旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blo...旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blot验证该重组蛋白能够与小鼠抗轮状病毒血清反应。将该重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性杂交瘤细胞,Western blot和间接免疫荧光验证阳性杂交瘤细胞分泌的单克隆抗体与RVA的反应性。经3轮亚克隆最终获得一株能稳定分泌针对VP6蛋白单克隆抗体的阳性杂交瘤细胞株5G9,其分泌的单抗亚类为IgG1,轻链类型为Kappa,制备的小鼠腹水效价可达1∶4096000,能够与不同基因型的RVA反应,同时不与常见的猪腹泻相关病毒反应,为未来猪RVA相关基础研究、临床血清学检测方法的开发奠定了基础。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
Grass carp reovirus(GCRV)is the causative agent of grass carp hemorrhagic disease,which has detrimental effects on the grass carp aquaculture industry.There are four known genotypes of GCRV and strains of different GC...Grass carp reovirus(GCRV)is the causative agent of grass carp hemorrhagic disease,which has detrimental effects on the grass carp aquaculture industry.There are four known genotypes of GCRV and strains of different GCRV genotypes differ greatly.In this study,the diversity of the protein VP6 from different GCRV stains and the effect of genotype GCRV 096 on replication was investigated in CIK cells.Our results showed that the VP6 protein of GCRV 096(genotypeІ)exhibited limited homology to that of GCRV GD108(genotypeШ),with few residues conserved in predicted protein-protein interaction domains.GCRV 096 VP6 protein was expressed and purified and an antiserum against it was characterized.Addition of purified VP6 protein or antiserum to culture media of CIK cells inhibited the replication of GCRV 096 in these cells.In contrast,replication of GCRV GD108(genotypeШ)was not affected in CIK cells under the same condition.Overall,our results indicated that the protein VP6 and VP6 antiserum did not provide cross-protection against GCRV strains and this can be attributed to differences among GCRV genotypes.It will be important to consider multiple GCRV genotypes in the development of effective GCRV vaccines and other therapies against grass carp hemorrhagic disease.In addition,bioinformatics analysis also suggested that the protein VP6 may have a role in the process of GCRV infection.This study lays the foundation for the prevention of grass carp hemorrhagic disease and further detailed studies on the pathogenesis of GCRV.展开更多
文摘旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blot验证该重组蛋白能够与小鼠抗轮状病毒血清反应。将该重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性杂交瘤细胞,Western blot和间接免疫荧光验证阳性杂交瘤细胞分泌的单克隆抗体与RVA的反应性。经3轮亚克隆最终获得一株能稳定分泌针对VP6蛋白单克隆抗体的阳性杂交瘤细胞株5G9,其分泌的单抗亚类为IgG1,轻链类型为Kappa,制备的小鼠腹水效价可达1∶4096000,能够与不同基因型的RVA反应,同时不与常见的猪腹泻相关病毒反应,为未来猪RVA相关基础研究、临床血清学检测方法的开发奠定了基础。
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.
基金This study was supported by National Natural Science Foundation of China(No.31602199)the Natural Science Foundation of Guangdong Province in China(No.2015A030313622)+3 种基金the Foundation of Guangdong Ocean University(K15246)the Foundation of Zhanjiang(No.2015A03027)the National 973 Plan Project in China(No.2009CB118704)and the project of Department of Education of Guangdong province in China(No.2012LYM_0075).
文摘Grass carp reovirus(GCRV)is the causative agent of grass carp hemorrhagic disease,which has detrimental effects on the grass carp aquaculture industry.There are four known genotypes of GCRV and strains of different GCRV genotypes differ greatly.In this study,the diversity of the protein VP6 from different GCRV stains and the effect of genotype GCRV 096 on replication was investigated in CIK cells.Our results showed that the VP6 protein of GCRV 096(genotypeІ)exhibited limited homology to that of GCRV GD108(genotypeШ),with few residues conserved in predicted protein-protein interaction domains.GCRV 096 VP6 protein was expressed and purified and an antiserum against it was characterized.Addition of purified VP6 protein or antiserum to culture media of CIK cells inhibited the replication of GCRV 096 in these cells.In contrast,replication of GCRV GD108(genotypeШ)was not affected in CIK cells under the same condition.Overall,our results indicated that the protein VP6 and VP6 antiserum did not provide cross-protection against GCRV strains and this can be attributed to differences among GCRV genotypes.It will be important to consider multiple GCRV genotypes in the development of effective GCRV vaccines and other therapies against grass carp hemorrhagic disease.In addition,bioinformatics analysis also suggested that the protein VP6 may have a role in the process of GCRV infection.This study lays the foundation for the prevention of grass carp hemorrhagic disease and further detailed studies on the pathogenesis of GCRV.
