臺灣博碩士論文加值系統

中文摘要
日本腦炎是由病媒蚊傳播的區域性流行疾病。日本腦炎病毒基因體全長約為11 kb的正股RNA，包含一個開放性讀架，能轉譯生成一段多蛋白，經切割後形成三個結構蛋白及七個非結構蛋白。病毒轉殖感染株(infectious clone)之建立，是研究病毒複製機轉及致病原因最有利之工具。然而，黃質病毒屬之轉殖感染株不易建立，可能是其基因產物會對細菌產生毒害，而導致病毒基因常會發生重組、突變或缺失的情形。本研究建立了一套簡單快速的方法，可以利用RT-PCR產生大量全長的JEV RP-9 cDNA。首先，在病毒基因體3’端設計兩種引子，以3JEV(-)引子（nt 10950-10976）進行反轉錄，再以3JEVXhoI引子(nt 10934-10976)和5’端包含T7 promoter之引子進行PCR反應，可複製出高濃度的11 kb片段。降低PCR的模板，可以有效地減少非專一性片段的產生，並增加11 kb基因體cDNA的合成量。利用T7 RNA polymerase，可將此cDNA經活體外轉錄產生11 kb 的RNA，此RNA轉染到細胞後，會使細胞產生病徵。以北方墨漬法及溶菌斑測試，證明此RNA具有感染能力。此外，利用各種不同載體與不同宿主組合以及降低培養溫度等各種條件，以選殖全長JEV cDNA至一穩定的載體中皆無法成功。目前可選殖到最長的片段為8.8 kb，包含了全部非結構蛋白基因和3’-untranslated region (UTR)，以及5’-UTR和C蛋白，證明日本腦炎病毒的prM和E蛋白結構基因是十分不穩定的區段。

Japanese encephalitis (JE) is a zoonotic viral disease that is a major public health problem in many Asian countries. Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus of approximately 11 kb in length, which encodes a single open reading frame. This polypeptide is then processed into three structural and seven nonstructural proteins. To understand the mechanisms of viral replication and pathogenesis, infectious clone methodology is a powerful tool of modern experimental virology. However, construction of infectious clones of flaviviruses is problematic due to instability, toxicity and recombination events during propagation in E. coli. Here a simple and rapid full-length long RT-PCR technique was established to produce genomic length cDNA from Taiwanese strain JEV RP-9. A system utilizing two antisense primers, 3JEV(-) (binds to nt 10950-10976) for reverse transcription, and 3JEVXhoI (binds to nt 10934-10976 with XhoI site) for long PCR, produced high yields of genomic length cDNA. Reducing the amount of RT products used for PCR efficiently minimized nonspecific amplification and increased the yield of genomic length cDNA. T7 RNA polymerase transcripts of the full-length cDNA template were infectious when transfected into BHK-21 cells. Infectivity was determined by cytopathic effect, northern analysis, and plaque assay of transfected cells. Extremely efforts were made to clone the full-length JEV genome cDNA into plasmid vectors. These include the use of different combinations of cloning vectors, bacterial host strains, and incubation temperature which have been successfully used to construct infectious clone in other flaviviruses. Unfortunately, none have worked to obtain a stable full-length 11-kb cDNA clone of JEV, yet the entire nonstructural genes and the 3’ UTR of 8.8 kb was successfully cloned. In addition, 5’UTR and C region was also stably maintained in E. coli vector. I have demonstrated that the unstable region in JEV is located within the prM and E proteins. This was derived from experiments demonstrating that when a 3.7 kb RT-PCR product covering the 5’UTR and NS1 gene was cloned into an E. coli plasmid, a fragment of 2.5-kb was invariably deleted.

