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研究生:郭瑞琳
研究生(外文):Kuo, Rei-Lin
論文名稱:B型肝炎病毒剪接轉錄物包裹之研究
論文名稱(外文):Study the encapsidation of spliced RNAs of hepatitis B virus
指導教授:蘇宗笙
指導教授(外文):Su, Tsung-Sheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
論文頁數:92
中文關鍵詞:微生物學免疫學B型肝炎病毒
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
相關次數:
  • 被引用被引用:1
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B型肝炎病毒感染細胞後,首先會把本身不完全雙股的DNAgenome修補成環形閉鎖DNA(closedc circular DNA)。在細胞中DNA經過轉錄做出許多種類的RNA,再經由轉譯作用做出core,polymerase Surface及X等蛋白。按著再由病毒的蛋白共同作用下把3.5-kb pregenomic RNA包入核心蛋白組成的外殼中,組合成一個新的病毒顆粒。在此過程中所需要的病毒產物包括被包褒RNA上的ε-sequence,與核心蛋白、DNA聚合酉每兩個蛋白。包入外殼中的pregenomic RNA經由反轉錄作用產生負股DNA,然後再合成五股DNA並釋放此病毒顆粒到細胞外。
B型肝炎病毒可以經過剪接作用產生數種剪接RNA,其中一種為2.2-kb單剪接RNA,上面有5個新的開放編列區:preC'',C'',C'',p'',p",S'',以及完整的x開放編列區。
我們利用聚合酉每鏈反應從一個HBeAg+,HBsAg+病人的血清中偵測到一種可能是包著單剪接RNA的病毒顆粒,DNA定序確定此種病毒顆粒,確實包裹的是2.2-kb單剪接RNA的反轉錄產物,我們稱它為一種缺陷性病毒顆粒。在in vitro轉染系統中我們利用B型肝炎痛毒的表現質體,pMH-9/3091,轉染人類肝癌細胞株,HuH.7,再自細胞培養液中收取病毒顆粒,同樣的,我們也發現了包含2.2-Kb單一剪接RNA的反轉錄產物病毒顆粒。
當我們把in vitro轉染系統中所得到的病毒顆粒經過CsC1密度梯度分層之後,我們發現B型肝炎病毒在CsC1密度分層中被分 成二群,一群已加上了由 HBsAg組成的套膜,浮力密度較小,稱Dane分層。另一群則未加上套膜,浮力密度較大,稱作core分層,而這種缺陷性病毒顆粒大多出現在core分層中。
為了進一步研究這種缺陷性病毒顆粒的特性,於是我們建立一個"trans"互補作用系統。我們把2.2-kb單剪接RNA的表現質體, pCH7-8T與B型肝炎病毒的DNA聚合酉每表現質體,pMT-P-Po共同轉染到HuH-7細胞中,再由細胞培養液中分離病毒顆粒。我們發現在B型肝炎病毒DNA聚合酉每幫助之下,2.2-kb單剪接RNA可以被包裹形成缺陷性病毒顆粒並釋放到細胞培養液中,即單剪接RNA在有野生種的B型肝炎病毒DNA聚合酉每幫助之下可以單獨包裹
此外,我們比較了B型肝炎病毒的pregenomic RNA和2.2-kb單剪接RNA的包裹效率,發現雖然2.2-kb單剪接RNA不能轉譯出完整的B型肝炎病毒DNA聚合酉每,但是其具有與pregenomic RNA相同的包裹效率。在此種剪接RNA的包裹過程中,B型肝炎病毒DNA聚合酉每的作用方式,以及剪接RNA本身的DNA聚合酉每產物P''扮演什麼樣的角色,則有待進一步的研究。
Hepatitis B virus (HBV) is a partially double-stranded DNA virus. The replication of HBV involves the packaging and reverse transcription of pregenomic RNA. Several species of spliced RNAs, i.e., 2.2-kb singly-spliced, 2.2-kb doubly-spliced and 2.6-kb singly-spliced RNAs, have been reported. All these RNAs are the spliced products of pregenomic RNA. A viral DNA derives from the reverse transcription of the 2.2-kb singly-spliced RNA was detected in serum of a HBsAg+ / HBeAg+ carrier by polymerase chain amplification. Similarly, this type of viral particle was also found in medium of HBV DNA- transfected HuH-7 cells. However, when one transfecting expression plasmid of spliced RNA into HuH-7 cells, no viral particle was produced, suggesting that the viral particle detected in vivo and in vitro is defected. Nevertheless, when co- expression with HBV DNA polymerase, the functional trans-complemeotation can be achieved that resulting in viral particle formation. Apparently, viral DNA polymerase, i.e., P protein, is required for either spliced RNA package or reverse transcription or both.
When viral particles from cultured medium of wild type HBV DNA-transfected HuH-7 cells were fractionated by CsCl density gradient, viral particles were found not only in Dane particle fraction, but also in core fraction, suggesting that a fraction of viral particles can be released into medium without envelope. However, these particles are rather immature as suggested by their shorter "plus" stranded DNA. The DNA can be further extended when repaired by DNA polymerase I, Klenow fragment. Interestingly, based on buoyant density, the defected viral particles were found mainly as core particles. By analyzing the distribution of viral particles in CsCl gradient, spliced RNA was shown not necessarily co-packed with pregenomic RNA. Furthermore, the packaging efficiency is about 30% for both pregenomic and spliced RNAs when viral RNAs inside cytoplasm and cytoplasmic core particles were quantified. Studies from several groups have indicated that P protein preferentially encapsidates pregenomic RNA from which P protein is synthesized, i.e., the action is in cis. How can spliced RNA presumably in trans to P protein be encapsidated so efficiently? Would the encapsidation of spliced RNA involve the truncated form of P product, i.e., P'' protein translated from spliced RNA? These are some important questions remain to be answered.
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