(3.238.130.97) 您好!臺灣時間:2021/05/18 10:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:楊智勇
研究生(外文):Yang, Zhi-Yong
論文名稱:D抗原的RNA結合區域在D型肝炎病毒複製的角色
論文名稱(外文):The role of RNA binding domains of delta antigen in genome replication of hepatits delta virus
指導教授:丁令白
指導教授(外文):Ting, Ling-Pai
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
中文關鍵詞:微生物學免疫學D型肝炎
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
相關次數:
  • 被引用被引用:0
  • 點閱點閱:73
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
D型肝炎病毒(HDV)是一種缺陷性的RNA病毒,其外套是由B型肝炎表面抗原所包覆,而位於病毒內部的成份主要有兩種:一為基因體(genome):這是一條單股,共價封閉的環形RNA;而另外一個成份就是D抗原(HDAgS)。D抗原同時也是日前所知此病毒基因體所轉譯的唯一的蛋白質。
目前已知,D抗原可分成大、小兩種,分別在HDV的基因體複製及組合的過程中扮演不同的角色。小D抗原是會促進HDV基因體的複製,而大D抗原則會抑制基因體的複製,且大D抗原是HDV基因體與B型肝炎表面抗原裝配組合時所不可或缺的。
利用細菌表現GST-DAg融合蛋白為抗原,製造抗D抗原的抗體,再以西方墨點分析免疫螢光染色來偵測D抗原和變異型在細胞內的大小和位置;為了研究小D及大D抗原上與調控基因體複製有關的功能區域,首先建立不會產生D抗原之HDV cDNA雙套體(defective HDV dimeric genomee)的複製互補系統(feplication complementation system)。
原型小D抗原主要在核仁(nucleolus),若去除二個RNA結合區域,則會在核質(nucleoplasm)。其中第一個結合區域(第104和105個股基酸)突變後,只會在核質;第二個結合區域(第139和140個股基酸)突變後,仍會在核仁。比顯示第一個RNA結合區域會影響小D抗原在細胞內的位置。在促進基因啦複製的功能上,第一個RNA結合區域很重要,而第二個RNA結合區域較不重要。此亦顯示一可能性,即第一個RNA結合區域突變失去促進基因體複製的功能可能與其無法位於核仁有關。
原型大D抗原位於核(nucleus)內,除在核質外亦會聚集在核仁。若突變第一個或第二個RNA結合區域或兩者,仍會在核質,但不會在核仁,可見RNA結合區域會影響大D抗原在細胞內的位置。在抑制基因體複製的功能上,無論是第一或第二個RNA結合區域皆不需要。
另外,大D抗原若刪減掉第89至162個胺基酸(NC變異型)或刪減掉第69至146個胺基酸(John Taylor et.al.),仍能完全抑制基因體的複製,但大D抗原刪減掉第56至162個股基酸(N'C變異型)則失去此抑制能力。此結果頭示:大D抗原第56至68個股基酸可能在抑制HDV基因體複製上扮演重要的角色。 Hepatitis delta virus (HDV ) is a defective RNA virus. The envelope of HDV is composed of the surface antigens of hepatitis B yirus ( HBV ). In side the HDV particle, there are the HDV single- stranded, covalently closed circular RNA genome with a size of 1.9kb and the only known virus-encoded proteins, the small and large delta antigens ( HDAgs ).
HDAgs have two forms, large delta antigen (L-HDAg; 214 amino acids, 27 kDa ) and small delta antigen ( S-HDAg; 195 amino acids, 24 kDa ). They display different roles during the genome replication and virion assembly. The small delta antigen is required to support the HDV genome replication. However, the large delta antigen acts as a trans- dominant negative regulator to suppress the genome replication and, on the other hand, promote the virus assembly in the presence of HBV surface and gens.
The anti-HDAg antibody was produced by immunizing rabbit with a bacterial expressed glutathione-S-transferase ( GST ) fusion protein GST-HDAg. The size and localization of the wild type or mutants of small and large HDAgs in human hepatoma HuH-7 cells are examined by Western blot analysis and immunofluoresence, respectively. In order to study the putative functional domains in HDAgs involved in the regulation of genome replication, the replication complementation system of the HDV dimeric genome with defective HDAgs is established.The small HDAg shows a strong staining in nucleolus as bright speckles. However, the mutant NC, which lacks both RNA binding domain I and II as well as the leucine zipper H ,has a nucleoplasmic staining. The mutant of RNA binding domain I also has a nucleoplasmic staining. In contrast, the mutant of RNA binding domain II has a nucleolar staining. These results indicate that the RNA binding domain I affects the localization of small HDAg in the nucleus. In the support of genome replication, the RNA binding domain I play a more important role than the RNA binding domain II. Together, these suggest that the support genome replication of small HDAg is closely related to its nucleolar localization.
The large HDAg stains the entire nucleus with the concentrated staining at nucleolus. Mutants of RNA binding domain I , II or both shows a strong staining only in the nucleoplasm. The results indicate that the RNA binding domains affect the localization of large HDAg in the nucleus. Both of the RNA binding doamin I and II are not required for trans-inhibition of genome replication.
Interestingly, we found that the large HDAg with deletions from amino acid 89 to 162 ( NC mutant ) or from 69 to 146 ( John Taylor et. al.) retained the full activity of trans-inhibition of genome replication, whereas a deletion from amino acid 56 to 162 ( N'C mutant ) failed to have this activity. This finding suggests that the subdomain between amino acid 56 to 68 of large HDAg may play an important role in the trans-inhibition.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