表現登革病毒2型PL046外膜蛋白以用於建構假型小鼠白血病病毒

Abstract

登革病毒（Dengue virus）屬於黃質病毒科（Flaviviridae），為一正向單股（positive single stranded）的RNA病毒，並具有外膜蛋白（E：envelope protein），是引起登革熱與出血性登革熱的病原體，且有日趨嚴重的現象，已漸漸成為世界性的公共衛生問題。研究顯示在登革病毒進入細胞的過程當中，其外膜蛋白扮演了一個重要的角色。它是一個由四百九十五個胺基酸所構成的醣蛋白，位於病毒脂質雙層膜的外側，是一個穿膜蛋白質。外膜蛋白外露在病毒膜外的部分，被認為可以和其受體結合。由於登革病毒感染細胞之後，細胞型態變化不大，因此對於感染成功與否與實驗的有效性多半靠溶斑試驗（plaque assay）來確認，且被感染的細胞株並非每一種細胞株都會產生溶斑（plaque），加上檢定plaque形成的時間需5~7天，造成研究登革病毒的困難度。因此本研究的主要目的，其一為表現登革病毒的外膜蛋白，其二為建立一帶有報導基因之假病毒（pseudotype virus）模式，以應用於登革病毒感染過程的相關研究。將登革病毒外膜蛋白(E)表現質體、外膜蛋白基因缺陷之小鼠白血病病毒（murine leukemia virus）複製及結構表現質體以及帶有β-galactosidase之複製缺失基因組表現質體共同轉染哺乳動物細胞株，使其組裝產生假病毒顆粒。本研究結果顯示：(一)登革病毒二型的全長外膜蛋白(DV-2 full-length E protein)在E.coli Novablue(DE3)之表現，可使用anti-His-HRP抗體藉由Western blot偵測到recombinant protein的表現，惟在哺乳類BHK21細胞株中無法偵測。(二)由穩定細胞株的細胞融合現象推測E protein會表現在細胞膜上。(三) 雖然在我目前的實驗條件下，還無法成功的產生假型病毒顆粒，但根據前人研究證明：小鼠白血病病毒（MLV）與C型肝炎病毒（HCV）組合成假型病毒顆粒是可行的，而C型肝炎病毒屬於黃質病毒科，其與登革病毒有相同的結構及組成，所以未來可繼續改善方法與技術以成功產生假型病毒顆粒。

Dengue virus (DV), a member of the family Flaviviridae, is an enveloped, single-stranded positive sense RNA virus. Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by the dengue viruses that represent a global public health problem. In the process of virus entry, the envelope protein (E protein) plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. The extracellular domain of E protein is thought to interact with the host cell receptor. There is no discernable change in the host cell that infected by dengue virus. In general, one can only use plaque assay to determine whether the propagation is successful. However, not every cell line infected can produce plaques and even if it can, the plaque formation needs 5~7 days. Those events make research in dengue virus difficult. The objectives of this study were to generate infectious pseudotyped-particles that were assembled by functional dengue virus E proteins onto retroviral core particles. In this assay, mammalian cells are cotransfected with 3 plasmids, one expressing the retroviral proteins—expect the envelope—from murine leukemia virus, another expressing the replication-defective retroviral genome with β-galactosidase gene marker, and the other expressing the E gene from dengue virus. In conclusion: First, the recombinant proteins of DV-2 E protein fused with His-tag can be detected in the E.coli expression system by Western blotting analysis but not in BHK21. Second, the DV E proteins can reach the cell surface because the stable cell lines transfected with the plasmid expressing DV E gene displayed syncytium. Third, in this study, no pseudotype viruses were detected. Further studies are required.