초록 ▼

1. HBV-X 발현하는 eukaryotic expression vector pMAMneo-X를 구축하였다.2. 제조된 HBV-X 단백질 발현 벡터를 간암세포 HepG2에 transfection시켜 HBV-X 발현 벡터 DNA가 숙주세포내의 염색체에 intergration되어 있으며 발현되는지의 여부를 Southern과 Northern Blot 분석을 통하여 확인하였다.3. 세포주에 도입한 X 유전자의 trans-activator로서의 활성을 특정하기 위해 CAT 분석을 실시하여 X 유전자의 전사가 효율적으로 일어남을 확인하였고 A

1. HBV-X 발현하는 eukaryotic expression vector pMAMneo-X를 구축하였다.2. 제조된 HBV-X 단백질 발현 벡터를 간암세포 HepG2에 transfection시켜 HBV-X 발현 벡터 DNA가 숙주세포내의 염색체에 intergration되어 있으며 발현되는지의 여부를 Southern과 Northern Blot 분석을 통하여 확인하였다.3. 세포주에 도입한 X 유전자의 trans-activator로서의 활성을 특정하기 위해 CAT 분석을 실시하여 X 유전자의 전사가 효율적으로 일어남을 확인하였고 AP-1에 HBV-X에 의해 발현이 증가됨을 gel retardation 분석을 통하여 확인하였다.4. X 단백질이 대량발현되는 system을 확립하고 발현된 X 단백질을 정제하여 western blotting을 실시하여 정확한 X 단백질의 발현을 확인하였다.

Abstract ▼

1. We established human hepatoma cell lines, which expressed the human hepatitis B virus-X protein. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene were constructed. 2. Transfected HBV-X gene integration an expression were detected by Southern and Norther

1. We established human hepatoma cell lines, which expressed the human hepatitis B virus-X protein. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene were constructed. 2. Transfected HBV-X gene integration an expression were detected by Southern and Northern blot analysis. Most of the clones showed X-gene integrations into the chromosome while HepG2 and Hep3B did not show any integrantions. HBV-X transfected HepG2 and Hep3B cell lines showed X transcripts expression; Untransfected HepG2 and Hep3B did not show any RNA transcripts.3. In order to evaluate the transcriptional transactivation actiity of the X-expressing cell lines, chloramphenical acetylase transferase(CAT) assay using some kinds of promoter CAT reporter systems were performed and showed strong induction in X-transfected cell lines compared to the control HepG2 and Hep3B cells.4. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. HBV-X expressing cell line, HepG2-4X, which showed the highest expression levels of X-transcripts. 5. These cell lines are useful in analyzing the biological function of HBV-X and possibly the mechanism of the causes of hepatocarcinogenesis caused by HBV-X. 6. We established the large scale expression system of X-protein in E. coli and we purified the X-protein using CM Sepharose column and performed western blot analysis.

목차 Contents

• 1. 서론...8
• 2. 실험재료 및 방법...16
• 3. 결과 및 고찰...17
• 참고문헌...20