Expressing dengue virus 2 E protein for pseudotype formation of murine leukemia virus with dengue virus envelope

• Main Authors: Po-Yin Lin, 林柏吟
• Other Authors: Yun-Liang Yang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/35241300985270384102

碩士 === 國立交通大學 === 生化工程研究所 === 94 === 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.