应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p Q...应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p QENS2转化 JM10 9菌株 ,诱导表达出 N端含有 6个 His的全长 NS2蛋白 ,用 Ni-NTA- agarose柱层析纯化 ,获得提纯的全长 NS2蛋白 .展开更多
The NS2 gene of Rice stripe virus(RSV) was amplified by RT-PCR,cloned into pGEM-T vector and sequenced.The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2.The ...The NS2 gene of Rice stripe virus(RSV) was amplified by RT-PCR,cloned into pGEM-T vector and sequenced.The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2.The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLysS.SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG.The recombinant NS2 protein was purified with Ni2+-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit.NS2 protein was successfully detected in small brown planthopper(Laodelphax striatellus) at 1∶1 600 dilution of the total protein of single planthopper and in infected rice(Oryza sativa) at 1∶800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.展开更多
文摘The NS2 gene of Rice stripe virus(RSV) was amplified by RT-PCR,cloned into pGEM-T vector and sequenced.The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2.The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLysS.SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG.The recombinant NS2 protein was purified with Ni2+-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit.NS2 protein was successfully detected in small brown planthopper(Laodelphax striatellus) at 1∶1 600 dilution of the total protein of single planthopper and in infected rice(Oryza sativa) at 1∶800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.