Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification...[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.展开更多
Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been re- ported to be associated with virus-induced cell apoptosis and pathogenicity,...Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been re- ported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral rep- lication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96―129 bp located between ORF A1and ORF A2 of genomic segment A of IBDV strain HZ2 were de- leted, and an Nhe I (GcTaGc) site was inserted at 96―102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chi- meric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the patho- genesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.展开更多
Dear Editor,Myristoylation is a naturally occurring post-translational modification for targeting cytoplasmic proteins to intracellular membranes.Unlike enveloped animal viruses,which enter host cells by membrane fusi...Dear Editor,Myristoylation is a naturally occurring post-translational modification for targeting cytoplasmic proteins to intracellular membranes.Unlike enveloped animal viruses,which enter host cells by membrane fusion,nonenveloped animal viruses must disrupt the cell membrane to initiate infection.Some animal viruses and several nonenveloped viruses展开更多
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
基金Supported by the National Natural Science Fundation Item of China(30970578,31070651)"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~
文摘[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.
文摘Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been re- ported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral rep- lication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96―129 bp located between ORF A1and ORF A2 of genomic segment A of IBDV strain HZ2 were de- leted, and an Nhe I (GcTaGc) site was inserted at 96―102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chi- meric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the patho- genesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.
基金supported in part by grants from the National Natural Science Foundation of China (31672693, 31372565, 31172434)
文摘Dear Editor,Myristoylation is a naturally occurring post-translational modification for targeting cytoplasmic proteins to intracellular membranes.Unlike enveloped animal viruses,which enter host cells by membrane fusion,nonenveloped animal viruses must disrupt the cell membrane to initiate infection.Some animal viruses and several nonenveloped viruses
