagga gtgacctggg gctggctggg ctaggcagca tcacagggtt 46440 ctgcagccca actgcacatc aggctggtga cagtcacaca gcctattact tcgtgtgtca 46500 tcagagcatc gccagaacgc agcacttcaa gtatgcagat ttagtgagcc atagtttaaa 46560 gacaaataga gccactgaaa tcctaaattt caa 46593 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SDHA siRNA sense <400> 3 gucuagagau guggugucu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SDHA siRNA antisense <400> 4 agacaccaca ucucuagac 19 <110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for diagnosing and treating anti-cancer drug resistance <130> PDPB194289 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 664 <212> PRT <213> Homo sapiens <400> 1 Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu 1 5 10 15 Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly 20 25 30 Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser 35 40 45 Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala 50 55 60 Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu 65 70 75 80 Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr 85 90 95 Arg Ser His Thr Val Ala Ala Gln Gly Gly Ile Asn Ala Ala Leu Gly 100 105 110 Asn Met Glu Glu Asp Asn Trp Arg Trp His Phe Tyr Asp Thr Val Lys 115 120 125 Gly Ser Asp Trp Leu Gly Asp Gln Asp Ala Ile His Tyr Met Thr Glu 130 135 140 Gln Ala Pro Ala Ala Val Val Glu Leu Glu Asn Tyr Gly Met Pro Phe 145 150 155 160 Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln 165 170 175 Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val 180 185 190 Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser 195 200 205 Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu 210 215 220 Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu 225 230 235 240 Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr 245 250 255 Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser 260 265 270 Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln 275 280 285 Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly 290 295 300 Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn 305 310 315 320 Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp 325 330 335 Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg 340 345 350 Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu 355 360 365 His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser 370 375 380 Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile 385 390 395 400 Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn 405 410 415 Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val 420 425 430 Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly 435 440 445 Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly 450 455 460 Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys 465 470 475 480 Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu 485 490 495 Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg 500 505 510 Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val 515 520 525 Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly 530 535 540 Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr 545 550 555 560 Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu 565 570 575 Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His 580 585 590 Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys 595 600 605 Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys 610 615 620 His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu 625 630 635 640 Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr 645 650 655 Val Pro Pro Ala Ile Arg Ser Tyr 660 <210> 2 <211> 46593 <212> DNA <213> Homo sapiens <400> 2 actgcagccc cgctcgactc cggcgtggtg cgcaggcgcg gtatcccccc tccccccgcca 60 gctcgacccc ggtgtggtgc gcaggcgcag tctgcgcagg gactggcggg actgcgcggc 120 ggcaacagca gacatgtcgg gggtccgggg cctgtcgcgg ctgctgagcg ctcggcgcct 180 ggcgctggcc aaggcggtga gtccgtgccg cggaccgggg cggggcaggc gggggccgag 240 gcggcggtag gagcgggacg gtccccagcg ggtccgagcg gagcgggcgc cgggtccctg 300 cgccctctgt cccgggatcg ggaaggggct gagagagccc tgggccggtg cgaggggaag 360 ccgcggggcg gactcgggga cccggggagc tcggtcctta gtagatagtc cgcgtccggg 420 tgaaggtcac aaccccgcgg gcttgctggg cgtcccctcc gccgccttgg tccgggcctg 480 gggtcctggg accccgcggg ctgaggtagc ccctcgcctc agtgcctggc aggtggactc 540 ggggaggagt cgtgtctgcc caaggtcacc cgggcggata gcggccggtg gccgccctgg 600 ctgggctggg cctctgccgc cctctgtgcg ggttgtcctg aggagcagcc cgcagcccgt 660 gggtggggcc ggcggggcgg gtgagaccgc ccgggtgggt gcgaggagtg gccgggctcg 720 gcccggtggg cgtccggtgg gaagcgtggc gcgcccgagc ttaggcttgc agttcgcctt 780 tccagaaagc gcaaatctgt ccatgtccac ttcgagacct tgtaagttaa gggcttctac 840 tttgggtcgt gtttggtggt ccttatgcca ccaaaaatgt gccagtgttt aaaagcagct 900 gtgccagttt ttaaaactca ggcggagagc tcagcgcact gaccgggcga ggacgccggg 960 acctgtgctt gcctgtgcgc tgagtgcctc tagggccggg ctcggcttag tccaggatgg 1020 tggtcagggt tatacttccc tgagcccttg ctctctgagt gtctgaatgt gccctctacg 1080 attgcatctt aagaatcggc cttctagggt tttatttaat caagctaaat tggataggtt 1140 tagttgtttg gttcttttaa atgaacttac ccacccacct ccttaattac aaagtaattt 1200 taaattgcag aataaaaatc tcaataggaa ccaaggcatt cagcaatatt gatttgaatt 1260 atgcctgtaa ttgtgcaatt ttctcctttt tgaaatactt attaaaaatc tcgttgagtt 1320 aaatgtgagg attagtcata cagccatcct gccaacatca gaaagcgtgt aaaccgttct 1380 agtgtgttgc tgtggttggt actgactgag cagagacccc tgccgcatct tgggctttta 1440 agagctgcta ggagggcgtc cacaagcagg aaggaaagcc catggtcagt ggggcttttt 1500 aggggaaatg gtagcttgtg attggagaaa agctagagct agtgcctttg tcctcaaccc 1560 agatggtgcc cggtgttcct tctgcagact aagaccctgc cagcggcgga ggcacctcag 1620 acgggatggc aagatagcaa aattgaacca aaatttagtc ttgggttttg taaaagtctt 1680 tttatcttga tgaagttagc ttttcctaca gaaattctgc ctgcaagcag catatttatc 1740 ggcatttcag ataggctttt tttcctgaag aattggagtt tacgaaggaa cagcataatc 1800 aaaaaattag cagaaaaaag aaagaaattc gtgatagcca actgagaaat gtgtttggaa 1860 aatagatgct gaggataatt tcagtaaagt ccagagagaa ttttttaacc tcattttggc 1920 cagttctgtg gtgttttgat cattctgtat ttacaactgc tttcaagtga cgaacttatc 1980 agacttattt tgcttttggt ttctttttat ggcccagtgc actgacacag cttgcaaact 2040 gttcgaaagc agtttaatca gaggaaaaag aaaaccaaga aacttcaata gttactggtt 2100 aactgcttac ttcaggtgaa gtttttcaaa tcccttaaca tgaacatcca atatattgtt 2160 ctaggacaaa ttgatttctt ttctttgaat ggattgttct gaatatgttt aatggtgttc 2220 tagaaataca ttatttaggc cattatagta gtaaaccttt gctttgcttc ttgggataat 2280 tgtaacaaca atggcattcc attctgcatt cctcatgatg tttttcctga tttatttgaa 2340 aaattgtgac ttgttttttg atatgaaacc tggatattaa tattagttgg gagtctcaga 2400 gatttttccc aagggtcttt agataggggg gcaagtccag gatccagagc caggccggcc 2460 gacttgaatc ctctctgagc tccgtctttc cgtggggtta gtgaggattc tgtgggtaat 2520 gtttgaaacc tttggtgatg ctcttgagaa ccagggctca cagcctatgc acagtaacca 2580 tgtgtgaact gaattttttt ttttttttga gacagagtct cactctgtca cccaggctgg 2640 agtgcagtgg cgcaatctcg gctcactgca agctctgcct cccgagttca cgccattctc 2700 ctgcctcagc ctcctgagta gctgggacta caggcacctg ccatcatgcc cggctaattt 2760 ttttgtgttt ttagtagaga cggggtttca ccatgttagc cagaatggtc tccatctcct 2820 gacctcgtga tccgcccgcc tcggtctccc aaagggctgg gattacaggt gtgagccacc 2880 acgcccggcc gaactgtatt ttaagtatcc ctgtttttaa aataataaac actaaaattc 2940 tagtgagaat aattacacgc agttgtaaaa gaatcaatga gtataccctc cctcacccag 3000 ttcattttcc atgaaagcaa ttcctgtttc tgatactgac tcctggcatt tgtaatacct 3060 ctgtttcttt agcggatatg caggtgcatt tttatcagtt ttagacttct tgcctagaga 3120 ggggatttag ttcacttaaa ctaccctgct tccgcttctt cctgatacag tcgtgcgttt 3180 ttaggttctt cgttggctgc ttttgtgatt gtgtaggttg tacatgcaga ttagtttctt 3240 gctcgtgatt tataggtgcg ttgtattaga tgaggacccc ttactttgct agatttcgga 3300 tatgaatgtc tctgcacttc ttacttttcc cctccacctc ctaattcagt catctgaaat 3360 tctgtattgt taagcaaggt ctaagtattc cttttagtta tatgttcccc attttttttc 3420 ttgaggaaat gtttgatagt ttctcctaaa aaaattaaca attggcacaa aagactagtt 3480 ttgtgtcaaa actagttttg agttttatct aaagactgaa attggcttaa agttgggctt 3540 tccaaattca aaaatctgcc ccagattaga tttagattga gagggttagt gtccttttcg 3600 ccagggggat ggcgtgataa tttgttcaag attgtgttat agtagctgcc ccttttaagc 3660 cagctctgtg tgtgtgtggt ggggggtggg cagtgggtct tccacatcaa catcctagaa 3720 agaacgaata aacattgagt gatctcactg tttctactta catttggtat aatatactgt 3780 tcttactggt gctattacta tgttaatagg gcactttaca acattttcaa gaacatttgt 3840 attaaaatta tttcaaagac ttctttctta aaatatgatt ttaccatgca aaaattttac 3900 taaggtagaa gaatatttgt tctttctcat tcattttctg aaaaaagaaa aaagctgaat 3960 tagcttatac caataaaaac agagactaga aattggagaa atgaagaata attttttatc 4020 ccacataata agtaatttct gaattgcaag tatttctaaa tacttgaaga cattcctcac 4080 atcccctctt ctgattgcat ttatttccta aagctttttt ttcttttctt tttttttttt 4140 ttggagacgt tgtctctctc tgttgccgag gccggagtgc agtggcacag tctaggctca 4200 ctgcagcctc tgccttctgg gttcaagcga ttctcctgcc tcagcctccc aagtagctgg 4260 gattacaggt gcccgccacg acgcccagct aattgttagt agagatgggg ttttgccatg 4320 ttggccagac tggtcttgaa ctcctgactt caggtgatcc acccaccttg gcctccccaa 4380 atgctgggat tacaggcatg agccaccgtg cccagcccct aaaactattc ttgatgatat 4440 ttctgagact attcagtggt cttctcaaat gtagccagca gaatggaaaa cgtatcccct 4500 aaatggctgg ccaaccttag catatggaca gtgtcacctc tctcacacag agccactaaa 4560 aactaaacac caaaaccagt tttcttgggt aaagttttct aagatggaaa atttaagcag 4620 caagatgtgt caagttgtag atgttggcca ggaaaaagcc agcagcagcc aggcagggga 4680 gagtgtgcat ccgacatcct cctgtgtgat gaagggatga cacctcgtcc ctctaggctg 4740 tcagctttta ctgttccagg atacagatct cctgattcaa tgttgagtgc cttttgaact 4800 gaccacaagc cctcctggac gattggaact gtaatgtgga aagggctatg atggagccag 4860 ttaaaatgct tcattacttg caaaatacca catacagtaa tacaatatac acgtctttcc 4920 ccttctccac taagtagcat acattaagac ttcacagagg aagtgtgcct ttttcatttc 4980 gtatctgagt cagtgagtat cctgtttgga aacaagcttc atcctggttt tctagagtgc 5040 caagtcaggg tggaaacgag gacctggggg ctcagtcctt ccttgcccct tgggctgcct 5100 tcagggttaa gtagagggtc ccagctgagc tctctggatg cacaggagca cctgggtaca 5160 taagaaggtg aacagtttgc aaggggaaat tactatcccc cacagcattt gttccttcag 5220 gacactaacc ctctggatct gtgtcttctg tgtctccagt ggccaacagt gttgcaaaca 5280 ggaacccgag gttttcactt cactgttgat gggaacaaga gggcatctgc taaagtttca 5340 gattccgtaa gttcatgctt tttgttccat