A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli.
Developing cost-effective methods for high throughput production of recombinant baculoviruses in insect cells is very challenging, because the baculovirus DNA preparation and the following transfection procedure are labour-intensive and time consuming. We developed a new method of introducing recombinant Bacmid DNA from bacteria into insect cells simply using invasive diaminopimelate (DAP) auxotrophic Escherichia coli to infectSpodoptera frugiperda 9 cells. The E. coli cells with recombinant Bacmids enter insect cells with the help of the invasion factor from Yersinia pseudotubercolusis. Without DAP in medium, the cell wall of DAP auxotrophic E. coli cannot be synthesized so that the bacterial cell will disrupt and release recombinant Bacmid. The released Bacmids will generate infective recombinant baculovirus particles in insect cells. We combined this E. coli invasion method with the zero background transposition system to generate recombinant baculovirus in a rapid and simple way. Without preparation and purification of recombinant Bacmids from E. coli and the labour-intensive and complex transfection procedure, this transfection reagent free method enables a convenient and economic high throughput production of recombinant baculoviruses.
Yao
Lun guang
Lg
China-UK, Nan Yang Normal University-Rothamsted Research Joint Laboratory of Insect Biology, People's Republic of China. lunguangyao@163.com
Sun
Jing chen
Jc
Xu
Hua
H
Kan
Yun chao
Yc
Zhang
Xiang man
Xm
Yan
Hui Chao
HC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification; D003960:Diaminopimelic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D018411:Spodoptera / Q000235:genetics*
D004274:DNA, Recombinant; D003960:Diaminopimelic Acid