论文部分内容阅读
为深入研究日本血吸虫细胞凋亡机制。利用PCR技术扩增得到Sjcaspase3的全长序列,其ORF含900 bp,编码299个氨基酸,理论分子量为33 509.7 Da,理论等电点为6.39。Real-time PCR分析表明该基因在日本血吸虫生长发育的各个时期均有表达,其中21 d表达量最高,42 d雌虫表达量高于42 d雄虫。成功构建了p XJ40-FLAG-Sjcaspase3重组质粒并转染到Hela细胞内,荧光定量PCR和Western blotting分析表明Sjcaspase3成功在Hela细胞中表达。酶活分析提示重组Sjcaspase3具有切割特异性底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)的活性。流式细胞术检测了Sjcaspase3可诱导Hela细胞发生早期细胞凋亡。研究结果为深入探讨Sjcaspase3的生物学功能及日本血吸虫细胞凋亡机制奠定了基础。
In-depth study of Schistosoma japonicum apoptosis mechanism. The full-length sequence of Sjcaspase3 was amplified by PCR. The ORF contained 900 bp, encoding 299 amino acids. The theoretical molecular weight was 33 509.7 Da and the theoretical isoelectric point was 6.39. Real-time PCR analysis showed that the gene was expressed at all stages of the growth and development of Schistosoma japonicum, with the highest expression level at 21 d and the higher expression level at 42 d than that at 42 d. The recombinant plasmid pXJ40-FLAG-Sjcaspase3 was successfully constructed and transfected into Hela cells. Fluorescence quantitative PCR and Western blotting analysis showed that Sjcaspase3 was successfully expressed in Hela cells. The enzyme activity analysis suggested that the recombinant Sjcaspase3 has the activity of cleaving the specific substrate aspartate-glutamic acid-valine-aspartic acid (DEVD). Flow cytometry detected that Sjcaspase3 induced early apoptosis in Hela cells. The results lay a foundation for further study on the biological function of Sjcaspase3 and the apoptosis mechanism of Schistosoma japonicum.