tataaatgat ttttttggct tagggggtaa 5400 ggatctatac cagtttgttt tcatatgagt catagacata agggaaaaat ttctcatagg 5460 tatccaatgc atgctgaaat tattttcagt gtaataatac ttaattgcaa gtacaaatat 5520 aaacatagat gtttacattt ttacttgtat ctgttatgta tctataaact agatttaaat 5580 tagatcaag taaagcaata aattaaaata aaatgaacag tgtctgctgt gatagatgat 5640 aaaattccta ctgaaaatag gacggtgggg ccagtgaaca gtatctgctg tgatagatga 5700 taaaatccta ctgaaaatag gatggtgggg cccctcgggt gtgggttgct ttgtatttag 5760 tgagcctatc tctgttgaaa aactgaaact tgctgagaag ttgttttctt ataagcccac 5820 aatagcgact gaatactcct tggcaccttc tcaggcataa gcataggcac ggccctgaag 5880 tagagttgtg gtcctcagtc tgaccccatg ggatagaccc tctaccattc gtagaatctg 5940 attgtcagtc ccttctccag gtgcgaatgt gcccacctct ccccgaactg ttcagggctc 6000 agccccagga caggatggag gccctgtgtg cccagcagtt gctcctttta tctttgtcaa 6060 gctctttcac tgtcacaaga ttcttcatgt ttggcatagt ggaacatgtg attgacaggt 6120 gaatttttct tttccagatt tctgctcagt atccagtagt ggatcatgaa tttgatgcag 6180 tggtggtagg cgctggaggg gcaggcttgc gagctgcatt tggcctttct gaggcagggt 6240 ttaatacagc atgtgttacc aagctgtttc ctaccaggtc acacactgtt gcagcacagg 6300 taagagaaag gtgccccact gtgctcccac tccgtgcagg tcccgcgcag cctcgcactt 6360 tctacctggg cagccccctg cctcctcccc ctgctccagc cacatggcct cttgctgtgc 6420 cttactcagc tcaccctctc aggggtctct ccctggagcc tcttccctgg ggactttgaa 6480 gggcgggaga cttgttgtca ctcctaattc agactccagt cacacttggg ttttctctga 6540 ccatcttgcc tactaccttc cccacctacc cccgccaccc caacacctta agaaaaggag 6600 atcacctaaa gaggaggaat cagaatttag gttggggaag aaaagggcaa gggtttcatt 6660 tgtccctgtg cttctgtctt ctgggactct ctgaggggta agacagtggt gggcacacac 6720 agccaaagga agctggggta caggggagtg cgactctgag tgtggagttt attacttggc 6780 aggaagcact tctagtcttt aacacatgcc cgtaaatgcc attgggaaga tttgttaata 6840 aaattatctg gaaagatttg ttgagtacct tttctgtgcc agataggtta ggttataagc 6900 atattacagg tagcctttca ctcactgctc cagtcagccc ttcctggagt tccctgtgtc 6960 tccaccacgc agatgaggag actgaggcta aggatggaat cactgggcga gtcggggagg 7020 ggttgtgatc tggaatctgt cgggtctcgc tgctcctctg ctgaggtcag ccctcactgg 7080 gagtcactgt gtgagtagtt ggctttctct gaatcccccc agcgggtgga tttgggcctg 7140 gaagacaaag ttggcgctcc tgtttgtggc ttgtaaggag tggttggtgt ttccagggag 7200 gaatcaatgc tgctctgggg aacatggagg aggacaactg gaggtggcat ttctacgaca 7260 ccgtgaaggg ctccgactgg ctgggggacc aggatgccat ccactacatg acggagcagg 7320 cccccgccgc cgtggtcgag gtgatgggcg ggaggctctg ggtgttctcg tggtctgttt 7380 ctagtacaaa agaatcctgg aaaaaaatgt aagcaattga ggcggatgtg gcagccaaaa 7440 gaatggtgat gagcaaagtt cacaagaaga gtctttttcc gttatgaact atgcattata 7500 tgtaacaaga aaacttctct ttgatgaagt gttgacattt tcataaaata ggttactttg 7560 ggtttgcaga tttgtgttaa acttgtttag tgtagattag ctgcgaatat cttgactcct 7620 ttaaggtgtt aaggtttttg tttgttttta tctttcacag ctagaaaatt atggcatgcc 7680 gtttagcaga actgaagatg ggaagattta tcagcgtgca tttggtggac agagcctcaa 7740 gtttggaaag ggcgggcagg cccatcggtg ctgctgtgtg gctgatcgga ctggccactc 7800 gctattgcac accttatatg gaagggtaag gccgcccccg tccacctgag acaggacacg 7860 tagtgctggg gcttatggtg acagtgggga atgggttagc gtgcccagtg agtcagccag 7920 agattacgta aaaagcaaca gagaacagca gcgtggggcg catgcagcga ctgtggatgt 7980 gacaggacca gacgtgtgcc ttgaggagct gtccctaagg cagtgtgtga cttcttgcat 8040 ctatgtcaga aagttgaatc gataatctta cataccaggt tttaacttgg gatatgtgac 8100 actcaacata caagagcaga gcaagcaggc caggcacagt ggctcaggtc tgtaatccca 8160 gcactttagg aggccaaggc aggaggatca cttgagacca gaagtttgag accagcctgg 8220 acaacatagc aagaccctgt ctctacaaaa agtttaaaaa ttagctgggc atggtggtac 8280 atgcttgtaa tcccagttac tcaggaggct gaggctcctg gatcacttga gaccaggagg 8340 ttgaggctac agtgagccat gttcgtacca ctgcactcca gcctgggcaa caggagaccc 8400 tgtctcaaaa aaagagaaag aacaggctgg gtgcggtggc tcacgcctgt aatcccagca 8460 ctttgtgagg cctaggcggg cagatcacaa ggtcaggagt ttgaggccat cttgcctaac 8520 actgtgaaac cccgtctcta ctaaaaatac aaaaaaatta gccaggcatg gtggcaggca 8580 cctgtagtcc cagctgcttg ggaggctgag gcaggagaat ggcgtgaacc caggaggcgg 8640 aacttgcagt gagccgagat cgcacccctg cactccagcc tgggcaacag aatgagactc 8700 catctcaaaa aaaaaaaaaa aaaaagaaca agtattttaa gtctctttta ccacctctga 8760 gttcctgaat ggattggttt tgtttgtttt gttttgtttt gtttttgaga cggagtctca 8820 ctctgtcacc caggctggag tgcagtagca caatctcagc tcactgctac ttccacctcc 8880 tggattcaag tgattctcct gcctcagcct cccgagtagc tgggactaca ggcgcacgcc 8940 accatgcctg gctaattttt tgtattttag tagagacagg gtttcccatg ttgcccaggc 9000 tggtcccgaa ctcctgagct caggcagtcc acctgccttg gcctccgaaa gtgctgggat 9060 tgcaggcgtg agccaccgca cccggccatg gattgtttta atattaactg ttaacactga 9120 agaaagactt gaggtgacaa tagttactgg gtaatcagag tcaactttgg catggccaaa 9180 taatattctg aacggtattg attcagagta atcaatattc tgaactttgt tgttttctga 9240 tgcatgggga cggatcgtta atttgcaggt tgttagaaca ccagtgactt ctctgtggct 9300 gagtgcatcg acaagtgtgt ggtgggagga gaccgcggct ccatctggag caggagctgt 9360 catgtgggga gctggcccag gcttacaaga gcgacttgtg ctggctgagg gaacggcagg 9420 tccaggcggg cagcgctgtc cggcgcctac ctttctgcgg tgccggaatc tgctcgtctg 9480 caaccgtccg ctttggtagc tgccagccac atggggctgt tgctaatgtg gcaggtgtag 9540 ctgaagagct gaatgttttg acttatttta attaattaag tggttatgtg ttgccagtag 9600 ctcccatctg ggctgtgact gcatggtctg cggacctcac tctggcacca gactccgagt 9660 ggagctgcat gcggccaccg gacagtgtgg agtgcctctt cgggttgtgt agaagtagga 9720 aatgtgtcac caaaatagga gctgttgctg ctgcgttctc tagcacacct gccttgtctg 9780 tactgctcgg cgtggaatgc ctctcgggct ctgacagtgt cactgacact gttgctgatc 9840 tccttggatt tacctggtcc atttggatca agttctttca cctattcaca tgagcagata 9900 ccaccttaaa accttaaagt ttggcttaac acttcttgcc cttttttttt cctttctttt 9960 agtctctgcg atatgatacc agctattttg tggagtattt tgccttggat ctcctgatgg 10020 agaatgggga gtgccgtggt gtcatcgcac tgtgcataga ggacgggtcc atccatcgca 10080 taagagcaaa gaacactgtt gttgccacag ggtaggaatc tcatttctac tttattttgt 10140 ttataaaaat gaataaattt catttagagt ttctttattt taatgaaaat agaggcattg 10200 tagaataacg gtttagacac aggccttgat ataaccatgt gagggtgatg gcctttccca 10260 gccgtggttc ctcacctgta aagggtgagg acaggagcac ctgcctcagg gtgagaaagc 10320 atggtcctca gtagatggta gtggttgccc tcaggttcac agcttacctc tcccaattta 10380 gatgagggaa ctgtggccca aagagtcaac atggggtttt ctggcaaaat ctctcttgtt 10440 ttagtgggta ctgtattgat actgattcct cgggtgggta agtccttctt ctccacatga 10500 tgaaaataag taactttaat tttatacact gtcagttact ttaaccattt taaaagttaa 10560 aaagtgtcag tacagccaaa caaaaaatca gcaaaactac aggttgggaa gaaatatttt 10620 ccaaaccata tgtgtaataa tatcttacta tctaaaatag caaaaaaaaa accctactaa 10680 aaacaaccta ctaaacccta ctaaaaaaca accctactaa aaatgggcaa aggacttgaa 10740 tagatatttt tctagagaag acatacaaat ggccaattga tgtatgaaaa aatgctcaac 10800 gtcactaagc accagagaaa tgcaaattaa aaccccaatg agatatcatc tcatctcgct 10860 ccagttagaa tgactgttac caagaggaca aaagagcaag tgttggtgag gatgtggaga 10920 aaagggaacc ctgtgtgctg ttggtgggaa tgtaaattag tacaactatt acggaaaact 10980 ctggaggttc ctcaaaattc ataggactac tatgtgctcc agcaacccca tttctgggtg 11040 tatatccaaa gggcatgaaa tcagaagctc aaagagacac ctggacccta tgttcattgc 11100 agcattattc acaatacccg agatatggaa acaacctaaa tatgtgttgg tgtttaatga 11160 caatgtggtg tgtatacaca actgaatatt attcggctat gaaaatgaag gaaatcctgt 11220 catgtgtgac aacgtggatg aacccaaagt cattatgtta agtgaaacga gccaggcaca 11280 gaaagacaga tactgcatgt cactcatgtg gaatctaaaa cagtcacaac tcacagaaac 11340 agagtaggac agtggttgcc aggggctggg ggaatgcaga ctgtggcgat gctgattaaa 11400 ggtgtaactt cccgttacaa ggtgaagttc tgaaggtcta atatacaaca tggtggctat 11460 agttaacatt atacagcatg gtggccagag ttaacattat acagcatggt ggctatagtt 11520 aacattatac agcatggtgg ttagagttaa cattatacag catggtggct agagttaaca 11580 ttatacagca tggtggctag agttaacatt atacagcatg ctggctatcg ttaacattat 11640 acagcatggt ggccagagtt aacattatac agcatggtgg ccagagttaa cattatacag 11700 cacggtggct agagttaata tacagcatgg tggctatcgt taatattata cagcatggtg 11760 gctagagtta acattataca gcatggtggc tagagttaat attatacagc atggtggcta 11820 tcgttaatat tataacttga aatttgcgaa gatagtagac gttaggcgtc tgtatctcca 11880 aaaaaaggat aattgtatga ggtgatagat gtgtattaac ttgattgtgt catcatttca 11940 caaaataaat catcacgctg tacaccataa atatatagtt ttttttttaa attcatcaat 12000 catacctcag taaatctggg ggagaaaaaa atccataaaa attttaaaat tttcattata 12060 aaagtagtat atgcttattg gggaaacctt ttgaacagca caaagctaaa atacaaatag 12120 ggccaggtgt gcagtggctc acgcctgtaa tcccggcact ttgggaggcc aaagtgggag 12180 gatcacttga ggtcaggagt tcgagaccag cctggccaac atggtgaaac cccatctcta 12240 ctaaaaatac aaaaattaac tgggcgtggt ggtgcacact gtaatcccag ctactcggga 12300 ggctgaggcg gaagaatcac ttgaacctgg gagccggagg ttgctgtgag ccgagatcgc 12360 accactctac tccagcctgg tcaacagagt tagatcctat ctcaaaataa ataaataaaa 12420 tacaaatgga agtcctttct ttgatcccag gcttctatcc tacctgtgca gggtagcagc 12480 accagtggcg cggcccctag tgatatgtgt ggggtgtgcg tgagtagggg gttgtgtgtg 12540 cacagcactg agaagacggt gctgggggct gccgtgtcca ttctgtgatc tcaccagata 12600 ggaggtccag atgtgggccg ctgtgtgcag tcactgctct ctattgtttc cagaggctac 12660 gggcgcacct acttcagctg cacgtctgcc cacaccagca ctggcgacgg cacggccatg 12720 atcaccaggg caggccttcc ttgccaggac ctagagtttg ttcagttcca ccctacaggt 12780 agggcaggac gccttgcccg gcaggtgttt ggcttgtgtg tgtcttgtaa gcatgtgatg 12840 cctactcatt gctcttccat agttttatgt aataacatgg ttttgaagat cagcttcctc 12900 agctctcagg tcctaacttc aacgtcattt acctaaggag acttttccca cactcccctt 12960 cccctaaggc agtttgggcc accgtcttat gtatttctca agagcgctaa accctgcctt 13020 ggtgatactt aagccagcag taaagcaggg attgaggccg agcacagtgg ctcactcctg 13080 tagtcccagc actttaggag gccaaggcgg gcggatcacc tgaggtcagg agttcgagac 13140 cagcctgacc aacatggtga cacctcgtct ctactaaaaa tacaaaaatc agctgggcgt 13200 gatggcgtgt acctgtaatc ccaggtactt gggaggctga ggtgggagga atgcttgaac 13260 ctgggaggca gaggttgcag tgagccgaga tcgcgccact gcactccagc ctgggcaaca 13320 gagtaagact ccgtctcaaa aaaaaaaaaa aaaaaagtaa agcagggatt gttcagtgtc 13380 cctcttcgca acgaggtcgt cagctcctcg ggcaggcagg tcttctcgtt agctgtggtg 13440 ctccatgccc agcacatggt agggtctcca ccggggttta ctgagtgaac attctgagag 13500 ctgggagaat gccatggaac cgagaagcag cacaggcaga cttcagcttt gtaggagaac 13560 acggagcttc tgtgacaatg ggatgtaaag ttaggacgca gccgtgacag aaccccatgt 13620 gacattgggc gctgggctca gcccacgtga ccactgagag agcttgtcgt ggggaagatg 13680 agctcgtctt ggggacgtct gacggttgag