Screening of the siRNAs for silencing inhibitor apoptosis protein in Schistosoma japonicum
LUO Rong,ZHAO Jiang-ping,HU Chao,CHENG Guo-feng
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences /Key Laboratory of Animal Parasitology, the Ministry of Agriculture, Shanghai 200241, China
摘要 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(12 d)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RT-PCR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。
Abstract：Our previous study suggests that the inhibitor apoptosis protein in Schistosoma japonicum (SjIAP) may play important roles in parasitic living and development as well as in the host parasite interactions. To obtain the best efficacy of small interfering RNA (siRNA) for silencing the SjIAP, three siRNA duplexes targeting SjIAP were bioinformatically designed and chemical synthesized. Then, each siRNA duplex and the recombinant plasmid for expressing SjIAP were co-transfected into human HEK293T cells. Real-time RT-PCR and Western blot analyses indicated that the siRNA-951 showed the best efficacy for silencing SjIAP expression in both mRNA and protein levels. Then, the siRNA-951 was further added into the medium of in vitro cultured schistosomula (12 days). Western blot analysis indicated that the expression of SjIAP to some extent inhibited at the protein level. In addition, the activities of Caspase 3/7 were also increased in the worms treated with siRNA-951. In conclusion, the best efficacy of siRNA for silencing SjIAP was obtained and the present study provides foundational information for the further investigation on SjIAP functions by using RNA interference.
罗荣,赵江平,胡超,程国锋. 干扰日本血吸虫凋亡抑制因子最佳siRNA分子的筛选[J]. 中国人兽共患病学报, 2013, 29(9): 846-849.
LUO Rong,ZHAO Jiang-ping,HU Chao,CHENG Guo-feng. Screening of the siRNAs for silencing inhibitor apoptosis protein in Schistosoma japonicum. Chinese Journal of Zoonoses, 2013, 29(9): 846-849.
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2013, Vol. 29 
