日本血吸虫凋亡基因Sjcaspase3的克隆、真核表达 及其功能分析

Cloning, eukaryotic expressing and function analysis of Schistosoma japonicum apoptosis gene Sjcaspase3

摘要:

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pXJ40-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。

Abstract:

For further research of the apoptosis mechanism of Schistosoma japonicum (S. japonicum). The cDNA encoding Sjcaspase3 of Schistosoma japonicum was amplified by polymerase chain reaction (PCR) technique, which contained 900 nucleotides and encoded 299 amino acids. The theory molecular weight and isoelectric point (PI) of the deduced protein is 33.5 kDa and 6.39, respectively. Real-time PCR was used to analyze the transcription profiles of Sjcaspase3 at different development stages of S. japonicum. The results showed that this gene was expressed in all stages of S. japonicum with the highest expression in 21d worms, and the level of gene transcription in 42 d female worms was higher than that of male worms. The recombinant plasmid pXJ40-FLAG-Sjcaspase3 was constructed and transfection into Hela cells successfully. Real-time PCR and Western blotting analysis showed Sjcaspase3 was successfully expressed in Hela cells. Enzyme activity analysis revealed that recombinant Sjcaspase3 possessed the activity to cut substrate DEVD. Flow cytometry proved that Sjcaspase3 could induce early apoptosis of Hela cells. The results provide the basis for proceeding further study on the biological function of Sjcaspase3 and better understand the apoptosis mechanism of S. japonicum.