請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75928
標題: | 促使外源基因在桿狀病毒表現載體系統中達到最高表現率之研究 Maximizing the Expression Leve of Foreign genes in Baculovirus Expression Vector System |
作者: | 呂健宏 |
出版年 : | 1992 |
學位: | 碩士 |
摘要: | 構築出一系列具有不同未轉譯引導序列的 HBsAg 重組病毒,以評估引導序列對外源基因表現的影響。其中的重組病毒 AcWt-HBVs ,其引導序列與野生型 AcNPV 的完全一樣,以期達到最高表現率。因為實驗設計的誤差,重組病毒 LCH-1 生產出融合性的 HBsAg 。利用 RIA 的測定,發現 LCH-1 的 HBsAg 產量最高,而不是 AcWt-HBVs 。分析細胞質 RNA ,證實多角體蛋白基因的未轉譯引導序列的完整性會影響到外源基因的轉錄。雖然 LCH-1 的蛋白質產量為最高,然其 RNA 的產量並不是。將外源基因換成 CAT ,則 CAT 的表現情形亦會因為 RNA 量的減少而下降。經由西方點墨法,發現昆蟲細胞所生產的 HBsAg ,其蛋白質的轉譯修飾與源自動物細胞的相類似。雖然 HBsAg 在 35S-Met 標定的三個小時內所得到的產量與 CAT 和多角體蛋白的相差不多,但在總產量上卻有極大的差異。根據各個實驗的結果,可以發現 HBsAg 的表現主要是控制在轉譯後的階段,包括蛋白質的加工修飾及穩定性等等。 A series of HBsAg recombinant baculoviruses, whose 5’ untranslated leader sequences were different from each other, were constructed in order to assess the influence of the leader sequence on gene expression. One of these viruses, AcWt-HBVs, which had exactly the same leader sequence of the HBsAg gene as for the polyhedrin gene, was expected to achieve maximal expression level. And due to the improper design of construction strategy, a fusion HBsAg protein was produced with one virus, LCH-1. HBsAg was detected with RIA, and the highest level of HBsA production was not obtained with AcWt-HBVs, but LCH-1. With the analysis of steady-state mRNA levels, it showed that the integrity of polyhedrin leader sequence could affect the transcription efficiency. And the RHA level of LCH-1 was lower than the others. The influences of those leader sequences were also investigated with CAT reporter gene. And the decreased level of protein synthesis was consistent with that of RNA. The HBsAg produced in insect cells was characterized with western blot and immuno-staining. Some degraded HBsAg protein could be observed. And part of post-translational modifications of insect cells-derived HBsAg were similiar to mammalian cells-derived. The HBsAg protein was the predominant 35S-Met-labelled band on autoradiogram by pulse-labelling for 3 hr, and could be compared to that of CAT and polyhedrin. But the amount of accumulated HBsAg was only up to the level of ug/ml. To sum up, the major determinants of the expression of HBsAg with baculovirus expression vector system were controlled at the post-translational levels, including modification, processing and stability of the protein. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75928 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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。