Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus.
The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.
Choi
Jae Young
JY
Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea.
Kim
Yang-Su
YS
Wang
Yong
Y
Kang
Joong Nam
JN
Roh
Jong Yul
JY
Shim
Hee Jin
HJ
Woo
Soo-Dong
SD
Jin
Byung Rae
BR
Je
Yeon Ho
YH
D000818:Animals; D001426:Bacterial Proteins; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018071:Polydnaviridae / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D014162:Transfection; D014863:Wasps / Q000821:virology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease