2003 Fiscal Year Final Research Report Summary
Expression of proteins using the HIV virus vectors
| Project/Area Number |
14570814
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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 |
Dermatology
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| Research Institution | Oita University Faculty of Medicine |
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Principal Investigator |
NAKASHIMA Kazutoshi Oita University Faculty of Medicine, Faculty of Medicine, Research Assistant, 医学部, 助手 (90305037)
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| Co-Investigator(Kenkyū-buntansha) |
UNOSHIMA Masako Oita University Faculty of Medicine, Faculty of Medicine, Research Assistant, 医学部, 助手 (90336256)
HIRAMATSU Kazufumi Oita University Faculty of Medicine, Faculty of Medicine, Research Assistant, 医学部, 助手 (80301381)
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| Project Period (FY) |
2002 – 2003
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| Keywords | HIV virus vectors / protease nexin- |
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| Research Abstract |
Establishment to clone the cells transfected with HIV virus vectors Cultured 293 cells were transfected with reconstructed HIV virus vectors containing GFP gene insert.
Cloning the expression cells has been established, maintaining their morphology and growth rate before being transfected. It has been confirmed that the GFP proteins, which were detected as fluorescent signals, were successfully expressed in almost all the cells, whereas efficiency of the expression decreased as passages were repeated.
Establishment to reconstruct HIV virus vectors carrying protease nexin-1 gene SIN vector plasmid was subcloned with a full length of protease nexin-1 (PN-1)cDNA.
The cultured 293 cells were transfected together with the SIN, package, Rev, and envelope plasmid, and then the supernatant containing the reconstructed HIV virus vectors were collected.
The titer of the HIV vectors was estimated by determining the quantity of p24 HIV proteins using sandwich ELISA method, showing 10 3 TU (transfection unit) / ml. Expression of PN-1 proteins in the cultured cells using the HIV vectors The 293 cells were transfected with the HIV vectors containing the PN-1 cDNAs. The efficiency of the PN-1 expression was estimated to be less than 10%. Preparation of anti-PN-1 antibody was set about to detect the PN-1 proteins expressed in the infected cells.
The serum obtained from rabbits, which have been immunized with PN-1 peptides, was purified and concentrated using protein-G column. No obvious band of PN-1 was detected in Western blotting method. To obtain more efficient expression of PN-1, the HIV vectors were concentrated by means of centrifuge, but the titer
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