gttacgaatg tgcaattcgg agacattaac 13740 tcagaaatga cagttgaagt cttgagtttg gaggaggttc ttcagaatga gtcagagaca 13800 gacacacacc tgcctcttgt ttgaggtgac ttgtcctata cttttctggg ttgcttttct 13860 acctgtggac gatggagacc tctgaagtgg tgccaaagaa ccagacctgt gtcttctctt 13920 tctctgtcag tgtcagcttt ctgatccctg gaagggatga aaataagaaa tgaatttgtt 13980 gtagtgtttt ttttttattt gtttcgagat ggggtcttgc tctgttgctc aggctggagt 14040 gcagtggagc aatcttgact cactgcagcc tttgtctccc aggctcaaac gatccttcca 14100 cctcagcctc ccaaacagct gggactacag gcatgtgtta ccatgcccag ctaattttcg 14160 agggtttttt cgtttttggt agagacggga tgtcaccgtt ttgcccaggc tggtctcaaa 14220 ctccagggct taggccatcc tcctgcctcg gcccctcagg gtcctgggat tatagccatg 14280 agccactgca cccggcctgt tgtatttttt attactgttt ttaatgaata aaatgtcact 14340 gagcccctga attccctctg taatttgctt agagctccac tgctcatgct ttgtcttcag 14400 tatgtgagaa gtaaccacag aaaaaagagc attgaaactt agaaaatcaa aagacagaca 14460 agaatcacat tcgtttttta aaagcaagat tgcccttttt gtatactaat aaaaattgta 14520 gctttcgaaa tacatttagt ttagggtttt tgatctcctt tgttaaaatt cgagagcttg 14580 gcatgccctg tttctcatcg cactgaggag tcacagagcc gctgtttgga gcacagaccg 14640 ccggactacc caggtttggg tttgagctct gtcctcagct gcatgactgg atgttaccaa 14700 gtgttaattt gcttgtcact gagaaagggg gtcgtactac ccagagttgt tgtaagactt 14760 aaacgagtta atatgtggaa agcagctaga actgcccata gcaagtgcag tgtaaatatt 14820 atctaaaata atcattatta ctgtccttgt cactgtttgg ataatttaga tatgaatact 14880 tcaagctagt attcttaact agtcaccaca gtatcatagt gcaaaagaat gttcaaaaat 14940 taaaacaaaa tttgaggcat ccaacgtaca ctgggctgta atcagagtat tggccagagg 15000 tctgtgggcc ggcctttctc ctctctgggg acaccgctct gccccagcct ctgctgcagc 15060 tgtcagcctt gtcagtgctt tttgttatcc agactttcta ctgtatctta actgtcttgc 15120 tgtgcagttt tgcacataat gccttctgct tatctttttc ctctttcccc caaaaaatgt 15180 cttgaaaaaa ataatgcatt tgaaatagag atctagcaat tgttaggtaa taaatatgtg 15240 tggttttttg caggcatata tggtgctggt tgtctcatta cggaaggatg tcgtggagag 15300 ggaggcattc tcattaacag tcaaggcgaa aggtttatgg agcgatacgc ccctgtcgcg 15360 aaggacctgg cgtctagaga tgtggtgtct cggtccatga ctctggagat ccgagaagga 15420 aggtgcgtgt gatttaccac cagcactgtc tgagcgggca cacgggccgg ggttgcttct 15480 gtgagtttca gcaccgctcg ccctcacctt cgtgtgcagg cgcatgtgca cagccacctc 15540 tcttagctgc tggcaggcgt ctgttagtct gcgatatttt cctaaagacc tacattttga 15600 aaattttagc cagtttcttt ctcaaatctg tggaacagag tttctcttag tgtgtgtgag 15660 tatgtgacgg agtatgggag agagagacac acacccaacc tgaagtcggc atgtgagcct 15720 tgggtgttgt gtctgatacc cacagatgtt ttttggcagc tttcaaagtg tgtgggttat 15780 ttggctttca gtaaaacagt ttgcagctct ttcattgcct gaccctgttc tttaatgtaa 15840 tgacatttgc taaatatctg ctggtatggc ctttagaggt tttacatttt tatattaaaa 15900 aaacagagaa gtcaggtggg gcgcagtggc tcacgcctgt aatcccagca ctttgggagg 15960 ctgaagcggg cagatcagga ggtcaggaga tcgagaccat cctggctaac acggtgaaac 16020 cccgtctcta ctaaaaatac aaaaaattag ccgggcgtga tggcaggtgc ctgtagtccc 16080 agctactcgg gaggctgagg caggagaatg gcgtgaacct gggaggcgga gcttgcagtg 16140 agctgagatc acaccactgc actccagcct gggcgacaga gcaacactct gtctcaaaaa 16200 aaaaaaaaaa aaaaaaaaaa acagagaagt caaatggttt tttggaatat ggtggccctc 16260 cgtacccatt ggttccacat gtgtggtttc agccaactat gtattgaaaa taaaattgca 16320 tccttacaaa tatgcagact ttttttcctt gtcattgttc ccttaacaat acagtgtaac 16380 agctatttac gtagcattta cattgtatta ggtactatga gtcatcctgg agttgctgta 16440 aaacttaaac gtaaaacttg aaatgaggat gattaaagt atagaggagg atgtgcatag 16500 gttatatgca aatactctcc cattttatat tagggacttg agcatccacg gattttggta 16560 tccgtggggg tcctggaccc aacctgccac ggataccgcag ggacgactgt atttggcata 16620 gaggcctttc taatgcactt accaaggaca cctgcagcag gctgtgatcc ctgagacgag 16680 cgtgagttta gtgagggcag agtttttgtt ctggttctca gctgtgtccc agcacctggg 16740 attgtccctg gcacacagta gctgcttaga aaagatttga tgagagggtg accatacatg 16800 aggggaaatt ttcctcagta tcaaaacatg ttgaaactca cacacttcca agatgacgta 16860 ttctcaggtc tcctgccgtt gccgttctct gccgtatgtg atggtgttct gtcttaccag 16920 aggctgtggc cctgagaaag atcacgtcta cctgcagctg caccacctac ctccagagca 16980 gctggccacg cgcctgcctg gcatttcaga gacagccatg atcttcgctg gcgtggacgt 17040 cacgaaggag ccgatccctg tcctccccac cgtgcattat aacatgggcg gcattcccac 17100 caactacaag gggcaggtga tggtgctggc tcctccccca cagctggaaa gaaggctggg 17160 acgacggggc ccacctcgca gttgtctctt tagatcttac aggaaaagat agatgtttcc 17220 ttcaagaaag tactgtattg ttttctagat tgcactttaa atttctatta ccggaggatg 17280 gagggggctt aataatttat tcctccttag taaattgtca gagatacatc atttgcagct 17340 ttttccattt tgtaattact ttcctatatg atcttgtgtt atttctaatg atcttacaca 17400 tcaagggatc tttataattc attcctttga gtggtttgtg gttcacacag agcttgtcag 17460 tcacttaggc tccttgttgg gcgaggtggg tggaagctgt tgctctccct gcgtagacga 17520 agaggtgaac ggggtagaac agtctggaac atcagtctcc cctgctgatg ttcctccacc 17580 tgccgtgctc ctgggtctga gccggagcac aggtggtgag ggccccggga acatgggaca 17640 cgggggacag tcgcagatgc tgacattgga ggtcctctga cctgcttgta acagcaggtg 17700 ctcaggggca gaggggaaac tgggggatac cttcggaagc ttccctctga agaagagtag 17760 ctatggtcct tacttccctc tagatacgg tctttacttc cctctctttt tttttctttgg 17820 agatgcagtc tcactctgtt gcttcggctg gagagcagtg gtgcgatctc agctcactgc 17880 aacctctgcc tcccaggttc aagtgatttt tccgcctcag cctccctagt agctgggatt 17940 acaggcaccc gccattatgc cccgctaatt tttgtatttt tagtagagat ggggtttcac 18000 cgtgttagcc agacaggtct tgaacccctg acctcaggtg atcacccacc tcagcctccc 18060 aaagtgctga gattacaggc gtgagccacc acgcctggcc tacttccctc tctctgacct 18120 gcagcacaga caccctgttg gggaaggtgg gctggtggag gcatgggcac cttgacattt 18180 cacctgaaat cttcctttcc acaggtcctg aggcacgtga atggccagga tcagattgtg 18240 cccggcctgt acgcctgtgg ggaggccgcc tgtgcctcgg tacatggtgc caaccgcctc 18300 ggggcaaact cgctcttgga cctggttgtc tttggtcggg catgtgccct gagcatcgaa 18360 gagtcatgca ggcctggtaa gtgttttctt caggagccag actatttgag aaggcgcagg 18420 acgttagaaa gtcttttttc ttttttttga gaaagggtca gcccaggctg gagtgcagtg 18480 gcacagtcat agcagcctca acctcccggg ctcaagcagt cttcaacacc tcaaccttca 18540 gagtcccaag tagctgggac tacagatgtg caccaccaca cctggctaat ttttaaaaaa 18600 atttattttg tagagacagg gtctcacaat attgcccagg ctggtcttga actcctagac 18660 tcaaacagtc ctgcctcagc ttcccaaagt atcgggatta caggcatgag ccactgcacc 18720 cagccaggtt acaaagcctt gatttcttac tggaaatttg cttagtgatc atatagaggt 18780 agtctgggtt tttcccccag aagtgattaa actgagaaat ccagaaatta tatggtggta 18840 atgttgagac tagatagagg ctggttgggg atcttaacag ttaaggtgac atttttgggg 18900 ttacattttt ttttaattat tttgcagtca ttatgttctg tttagaaaaa gcactattag 18960 aaagttgtta tttttagggg aatcattaca tattacttgc ctgataaaaa tcacttattt 19020 gcaatgaaat attttaagta gttggcatga atgaatatgt aacttcttgg tacttagaaa 19080 agtaatttag gccattctag aaacaaagta cagctagcct ctattagagg agaagggatg 19140 acttacagtg aacaggattc ccacccttta cggacagatt ggatttcact tgctggtttt 19200 cttttcatga tagcatcaaa taatgtgcag taaaggaaat accatgtgtg ggagtgtgag 19260 tcttacgtgc actaagaacg gggcagttag catctctccc acctccagac atcctcacgg 19320 tggttatcca gcctcgtgtg ctcagaacag tgtgaggtgg atgaggcact ggtggatgtt 19380 tgcgtggcaa ggatggtggg accccaggcc catgttcttc ccgttacctt tctctggtgt 19440 taactgttag catcatttct gctgttttta tacaacaggt gctttttgta tggattcaag 19500 tgaaataaaa actagcacgg ctgtaactta taaacgtgcc cccttttgta tctgtagtta 19560 gaaaggtgca gatagtatta aaagggtagc ttacttcaga cactctgtct ctggatctga 196220 ccaccgctcg ggaggccagc acacgcagag ctggcgtctc atccccagcc attggtgatc 1980 atcggcgaag gcggagttca ggtccatcgt tcctgacgcc gcaggtagtg cttgtctcac 197440 tccatagccc tgcactttgt cgcagtgagg actgatacca cttctctcag agcaatgtag 1800 aaattttgag ctgctctttc tttgaaaatg caaaaaagaa cattttgtaa gaatacccta 19860 tactttacat ctgagaaacc gcccacgcat gcagcatctc acgcagaatg ctgtggagtc 19920 agactcaaaa ggctgcatgc ctgtggttct gttgatacga cattctggaa aaggcacatc 19980 tagggaagac aagggattgg tggttgccag aggctgcttc ctgattgtgc tgagacttac 20040 agacacaact ctgtgtgtgt caaaatttga aaaaccctac actaaaaatg atgagtttat 20100 tttactgtat ctttatgctt taattttcaa aaatgaaaag gaaagaaaaa atgcttgtag 20160 catcactatt ctcccccaaa accccctgca aaaaaaaata catatatata cacatatata 20220 tgtatttttt ttttttaaga gatagtctcc ctctgtcgcc caggctgtag tgcagtggta 20280 cgatcaggtg cacaccacca cacctggcta atttttaaaa atgttttgta gggacagggt 20340 ctccctgtgt tgcccaggat gggcttgaac tcctggcctc aagtgttcct cctgtctcag 20400 cctcccgaag tggttacatg cgcctataca tgtgttaaaa ttggtagaac tgaggctggg 20460 tgcagtggct cacgcctata atcccaccgc tttgggaggc cgaggcaggc agattgcttg 20520 agctcaggag ttcgagacca gcttgggcaa cgtggtgaaa ccccgtctct accaaaaata 20580 caaaaattag ctgggcatgg tggctcacac ctatgtagtc ccagctactt gggaggctga 20640 ggttggagga ttgctggagc ctggaaggca gaggttgcag tgagccaaga tcacaccact 20700 gtactccagc ctgggcaaga gagggaggag acactgtctc aaaaaaaaga aaaagtaaat 20760 tgtagaactg tccacctaaa gaaaaaagtc aattttactg aatgatcaat ttttaaagca 20820 ttattatcaa aaggaagaga aacaccagcg agcctagaag catttgagca gaccctcaag 20880 agacccatag cctggtcctg tggagaggtg gtgggcgggg tggggcctgt ttgactcctg 20940 catttcatac atcctacctc ctgcatgtat tacctgttga agaaaatata tataacgtta 21000 taaaaaaaac ttaaaaactt ttttcaagac atcgtagaaa cacaagagtt gcaaatcttg 21060 gctgtgcgca gtggctcaca cgtgatccca gcactttggg aagccaaggc aggtggctca 21120 cctgaggtca ggagttctag actggccaac atggtgaaac ccatttctac tgaaaataca 21180 aaaaattagc caggtatggt ggcatattcc tgtagtccca gctactctgg aggctgaggc 21240 aggaggcttg cttgaacccg gagatggagg ttgcagtgag ccgagatggt gccactgact 21300 gcactccaac ctgggctaca gaacaaaatt ccatctcaaa aaaaaaaaaa ttgctaatct 21360 tgaagtatag ttgagagcac ataagagtcc aaatcaacag gtgactttca agcacacagc 21420 agccaccttc cccccgctga tgtgaagggt ggccggcccc ttgggaccac catctggaag 21480 gtgtctcttt tttcccttag tggagtgaca tttatataca cttaatatat ataaatttgt 21540 atacatttaa tttttttttt ttttaagaca gggtctcgct ctgttgccca ggctggagtg 21600 cagtggcgcg atctcggctc actgcaacct ccacctttag ggttcaagca gttctcatgc 21660 ctcagcctcc cgagtagctg ggattataga cgcgtgccac catacccaga taatttttgt 21720 gtttttagtg aaacgaggtt ttgccatgtt ggccaggctg ctcttgaatt cctgacctca 21780 agtgatccac ctaccttggc ctcgtaaagt gctgggatta caggcgtgag ccaccgcacc 21840 cggcctacat ttaatttttt aattttagag atgatttcta gtttattcac tctaagatca 21900 cttaatggat atctactgtg tgccaacaat tttgccttta tgttctttaa aattggcccc 21960 aactcaacag atggcctcag ctgtagggtg