We have now construted a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) Cry1Ac crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Suprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus paticles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantrea cunea, was strikingly improved in comparison with the wild-type AcNPV.
Bacillus thuringiensis* ; Bacillus* ; Baculoviridae* ; Insects ; Nucleopolyhedrovirus

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Baculoviridae ; Gene Expression ; Insects* ; Nucleopolyhedrovirus* ; Sf9 Cells

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Baculoviridae ; Gene Expression ; Insects* ; Nucleopolyhedrovirus* ; Sf9 Cells

No abstract available.
Baculoviridae* ; Glycoproteins* ; Insects*

No abstract available.
Baculoviridae* ; Bombyx* ; Coinfection*

No abstract available.
Baculoviridae* ; Herpesvirus 1, Suid* ; Pseudorabies*

Bovine growth hormone (bGH) gene was expressed in an insect spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into s. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication patters of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern lot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml (106 cells) by radioimmunoassay.
Baculoviridae* ; Cell Line ; Clone Cells ; DNA ; DNA, Complementary ; Growth Hormone* ; Insects ; Nucleopolyhedrovirus* ; Plasmids ; Polymerase Chain Reaction ; Radioimmunoassay ; Spodoptera

No abstract available.
Animals ; Baculoviridae* ; Newcastle disease virus* ; Newcastle Disease*

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the peal-pol clone (Lee et al 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind 75 phage promoter and the 6x His region of the pQE-30 expression vector, and it was called pQEVP1. Again, the 6xHis-tagged VP1 DNA fragment in the pOEVPl was cleaved with EcoRl and transferred into the VP1 site of the pBacVPl, resulting pBacHis-VPl recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (Lacz-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay, The recombinant virus was named VP1-HcNPV-1. The 6xHis-tagged VP1 protein produced by the pQEVPl was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was 2.0x10(5) pfu/ml at 7 days postinfection.
Bacteriophages ; Baculoviridae ; Blotting, Western ; Chromatography ; Clone Cells ; DNA ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Genome ; Inclusion Bodies ; Infectious pancreatic necrosis virus* ; Molecular Weight ; Nucleopolyhedrovirus* ; Recombination, Genetic* ; RNA

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.
Animals ; Baculoviridae ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Larva ; genetics ; virology ; Nucleopolyhedrovirus ; genetics ; Spodoptera ; genetics ; virology