초록 ▼

1. 국내 분리EMC 바이러스와 표준 strain과의 성상 비교Line cell의 계대배양기술을 확립하고 이를 이용하여 바이러스를 증식하였음. 증식된 바이러스를 순수정제하여 자외선에 대한 흡광도, polypeptide profile 및 antibody에 대한 반응도등의 시험을 실시함2. Viral RNA의 분리 및 cDNA 제조EMC 바이러스의 RNA를 정제하고 전기영동으로 크기를 확인하였으며 oligo dT primer 및 AMV reverse transcriptase cDNA를 합성하고 λgtll으로 클로닝함.3. Probe의

1. 국내 분리EMC 바이러스와 표준 strain과의 성상 비교Line cell의 계대배양기술을 확립하고 이를 이용하여 바이러스를 증식하였음. 증식된 바이러스를 순수정제하여 자외선에 대한 흡광도, polypeptide profile 및 antibody에 대한 반응도등의 시험을 실시함2. Viral RNA의 분리 및 cDNA 제조EMC 바이러스의 RNA를 정제하고 전기영동으로 크기를 확인하였으며 oligo dT primer 및 AMV reverse transcriptase cDNA를 합성하고 λgtll으로 클로닝함.3. Probe의 제조제조한 cDNA를 pUC19 및 M13mp18으로 subcloning하고 DNA sequencing으로 염기서열을 확인한 후 random priming method로 probe를 제조함4. Hybridization에 의한 항원 검색 system개발EMC viral nucleotide와 제조한 probe와의 slot blot 및 northern hybridization을 시도하여 신속한 항원검색 system을 확립함

Abstract ▼

Encephalomyocarditis(EMC) virus isolated from Korea(K3 strain) was investigated for the characterization and was applied for the synthesis and cloning of cDNA with purified viral RNA to development of diagnostic method for viral disease using genetic engineering techniques. The nucleotide sequencing

Encephalomyocarditis(EMC) virus isolated from Korea(K3 strain) was investigated for the characterization and was applied for the synthesis and cloning of cDNA with purified viral RNA to development of diagnostic method for viral disease using genetic engineering techniques. The nucleotide sequencing of cDNA was performed and analysis of DNA homology compared to other EMC virus strains. The cDNA probe was produced and applied for the slot blot hybridization and northern hybridization to monitor the quantitation, specificity and detection of viral nucleotide in blood and tissue samples. The cytopathic effects were observed with cell rounding, pyknosis and necrosis after 36∼48 hrs' postinoculation of EMC K3 strain. The buoyant density of the virus was 1.30 and 1.35g/ml respectively. The size of purified viral RNA was 7.7Kb in molecular weight. The protein profiles of EMC virus K3 strain showed three kinds of polypeptide fragments with 26kd, 32kd, 34kd in molecular weight. The cDNA synthesized from EMC virus K3 strain showed variable size form 0.3Kb to 2Kb. The sequence of partial cDNA(392 bp) showed 99.2% homology to EMC Palmenberg strain and 89.5% homology to strain.Random labelled cDNA probe showed high sensitivity to detect EMC viral nucleotide more than 1ng level with slot blot hybridization and showed high sensitivity to detect the viral RNA with northern hybridization. The practical results were obtained to detect viral nucleotide in blood and tissue samples with cDAN probe manufactured from EMC virus K3 strain.

목차 Contents

• 제1장 서 론...21
• 제2장 재료 및 방법...25
• 1. 바이러스...25
• 2. 항 혈 청...25
• 3. 세균주, 플라스미드, 파아지...25
• 4. 배 지...26
• 5. 바이러스 증식, 정제 및 적혈구응집반응...26
• 6. 바이러스 단백질 분석...28
• 7. RNA의 확인...28
• 8. cDNA의 제조...29
• 9. Recombinant phage DNA의 정제...33
• 10. cDNA subcloning...34
• 11. DNA sequencing...35
• 12. DNA-RNA hybridization...36
• 제3장 결 과...39
• 1. EMC 바이러스의 성상...39
• 2. EMC 바이러스의 polypeptide profile...43
• 3. EMC viral RNA의 분자량...43
• 4. EMC viral RNA로 부터 합성한 cDNA...47
• 5. λgt11으로 클로닝한 cDNA의 확인...47
• 6. cDNA의 pUC19 및 M13mp18을 이용한 subcloning...48
• 7. cDNA의 염기서열...50
• 8. EMC viral RNA에 대한 cDNA probe의 특이성...52
• 제4장 고 찰...55
• 참고문헌...61