Optimization of Environmental Factors in Different Mammalian Cells for Transduction of Genetic Recombinant Baculoviruses into Various Mammalian Cell Lines and Development of a BEFV G Subunit Vaccine by a Novel Baculovirus Display System

• Main Authors: Yu-Chieh Chen, 陳宥婕
• Other Authors: Jue-Liang Hsu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/96673603422221499875

碩士 === 國立屏東科技大學 === 生物科技研究所 === 100 === Baculovirus is a novel vector that as a tool of gene therapy, and provides a useful system for expression of recombination protein in insect and mammalian cells. In this study, we established a transduction protocol using a novel baculovirus display system for efficient gene delivery into various cell lines as a tool in developing subunit vaccines or gene therapy tools. We used newly developed vector pBacCE that including a reporter gene, enhanced green fluorescent protein (EGFP) gene under the CMV promoter and polyhedron promoter, and then clone vesicular stomatitis virus (VSV) G protein gene under the p10 promoter called pBacVCE. Next HA of the membrane protein of avian influenza virus (AIV-HA) constructed to pBacVCE vector, called pBacVCE-HA2. The pBacVCE-HA2 applies to transduction protocol. The HA protein, a foreign protein expressed in mammalian cells, we assay the protein by Western blotting. We have successfully established a novel expression vector and transduction protocol for high level of protein expression in various mammalian cell lines when the genetic recombinant baculovirus transduction occurred at 25℃ for 4 h by using Dulbecco’s phosphate-buffered saline (D-PBS) as the transduction solution. The bovine ephemeral fever virus (BEFV) G protein contains type-specific and neutralizing antigenic sites that can induce protective immunity in cattle. Therefore, we also construct a genetic recombinant baculovirus expressing the bovine ephemeral fever virus (BEFV) G protein for producing subunit vaccine for BEF. To achieve high level expression of BEFV G protein, we have also constructed a novel expression vector that includes quadruple promoters for expressing the BEFV G gene to enhance the production of BEFV G protein. Further, the novel recombinant baculovirus was provided with high level of BEFV G protein by Western blotting. Taken together, these date indicate that the baculovirus vector includes quadruple promoter for expressing BEFV G protein should be a useful subunit vaccine that has some characteristics about high expression, high efficient and lower cost.