ggctggcagt gtgttagctc aggagactta 22020 cagagtttcc aagctccttg agtggctgtg ctacatgttt gtgtgtcatt ctaaatccat 22080 ttggtttttt aaaacggttt tcaaaagtta aattctagct tttttttgtt ttaggagata 22140 aagtccctcc aattaaacca aacgctgggg aagaatctgt catgaatctt gacaaattga 22200 gatttgctga tggaagcata agaacatcgg aactgcgact cagcatgcag aaggtaagag 22260 cctggactcg ctctggagtg agcaggaggg ctgcatacct ggccctgcac tggttttgtt 22320 ttttaaaaac tagatctagg gggatgcagg tgcagttttg tgtggatgta ctgggaggtg 22380 gtggagtctg ggcttttcat gtacccgtca cacaagtcgt gtgtgttgta cccagtaggt 22440 aattgttcaa ccccacccct cccgcttttt ggagctccca gtctctgcta ctccactcca 22500 tgtgtccatg tgtactcacc attcagcttc cacttccaag tgagaatgtg tgacacttga 22560 ccttctgagt cacttcactt aggatagtga cctcctgttc catccgtgtg gctgcagaag 22620 acatgattgc gttctttttt tatggccgag tagtatttca tggtatatat gtaccacatt 22680 ttcttcatct ggtcatccgt tgatgggcac ttaggttgat tccatgactt tgctgttgtg 22740 actagtgctg cgataaatac ataaggctgc accagtatct ggaggtaaac agcggtagga 22800 cgtactcctc accgtatcaa gaatatgaaa gagaccagga ggcctgaact atacagaagt 22860 gcacttcttt tccacataga aggtcggcag actagggcag aattagtgac tgcttggcat 22920 ccaggacagc cttctgtggt tcactcgtgt gtgcttgggt gtgacctccg tgacctgacc 22980 attgctggct gtcatggatg agtcacagcg tggaggagag ggaactgcag gaccgctgga 23040 gaagctcagt ccccagcagg gcagctttct cttagaggtt tcctggagtt caacacaaca 23100 ctggtgctta catctcaggc ccggatatcg atcatgtcat catgcctggc ttccagcagc 23160 tcggaaatgt ctttaagctg gacttgttgc tgcctctaaa tatactcgga agagagggag 23220 agtgggcggc aggtggaaac tgatctgtgg cctgtgctgc tgggagtcgg tccagtagga 23280 ccatctgtga tgatggaaat gttccgagtg tttgccatcc agtatggcag ccattagcca 23340 tggggccgtg gagaacctga tacatggtca gtctaagaaa ctgaattcct ctaataacaa 23400 ggattcttgt ccatgaatga gatctcttgc ctactatttg caagaatttc tgtgtatttt 23460 ctaaaaagtc cattgcttta gtcctattct gaaataggtg tctaacacct atgtggtgtt 23520 agacacagga gaacaggttc cctgctgaca ttttcagagg cctgtgccct tcagtcttca 23580 agtgaagctg ggcttcaggg aggctttgtg gaatggtgag aagaacagtg tgactaaggc 23640 acagaaggct gagtgatgcc ctgcagtact attgtagggt tggaggccag ctgggaaaga 23700 aagaaccatt gcattagaga atgggaacat gccttcggat atagaaatgg caaatccatg 23760 agatagttta aagtgagaat actagaagca ttcccaccaa acatggtgtg tcttggtgcc 23820 tgctgtatcc caggctccac gagatgctgg agtcagcact gaacaaacag agtttcctct 23880 gctcgtggaa atgcgttccg ttggggaagg gatttttctg ctgagtctgg ctattcatag 23940 taagaagcaa aaaaaagaaa tgaggtaaat ttttgggaac aggcccccaa atgtggccca 24000 aaactgacca taaacaaaat ctctgcagca ctgtgacatg ctcttgatgg ccatgacgcc 24060 cacgctagaa ggctgttggt ttaccagaat gagggcaagg gacacctggc ccacccaggg 24120 tggaaaaccg cttaaggcat tcttaaacca caaacaatag catgagcgat ctgtgcctta 24180 aggacatgtt cctgctgcag ataactagcc agagcccata cctttgtttc ccgtaaggaa 24240 tacttttagt aaatcttatg actggcttgc tctcaataaa tatgtgggta aatctctgtt 24300 caaggctctc aactctgaag gctgtgagac ccctgatttc ccactccaca cactatattt 24360 ctgtgtgtgt gtctttaatt cctctagcgc cgctgggtta gggtctccat gaccgagctg 24420 gtctcggcaa atggcgccca tatatggggg ctcgaatcta ggtcgaaggg tcaccggagc 24480 gatggagaac atggaactaa gctggaggac acctgagtac tcttacgcag tccctgtggt 24540 gagtaagacg ggtagctcag aagcatcagg gtaacaatgg gacacgtgtg ggctctggtt 24600 cgttccacct tggaaccttt tcatgctaat aataaggggg aaggagagta taacgaagta 24660 acagaagaaa ttacagagca ggtttgtttg ccaactaaag ctaaagcggc aaaggaggga 24720 gaggttcatc cctacccttc tgcaccccct ccttattttg aagaaaaaga gtggcctgac 24780 cctccagatc tttcttttcc agaggacagt gggcgaaaag tagttgctcc agtgactgtc 24840 cgagcagcac cttgagcgac ccctctcagt tctattcagg caggaattca gcaagctaga 24900 tgagagggtg atttagaggc ttggcagttc cctgttagaa tacaccctgc agatcaacag 24960 ggaaatatta cagctacatt tgagcctttt ccctttaaat tacttaagga gtttaaacac 25020 gctatcaatc agtatggacc aggttctcct tttgtaacag gactgttaaa gaatgttgtt 25080 gtttccagtc ggatgattcc tactgacttg gacgctctta cttgagcttg tctaactcct 25140 gctcagttct tacaatttaa aacttggtgg gcagttgtag cttccattca gcctacttgc 25200 agcgcccagg cccaacctca aattaatata actgcagacc agcttttggg ggtcggcagc 25260 tgggctggtt tagatgcaca agtggtcgtg caggatgatg ccgtagaaca gcttagagga 25320 gtgtgcatta gagcttggga aaaaatcact tcaggtagag aacaataccc ttcctttagt 25380 gcagtaaaac agggaccaaa agaaccgtac gctgatttta tagctcggtt acaggagtct 25440 cttaaaaagg tgatcgcaga ttcggctgct caggatatag tgctgcagtt atcagctttc 25500 gacaatgcta atcccgattg ccaggctgct ctgtgaccta tcagagggaa agcacactta 25560 gttgattata tcaaggcctg tgatggtatc agaggtactc tgcataaagc gactttgttg 25620 gcacaagcta tggcaggact gaaagtgagt aaaggaaata ctccgtttcc tggaggttgt 25680 tttaactgtg ggaagcatgg tcatactaaa aaagaatgta gaaaaaaatc agtgagtgag 25740 ggtgccagat gggggaagaa agaaaactgc taagcctgaa atatgtccaa aatgtaaaaa 25800 aggaaaactt tgggctaatc agtgtcactc taagtttgat aaagatggga accccatttt 25860 gggaaatgcc atgaggggcc cgtcctgggt cccgttccaa accggggcat ttccggctca 25920 ggccactccc tcacccctgt acaatgtctg ttccccgcca cagccggtag tgccacagta 25980 gattatgct gcacagtagc tgtgagtctt ctgcttgggg aacccccacc aaaggtccca 26040 gcaggagtct gtggaccctt gcaagcaggg acgataggat tactcctagg caggtctagt 26100 ttaagtttaa aagggtacaa atacatagag gagtcattga ttcagattac aatgaggaaa 26160 ttcaaattgt tacatctact tctgttccct ggaaagcaga gccaggagag tgcatagcac 26220 agctcctgat tgtgccatat gtggaaatgg ggaaaagtga aattaaatga acaaaaggat 26280 atggaagcac agataaacaa ggcaaagcag cctattgggt gaatcaaatt actgataaac 26340 gtcctacctg tgaaataact attcagggaa agaaatttaa aggtttggta gatgcaggag 26400 cggacatttc aatcatttct ctacagcact ggccgtctgt gtggccaatt caacctgctc 26460 aatttaacat agttggagtt ggtaaagccc ctgaagtata tcaaagtagt tatatttcca 26520 ttgtgaaggg cccaatggac aacctgggac tattcaatga taacttctgt acctataaat 26580 ttatggggaa gagatttatt acaacgatgg ggagcacaag ttctaattcc agagcaatta 26640 tatagccctc aaagtcaaca tatgcatgaa atggggtatg tccctggtat gggactagga 26700 aaaaatttgc agggtttgaa agaagtgctt caagtggaaa gacaaaagtt cctgccaagg 26760 tttaggatat tatttttgat ggcggccatt gttaagcttc cagaacctat acctttaaaa 26820 tggttaacat ataagccaat ttggatagaa caatggccac tgagtaaaga gaaactggag 26880 gctttagagg acttagttaa tgaacaatta gaaaagggac acatagctcc aacattttcc 26940 ccttgtaatt ctttggtttt cttaattaag aaaaaatcag gtaaatggag aatgttaact 27000 gacttaagag ccattaattc agttatacaa cctacgggga cattacagcc aggattgcct 27060 tctcctgcta tgattccaaa aaattggcct ttaatagtca tagatttaaa agactgtttc 27120 tttactatcc ccttagctga gcaagactgt gaacggtttg catttacaat tcgtgcagta 27180 aacaacctgc agcctgctaa gcgttttcat tggaaagtgt tgcgacaagg catgttaaac 27240 agtccaacga tttgccagac ttgtgtaggg caagcaattg aacctactca taaaacattt 27300 tcacagtgtt acattatca ttatatggat gatatacttt gtgctgcccc tactcgagaa 27360 atattactcc aatgttatga tcatttgcaa aattcaattt ctggtgctgg tttaattata 27420 gctcctaaca aaatccagat tactactctt tactcctact tgggaacctt agtaaatgac 27480 attaccattg tgctacagaa agtagccata cgtagggatc aattgaaaac attaaatgag 27540 actttcaaaa attactgggg acattaactg gatacaccct gctctaggca ttcctaccta 27600 tgccatgagt aatctatttt ctatccttag aggagatcct agtctcacta gccctcggct 27660 tcctacctat gccatgagta atctattttc tatccttaga ggagatccta gtctcactag 27720 ccctcggcta tgccatgagt aatctatttt ctatccttag aggagatcct ggtctcacta 27780 gccctcagca attaacaaaa gaaactaagg gagtgcctca gctgatcgaa aagcaagtcc 27840 ataaagccca aataaataga acagatccag agaagactcg agctgcagct gatcgaaaag 27900 caaggccata aagcccaaat aaatggaaca gatccagaaa agactcaagc tgcagccgat 27960 cgaaaagcaa ggccataaag cccaaataaa tagaatcaat gcagagaaga ctcaagctgc 28020 agctgatcga aaagcaaggc cataaagccc aagtaaatag aatcgatcca gagaagactc 28080 gagctgcagc tgatcgaaaa gcaaggccat aaagcccaag taaatagaat cgatccagag 28140 aagacttgag ctgcagctga tcgaaacgca aggccgtaaa gcccaaatca atagaatcga 28200 tccagagaag actcgagctg cggctgatca aaaagcaagg ccgtaaagct caaatcaata 28260 gaatagatcc agagaagact tgagctgcag ctgatcgaaa agcaaggcca taaagcccaa 28320 gtaaatagaa tagttccaga gaaggctcta gatttgctaa tttttccaac tccacattca 28380 cctactggtg ttattgttca agagcaagat tttgtagaat ggctttttct tccacatact 28440 aattcatgga ctctagagaa ggctctagat ttgctaattt ttccaactcc acattcagct 28500 actggtgtta ttgttcaaga gcaagatctt gtagaatggc tttttcttcc acatactaat 28560 tcatggactc taactcctta tttggatcaa attgctacta tgataggaaa tgggagaact 28620 tggattgtta aattacatgg atatgatcct gaaaaaattg tccctctcac gaaggcacaa 28680 atacagcaag attttttaaa tagtcttact tggcaaaccc atttagctga ctttgtgggt 28740 attcttgata ttttcctaaa atgaaactgt ttcaattttt gaaattaact aattggattc 28800 tccctaaaat aactaaattt aaaccaaatg aagatgcaga gaatgttttt acagatgggt 28860 ctagtaatgg taaagcttct tactctggct cgaaaggtaa agttttctag acaccctatg 28920 cttcagctca aaaagtggag cttgtagctg taattgaggt attgactgct tttaatatgc 28980 ttattaatgt gatttctggt tcttcataca tggttcattc cacacaatta attgaaaatg 29040 ctgagttacg atttcataca gatgaacaac tgatgacttt atttatgcaa ttgcaaacag 29100 cagttaggag tagaatgcac cctttttaca tcactcacat tagggctcat acacctcttc 29160 caggaccttt gactgcaggg aatcaaatgg ctgatcgcct agttgctact gcaatatcta 29220 atgctagaca ctttcacaat ttaacccgtg ttaatgcctc tggtctcaaa cgcagataca 29280 gcagtacccg gaaagaagct aaagctatta tccagcgatg cccaacttgc caaatggtac 29340 attcctcatc ttttacagga ggagttaatc ctcgaagatt ggaacctaat tctctttggg 29400 aaatggatgt cacacatgtt ccctcgtttg ggagactagc ttatgtacat gcatgtgtgg 29460 acaccttttc actttgggct gcatgccaat caggagagtc ttctgcctac gttaaacgtc 29520 accttttgca gtgttttgtg gtaattggca ttctagcttc tattaaaaca gataacgccc 29580 caggctatac tagccaagct ctagctacat ttttctctat acggaatatt aaacacatta 29640 ctggtatccc ttataattct caaggacaag ccatagtgga aagaatgaat ctttccctga 29700 aacagcagct gcaaaagtaa aagggggaaa acagggacta cgggacaccc catatgcaat 29760 tgaatcgcat tatttaaatt ttttgagcct gcctaaaggc cagatcttat cagcagctga 29820 acagcatcta cagaaaccag ctgcaaagac agaagcagaa caactggttt ggtggagaga 29880 cctgataata aaaagttggg aaataggtaa aataataact tggggtagag gttatgctta 29940 tgtttctcca ggacggaacc atcaagacac ctgaaacctt atgagctgac actgaggaag 30000 agattctggg aggatcccga ggacccctcg gttgcagcca tgtcaagact gatgctgagg 30060 aggaccccaa ctgtcatgag caacacccgt cgaacacagc cacccacctg aggtcagatc 30120 aagaagctgt cacagatggc agaagaaaac ctgaggaaag cgggacaacc agtcacaatg 30180 agtaatttaa tggtagctgt gatagcagtg atcaccattg ccatgagtat tccttcaaca 30240 agggctgaca cagagaacaa ttatacttat