An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was trans...An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.展开更多
番茄褐色皱纹果病毒(tomato brown rugose fruit virus,ToBRFV)于2015年在约旦被首次报道,随即迅速在全世界蔓延。本试验对北京南口、山东寿光及陕西白水出现的疑似ToBRFV典型症状的感病植株的保守结构域进行RT-PCR扩增检测,结果表明ToB...番茄褐色皱纹果病毒(tomato brown rugose fruit virus,ToBRFV)于2015年在约旦被首次报道,随即迅速在全世界蔓延。本试验对北京南口、山东寿光及陕西白水出现的疑似ToBRFV典型症状的感病植株的保守结构域进行RT-PCR扩增检测,结果表明ToBRFV已经在北京、山东和陕西发生。对ToBRFV保守基因RdRP测序发现,北京、山东、陕西感病材料的ToBRFV RdRP基因遗传距离较近,与其他国家的ToBRFV RdRP基因遗传距离较远。展开更多
目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体...目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体中,并在其3′端插入EGFP报告基因,构建融合表达质粒pcDNA3.0-RdRp-EGFP,脂质体法转染PLC/PRF/5、A549和HepG2细胞,荧光显微镜观察报告基因的表达,RT-nPCR检测RdRp基因mRNA的转录情况,Westernblot检测RdRp蛋白的表达。结果重组表达质粒经酶切鉴定和测序证实构建正确;RdRp基因和蛋白在A549和HepG2细胞中可高效、特异性表达。结论成功构建了RdRp基因真核表达质粒,并在A549和HepG2培养细胞中大量表达,为进一步研究RdRp在HEV复制过程中的功能奠定了基础。展开更多
文摘An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.
文摘番茄褐色皱纹果病毒(tomato brown rugose fruit virus,ToBRFV)于2015年在约旦被首次报道,随即迅速在全世界蔓延。本试验对北京南口、山东寿光及陕西白水出现的疑似ToBRFV典型症状的感病植株的保守结构域进行RT-PCR扩增检测,结果表明ToBRFV已经在北京、山东和陕西发生。对ToBRFV保守基因RdRP测序发现,北京、山东、陕西感病材料的ToBRFV RdRP基因遗传距离较近,与其他国家的ToBRFV RdRP基因遗传距离较远。
文摘目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体中,并在其3′端插入EGFP报告基因,构建融合表达质粒pcDNA3.0-RdRp-EGFP,脂质体法转染PLC/PRF/5、A549和HepG2细胞,荧光显微镜观察报告基因的表达,RT-nPCR检测RdRp基因mRNA的转录情况,Westernblot检测RdRp蛋白的表达。结果重组表达质粒经酶切鉴定和测序证实构建正确;RdRp基因和蛋白在A549和HepG2细胞中可高效、特异性表达。结论成功构建了RdRp基因真核表达质粒,并在A549和HepG2培养细胞中大量表达,为进一步研究RdRp在HEV复制过程中的功能奠定了基础。
