以多效型桿狀病毒載體系統表現類輪狀病毒粒子及其特性分析

類病毒顆粒(Virus-like particle，VLP)的應用相當廣泛，除了可作為次單位疫苗外，亦可做為基因或藥物的載體以及奈米材料之模板。本實驗利用多效型桿狀病毒表現系統，生產G9輪狀病毒 (G9 rotavirus)主要三個結構蛋白質VP2、VP6及VP7，這些結構蛋白將會自我組裝形成50~70奈米的類病毒顆粒。我們利用桿狀病毒雙效表現載體於Sf21昆蟲細胞中單獨表現VP2、VP6及VP7結構蛋白，以IRES轉譯機制同時表現綠螢光蛋白(enhanced green fluorescent protein，EGFP)，以利重組桿狀病毒的篩選，並可觀察在表現VP2、VP6及VP7結構蛋白時細胞型態是否會變化。以goat輪狀病毒血清進行西方墨點分析，可產生綠螢光的桿狀病毒都可於胞內表現VP2和VP6蛋白，但卻無法表現VP7蛋白。有趣的是，當VP7第223胺基酸由K突變為E（K223E）時卻能大量的表現VP7蛋白，並分泌至細胞外。表現VP6蛋白時，於螢光顯微鏡下可觀察到Sf21細胞呈現不規則多角型的型態，推測有可能是VP6蛋白形成管柱狀結構並包埋著EGFP蛋白，而形成類似奈米螢光管柱之結構，此現象可直接於顯微鏡平台上觀察到其Sf21細胞是呈現不規則多角型的型態。於電子顯微鏡觀察結果，發現此管柱狀結構有整束形狀或纖維狀型態呈現，當此結構組裝同時可能將綠螢光蛋白導入這些結構之空隙，並皆分佈於細胞質中。此外，由蔗糖梯度結果可粗略推測此管柱狀結構為開放式型態。當我們進一步利用多效基因表現載體，共同表現VP2、VP6結構蛋白時細胞型態，則由多角型改變成圓形，此結果顯示VP2和VP6結構蛋白間可能存在交互作用，使得管柱狀結構的VP6無法形成，並結合VP2/6 (2/6 double layered VLP) 約60奈米大小的雙層病毒粒子。

關鍵字

輪狀病毒；桿狀病毒；類病毒顆粒

並列摘要

Abstract Virus-like particles have a wide range of application, for instance, subunit vaccine production, gene/drug-delivery systems, and even as nanomaterial templates. We utilized the polycistronic baculovirus expression system to produce G9 strain rotavirus which contain three major structural proteins VP2, VP6 and VP7. Such viral structural proteins self assemble to form 50~70 nm virus-like particle. Initially, VP2, VP6 and VP7 structural proteins were expressed in an insect cell using bicistronic vector which employed IRES translation mechanism that co-expressed EGFP. The beneficial recombination virus selection facilitated the detection of change in cell morphology when VP2, VP6 and VP7 structural proteins were expressed in the cell. Western blot analysis of recombinant virus-like particles using goat antiserum against rotavirus revealed the potentiality of baculovirus to express VP2 and VP6 proteins along with EGFP but not VP7 protein. However, when the VP7 mutation at 223 residue (K223E) was expressed; it was over expressed and secreted into the medium. Moreover, when VP6 protein expression was observed in fluorescence microscope, irregular morphology of Sf21 cell was noticed. We assumed that tubular structure of VP6 protein is due to the packaging of EGFP which resembles fluorescence nanotubes morphology. Observation of cells on transmission electron microscope showed that tubular structure was distributed in cytoplasm as singular or complex form and some of these tubular structure was found to be in open form. Furthermore, when polycistronic gene expression vector was used to co-express VP2 and VP6 proteins, the morphology of the cells appeared to be circular and 60 nm size double-layered VLP were observed in the cytoplasm. Taken together, these results suggest that the new conformation due to the possible interaction between VP2 and VP6 structural proteins.

並列關鍵字

VLP ； Baculovirus ； Rotavirus

參考文獻

Adams, W.R., and Kraft, L.M. (1963). Epizootic diarrhea of infant mice： indentification of the etiologic agent. Science 141, 359-360.

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.

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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.

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以多效型桿狀病毒載體系統表現類輪狀病毒粒子及其特性分析

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