Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum...Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism展开更多
The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by ...The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.展开更多
为探讨腺病毒介导的基因操作技术在血吸虫基因功能中的应用,本研究构建了表达日本血吸虫凋亡抑制因子(aninhibitor apoptosis protein of Schistosoma japonicum,SjIAP)的腺病毒,免疫印迹分析表明获得了表达SjIAP的重组病毒,并对重组病...为探讨腺病毒介导的基因操作技术在血吸虫基因功能中的应用,本研究构建了表达日本血吸虫凋亡抑制因子(aninhibitor apoptosis protein of Schistosoma japonicum,SjIAP)的腺病毒,免疫印迹分析表明获得了表达SjIAP的重组病毒,并对重组病毒的滴度进行了测定。本研究为进一步利用腺病毒对血吸虫基因操作相关研究及SjIAP的功能奠定了基础。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
基金国家高技术研发 863项目 (No 2 0 0 1AA2 15 15 1)上海市重大科技攻关项目 (No 0 3DZ192 3 1)资助~~
文摘Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism
文摘The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.
文摘为探讨腺病毒介导的基因操作技术在血吸虫基因功能中的应用,本研究构建了表达日本血吸虫凋亡抑制因子(aninhibitor apoptosis protein of Schistosoma japonicum,SjIAP)的腺病毒,免疫印迹分析表明获得了表达SjIAP的重组病毒,并对重组病毒的滴度进行了测定。本研究为进一步利用腺病毒对血吸虫基因操作相关研究及SjIAP的功能奠定了基础。
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。