tgggcatatt tatcaatctt ggctggcaag 30300 aatgcctgga tgtaatcact ctatgatgca gttacacatg ctttctgatc tcagtattta 30360 ccatgataaa tctgcttcta taattgaggc ataccgccct caaaaaccta tctgtaaaca 30420 ggattggacc cagtcagaga aaatgaacgt acatgtttgg gaagattgca ttgcagaaca 30480 ggcaggggtg ctggcaacga ttcctatgga atcattattg attgatctcc taaggggatg 30540 tttagcttga attgcacctc tctgtctgcg tgccacggcc acactatgtt cagctggtct 30600 gaacaaaatt gtcagatggt agaaatgata agaaatacgg caagggttcc tattatctgg 30660 aaccatggcg gtgtagtggc acctcaacct caaatggtat ggccctttgt aggagctaaa 30720 cataaggatt tgtggaaact gttaatagct cttaataaga tcaaaatttg ggaaagaata 30780 aaaaagcatc tagaaggaca ccgtacaaac ttgtctttgg atattgcaaa attgaaagaa 30840 cagatatttg aagcatccca gcacacctga ccttaatgcc aggaactgga gtgcttgaag 30900 gagctgcaga cagattagca gctggtaatc cattaaaatg gataaaaaca cttggaagct 30960 ctgtgatttc aatgaagatt gtgcttttaa tctgtgttat ttgtatagtc tgcagatgtg 31020 gatcctgact ctgcaagaag tagctcaccg taacaaagct gcctttgctt ttatcacttt 31080 gcaaaacaaa taaaggggac acgttgggaa caggcccccc aatgtggcca taaactgtcc 31140 ccaaaactgg ccataaacaa aatctctgca gcactctgac atgctcttga tggccatgat 31200 gcccacgctg gaaggttgtc ggtttactgg aatgagggca agggacacct ggccccaccca 31260 gggtggaaaa ccgtttaagg cattcttaaa ccacaaacaa tagcatgagc gatctgtgcc 31320 ttaaggacat tttcctgctg cagataacta gccagagccc atccttttgt ttcccgtaag 31380 gaatactttt agtaaatctt atcactggct tgctgtcaat aaatatgtgg gtaaatctct 31440 gttcaaggct ctcagctctg aaggctgtaa gacccctgat ttcccacttc acatgctgta 31500 tttctgtgtg tgtgtcttta attcctccag caccgctggg ttagggtctc cacaaccgag 31560 ctggtctcag cagtaaatat tgaaaaggaa tagatacaat tgtcattatt tacagataga 31620 atttccaaac aattcccagg gaataaactg aaaaactaac agaaacaaaa caagaatgta 31680 gtaaggtgtc tggataagag atttgtatct aaaaatcagt agctttacaa tgtgccagca 31740 gtagtctgct cagacatcag taaatatctc attcacattt caaaaaaaat tgaaaatgcc 31800 ctgaaataat gtaaccagaa atacgaaaaa aaggatatga aaacatgtgg ctgctaatgg 31860 acatgaaaga atgtaatact agattctgag atgcaatgtt tttcatttgt tcttcctgaa 31920 aaaccattag gttgatgtgc attacagtgt tacgattatg tatgagtcta aggaaaatca 31980 gatgaaatgt ccaaattaaa ccatgaaggt gcattggtag aggaagagaa aattagggtc 32040 agtggagcaa agcacagtta gagggagaag caaggaggag gagggatcac tgaggtggtg 32100 cctgtcccac aggaagcaaa agctgacgcc tagttcccag catacctaca gtaaacttca 32160 ggtccactgc atagcacgtc tctcatcaca gtaaaactat gaaggaactc agtgtacaag 32220 gagcttctac aagataggca gaagacagta gccagatggg ccaagggccc cagccaccca 32280 cgcccctccc tctccttgaa gacgtgcagc tccaacacca ccatcaccag ggctctgctc 32340 agctcctcct agtgtgtatc accacagggc tgctggcttg tgtcacgttc accaccagac 32400 cccgcgtcag gagtcccgcc aggggtgtgg agaggcagcg ctgcctggtt ggccgtggag 32460 ctgtatggaa catggtgcct cacaggcagt ctgcttggag tcctggaccc tggctgtatc 32520 ctgctggaaa ggatgtgtgt gggtctaaga tgtgtatgta atagaaacat ttatttattc 32580 agaagcttta gtcaaaactt catttttaag tttggagtaa taaactcata gtctgaattt 32640 cctaattttt ctgtttaatt tatgtaactt ttaagtgaaa tgcaaaacaa caggtctaaa 32700 agttaagcag ttcttggtat ggctgcttct atggattaaa agtttacaaa taatattttg 32760 tgccacagtc aatgcaaaat catgctgccg tgttccgtgt gggaagcgtg ttgcaagaag 32820 gttgtgggaa aatcagcaag ctctatggag acctaaagca cctgaagacg ttcgaccggg 32880 gtgagcagac agtgggctct gtgcacactg ttgggccctt ccttctgcag ggtgggctgg 32940 tgtctgtccc gtcagtgctg acttagttcc gtgcttgctg tctggatggg tcctggccca 33000 cagctataaa gccacaacca gtgactccat ggactagcag gcccaggctg acagctcgga 33060 gggcccatgt gactgggtcc cgcctgcccc tgatggaact ttttgtgtcc ccaggaatgg 33120 tctggaacac ggacctggtg gagaccctgg agctgcagaa cctgatgctg tgtgcgctgc 33180 agaccatcta cggagcagag gcacggaagg agtcacgggg cgcgcatgcc agggaagact 33240 acaaggtggg ccttctcacc acgcccacct gcacctgcct tttcctgcca cctggtggga 33300 ctcagcccca cccctgcatt ttctctgcat tttctttcgt tgccccaaaa gtaaatccaa 33360 aaaatgcctt tttcccccct ggtaactttg atccctgggt tctcgccatc ttctggatca 33420 ctgtgacctt ttccttgctt tgggtcggca tccactgatg ccagcagtgg catctccaag 33480 ccaatgtgct ttgctgttag aaggccaagg ttagaagtgc agctagagtg gcaagaccag 33540 gaaataaatg ccagtttatt aaataacgag taagccatca tttcaagcct gccctgtgga 33600 ggaaatgcca gtttattaaa taacgagtaa gccaccgttt caaacctgcc ctgtggagga 33660 aatgccagtt tattaaataa cgagtaagcc accgtttcaa acctgccctg tggaggaaat 33720 gccagtttat taaataacga gtaagccacc gtttcaagcc tgccctgtgg aggaaatgcc 33780 agtttattaa ataacgagta agccaccgtt tcagacctgc cctgtggtgg aaatgccagt 33840 ttattaaata acgagtaagc cactgtttca aacctgccct gtggaggaaa tgccagttta 33900 ttaacgagta agtcactgtt tcaaacctgt cctgtggagg aaatgccagt ttattaacga 33960 ataagccacc gtttcaaacc tgccctgtga aggaaatgcc agtttattaa ataaggagta 34020 agccaccgtt tcaagcctgc cctgtggagg aaatgcccgt ttattaaata acgagtaagc 34080 caccgtttca agcctgccct gtggaggaaa tgcccgttta ttaaataagg agtaagccac 34140 catttcaagc ctgccctgtg gaggaaatgc cagtttatta aataacgagt aagccaccgt 34200 ttcaagcctg ccctgtggag gaaatgccag tttattaaat aacgagtaag ccaccatttc 34260 aaacctgccc tgtggaggaa atgccagttt attaacgagt aagtcaccgt ttcaaacctg 34320 ccctgtggag gaaatgccag tttagtaacg agtaagtcac cgttttaagc ctgtcctgtg 34380 gaggaaatgc cagtttatta acgagtaagc caccgtttca aacctgccct gtggaggaag 34440 tgccagttta ttaaataatg agtaagtcac cgtttcaagc ctgccctgtg gaggaaatgc 34500 cagtttatta aataacgagt aagtcaccgt ttcatacctg ccctgtggag gaaatgccag 34560 tttattaacg agtaagccac cgtttcagac ctgccctgtg gaggaaaatg ccagtttatt 34620 aacaagtaag tcactgtttc agacctgccc tgtggatgaa atgccagttt attaatgagt 34680 aagtcaccat ttcagacctg cgctgtggag gaaatgccag tttattaaat aaggagtaag 34740 ccaccatttc agacctgtcc tgtggtttgg aaaaggtatt atagagcctg ccctgtggtc 34800 acttgttctt cagatgaact gatttttgtg cagagcgcac gtgttggatt ctgcctggta 34860 agagtttttc ccatatgata gcaaaaaacg acggaaaggg aagcttggga tgcaaatgca 34920 agttcaggat aaaccacatc agcaaaagga caaaggctcc acaaggcagg cgcacaggct 34980 ggttcaggac cgtgtgtagg cggctggtgg cagcctttcc agtcagctga acatggtgaa 35040 tgggaaaatc atttttattc accatgaaat tttactgatt taccctccac tagaatatgc 35100 tgatggctgt gatcactgct cagaacttgc tcttgtctcc ttgtatgtat taagagtttc 35160 ctgcaaagta tatgaatccg tgtttgccag aatacagaat aatagtaaat ttattatttt 35220 tatttttatt tttttgagaa ggaatctcac tgtcccccag gctggagtgc agtggcgtga 35280 tctcagctca ctgcaacctc tgcctcccgg gttcaagtga ttctcctgcc tcagcctccc 35340 aagcagctgg gattacagac acactgccac catgcctaat tttgtatttt cagtagagat 35400 ggggtttcac catattggcc gggctggtct cgaactcctg acctcaagtg aaccacccac 35460 ctcggcctcc caaagtgcta gggttacagg catgagccac tgtgcctggc cagtgcataa 35520 atttagttgg tcacagtcag ttttaattag aatagaagcc aggtgcagtg cctcacacct 35580 gtaatcccag catttgggag gctgaggcag gccaatgact tgagccccag agttgtagac 35640 cagcctgggc aacatggcga aacgtgtctc tacaaaaaca taaaaaatga gccaggtgtg 35700 gtggtagcac aggaggctga ggcaggagga ttgattgagc ctgggaagtc aaggctccgg 35760 caagctgtga tcacaccact gcactccagc ccaggtgaca tagccagacc ctgtctcaaa 35820 aaaaaaattt ttttttaata aaaacaggct gaaagaaaag attgaggtag tctcccagca 35880 catggagcaa aaagacaaag tatttgataa actcttaggt acataaagga tgcttaaggg 35940 aacatgcgga catggattac tgtggactca ctgctggctg cacaccccct ggccagccat 36000 gcggcctccg tgggttctga acatgttgat ggtgccaacc tcctgggctg aagtggaaat 36060 ggaatgagtt ctagggcatc tgtctcttag atcatgttaa tgtctgctgt gttttttctg 36120 tattgctctg ttagagtaat aagaaacgtg atggtgtttc tggcctcagg tgcggattga 36180 tgagtacgat tactccaagc ccatccaggg gcaacagaag aagccctttg aggagcactg 36240 gaggaagcac accctgtcct atgtggacgt tggcactggg aaggtcagtg tggagctcgt 36300 tctcaccaca gcccagcacc cacacggccc cgcccaggcc tgcgggctgg ccttgctgat 36360 ggtgaacggg gaagagcagg ccagatttaa atcaactccc gacagattcg aggcaccgct 36420 gaaaaaggca ctccgacagc agtcgggctt cgggctggaa acagaatcca gttcctgcag 36480 gtggttcaga ggagccttaa ggaagggttg ctccgtggtg tgcgccagat ggacgtcact 36540 gggcaggagc aagtgtccaa ggcctggtgg taggggagga gatgatgatt gtggacctag 36600 cgagaaagca tctgtggggt ggggacacac agccattacc agaaaccagt cccacgttaa 36660 gggagcccag aagagacacc tcccctctcc tccccacgggc tgggccaact ggaagcgtct 36720 gcaggggagc tgaggggatg tggtgcagcc tttagcatcc cctgggcact gagcaagcag 36780 agaagggcag aaatggaggt ggggttgggg tgagcagtgt cctgggaaca gccagccgag 36840 ggtgtggtag ggggtcgcag cctttttcca cacacggtga gcagcgtcct gggaacagcc 36900 agccgagggt gtggtagggg ggtcgcagcc ttgttccaca caagcacagt tcacctgtgt 36960 ggcatttccg ctgggcatta agattcagaa ataatgaaga tagaaagctt ttaccccttaa 37020 acttttcata gcttgtaaga gtcgtataat cacttagctg tgtctgtgga agttaccttt 37080 ggactctcac tatcatctag tgtgtctgtg attcaggcag taggtcattt tcaggacttc 37140 tcatgaaggc catttcttga tagtgtatag aaatcacact tcactcgctt agcacaagtc 37200 tatttttaat gtttccgggt tcgggttttt tggttttttt ttgtttttct gagatagagt 37260 ctcatctctg ttgcccaggc tgatatgtgt tggcttgatc tcgtctcact gcagcctcaa 37320 cctcccccag gctcaggtga tcctcccacc tcagcttcct gggcacatgg caccatgcct 37380 ggctaatttt tatatgtttt gtagagatgg ggtttctcca cgttgcccag gctagtcttg 37440 aactcctgcg ctcaagtgat ccacctgtct tggcctccca acaggcatga accaacaccc 37500 ccagccagtt ttagtgattt ttcaagaaat acatactcat tttagaaagt acaaagagat 37560 tgaaataaga gctcactaac cagagacgac ccattatggt ttaaatttct ttgtatatgt 37620 gcctaccttt tcctgtgtgt gcatatttaa tacacagctt gagtattcct tctctgaaat 37680 ccttgggacc agaagcgttg tggatttcag gcttgttcag actttggaat atttgcatta 37740 tacttactgt ctgagcatcc ctaatcggaa gatctgactc catagcacat ttcttttgaa 37800 tgtcatgctg gtgctcagaa agtttcagat tttagagaat ttcagatttt tggattaggg 37860 atgttcaacc tatataaata tttactttga agtagaaaaa ctggaagtag atggtttaaa 37920 cataaagtgt cttggtatag acaagggttc tcccacttta catgatggga gcatttttgt 37980 aaagcactga gaatcttaaa gttcacatgc cataaatcta cttttatagt taaaattttt 38040 caaaaggaac acaagagatg gctttttgta catttttgtg cttaacttac cactgactct 38100 tcttttcaag gtcactctgg aatatagacc cgtgatcgac aaaactttga acgaggctga 38160 ctgtgccacc gtcccgccag ccattcgctc ctactgatga gacaagatgt ggtgatgaca 38220 gaatcagctt ttgtaattat gtataatagc tcatgcatgt gtccatgtca taactgtctt 38280 catacgcttc tgcactctgg ggaagaagga gtacattgaa gggagattgg cacctagtgg 38340 ctgggagctt gccaggaacc cagtggccag ggagcgtggc acttaccttt gtcccttgct 38400 tcattcttgt gagatgataa aactgggcac agctcttaaa taaaatataa atgaacaaac 38460 tttcttttat ttccaaatcc atttgaaata ttttactgtt gtgactttag tcatatttgt 38520 tgacctaaaa atcaaatgta