Expression and Characterization of RSV fusion protein

• Main Author: 陳孟辰
• Other Authors: 周彥宏
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/29590218293790779414

碩士 === 國立清華大學 === 生物科技研究所 === 99 === Respiratory syncytia virus (RSV) has been recognized as the leading cause of lower respiratory infections in children and in elderly. Human RSV occurs in yearly outbreaks and is highly contagious. As a result, it causes mild cold symptoms following virus exposure could often progress to more serious lower respiratory tract diseases such as bronchilitis and pneumonia. Currently no effective vaccine is available. Previous studies have shown that RSV fusion (F) protein elicits neutralizing antibody, induces CTL responses and stimulates an increased production of Th1 cytokines, IL-2, IL-12 and IFN-γ raised against live RSV infection and importantly does not induce vaccine-enhanced diseases. In this rationality, full length RSV fusion protein (F) and transmembrane domain truncated F protein (F0ΔTM) are chosen as target antigens of RSV vaccine development. To investigate the antigenicity of F0 and F0ΔTM in animal immunization study, expression of His-tagged F and F0ΔTM in E. coli BL21 (DE3) cells, baculovirus/Sf9 insect cells, and mammalian 293 cells, which possess the different mechanisms of protein expression and translational modification, were performed. Recombinant protein expressed in three different systems were examined by performing SDS-PAGE, immunoblotting and peptide N-terminal sequencing. According to the results, recombinant fusion protein F0ΔTM expressed by mammalian 293 cells correspond to our original recombinant protein design. Therefore, this recombinant fusion protein F0ΔTM was purified and injected into BALB/C mice for animal immunization study compared with heat inactivated RSV B1 (HI-RSV B1). Mouse serum was performed with a series of assay including titer of antibody IgG, IgG1, IgG2a determination and plaque-reduction neutralization test. As the result, titer of anti-virus antibody raised by fusion protein F0ΔTM was same with HI-RSV B1 and also the ability of live RSV neutralization. Moreover, this antigen stimulates a immune response toward TH1 pathway. From the indications of these consequence, we could say that recombinant fusion protein F0ΔTM expressed by 293A cells is a good protective antigen for raising correct and strong humorial immunity.