Objective: To study the effect of RNA interference(RNAi) on WD101 gene and its effect on the expression of WD101 mRNA and protein in Schistosoma japonicum.Methods: Doublestranded RNA(dsRNA) WD101 gene and control gene...Objective: To study the effect of RNA interference(RNAi) on WD101 gene and its effect on the expression of WD101 mRNA and protein in Schistosoma japonicum.Methods: Doublestranded RNA(dsRNA) WD101 gene and control gene(lacZ) were generated by in vitrotranscription and transfected into mechanically transformed schistosomula.The total RNA and protein were isolated simultaneously using TRIzol reagent.The expression levels of mRNA and the protein were determined by quantitative real-time PCR(qPCR) and Western blotting, respectively.After injected dsRNA-electroporated schistosomula into BALB/c mouse six weeks, the male and female reproductive organs were observed and measured under the confocal laser scanning microscope.Results: After 1, 3 and 5 d of RNAi, WD101 mRNA level was decreased by 15%, 39%, and 58% in experiment group compared to that in control group; meanwhile, WD101 protein level was decreased by 11%, 28%, and 43% in experiment group compared to that in control group.There were significantly more sperms in testicular lobes in experiment group than that in control group, while there were no significant differences in terms of ovary and vitelline glands between two groups.Conclusions: The ds WD101-RNAi can effectively induce suppression of WD101 gene expression at both mRNA and protein levels.WD101 gene might be a reproduction-related gene in Schistosoma japonicum.展开更多
应用异硫氰酸胍-酚-氯仿一步法抽提日本血吸虫成虫RNA,能够在数小时内抽提出完整的成虫RNA。此法对于分离数量很少的寄生虫材料中的RNA尤为有效。通过对日本血吸虫成虫RNA变性琼脂糖凝胶电泳分析,发现其核糖体大亚基RNA(L-rRNA)体内切口...应用异硫氰酸胍-酚-氯仿一步法抽提日本血吸虫成虫RNA,能够在数小时内抽提出完整的成虫RNA。此法对于分离数量很少的寄生虫材料中的RNA尤为有效。通过对日本血吸虫成虫RNA变性琼脂糖凝胶电泳分析,发现其核糖体大亚基RNA(L-rRNA)体内切口(in vivo nick)现象的存在。并随核糖体小亚基RNA(S-rRNA)一道电泳迁移。反映了L-rRNA的后转录加工。展开更多
目的筛选干扰日本血吸虫凋亡抑制因子(Inhibitor of apoptosis protein of Schistosoma japonicum,SjIAP)较好的siRNA分子。方法应用生物信息学设计了针对SjIAP的3对siRNA分子。利用脂质体将每对siRNA分子和表达IAP的重组质粒传染到HEK2...目的筛选干扰日本血吸虫凋亡抑制因子(Inhibitor of apoptosis protein of Schistosoma japonicum,SjIAP)较好的siRNA分子。方法应用生物信息学设计了针对SjIAP的3对siRNA分子。利用脂质体将每对siRNA分子和表达IAP的重组质粒传染到HEK293T细胞中,利用实时定量RT-PCR和免疫印迹检测IAP的表达。将siRNA分子与日本血吸虫童虫(12d)进行体外共培养,利用实时定量RT-PCR和免疫印迹检测虫体内的IAP表达。结果细胞转染实验的实时定量RTPCR和免疫印迹分析表明本研究获得了2个干扰SjIAP较好的siRNA分子,且其中1对siRNA分子可显著抑制日本血吸虫虫体内IAP蛋白的表达,同时虫体Caspase活性有所上升。结论本研究获得了2个干扰日本血吸虫IAP较好的siRNA分子,为进一步利用RNA干扰研究血吸虫IAP的功能奠定了前期基础。展开更多
