Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector
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Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector. / Zuidema, D.; Schouten, A.; Usmany, M.; Maule, A. J.; Belsham, G. J.; Roosien, J.; Klinge-Roode, E. C.; Van Lent, J. W.M.; Vlak, J. M.
In: Journal of General Virology, Vol. 71, No. 10, 1990, p. 2201-2209.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector
AU - Zuidema, D.
AU - Schouten, A.
AU - Usmany, M.
AU - Maule, A. J.
AU - Belsham, G. J.
AU - Roosien, J.
AU - Klinge-Roode, E. C.
AU - Van Lent, J. W.M.
AU - Vlak, J. M.
PY - 1990
Y1 - 1990
N2 - 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 Escherichia coli β-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene was transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (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 M(r) 46,000. 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.
AB - 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 Escherichia coli β-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene was transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (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 M(r) 46,000. 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.
UR - http://www.scopus.com/inward/record.url?scp=0025146897&partnerID=8YFLogxK
U2 - 10.1099/0022-1317-71-10-2201
DO - 10.1099/0022-1317-71-10-2201
M3 - Journal article
C2 - 2230725
AN - SCOPUS:0025146897
VL - 71
SP - 2201
EP - 2209
JO - Journal of General Virology
JF - Journal of General Virology
SN - 0022-1317
IS - 10
ER -
ID: 381227868