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사람 표피 각질 형성 세포에서 사람 유두종 바이러스 16형의 E6와 E7 Open Reading Frame 단백질의 표현

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dc.contributor.author김건홍-
dc.date.accessioned2015-11-20T04:32:39Z-
dc.date.available2015-11-20T04:32:39Z-
dc.date.issued1992-
dc.identifier.urihttps://ir.ymlib.yonsei.ac.kr/handle/22282913/115127-
dc.description의학과/박사-
dc.description.abstract[한글] Human papillomavirus(HPV)는 최근가지 약 60가지 형이 보고되었으며, 이들은 HPV와 연관된 병변의 악성 종양으로의 이행 빈도에 따라 저위험군 및 고위험군으로 분류되고 있다. 고위험군에 속하는 HPV로 부터는 감염된 세포의 종양 형성에 중요하게 관여하는 E6 및 E7 두개의 종양단백질이 표현되고 있으며, 세포 형질 변환 기전과 연관하여 이들 단백질의 세포내 존재 위치에 대한 연구가 여러 연구자들에 의해 보고되었으나 서로 다른 결과를 제시하고 있어 본 실험에서는 HPV 16형의 E6 및 E7 종양단백질의 세포내 존재 위치를 확인하고자 본 실험에 착수하여 다음과 같은 결론을 얻었다. DNA 합성기를 이용하여 HPV 16형의 E6 및 E7 open reading frame(ORF)의 단백질 합성(translation) 시작 codon(ATG)부터 단백질 합성 종료 codon(TAA)가지를 포함하는 두쌍의 primer를 각각 제조하고 polymerase chain reaction(PCR) 방법으로 505bp 크기의 E6 ORF와 337bp 크기의 E7 ORF DNA를 얻었다. 세포내에서 E6 및 E7 단백질을 표현시키기위한 표현 체계로서 vaccinia virus를 이용하였으며 vaccinia virus의 DNA내로 E6 및 E7 ORF DNA를 삽입시키기위해 pSCII transfer plasmid에 E6 및 E7 ORF DNA를 subcloning하고 각각을 p SCII-E6 및 pSCII-E7이라고 명명하였다. 자연형의 vaccinia virus를 감염시킨 CV-1세포에 pSCII-E6 및 pSCII-E7을 transfection하여 세포질내에서 homologous recombination을 유도하고 CV-1 세포에서 virus를 유리한 뒤 Hu TK-143 B 세포에 감염시켜 bromodeoxyuridin e(BrdU)과 X-gal이 포함된 선별 배양액으로 recombinant vaccinia virus pleaque를 선별하였으며, 각각의 plaque로 부터 유리된 recombinant vaccinia virus를 VV-E6 및 VV-E7으로 명명하였다. HPV의 숙주 세포인 사람 표피 각질 형성 세포(RHEK-1)에 VV-E6 및 VV-E7 을 각각 감염시키고 12시간 후에 paraformaldehyde로 세포를 고정한 후 1차 항체로서 마우스 항 E6 ORF 단클론 항체 또는 마우스 항 E7 ORF 단클론 항체를 처리하고 2차 항체로서 염소 항 마우스 IgG-FITC 결합 항체를 처리한 후 간접 형광 현미경에서 400배 시야로 관찰한 결과 RHEK-1 세포에서 표현된 HPV 16형의 E6 ORF 및 E7 ORF 단백질은 모두 세포질내에 존재하는 단백질임을 확인하였다. [영문] Over 60 different types of human papillomavirus(HPV) have been identified, and they are classified into high risk and low risk groups based on the risk for malignant progression of HPV associated lesion. HPVs belonged to a high risk group have been known to express two major transforming proteins, E6 and E7. In association with the transforming activities of E6 and E7proteins, many investigators have reported the location of these proteins in the cell, but their results were still controversial. This study was designed to confirm the location of these proteins in the cell and following results were obtained. Two sets of primers were synthesized by DNA synthesizer in order to amplify coding region of E6 and E7 proteins. Using these primers. polymerase chain reaction (PCR) was performed and amplified 505 bp E6 ORF DNA fragment and 337bp E7 ORF DNA fragment were obtained. In order to express the E6 or E7 ORF DNA in the human epidermal keratinocytes, vaccinia virus expression system was employed. For preparation of recombinant vaccinia virus containing E6 or E7 ORF DNA, amplified E6 or E7 ORF DNA was subcloned into pSCII vaccinia virus transfer plasmid, and each subctoned plasmid was named as pSCII 1-E6 and pSCII 1-E7, respectively. To in-duce the homologous recombination in the cytoplasm of the cell, CV-1 cells which were infected with wild type vaccinia virus were transfected with subcloned pSCII 1-E6 or pSCII 1-E7 using calcium phosphate coprecipitation technique, and then recombinant vaccinia viruses were selected by plaque media containing bromodeoxy uridine(BrdU) and X-gal and named as VV-E6 and VV-E7,respectively. To identify the subcellular location of E6 and E7 proteins in the cell, RHEK-1,human epidermal keratinocytes that are natural host for HPV were infected with VV-E6 or VV-E7 and then fixed with 4% paraformaldehyde at 12 hours after infection. The fixed cells were in-cubated with mouse anti-E6 ORF monoclonal antibody or mouse anti-E7 ORF monoclonal antibody followed by incubation with FITC conjugated goat anti-mouse 19G antibody. Indirect fluorescence microscopy of immunostained cells revealed that E6 and E7 proteins encoded by E6 and E7 ORF DNA of HPV type 16 were located only at the cytoplasm of RHEK-1 cells.-
dc.description.statementOfResponsibilityrestriction-
dc.publisher연세대학교 대학원-
dc.rightsCC BY-NC-ND 2.0 KR-
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/2.0/kr/-
dc.title사람 표피 각질 형성 세포에서 사람 유두종 바이러스 16형의 E6와 E7 Open Reading Frame 단백질의 표현-
dc.title.alternativeExpression of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte-
dc.typeThesis-
dc.contributor.departmentDept. of Biochemistry and Molecular Biology (생화학-분자생물학교실)-
dc.contributor.departmentDept. of Biochemistry and Molecular Biology (생화학-분자생물학교실)-
dc.contributor.localIdA00289-
dc.identifier.urlhttps://ymlib.yonsei.ac.kr/catalog/search/book-detail/?cid=CAT000000005526-
dc.contributor.alternativeNameKim, Kun Hong-
dc.contributor.affiliatedAuthor김건홍-
dc.type.localDissertation-
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Biochemistry and Molecular Biology (생화학-분자생물학교실) > 3. Dissertation

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