請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76093
標題: | 梨形鞭毛蟲病毒雙股核醣核酸四個末端的二級結構及序列之決定 Determination of structure and nulceotide sequence at the four termini of giardiavirus double-stranded RNA |
作者: | 吳治宏 |
出版年 : | 1994 |
學位: | 碩士 |
摘要: | 梨形蟲病毒(Giardiavirus, GLV)是寄生在梨形蟲(Giardia lamblia)的病毒。它是由主要蛋白p100(gag protein)、次要蛋白p190(gagpol fusion protein)及雙股核醣核酸(dsRNA)所構成。根據前人的研究得知:以電穿孔法(electroporation)將病毒的單股核醣核酸(ssRNA)送入未感染病毒的梨形蟲中就會完成整個複製過程並產生病毒子代,所以此ssRNA在病毒的複製過程中扮演複製中間體(replicative intermediate)及訊息RNA (mRNA)的角色。如同噬菌體可做為外來基因進入細菌的載體(vector),將來此梨形蟲病毒亦可發展成為外來基因進入梨形蟲的載體,如此對於梨形蟲分子生物學上的研究,將是一大進步。欲瞭解此病毒的複製機轉及建立梨形蟲的外來基因載體系統,得到完整病毒的互補去氧核醣核酸(complementary DNA,cDNA)是不可或缺的。雖然前人已得到6.1kb( kilobase)的病毒cDNA,但此cDNA的長度與先前所估計病毒基因組(genome)的長度7kb並不一致。在本篇論文中証明瞭先前6.1kb的病毒cDNA並非是完整的病毒cDNA,並在其末端增加了177nucleotides,且以數種方法?3’-RACE (Rapid Amplification of cDNA End)、5’-RACE及reverse transcriptase定序法?相互比對,確認了我們已要得到完整的病毒cDNA。並利用電腦程式將GLV、酵母菌殺手病毒(Yeast killer virus, ScV)、利什曼原蟲病毒(Leishmania RNA virus, LRV)的ds RNA 5’ 端及 3’ 端的序列做折疊(folding),以比較他們端點RNA的二級結構(secondary structure)之異同點。藉此預測GLV dsRNA 5’端及3’端在病毒複製過程中所可能扮演的角色。 Giardiavirus(GLV) lives in parasitic protozoa Giardia lamblia. The virus contains double stranded RNA, major capsid protein pl00 (Gag) and minor protein p190 (Gag-Pol). The plus stranded RNA is infectious, serving both as the replicative intermediate and mRNA since electroporating this RNA into uninfected host yielded infectious virion from the culture media. Because of its simplicity, the virus is ideal to be used as vector for introduction of foreign DNA into Giardia which will have great impact and will facilitate the study of the molecular biology of this importment and interesting protozoan parasite. To understand replication mechanism in detail and to establish the vector system, the determination of the four terminal nucleotide sequences is a critical and essential step. However, the previously published 6.1 kb cDNA of the RNA genome showed certain discrepancy to the 7.0 kb RNA genome size estimated from agarose gel. In this report, additional DNA sequence of 177 bp is found to be present at the 3’ terminus of GLV cDNA. The conclusion that this new addition completes the cDNA of the full length RNA genome is verified by the methods of 3’-RACE (rapid amplification of cDNA end), 5’-RACE and reverse transcriptase sequencing. The secondary structures of the 5’ and 3’ termini are folded using computer program Mulfold. The possible role of the termini during viral replication is deduced by comparing the structure with those of yeast killer virus, ScV, and of Leishmania RNA virus, LRV. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76093 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。