本研究利用CTAB法提取感染枣疯病的枣树叶片总DNA,再通过氯化铯-双苯酰亚胺密度梯度离心从总DNA中富集纯化枣疯病植原体DNA。利用枣树26S r DNA基因和枣疯病植原体16S r DNA基因的特异引物,分别对富集纯化前后感病枣树叶片总DNA进行PCR...本研究利用CTAB法提取感染枣疯病的枣树叶片总DNA,再通过氯化铯-双苯酰亚胺密度梯度离心从总DNA中富集纯化枣疯病植原体DNA。利用枣树26S r DNA基因和枣疯病植原体16S r DNA基因的特异引物,分别对富集纯化前后感病枣树叶片总DNA进行PCR扩增。结果表明,模板浓度为1 ng/μL时,富集后的总DNA模板扩增枣树26S r DNA的电泳条带亮度低于富集前,扩增枣疯病植原体16S r DNA电泳条带亮度明显高于富集前。Real-time PCR检测结果表明,模板浓度为1 ng/μL时,纯化后的枣疯病植原体DNA中26S r DNA相对纯化前的量为0.0638,而JWB相对纯化前的量为0.5035,这说明采用氯化铯-双苯酰亚胺密度梯度离心法可以有效地从感染枣疯病枣树总DNA中富集纯化枣疯病植原体DNA。展开更多
An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach...An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.展开更多
文摘本研究利用CTAB法提取感染枣疯病的枣树叶片总DNA,再通过氯化铯-双苯酰亚胺密度梯度离心从总DNA中富集纯化枣疯病植原体DNA。利用枣树26S r DNA基因和枣疯病植原体16S r DNA基因的特异引物,分别对富集纯化前后感病枣树叶片总DNA进行PCR扩增。结果表明,模板浓度为1 ng/μL时,富集后的总DNA模板扩增枣树26S r DNA的电泳条带亮度低于富集前,扩增枣疯病植原体16S r DNA电泳条带亮度明显高于富集前。Real-time PCR检测结果表明,模板浓度为1 ng/μL时,纯化后的枣疯病植原体DNA中26S r DNA相对纯化前的量为0.0638,而JWB相对纯化前的量为0.5035,这说明采用氯化铯-双苯酰亚胺密度梯度离心法可以有效地从感染枣疯病枣树总DNA中富集纯化枣疯病植原体DNA。
文摘An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.