目的 构建日本血吸虫 RNA聚合酶 转录延长因子 S - p15真核正反义表达载体 ,为进一步鉴定该因子的生物学功能奠定基础。方法 采用表达序列标签法从日本血吸虫成虫 c DNA文库中获得了 1个 RNA聚合酶转录因子 (S - p15 )全长 c DNA序...目的 构建日本血吸虫 RNA聚合酶 转录延长因子 S - p15真核正反义表达载体 ,为进一步鉴定该因子的生物学功能奠定基础。方法 采用表达序列标签法从日本血吸虫成虫 c DNA文库中获得了 1个 RNA聚合酶转录因子 (S - p15 )全长 c DNA序列 ,克隆及序列测定后 ,将该片段正向及反向插入到真核表达载体 pc DNA 3中。重组载体采用氨苄青霉素 L B培养基筛选、双酶切、PCR及测序鉴定。结果 经鉴定 ,S - p15 - pc DNA3真核正反义表达载体构建成功。结论 构建成功 S -p15 - pc DNA3真核正反义表达载体 ,为下一步鉴定其生物学功能奠定基础。展开更多
基金supported by Natural Science Foundation of Anhui Education Department(KJ2013B318)
文摘Objective: To study the effect of RNA interference(RNAi) on WD101 gene and its effect on the expression of WD101 mRNA and protein in Schistosoma japonicum.Methods: Doublestranded RNA(dsRNA) WD101 gene and control gene(lacZ) were generated by in vitrotranscription and transfected into mechanically transformed schistosomula.The total RNA and protein were isolated simultaneously using TRIzol reagent.The expression levels of mRNA and the protein were determined by quantitative real-time PCR(qPCR) and Western blotting, respectively.After injected dsRNA-electroporated schistosomula into BALB/c mouse six weeks, the male and female reproductive organs were observed and measured under the confocal laser scanning microscope.Results: After 1, 3 and 5 d of RNAi, WD101 mRNA level was decreased by 15%, 39%, and 58% in experiment group compared to that in control group; meanwhile, WD101 protein level was decreased by 11%, 28%, and 43% in experiment group compared to that in control group.There were significantly more sperms in testicular lobes in experiment group than that in control group, while there were no significant differences in terms of ovary and vitelline glands between two groups.Conclusions: The ds WD101-RNAi can effectively induce suppression of WD101 gene expression at both mRNA and protein levels.WD101 gene might be a reproduction-related gene in Schistosoma japonicum.
文摘应用异硫氰酸胍-酚-氯仿一步法抽提日本血吸虫成虫RNA,能够在数小时内抽提出完整的成虫RNA。此法对于分离数量很少的寄生虫材料中的RNA尤为有效。通过对日本血吸虫成虫RNA变性琼脂糖凝胶电泳分析,发现其核糖体大亚基RNA(L-rRNA)体内切口(in vivo nick)现象的存在。并随核糖体小亚基RNA(S-rRNA)一道电泳迁移。反映了L-rRNA的后转录加工。
文摘目的筛选干扰日本血吸虫凋亡抑制因子(Inhibitor of apoptosis protein of Schistosoma japonicum,SjIAP)较好的siRNA分子。方法应用生物信息学设计了针对SjIAP的3对siRNA分子。利用脂质体将每对siRNA分子和表达IAP的重组质粒传染到HEK293T细胞中,利用实时定量RT-PCR和免疫印迹检测IAP的表达。将siRNA分子与日本血吸虫童虫(12d)进行体外共培养,利用实时定量RT-PCR和免疫印迹检测虫体内的IAP表达。结果细胞转染实验的实时定量RTPCR和免疫印迹分析表明本研究获得了2个干扰SjIAP较好的siRNA分子,且其中1对siRNA分子可显著抑制日本血吸虫虫体内IAP蛋白的表达,同时虫体Caspase活性有所上升。结论本研究获得了2个干扰日本血吸虫IAP较好的siRNA分子,为进一步利用RNA干扰研究血吸虫IAP的功能奠定了前期基础。
文摘目的 构建日本血吸虫 RNA聚合酶 转录延长因子 S - p15真核正反义表达载体 ,为进一步鉴定该因子的生物学功能奠定基础。方法 采用表达序列标签法从日本血吸虫成虫 c DNA文库中获得了 1个 RNA聚合酶转录因子 (S - p15 )全长 c DNA序列 ,克隆及序列测定后 ,将该片段正向及反向插入到真核表达载体 pc DNA 3中。重组载体采用氨苄青霉素 L B培养基筛选、双酶切、PCR及测序鉴定。结果 经鉴定 ,S - p15 - pc DNA3真核正反义表达载体构建成功。结论 构建成功 S -p15 - pc DNA3真核正反义表达载体 ,为下一步鉴定其生物学功能奠定基础。
