The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian...The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian sheep blowfly Lucilia cuprina(LcEcR). pSXIVVI +X3/2 baculovirus transfer vector was chosen for a 2.8kb LcEcR cDNA subcloning since pSXIVVI +X3/2 contains an efficient translational initiation signal (ATG) and it allows the LcEcR cDNA fusion to N-terminal codons in the correct reading frame. The resulting transfer plasmid pSXIVVI +X3-LcEcR was cotransfected into BT1-Tn-5B1-4 cells with the parental virus TnNPV-SVI -G minus polyhedral inclusion body, which expresses β-galactosidase gene. After 3～4 runs of plaque purification, three TnNPV-LcEcR clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters. These three TnNPV-LcEcR clones all showed white phenotype when stained with X-gal. Western blot analysis showed 2～3 specific polypeptides with molecular weight ranging from 70～90kD. Three TnNPV-LcEcR clones expressed different level of LcEcR polypeptides in BT1-Tn-5B1-4 cells. The TnNPV-LcEcR-1 clone expressed the highest level of LcEcR polypeptides in BT1-Tn-5B1-4 cells 48～72h post infection.展开更多
文摘The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian sheep blowfly Lucilia cuprina(LcEcR). pSXIVVI +X3/2 baculovirus transfer vector was chosen for a 2.8kb LcEcR cDNA subcloning since pSXIVVI +X3/2 contains an efficient translational initiation signal (ATG) and it allows the LcEcR cDNA fusion to N-terminal codons in the correct reading frame. The resulting transfer plasmid pSXIVVI +X3-LcEcR was cotransfected into BT1-Tn-5B1-4 cells with the parental virus TnNPV-SVI -G minus polyhedral inclusion body, which expresses β-galactosidase gene. After 3～4 runs of plaque purification, three TnNPV-LcEcR clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters. These three TnNPV-LcEcR clones all showed white phenotype when stained with X-gal. Western blot analysis showed 2～3 specific polypeptides with molecular weight ranging from 70～90kD. Three TnNPV-LcEcR clones expressed different level of LcEcR polypeptides in BT1-Tn-5B1-4 cells. The TnNPV-LcEcR-1 clone expressed the highest level of LcEcR polypeptides in BT1-Tn-5B1-4 cells 48～72h post infection.
