1887

Abstract

An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the β-galactosidase gene () as a genetic marker for positive recombination between wt nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, is transcribed from the heat-shock promoter (hsp70), which is constitutively expressed in baculovirus-infected (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of 46000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.

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/content/journal/jgv/10.1099/0022-1317-71-10-2201
1990-10-01
2019-11-21
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http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-10-2201
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