以多效型桿狀病毒載體系統表現輪狀病毒VP4及VP7表面蛋白之研究

Expression of Rotavirus VP4 and VP7 Proteins by a Polycistronic Baculovirus Expression Vector

葉舒平(Shu-Ping Yeh)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2011年)

https://doi.org/10.6840/cycu201100795

摘要

摘要
類病毒顆粒 (Virus-like particle，VLP) 的應用相當廣泛，可作為次單位疫苗之外，亦可做為基因或藥物的載體以及奈米材料之模板。本實驗可分成兩個部分: (i) 利用多效型桿狀病毒表現系統，生產G9輪狀病毒 (G9 rotavirus) 中主要的外層結構蛋白質 (outer structure protein) VP4和VP7，以及將這兩種結構蛋白和內核的結構蛋白VP2、內層的結構蛋白VP6共同表現，預期這些結構蛋白會自我組裝形成50~70奈米的類病毒顆粒。(ii) 先前本實驗室人員已成功利用桿狀病毒雙效表現載體於Sf21昆蟲細胞中單獨表現生單一VP2、VP6、VP7結構蛋白，VP2和VP6蛋白會在細胞內表現，但VP7第223胺基酸由K突變為E （K223E）時才會大量的表現，並分泌至細胞外。本實驗目的為共同表現VP2/7、6/7、2/6/7，再以anti- goat之輪狀病毒血清進行西方墨點分析，可觀察到雖然胺基酸從K變成E的VP7會大量分泌至胞外、但若組合成DLP (double-layer particle) 或完整的TLP (triple-layer particle) ，則上述三種組合都會在胞內表現。

關鍵字

桿狀病毒表現系統 ； 輪狀病毒

並列摘要

Abstract
Virus-like particles (Virus-like particle, VLP) is applied extensively, for instance, subunit vaccine production, gene/drug-delivery systems, and even as nanomaterial templates. The experiment can be divided into two parts: (I) the use of multi-effect-type baculovirus expression system to produce (G9 rotavirus) major structural protein of the outer layer (outer structure protein) VP4 and VP7. Also, the common performances of two structures protein and core structural proteins VP2 and VP6 inner structure of proteins. The mutual presentation of VP2 and VP6 is predicted to self-assemble into 50 to 70 nm virus-like particles. (II) laboratory personnel have previously successfully used the baculovirus expression vector dual effect on the performance of Sf21 insect cells alone single VP2, VP6, VP7 structural proteins, VP2 and VP6 proteins perform in the cell, but the VP7 amino acid will not be showed massively until mutations from K into E (K223E), and secreted to the extracellular. The purpose of this study is to show VP2/7,6/7, 2/6/7, and then analysis by the use of rotavirus goat serum Western blot to analysis. Although the amino acids can be observed from K into E will be a substantial secretion of VP7 to extracellular. However, both double-layer particle (DLP) and triple-payer particle (TLP) can be performed in the cell.

並列關鍵字

Baculovirus expression system ； Rotavirus

參考文獻

Angel, J., Franco, M.A., and Greenberg, H.B. (2007). Rotavirus vaccines： recent developments and future considerations. Nat Rev Microbiol 5, 529-539.

Arnoldi, F., Campagna, M., Eichwald, C., Desselberger, U., and Burrone, O.R. (2007). Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2. J Virol 81, 2128-2137.

Beards, G.M., and Brown, D.W. (1988). The antigenic diversity of rotaviruses： significance to epidemiology and vaccine strategies. Eur J Epidemiol 4, 1-11.

Bertolotti-Ciarlet, A., Ciarlet, M., Crawford, S.E., Conner, M.E., and Estes, M.K. (2003). Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice. Vaccine 21, 3885-3900.

Bishop, R.F. (1996). Natural history of human rotavirus infection. Arch Virol Suppl 12, 119-128.