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Arch Virol. 2008;153(9):1667-75. doi: 10.1007/s00705-008-0165-z. Epub 2008 Jul 25.

Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector.

Author information

1
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan.

Abstract

A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3-1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-gamma) and successfully utilized to produce HuIFN-gamma in both BY-2 protoplasts and transgenic BY-2 cells.

PMID:
18654737
DOI:
10.1007/s00705-008-0165-z
[Indexed for MEDLINE]

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