| Summary: | 碩士 === 國立中興大學 === 昆蟲學系 === 82 === For comparing the expression level of Bombyx mori nuclear
polyhedrosis virus expression vectors, chloramphenicol
acetyl transferase gene (abbreviated cat) was inserted into
each expression vector as reporter gene. The p10 promoter was
utilized in the transplacement plasmids, pBmp10MZ.CAT and pBmp10
M.CAT, and polyhedrin promoter was constructed in the other
two, pCM101.CAT and pCM114.CAT. Except pBmp10M.CAT, each of
the other three transplacement plasmids carries an Escherichia
coli lacZ gene as marker gene driven by Drosophila melanogaster
heat shock promoter. An SV40 transcription terminator is
included in this gene cassette downstream of the lacZ gene. In
pCM114.CAT, the reporter gene is transcribed in the same
direction as the marker gene. The former is upstream. In pBmp10
MZ.CAT and pCM101.CAT, the two genes are transcribed oppositely
toward each other. These three transplacement plasmids with the
lacZ gene cassette undergo homologous recombination with wild
type virus genome and gave rise to recombinant viruses, Bmp10MZ.
CAT, BmCM101.CAT and BmCM114.CAT. These recombinant viruses can
be distinguished from wild type virus by their ability to form
blue plaques on plates containing X-gal. The lacZ gene cassette
was not present in the transplacement plasmid pBmp10M.CAT.
Recombinant virus Bmp10M.CAT was obtained by cotransfecting BmN
cells with pBmp10M.CAT and the virus genome from a virus Bmp10
MZ that carries the lacZ gene cassette, and selected as
colorless virus that can form polyhedra upon infection. All
four purified recombinant viruses were used to infect BmN
cells. The production of the CAT protein and mRNA were analyzed
via ELISA, SDS-PAGE, Western blot and slot blot. The results
indicated that the RNA expression level from p10 promoter of
Bmp10MZ.CAT is approximately 1.5 times from polyhedrin promoter.
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