本研究旨在表达口蹄疫病毒(FMDV)的VP1全基因并制备特异性的多克隆抗体。利用PCR方法扩增Asia 1 IND 49197株VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6ku,以包...本研究旨在表达口蹄疫病毒(FMDV)的VP1全基因并制备特异性的多克隆抗体。利用PCR方法扩增Asia 1 IND 49197株VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6ku,以包涵体的形式存在。通过Ni-NTA Purification System纯化后进行western blot和间接ELISA分析,结果显示重组蛋白能够被FMD阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,ELISA测定抗体效价为1∶20480,病毒中和试验测定抗体效价为1∶64。本研究所表达的VP1蛋白可用于开发检测Asia1口蹄疫抗体的诊断试剂,所制备的多克隆抗体为进一步研究VP1的结构、功能以及抗原表位的鉴定提供了条件。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.
