碩士 / 國立臺灣大學 / 漁業科學研究所 / 85 / In order to understand the physiology of the fish vision, we
constructed a plasmid, pET32-Rho, which encoded the coding
region of rhodopsin cDNA and was driven by T7 promoter. The
recombinant rhodopsin is a fusion protein with thioredoxin and 6
histidine residues at the amino terminus. After IPTG induction,
the cell extracts of Escherichia coli BL21(DE3) containing
pET32-Rho was collected and run through the Ni2+ affinity column
chromatography, and then their proteins were analyzed by SDS-
polyacrylamide gel electrophoresis. Results demonstrated that a
band with molecular mass of 55 kD was shown on the gel, but no
positive reaction for western blot analysis by using anti-bovine
rhodopsin polyclonal antibody(CERN886). The coding region of
rhodospin cDNA was inserted into the baculovirus transfer
vector, pBAC-Rho, under the control of the polyhedrin promoter.
Linearlized virus DNA and the construct plasmid were co-infected
Sf9 cells. Cells infected by recombinant virus were screened,
purified and amplified. Extracts of insect cells infected with
the recombinant virus at MOI of 5 after 72 hours. postinfection
we recollected and treated with histidine-bind column.. Three
major bands of 58 kD, 40 kD, and 36 kD were shown on the gel.
However ,they were all immunologically positive with the
polyclonal antibody (CERN886) in which the 40 kD band was
excepted as recombinant rhodopsin. Further confirmation study
remains to be investigated.
| Identifer | oai:union.ndltd.org:TW/085NTU00451014 |
| Date | January 1997 |
| Creators | Jeng, Juinn-ho, 鄭俊和 |
| Contributors | Tsai Huai-Jen, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 66 |
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