目 錄


中文摘要……………………………………………………2
英文摘要……………………………………………………4
第一章 研究背景及重要性………………………………6
第二章 材料與方法……………………………………..15
第三章 結果……………………………………………..23
第四章 討論……………………………………………..30
第五章 參考文獻…………………………..……………35
第六章 圖表……………………………………………..38

1.Burks, D. S., and C. J. Leake. 1988. Japanese ecephalitis, p. 63-92. In T. Monath (ed.), The arboviruses: epidemiology and ecology, vol. 3. CRC Press, Inc., Boca Raton, Fla.
2.Campbell, M. S., and A. G. Pletnev. 2000. Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence. Virology. 269:225-37.
3.Cui, T., R. J. Sugrue, Q. Xu, A. K. Lee, Y. C. Chan, and J. Fu. 1998. Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology. 246:409-17.
4.Gritsun, T. S., and E. A. Gould. 1998. Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy. J Virol Methods. 76:109-20.
5.Gritsun, T. S., and E. A. Gould. 1995. Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR. Virology. 214:611-8.
6.Gupta, S., and R. M. Anderson. 1999. Population structure of pathogens: the role of immune selection. Parasitol Today. 15:497-501.
7.Hahn, C. S., Y. S. Hahn, C. M. Rice, E. Lee, L. Dalgarno, E. G. Strauss, and J. H. Strauss. 1987. Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol. 198:33-41.
8.Hurrelbrink, R. J., A. Nestorowicz, and P. C. McMinn. 1999. Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA. J Gen Virol. 80:3115-25.
9.Kapoor, M., L. Zhang, P. M. Mohan, and R. Padmanabhan. 1995. Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain). Gene. 162:175-80.
10.Khromykh, A. A., and E. G. Westaway. 1994. Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA. J Virol. 68:4580-8.
11.Mandl, C. W., M. Ecker, H. Holzmann, C. Kunz, and F. X. Heinz. 1997. Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J Gen Virol. 78:1049-57.
12.Markoff, L., B. Falgout, and A. Chang. 1997. A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein. Virology. 233:105-17.
13.Mishin, V. P., F. Cominelli, and V. F. Yamshchikov. 2001. A 'minimal' approach in design of flavivirus infectious DNA. Virus Res. 81:113-23.
14.Polo, S., G. Ketner, R. Levis, and B. Falgout. 1997. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast. J Virol. 71:5366-74.
15.Pur, B., S. Polo, C. G. Hayes, and B. Falgout. 2000. Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain. Virus Genes. 20:57-63.
16.Rauscher, S., C. Flamm, C. W. Mandl, F. X. Heinz, and P. F. Stadler. 1997. Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities. Rna. 3:779-91.
17.Ravi, V., A. S. Desai, P. K. Shenoy, P. Satishchandra, A. Chandramuki, and M. Gourie-Devi. 1993. Persistence of Japanese encephalitis virus in the human nervous system. J Med Virol. 40:326-9.
18.Rice, C. M., A. Grakoui, R. Galler, and T. J. Chambers. 1989. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol. 1:285-96.
19.Ridpath, J. F., S. R. Bolin, and E. J. Dubovi. 1994. Segregation of bovine viral diarrhea virus into genotypes. Virology. 205:66-74.
20.Sriburi, R., P. Keelapang, T. Duangchinda, S. Pruksakorn, N. Maneekarn, P. Malasit, and N. Sittisombut. 2001. Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid. J Virol Methods. 92:71-82.
21.Sumiyoshi, H., C. H. Hoke, and D. W. Trent. 1992. Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. J Virol. 66:5425-31.
22.Wengler G, W. G., Gross HJ. 1978. Studies on virus-specific nucleic acids synthesized in vertebrate and mosquito cells infected with flaviviruses. Virology. 89:423–437.
23.Yamshchikov, V., V. Mishin, and F. Cominelli. 2001. A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli. Virology. 281:272-80.
24.Yamshchikov, V. F., G. Wengler, A. A. Perelygin, M. A. Brinton, and R. W. Compans. 2001. An infectious clone of the West Nile flavivirus. Virology. 281:294-304.
25.Zhang, F., Q. Huang, W. Ma, S. Jiang, Y. Fan, and H. Zhang. 2001. Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector. J Virol Methods. 96:171-82.