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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 呂健宏 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:16:31Z | - |
| dc.date.available | 2021-07-01T08:16:31Z | - |
| dc.date.issued | 1992 | |
| dc.identifier.citation | Adang, M.J. and Miller, L. K. 1982. Molecular cloning of DNA complementary to mRNA of the baculovirus Autorapha californica nuclear polyhedrosis virus: location and gene products of RHA transcripts found late in infection. , J. Virol. 44:782—793.
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Malitschek, B. and Schsrtl, M. 1991. Rapid identification of recombinant baculovirus using PCR. Biotechniques 11:177—178. Martin, B. M., Tsuji, S., LaMarca, M. E., Maysak, K. and Eliason, W. 1988. Glycosylation, and processing of high levels of active human glucocerebrosidase in invertebrate cells using a baculovirus expression vector. DNA 7:99—106. Matsuura, Y., Possee, R. D., Overton, H. A. and Bishop, D. H. 1987. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J. Gen.Virol. 68:1233-1250 Mm, Mi—Kyung and Bishop, D. H. L. 1991. Transcriptional analyses of baculovirus polyhedrin and foreign gene expression relative to baculovirus p10 mRNA levels. J. Gen. Virol. 72:2551-2556. Miller, L. K. 1988. Baculovirus as gene expression vecto rs. Ann. Rev. Microbiol. 42:177-199. Miller, L. K. 1989. Insect baculoviruses: powerful gene expression vectors. Bioessay 4:91-95. 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Translational selection in the expression of hepatitis B virus envelope proteins. DNA 7:417-422. Rankin, C., Ooi, B. G. and Miller, L. K. 1988. Eight base pairs encompassing the transcriptional start point are the major determinant for baculovirus polyhedrin gene expression. Gene. 70:39—49. Rohrmann, G. F. 1986. Polyhedrin structure. J. Gen. Virol. 67:1499-1513. Sanibrook, J., Fritsh, E. F. and Maniatis, T. 1989. Molecular cloning: A laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. Smith, G. E., Fraser,M. J. and Summers, M. D. 1983a. Molecular engineering of the Autographa californica nuclear polyhedrosis vorus genome: deletion mutations within the polyhedrin gene. J. Virol. 46:584—593. Smith,G. E., Ju, G., Ericson, B. L., Mosehera, L. and Laha, H .—W. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc. Natl. Acad.Sci. USA 82:8404-8408. Smith, G. E., Summers, M. D. and Fraser, M. J. 1983b. Production of human beta inteferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3:2156-2165. Smith, U. E., Vlak, J. M. and Summers, M. D. 1983c. Physical analysis of Autographa californica nuclear polyhedrosis virus transcripts for polyhedrin and 10,000-molecular weight protein. J. Virol. 45:215-225. Stewart, L.M. D., Hirst, M., Ferber, M. L., Merryweather, A. T., Cayley, P. J. and Possee, R. D. 1991. Construction of an improved baculovirus insecticide containing an insect— specific toxin gene. Nature 352:85-88. Tessier, D. C. Thomas, D. Y., Khouri, H. E., Lalibert?, F. and Vernet, T. 1991. Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177-183. Takehara,K., Ireland, D. and Bishop, D. H. L. 1988. Co- expression of hepatitis B surface and core antigens using baculovirus multiple expression vectors. J. Gen .Virol. 