atctttgtat tgtgttacat caaaatccag atattttgta 38580 tagtttcttt tttctttttc ttttcttttt tttttttgag acaggatcgg tgcagtagta 38640 caatcacagc tcactgcagc ctcaaactcc tgggcagctc aggtgatctt cctgactcag 38700 ccttctgagt agttggggct acaggtgtgc accaccatgc ccagctcatt tattttgtaa 38760 ttgtagggac agggtctcac tgtgttgcct aggctggtct caagtgatcc tccctccttg 38820 gcctcccaag gtgctggaat tataggtgtg aacaaaccac catgcctggc cttgtagttt 38880 atttctaagt tcaaattaat gttggtgcct gagaacgaat ggagaaaact aacatttcct 38940 cctttttctt ggcagacggt tactaggtg agtgtgtcct tgattattca aatcaggggt 39000 ccccaatccc caggccacag attgttacca gtccatggcc tgttaggaac caggccgcac 39060 agtaggaggt gagcagtggg ccggtgagct actgtgtgag ctccaccccc tgccagagca 39120 ttactgtgaa ctccgccacc tgtcagagca ttactgtgtg agctccgccc cctgccagag 39180 cattactctg tgaacaccac ctgtcagagc attaccgtgt gagctccgcc tgctgccaga 39240 gcattactct gtgagctccg cctcctgtca gagcgttacc gtgagctccg ccccctgttg 39300 gagcattact gtgtgagctc cgcgccctgc cagagcatta ctgtgtgagc tccgcccctg 39360 ccagagcatt accttgtgag ctccaccccc tgccagagca ttactgggtg agctccaccc 39420 cctgccagag cattactggg tgagctccac cccctgccag agcattactg tgagctccgc 39480 ctccccgacag cattaccgtg tgagctccgc gccctgccag agcattactg tgtgagctcc 39540 gcccctgcca gagcattacc ttgtgagctc caccccctgc cagagcatta ctgggtgagc 39600 tccaccccct gccagagcat tactgtgtga gctccgcctc ctgccatagc attactgtga 39660 gctccgcctc ccgacagcat taccgtgtga gctccgcccc cgttagagca ttactgtgtg 39720 agctccaccc cctgccagag cattactggg tgagctccgc ccctgccaga gcattacctt 39780 gtgagctcca ccccctgcca gagcattact gggtgagctc caccccctgc cagagcatta 39840 ctgtgtgagc tccgcctcct gccatagcat tactgtgagc tccgcctccc gacagcatta 39900 ccgtgtgagc tccgcccccg ttagagcatt actgtgtgag ctccaccccc tgccagagca 39960 ttactgggtg agctccaccc cctgccagag cattaccgtg agctccgccc cctgtcagag 40020 cattactgtg agctccgccc catgttagag cattactatg tgagctccgc cccctgccag 40080 agcattactg tgagctccgc ccccctgtca gagcattagt gtgtgagctc ctcctcctgt 40140 cagagcctta ccgtgagctc cgccccctgc cagagcatta ccgtttgagc tccgcctcct 40200 gtcagagcat tactgtgagc tccatctctt gtcagagcat taccgtgtga gctccacccc 40260 ctgccagagc attaccgtgt gagctccgcc ccctgccaga gcattactgt gagctccttc 40320 tcctctcaga gccttgccgt gagctccgcc ccccgccaga gcattaccgt gtgagctccg 40380 cctcctgcca gagcattacc gtgtgagctc cgcctcccgc cagagcatta ccgtgtgagc 40440 tccgcctcct gtcacagcat taccgtgtga gctccgcctc ccgccagagc attaccgtgt 40500 gagctccgcc tcccgtccga gcattaccgt gtgagctccg ccccccgcca cagcattacc 40560 gtgtgagctc cgcctcccgt cacagcatta ccgtgtgagc tccgccccct gccagagcat 40620 tactgtgagc tccttctcct ctcagagcct tgccgtgagc tccgccctcc gccagagcat 40680 taccgtgtga gctccgcctc ccgtccgagc attaccgtgt gagctccgcc tcccgtccga 40740 gcattaccgt gtgagctccg cctcccgtca cagcattacc gtgtgagctc cgcctcccgc 40800 cagagcatta ccgtgtgagc tccgcctccc gccacagcat taccgtgtga gctccgcctc 40860 ccgtcagagc attaccgtgt gagctccgcc tcccgtccga gcattaccgt gtgagctccg 40920 cctcccgtca cagcattacc gtgtgagctc cgcctcccgt cagagcatta ccgtgtgagc 40980 tccgcctccc gtccgagcat taccgtgtga gctccgcctc ccgtcacagc attaccgtgt 41040 gagctccgcc tcccgccaga gcattaccgt gtgagctccg ccccccgcca gagcattacc 41100 gtgtgagctc cgccccccgc cagagcatta ccgtgtgagc tccgcccccc gccacagcat 41160 taccgtgtga gctccgcctc ccgtcacagc attaccgtgt gagctccgcc tcccgacaga 41220 gcattaccgt gtgagctccg cctcctgcca gagcattacc gtgtgagctc cgcctcctgt 41280 cacagcatta ccgtgtgagc tccgcctccc gccagagcat taccgtgtga gctccgcctc 41340 ccgtcacagc attaccgtgt gagctccgcc tcccgccaga gcattaccgt gtgagctccg 41400 cctcccgtcc gagcattacc gtgtgagctc cgcctcccgc cagagcatta ccgtgtgagc 41460 tccgcccccc gccacagcat taccgtgtga gctccgcctc ccgtccgagc attaccgtgt 41520 gagctccgcc tcccgtccga gcattaccgt gtgagctccg cctcccgcca gagcattacc 41580 gtgtgagctc cgcctcccgt ccgagcatta ccgtgtgagc tccgcctccc gtcacagcat 41640 taccgtgtga gctccgcctc ccgtcacagc attaccgtgt gagctccgcc tcccgtcaga 41700 gcattaccgt gtgagctccg cctcccgcca gagcattacc gtgtgagctc cgcctcccgc 41760 cagagcatta ccgtgtgagc tccgcctccc cacagagcat taccgtgtga gctccgcctc 41820 ccggcagagc attaccgtgt gagctccgcc tcccgtcaga gcattaccgt gtgagctccg 41880 cctcccgtca gagcattacc gtgtgagctc cgcctcccgt cagagcatta ccgtgtgagc 41940 tccgcctccc gtcagagcat taccgtgtga gctccgcctc cgtcagagca ttaccgtgtg 42000 agctccgcct cccgccagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 42060 tgtgagctcc gcctcccgcc agagcattac cgtgtgagct ccgcctcccg tcagagcatt 42120 accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg agctccgcct cccgacagag 42180 cattaccgtg tgagctccgc ctcccgccag agcattaccg tgtgagctcc gcctcccgtc 42240 acagcattac cgtgtgagct ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc 42300 cgtcacagca ttaccgtgtg agctccgcct cccgtcacag cattaccgtg tgagctccgc 42360 ctcccgtcac agcattaccg tgtgagctcc gcctcccgtc agagcattac cgtgtgagct 42420 ccgcctcccg tcagagcatt accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg 42480 agctccgcct cccgacagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 42540 tgtgagctcc gcctcccgac agagcattac cgtgtgagct ccgcctcctg ccagagcatt 42600 accgtgtgag ctccgcctcc cgccagagca ttaccgtgtg agctccgcct cccgtccgag 42660 cattaccgtg tgagctccgc ctcccgccag agcattaccg tgtgagctcc gccccccgcc 42720 acagcattac cgtgtgagct ccgcctcccg tccgagcatt accgtgtgag ctccgcctcc 42780 cgtcagagca ttaccgtgtg agctccgcct cccgccagag cattaccgtg tgagctccgc 42840 ctcccgtccg agcattaccg tgtgagctcc gcctcccgtc acagcattac cgtgtgagct 42900 ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc cgtcacagca ttaccgtgtg 42960 agctccgcct cccgtcagag cattaccgtg tgagctccgc ctcccgtcac agcattaccg 43020 tgtgagctcc gcctcccgtc acagcattac cgtgtgagct ccgcctcccc gcagagcatt 43080 accgtgtgag ctccgcctcc cggcagagca ttaccgtgtg agctccgcct cccggcagag 43140 cattaccgtg tgagctccgc ctcccggcag agcattaccg tgtgagctcc gcctcccgtc 43200 agagcattac cgtgtgagct ccgcctcccg gcagagcatt accgtgtgag ctccgcctcc 43260 cgccagagca ttaccgtgtg agctccgcct cccgtcacag cattaccgtg tgagctccgc 43320 ctcccgtcag agcattaccg tgtgagctcc gcctcccgcc agagcattac cgtgtgagct 43380 ccgcctcccg tcacagcatt accgtgtgag ctccgcctcc ccacagagca ttaccgtgtg 43440 agctccgcct cccgtcacag cattaccgtg tgagctccgc ctccccggcag agcattaccg 43500 tgtgagctcc gcctcccggc agagcattac cgtgtgagct ccgccccccg ccagagcatt 43560 accgtgtgag ctccgccccc cgacagagca ttaccgtgtg agctccgccc tcccgtcaga 43620 gcattaccgt gtgagctccg cctcccgtca gagcattacc gtgtgagctc cgcctcccgt 43680 cacagcatta ccgtgtgagc tccgcctccc gtcacagcat taccgtgtga gctccgcctc 43740 ccgtcacagc attaccgtgt gagctccgcc tcccgtcaca gcattaccgt gtgagctccg 43800 cctcccgtca cagcattacc gtgtgagctc cgcctcccgt cacagcatta ccgtgtgagc 43860 tccgcctccc gtcacagcat taccgtgtga gctccgcctc ccggcagagc attaccgtgt 43920 gagctccgcc tcccgtcaga gcattaccgt gtgagctccg cctcccgcca gagcattacc 43980 gtgtgagctc cgcctcccga cagagcatta ccgtgtgagc tccgcctccc gtcacagcat 44040 taccgtgtga gctccgcctc ccgacagagc attaccgtgt gagctccgcc tcccgtcaga 44100 gcattaccgt gtgagctccg cctcccggca gagcattacc gtgtgagctc cgcctcctgt 44160 ccaagcatta ctgtgtgaga tccgccccct gtcatatcat tactgggcga gctccgcctc 44220 ctgccagatc agtggtggca ttagattctc ataggagtgg aaccctgttg tgaactgcac 44280 atgcgaagga tctaggttat gcgccgctta tgaaaatcta atgctgatga tctgagatgg 44340 aaccgtttca tctccaaacc atccccacca tccccacact tgtccgtgga aaaagtgtct 44400 tccatgaaac cagtccctgg tgccaacaag gttagggacc accagtttaa ataaccaaat 44460 actaaaagaa ctggcataga agtaaatggg ctgctgcttt atttttaggc tgttcttttt 44520 agagaacaat gacaattatt tccaagtttg tcattagaaa gtaatattag gttggtgcaa 44580 aagtaattgc agtttttgcc attactttca atggtaaaag ccacgattac ttttgcacca 44640 acttaatatg ataaatttgt tccttaaagt gtatttttgg taagaaaaac cttttgtttt 44700 tccttatatt aattttttgt tttttttctt ggtagagaca gagtcttgcc atgctgccca 44760 ggctggagtg cagtgctgtg atctcggctc actgcaacct ccacctccgg ggctccagca 44820 atcctcccac ctcagcctcc caaggagctg agactacagg tgtgagccac catgcctggc 44880 taatttttat attttttaca gagacagggt ttcaccatct tgcccaggct ggtctcaaac 44940 tcctgggctc aagcagtcct cctgcctcag cctcccagag tattgggatt ataggtgtga 45000 gccactgcca gaaaaacttt tcctaagaca aggcaggttt tacattatat ttagattttt 45060 tttttaatga tgtctttttt ggcagtgcac agccagagaa caacacatca cacacaggaa 45120 acagttgtgc tcatgtgatg ggggcctcag cactaggaag gagcggacta ttggtgcacg 45180 cagcagcttg aataaatctg aaagtcacta tgctgtgtaa gagaagccaa attttaaaag 45240 tgcatactgt gtacagaggg tgtcgagaat gcctcctacg tgacggaaag cagatccgtg 45300 gttccctgca gactggcagg agcagattcc aaaggcacag gaagaagctt gtgggtagag 45360 tgtgttcatt atcttctgtg cattacacca taaaaaagct ggtcataaaa atgcgaacca 45420 aaaacaaagg tgaaactagg ataaaatttc tcacctgtgt gattggtaaa tgtgcaggtt 45480 tgacatcatg ctttgtttat gaagctgtgg gatacaagga ctctcatacg tcactgtgga 45540 atgcagaaca ttgcaacctc atggaagagg atttggcagc atctaacaaa agtatgtggc 45600 atttgccctt tgactcagca attctagact gcctcaaaaa aaactctggc aaaaaaatga 45660 aaggacttta ctcacagagt tcttttcacg cctaaatatg tttgcaacca agttcttcac 45720 tgtggcattt gtaaaactgg aaacaatcaa aatgtccatc agtaggggat taggaacatt 45780 aattcgtaca gtggggaact cagtaccaga aggagggatg aggaagacct attgataagg 45840 ggcagagtac atatcatata atgcaaatat atatttgctt tttcttaaaa cagtacaaag 45900 ataaaaaact aaagtggttg ctgtggagga caggggtcaa tggtggaagt gagactgaaa 45960 tagactctga agtaatatct ggactttgaa attataagtg ttttacatat taccaaacta 46020 agttttaaga tagtccctaa aattgaaaga atagtatctg aaatgaatga atctaaattc 46080 cttggattgt cttctgcagg tgccaaccct gagaccaaaa tttggaaggt ggccctgagc 46140 agcagctgaa gggaagcggg aggtaagaca ggaaagaggt gagggcacag ggtgtctggg 46200 agccggatcc cacgtgggca gctgggcccg tgctcattgt gggagctggt gcattccttc 46260 accagcccac gctccacagg gtcaggatgg tcaattccgg gcaccccggc ctaatccagg 46320 acatgctcct gccaccagag aaagccccta ggcagcatcc cgggtgctgg tggtgtcaga 46380 atcaagtttg agtctgagga gtgacctggg gctggctggg ctaggcagca tcacagggtt 46440 ctgcagccca actgcacatc aggctggtga cagtcacaca gcctattact tcgtgtgtca 46500 tcagagcatc gccagaacgc agcacttcaa gtatgcagat ttagtgagcc atagtttaaa 46560 gacaaataga gccactgaaa tcctaaattt caa 46593 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SDHA siRNA sense <400> 3 gucuagagau gugugucu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SDHA siRNA antisense <400> 4 agacaccaca ucucuagac 19

