th instar silkworms were infected with recombinant baculovirus containing phytase gene or wild type BmNPV at 48hr after ecdysis,then treated with 100ppm Juvenile hormone.It showed that the expression level of phytase ...th instar silkworms were infected with recombinant baculovirus containing phytase gene or wild type BmNPV at 48hr after ecdysis,then treated with 100ppm Juvenile hormone.It showed that the expression level of phytase gene and polyhedrin gene per silkworm was increased by 30% and 40%,respectively.The LT 50 was lengthened for more than 4h,and the average weight of sick silkworm was increased by 10%.The results indicated that the improvement of expression efficiency of phytase gene and polyhedrin gene was mainly caused by longer time of virus replication in silkworm after the treatment of Juvenile hormone.展开更多
Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inne...Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.展开更多
文摘th instar silkworms were infected with recombinant baculovirus containing phytase gene or wild type BmNPV at 48hr after ecdysis,then treated with 100ppm Juvenile hormone.It showed that the expression level of phytase gene and polyhedrin gene per silkworm was increased by 30% and 40%,respectively.The LT 50 was lengthened for more than 4h,and the average weight of sick silkworm was increased by 10%.The results indicated that the improvement of expression efficiency of phytase gene and polyhedrin gene was mainly caused by longer time of virus replication in silkworm after the treatment of Juvenile hormone.
基金National Natural Science Foundation of China (Grant Nos 30470074,30671615)Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-N-021).
文摘Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.