69:2783—2777. Thiem, S. M. and Miller, L. K. 1989. Identification, sequence, and transcriptional Mapping of the major capsid protein gene of the baculovirus Autogranpha californica nuclear polyhedrosis virus. J. Virol. 63:2008—2018. Tiollais, P., Charnay, P. and Vyas, G. N. 1981. Biology of hepatitis B virus. Science 213:406-411. Vernet, T., Tessier, D. C., Richardson, C., Lalibert?, F., Khouri,H. E., Bell, A.W., Storer, A. C. and Thomas, D. Y.1990. Secretion of functional papain precursor from insect cells: requirement for N—glycosylation of the pro—region. J. Biol. Chem. 265:18661—10666. Webb, A. C., Brably, M. K., Phelan, S. A., Vu, J. Q. and Gehrke, L 1991. Use of the polymerase chain reaction for screening and evaluation of recombinant baculovirus clones. Biotechniques 11:512-519. Vilson, M. E., Mainprize, T. H., Friesen, P. D. and Miller, L. K. 1987. Location, transcription, and sequence of a baculovirus gene encoding a small arginine-rich polypeptide. J. Virol. 61:661-666. Yang, C. L.., Stetler, D. A. and Weaver, R. F. 1991. Structural comparison of the Autographa californica nuclear polyhedrosis virus - induced RNA polymerase and the three nuclear RNA polymerase from the host, Spodoptera frugiperda. Virus Res. 20:251-264. 楊蓓蘭．1991 . B 型肝炎表面抗原基因轉譯起始與終止點附近核?酸序列對其在桿狀病毒表現載體系統中表現率的影響．私立輔仁大學生物學研究所碩士論文． | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75928 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:16:31Z (GMT). No. of bitstreams: 0 Previous issue date: 1992 | en |
| dc.description.tableofcontents | 壹．緒言 . . . . . . . . 1 貳．材料與方法. . . . . . . . . . . . . . . . 9 －．供試的細胞株及病毒. . . . . . . . . . . 9 二．各式質體的來源. . . . . . . . . . . . . . 9 三．DNA 的剪接及操作. . . . . . . . . . . 10 四．引子的來源及目的. . . . . . . . . . . . 10 五．各種重組載體之構築. . . . . . . . . . . 12 1. pLCH－1之構築. . . . . . . . . . . . 12 2. pLCH－104之構築. . . . . . . . . . . 16 3. pLCH－T5之構築. . . . . . . . . . . 16 4. pAcWt－HBVs之構築. . . . . . . . . . 18 5. pT7－CAT672 及 pT7－CAT676 之構築. . . . . 21 6. pAWt－CAT 之構築. . . . . . . . . . . 24 六．質體構築時的各種 PCR 條件. . . . . . . . 27 七．質體 DNA 的定序. . . . . . . . . . . . . 27 八．共轉型感染及重組病毒之純化. . . . . . . . 27 九．利用 PCR 進行重組病毒的鑑定及其 DNA 的定序. . . . . 29 十．利用 RIA 或 EIA 進行 B 型肝炎表面抗原之測定. . . . . 30 十一．利用西方點墨法分析昆蟲細胞生產的 HBsAg . . . . . . 30 十二．Tunicamycin 對昆蟲細胞生產之 HBsAg 的影響. . . . . . 31 十三．在被感染的 SF－21AE 細胞中進行蛋白質的放射性標定. . . . . .31 十四．細胞質 RNA 的分離. . . . . . . . . . . . 32 參．結果. . . . . . . . . . . . . . . . . . . 33 －．各種不同重組質體之構築. . . . . . . . . 33 二．重組病毒之純化及鑑定. . . . . . 35 三．B 型肝炎表面抗原基因的 ATG 的前端序列對其在桿狀病毒表現載體系統中表現的影響. . . . . . . . . . . 42 四．各不同病毒株間的 mRNA 的分析. . . . . . 45 五．利用西方點墨法分析昆蟲細胞所生產的 B 型肝炎表面抗原. . . . . . 48 六． B 型肝炎表面抗原在昆蟲細胞中形成聚合體. . . . . . 51 七． CAT 基因的 ATG 的前端序列對其在桿狀病毒載體中表現的影響. . . 53 八．利用放射性標定進行重組蛋白的研究. . . . 58 肆．討論. . . . . . .. 61 伍．中文摘要. . . . . . . 69 陸．英文摘要. . . . . . 70 柒．參考文獻. . . . . . . . . . . . . . . .71 | |
| dc.language.iso | zh-TW | |
| dc.title | 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 | zh_TW |
| dc.title | Maximizing the Expression Leve of Foreign genes in Baculovirus Expression Vector System | en |
| dc.date.schoolyear | 80-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 80 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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