Claims (27)

SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질, 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 제제를 포함하는, 항암제 내성 진단용 키트. A kit for diagnosing anticancer drug resistance, comprising an agent for measuring the expression level of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA), or a gene encoding the same. 제 1항에 있어서,
상기 SDHA 단백질은 서열번호 1과 80% 이상의 상동성을 가지는 서열로 표시되는 것인, 항암제 내성 진단용 키트.The method of claim 1,
The SDHA protein is represented by a sequence having at least 80% homology with SEQ ID NO: 1, anticancer drug resistance diagnostic kit. 제 2항에 있어서,
상기 SDHA 단백질은 서열번호 1로 표시되는 것인, 항암제 내성 진단용 키트.3. The method of claim 2,
The SDHA protein is represented by SEQ ID NO: 1, anticancer drug resistance diagnostic kit. 제 1항에 있어서,
상기 SDHA 유전자는 서열번호 2와 80% 이상의 상동성을 가지는 서열로 표시되는 것인, 항암제 내성 진단용 키트.The method of claim 1,
The SDHA gene is represented by a sequence having at least 80% homology with SEQ ID NO: 2, anticancer drug resistance diagnostic kit. 제 4항에 있어서,
상기 SDHA 유전자는 서열번호 2로 표시되는 것인, 항암제 내성 진단용 키트.5. The method of claim 4,
The SDHA gene is represented by SEQ ID NO: 2, anticancer drug resistance diagnostic kit. 제 1항에 있어서,
상기 항암제는 OXPHOS 억제제인, 항암제 내성 진단용 키트.The method of claim 1,
The anticancer agent is an OXPHOS inhibitor, a kit for diagnosing anticancer drug resistance. 제 6항에 있어서,
상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제(ubiquinone reduction activity inhibitor), 미토콘드리아 전자 수송 사슬 Ⅰ 억제제(mitochondrial electron transport chain I inhibitor) 또는 철 킬레이터(iron chelator)로 이루어진 군에서 선택된 1종 이상을 포함하는 것인, 항암제 내성 진단용 키트.7. The method of claim 6,
The OXPHOS inhibitor is a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator It contains at least one selected from the group consisting of Phosphorus, anticancer drug resistance diagnostic kit. 제 7항에 있어서,
상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156인, 항암제 내성 진단용 키트.8. The method of claim 7,
The mitochondrial electron transport chain I inhibitor is IM156, anticancer drug resistance diagnostic kit. 제 1항에 있어서,
상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함하는, 항암제 내성 진단용 키트.The method of claim 1,
The agent for measuring the expression level of the protein comprises at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to the protein, anticancer agent Kit for tolerance diagnosis. 제 1항에 있어서,
상기 유전자의 발현 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함하는, 항암제 내성 진단용 키트.The method of claim 1,
The agent for measuring the expression level of the gene comprises at least one selected from the group consisting of primers, probes and antisense nucleotides specifically binding to the gene, anticancer drug resistance diagnostic kit. 제 1항에 있어서,
상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트인, 항암제 내성 진단용 키트.The method of claim 1,
The kit is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit or a multiple reaction monitoring (MRM) kit, a kit for diagnosing anticancer drug resistance. 제 1항에 있어서,
상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인, 항암제 내성 진단용 키트.The method of claim 1,
The anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, A kit for diagnosing anticancer drug resistance, for the treatment of cancer selected from the group consisting of liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymus cancer, squamous cell carcinoma, adenocarcinoma, or a combination thereof. 목적하는 개체로부터 분리된 생물학적 시료에서 SDHA의 단백질 또는 이를 코딩하는 유전자의 발현 수준을 측정하는 단계를 포함하는, 항암제 내성 진단을 위한 정보를 제공하는 방법.A method for providing information for diagnosing anticancer drug resistance, comprising measuring the expression level of a protein of SDHA or a gene encoding the same in a biological sample isolated from a subject of interest. 제 13항에 있어서,
상기 SDHA의 단백질 또는 이를 코딩하는 유전자의 발현 수준이 대조군 시료에 비하여 증가된 것으로 나타난 경우, 상기 항암제 내성으로 인하여 치료 예후가 좋지 않을 것으로 예측하는, 항암제 내성 진단을 위한 정보를 제공하는 방법.14. The method of claim 13,
When the expression level of the SDHA protein or the gene encoding it is shown to be increased compared to the control sample, predicting that the treatment prognosis will be poor due to the anticancer drug resistance, a method of providing information for diagnosing anticancer drug resistance. 제 13항에 있어서,
상기 SDHA 단백질은 서열번호 1과 80% 이상의 상동성을 가지는 서열로 표시되는 것인, 항암제 내성 진단을 위한 정보를 제공하는 방법.14. The method of claim 13,
The SDHA protein is a method of providing information for diagnosing anticancer drug resistance, which is represented by a sequence having 80% or more homology with SEQ ID NO: 1. 제 13항에 있어서,
상기 SDHA 유전자는 서열번호 2와 80% 이상의 상동성을 가지는 서열로 표시되는 것인, 항암제 내성 진단을 위한 정보를 제공하는 방법.14. The method of claim 13,
The SDHA gene is a method of providing information for diagnosing anticancer drug resistance, which is represented by a sequence having 80% or more homology with SEQ ID NO: 2. 제 13항에 있어서,
상기 항암제는 OXPHOS 억제제인, 항암제 내성 진단을 위한 정보를 제공하는 방법.14. The method of claim 13,
The anticancer agent is an OXPHOS inhibitor, a method of providing information for diagnosing anticancer drug resistance. 제 17항에 있어서,
상기 OXPHOS 억제제는 유비퀴논 환원 활성 억제제(ubiquinone reduction activity inhibitor), 미토콘드리아 전자 수송 사슬 Ⅰ 억제제(mitochondrial electron transport chain I inhibitor) 또는 철 킬레이터(iron chelator)로 이루어진 군에서 선택된 1종 이상을 포함하는 것인, 항암제 내성 진단을 위한 정보를 제공하는 방법.18. The method of claim 17,
The OXPHOS inhibitor is a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator It contains at least one selected from the group consisting of Phosphorus, a method of providing information for diagnosing anticancer drug resistance. 제 18항에 있어서,
상기 미토콘드리아 전자 수송 사슬 Ⅰ 억제제는 IM156인, 항암제 내성 진단을 위한 정보를 제공하는 방법.19. The method of claim 18,
The mitochondrial electron transport chain I inhibitor is IM156, a method for providing information for diagnosing anticancer drug resistance. 제 13항에 있어서,
상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인, 항암제 내성 진단을 위한 정보를 제공하는 방법.14. The method of claim 13,
The anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, A method of providing information for diagnosing anticancer drug resistance for treatment of a cancer selected from the group consisting of liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, or a combination thereof. SDHA(succinate dehydrogenase complex flavoprotein subunit A)의 단백질의 활성, 또는 이를 코딩하는 유전자의 발현 수준을 감소시키는 제제를 포함하는, 항암제 내성 치료용 조성물.A composition for treating anticancer drug resistance, comprising an agent that reduces the activity of a protein of succinate dehydrogenase complex flavoprotein subunit A (SDHA), or the expression level of a gene encoding it. 제 21항에 있어서,
상기 제제는 3-니트로 프로피온산(3-nitropropionic acid; 3NPA) 또는 아트페닌 A5(atpenin A5)인, 항암제 내성 치료용 조성물.22. The method of claim 21,
The agent is 3-nitropropionic acid (3-nitropropionic acid; 3NPA) or atpenin A5 (atpenin A5), anticancer drug resistance treatment composition. 제 21항에 있어서,
상기 제제는 SDHA 활성 억제제 또는 SDHA 발현 억제제인, 항암제 내성 치료용 조성물.22. The method of claim 21,
The agent is an SDHA activity inhibitor or SDHA expression inhibitor, anticancer drug resistance treatment composition. 제 23항에 있어서,
상기 활성 억제제는 SDHA에 특이적으로 결합하는 항체인, 항암제 내성 치료용 조성물.24. The method of claim 23,
The activity inhibitor is an antibody that specifically binds to SDHA, anticancer drug resistance treatment composition. 제 23항에 있어서,
상기 발현 억제제은 서열번호 3 및 서열번호 4로 표시되는 siRNA를 포함하는, 항암제 내성 치료용 조성물.24. The method of claim 23,
The expression inhibitor is a composition for the treatment of anticancer drug resistance, comprising siRNA represented by SEQ ID NO: 3 and SEQ ID NO: 4. 제 21항에 있어서,
상기 항암제는 IM156인, 항암제 내성 치료용 조성물.22. The method of claim 21,
The anticancer agent is IM156, anticancer agent resistance treatment composition. 제 21항에 있어서,
상기 항암제는 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선종, 흉선암, 편평상피세포암, 선암종 또는 이들의 조합으로 구성된 군으로부터 선택된 암의 치료용인, 항암제 내성 치료용 조성물.22. The method of claim 21,
The anticancer agent is breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, A composition for treating cancer selected from the group consisting of liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymoma, thymic cancer, squamous cell carcinoma, adenocarcinoma, or a combination thereof, anticancer drug resistance treatment composition.
KR1020200041598A 2020-04-06 2020-04-06 Composition for diagnosing and treating anti-cancer drug resistance KR102393682B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020200041598A KR102393682B1 (en) 2020-04-06 2020-04-06 Composition for diagnosing and treating anti-cancer drug resistance
PCT/KR2021/004253 WO2021206406A1 (en) 2020-04-06 2021-04-06 Composition for diagnosis or treatment of anticancer drug resistance

