Equine infectious anemia virus (EIAV) causes equine infectious anemia and persistent infection as well as repeated high-titer viremia in horses.The long-terminal repeat (LTR) of EIAV proviral genome contains an eukary...Equine infectious anemia virus (EIAV) causes equine infectious anemia and persistent infection as well as repeated high-titer viremia in horses.The long-terminal repeat (LTR) of EIAV proviral genome contains an eukaryotic promoter which plays an important regulation role on transcription and replication of the virus.In the study,the LTRs of an infectious molecular clone derived from EIAV donkey leukocyte-attenuated vaccine virus (DLA) were substituted with those of EIAV L strain,a virulent EIAV from which DLA strain was derived,resulting in a chimeric plasmid pOK-LTR DLA/L.The purified pOK-LTR DLA/L was used to transfect leukocyte cultures from EIAV-negative donkey.Reverse transcriptase (RT) activity was detectable by the 7th passage in cell cultures of the transfection products.The cytopathogenic effects typical for EIAV infection were developed since the 8th passage of cell cultures,and virions with cone-shaped core could be observed under electron microscope.It proved that the constructed chimeric pOK-LTR DLA/L is infective and it may be used in future for exploring of molecular mechanism of pathogenesis and immunity of EIAV.展开更多
为构建Asia1型口蹄疫病毒Asia1/YS/CHA/05株的感染性克隆,根据该毒株的全基因组序列,将其分为6段进行RT-PCR扩增,并拼接克隆至pBluescript II SK(+)载体中,构建了全长基因组的cDNA克隆pAsi。用NotⅠ将质粒线性化后体外转录并转染BHK-21...为构建Asia1型口蹄疫病毒Asia1/YS/CHA/05株的感染性克隆,根据该毒株的全基因组序列,将其分为6段进行RT-PCR扩增,并拼接克隆至pBluescript II SK(+)载体中,构建了全长基因组的cDNA克隆pAsi。用NotⅠ将质粒线性化后体外转录并转染BHK-21细胞,转染细胞中出现明显的细胞病变。RT-PCR结合序列分析表明,拯救病毒中作为人工设计的分子标记PstⅠ酶切位点被消除,因此,排除了亲本病毒污染的可能。该感染性克隆的构建,为深入探讨Asia1型口蹄疫病毒的致病机理以及新型疫苗的研制奠定了基础。展开更多
为检测高致病性猪繁殖与呼吸综合征病毒(PRRSV)感染的猪胸腺中凋亡相关因子的变化,本研究针对猪TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因及内参β-actin基因设计特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标...为检测高致病性猪繁殖与呼吸综合征病毒(PRRSV)感染的猪胸腺中凋亡相关因子的变化,本研究针对猪TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因及内参β-actin基因设计特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因的SYBR Green I qRT-PCR方法。结果显示,该方法线性关系好(R2≥0.998),敏感性高,初始模板的检出下限为10拷贝/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,批内、批间重复试验的变异系数均小于3%,具有良好的重复性。本研究为细胞凋亡相关因子变化趋势的研究提供方法。展开更多
文摘Equine infectious anemia virus (EIAV) causes equine infectious anemia and persistent infection as well as repeated high-titer viremia in horses.The long-terminal repeat (LTR) of EIAV proviral genome contains an eukaryotic promoter which plays an important regulation role on transcription and replication of the virus.In the study,the LTRs of an infectious molecular clone derived from EIAV donkey leukocyte-attenuated vaccine virus (DLA) were substituted with those of EIAV L strain,a virulent EIAV from which DLA strain was derived,resulting in a chimeric plasmid pOK-LTR DLA/L.The purified pOK-LTR DLA/L was used to transfect leukocyte cultures from EIAV-negative donkey.Reverse transcriptase (RT) activity was detectable by the 7th passage in cell cultures of the transfection products.The cytopathogenic effects typical for EIAV infection were developed since the 8th passage of cell cultures,and virions with cone-shaped core could be observed under electron microscope.It proved that the constructed chimeric pOK-LTR DLA/L is infective and it may be used in future for exploring of molecular mechanism of pathogenesis and immunity of EIAV.
文摘为构建Asia1型口蹄疫病毒Asia1/YS/CHA/05株的感染性克隆,根据该毒株的全基因组序列,将其分为6段进行RT-PCR扩增,并拼接克隆至pBluescript II SK(+)载体中,构建了全长基因组的cDNA克隆pAsi。用NotⅠ将质粒线性化后体外转录并转染BHK-21细胞,转染细胞中出现明显的细胞病变。RT-PCR结合序列分析表明,拯救病毒中作为人工设计的分子标记PstⅠ酶切位点被消除,因此,排除了亲本病毒污染的可能。该感染性克隆的构建,为深入探讨Asia1型口蹄疫病毒的致病机理以及新型疫苗的研制奠定了基础。
文摘为检测高致病性猪繁殖与呼吸综合征病毒(PRRSV)感染的猪胸腺中凋亡相关因子的变化,本研究针对猪TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因及内参β-actin基因设计特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因的SYBR Green I qRT-PCR方法。结果显示,该方法线性关系好(R2≥0.998),敏感性高,初始模板的检出下限为10拷贝/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,批内、批间重复试验的变异系数均小于3%,具有良好的重复性。本研究为细胞凋亡相关因子变化趋势的研究提供方法。
