摘要
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.
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.
出处
《中国病毒学》
CSCD
2007年第5期397-404,共8页
Virologica Sinica
基金
National Natural Science Foundation of China (Grant Nos 30470074,30671615)
Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-N-021).
关键词
草鱼呼肠孤病毒
VP6蛋白
增强绿色荧光蛋白
杆状病毒表达系统
共表达
Grass carp reovirus （GCRV）
Inner capsid protein VP6
Enhanced green fluorescence protein （ eGFP）
Baculovirus expression system
Co-expression