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020200041598A KR102393682B1 (en) 2020-04-06 2020-04-06 Composition for diagnosing and treating anti-cancer drug resistance

Publications (2)

Publication Number Publication Date
KR20210123861A true KR20210123861A (en) 2021-10-14
KR102393682B1 KR102393682B1 (en) 2022-05-03

Family

ID=78023576

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020200041598A KR102393682B1 (en) 2020-04-06 2020-04-06 Composition for diagnosing and treating anti-cancer drug resistance

Country Status (2)

Country Link
KR (1) KR102393682B1 (en)
WO (1) WO2021206406A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019084313A1 (en) * 2017-10-27 2019-05-02 Board Of Regents, The University Of Texas System Use of sdha as a prognostic marker and therapeutic target in uveal melanoma

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019084313A1 (en) * 2017-10-27 2019-05-02 Board Of Regents, The University Of Texas System Use of sdha as a prognostic marker and therapeutic target in uveal melanoma

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHANDRANI CHATTOPADHYAY et al., ‘Elevated endogenous SDHA drives pathological metabolism in highly metastatic uveal melanoma’, Invest Ophthalmol Vis Sci., 2019, Vol. 60, pp 4187-4195. 1부.* *
PAOLA GOFFRINI et al., ‘Functional study in a yeast model of a novel succinate dehydrogenase subunit B gene germline missense mutation (C191Y) diagnosed in a patient affected by a glomus tumor’, Human* *
이뮤노멧, OXPHOS 억제제 IM156 관련 1단계 임상 연구 시작 발표, 2017, 인터넷 입수: https://www.yna.co.kr/view/AKR20171003054000009. 1부.* *

Also Published As

Publication number Publication date
WO2021206406A1 (en) 2021-10-14
KR102393682B1 (en) 2022-05-03

Similar Documents

Publication Publication Date Title
KR102657306B1 (en) Use of markers including filamin a in the diagnosis and treatment of prostate cancer
AU2020267282B2 (en) Compositions and methods for decreasing tau expression
AU2021200783B2 (en) Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (LTBP4)
KR20210046031A (en) Diagnosis and treatment methods for breast cancer treatment
CN115181778A (en) Method for selecting therapeutic molecules
KR20160079824A (en) C/ebp alpha short activating rna compositions and methods of use
KR20070092737A (en) Nucleic acids and polypeptides useful for diagnosing and treating complications of pregnancy
TW201632629A (en) Methods for cancer diagnosis and prognosis
KR102585973B1 (en) Oligonucleotides to regulate tau expression
KR20150023904A (en) Use of markers in the diagnosis and treatment of prostate cancer
KR20230124915A (en) Treatment of liver disease with apoptosis-inducing DFFA-like effector B (CIDEB) inhibitors
CN102459645B (en) Phosphodiesterase 9A as a marker for prostate cancer
KR102183300B1 (en) Pharmaceutical composition for overcoming resistance to photodynamic therapy against cancer
DK2951317T3 (en) PROCEDURE FOR PREDICTING THE BENEFIT OF INCLUSING TAXAN IN A CHEMOTHERAPY PLAN FOR BREAST CANCER PATIENTS
KR102393682B1 (en) Composition for diagnosing and treating anti-cancer drug resistance
KR102307590B1 (en) Biomarker composition for drug resistance diagnosis and diagnostic kit using the same
US20220265798A1 (en) Cancer vaccine compositions and methods for using same to prevent and/or treat cancer
KR102353374B1 (en) Marker gene for colorectal cancer classification, method for determining lymph node metastasis for prognosis of colorectal cancer, and kit therefor
US20090258344A1 (en) Methods for identifying risk of breast cancer and treatments thereof
KR20220138230A (en) Biomarkers for predicting prognosis of cancer
TWI834177B (en) Compositions and methods for decreasing tau expression
RU2777570C2 (en) Compositions and methods for reducing tau expression
KR20050008644A (en) Gene expression profiles in stomach cancer
KR20130048240A (en) Monoclonal antibodies against pcbp-1 antigens, and uses therefor
KR20230052771A (en) Biomarker for Pseudoexfoliation glaucoma

Legal Events

Date Code Title Description
E90F Notification of reason for final refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant