Comparison of Bombyx mori polyhedrosis virus expression vectors utilizing chloramphenicol acrtyl transferase as reporter gene

• Main Authors: Wen-Hui Su, 蘇文慧
• Other Authors: Roger F. Hou, Nien-Tai Hu
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/03355402786293119560

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.