| Project/Area Number |
17560690
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | Kobe University |
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Principal Investigator |
YAMAJI Hideki Kobe University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (40283874)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Insect cell / Baculovirus / Antibody |
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| Research Abstract |
Display of foreign peptides or proteins on the surface of virus particles has proven to be valuable for selection of genes encoding novel functions from diverse libraries. Recently, baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) has been successfully used for displaying heterologous proteins on the surface of viral particles by fusing the protein to the major baculoviral envelope glycoprotein gp64. On infection of insect cells with such a recombinant baculovirus, gp64-fusion proteins as well as gp64 are expressed and transported to the cell membrane where they are picked up by progeny viruses during the budding process. In this study, generation of recombinant baculovirus displaying an antibody Fab fragment on its surface was investigated. Recombinant baculovirus was designed so that the gene encoding the light chain of the Fab fragment was expressed as a fusion to the N-terminus of gp64, while at the same time the gene of the Fd fragment of the Fab fragment was expressed as a secretion protein. Following infection of Sf9 insect cells with the recombinant baculovirus, culture broth of infected cells was analyzed by enzyme-linked immunosorbent assay (ELISA), indicating that the culture broth has an antigen-binding activity. The Fab fragments were successfully detected on the surface of Sf9 cells infected with the recombinant baculovirus by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated antibody specific to the Fab fragment. These results suggest that antibody Fab fragments can be displayed on the surface of baculovirus particles in an active form and that baculoviruses displaying desired Fab fragments can be selected by the use of fluorescence-activated cell sorter (FACS) with a fluorescence-labeled antigen. The successful display of a functional antibody Fab fragment may offer a novel approach for efficient selection of specific antibodies.
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