Comparison Of Differential Expression Of Apoptosis Associate Genes Of Schistosoma Japonicum From Rats And Mice And Cloning, Eukaryotic Expression And Functional Analysis Of Sjcaspase3/7

Schistosomiasis is a serious parasitic zoonosis, which is still one of the important public health problems in our country. Until the end of 2013, China still estimated more than 18 million people of schistosomiasis. At present, no ideal vaccine available for prevention of schistosomiasis, only rely on mass chemotherapy with praziquantel(PZQ). Therefore, it is urgent to screen new drug targets for the treatment of schistosomiasis. Apoptosis is one of the main pattern of programmed cell death. Study of apoptosis of Schistosoma japonicum(S. japonicum) may provide new ideas for screening new drug target for schistosomiasis.In this study, we obtained 15 apoptosis associate genes of S. japonicum by digging the whole genome of S. japonicum. The expression levels of these apoptosis associate genes in S. japonicum from different hosts and different stages of were analyzed by RT-PCR. The results showed that many apoptosis associate genes had different expression levels in S. japonicum from rats and mice. We also clone, eukaryotic expression and functional analysis of important differentially expressed genes Sjcaspase3/7. Our research provides the foundation of proceeding research on apoptosis and new drug target identification of S. japonicum. 1. Analysis of the expression level of apoptosis associate genes of S. japonicum from rats and miceThis study collected 14 d, 23 d, 32 d, 42 d S. japonicum worms from Wistar rats and BALB/c mice and used Trizol regent extraction total RNA of these eight groups of worms and reversed transcription into cDNA. We searched 15 apoptosis associate genes of S. japonicum totally using the local BLAST search of S. japonicum hole genome database. The expression of 15 apoptosis associate genes in different host and different stages were analyzed by RT-PCR. Preliminary analysis showed that the expression levels of these apoptosis related genes in different hosts were different. It showed little difference in early stages of 14 d worms. However, since 23 d the apoptosis associate genes were highly expressed in worms from rats than those from mice. This might be one of the important factors affecting the growth and development of S. japonicum wrom in less-susceptible host rat. Further analysis indicated that S. japonicum has at least one complete but relatively simple endogenous apoptosis pathway(mitochondrial pathway). The study provided foundation for further research on the host parasite interaction and the apoptosis mechanism of S. japonicum. 2. Cloning, eukaryotic expressing and function analyzing of S. japonicum apoptosis gene sjcaspase7The study cloned the apoptosis gene Sjcaspase7 of S. japonicum and constructed eukaryotic expressing plasmid pXJ40-FLAG-Sjcaspase7. IFA showed that recombinant Sjcaspase7 protein was successfully expressed in Hela cell. Western Blotting analysis showed recombinant Sjcaspase7 protein weight 36 KDa and could be cleavage out a 20 KDa subunit at its N-terminal. Enzyme activity analysis revealed that recombinant Sjcaspase7 possessed the activity to cut substrate DEVD and the enzyme activity could be inhibited by Z-DEVD-FMK. Flow cytometry proved that Sjcaspase7 could induce early apoptosis of Hela cells. Using RNA interference technology, we screen an effective small interfering RNA(SiRNA-883) of Sjcaspase7 gene in vitro. In vivo, SiRNA-883 also showed interference effect of Sjcaspase7 gene. Compared to control group, the expression of Sjcaspase7 gene down regulated 32.59%; worm burden and hepatic eggs reduced by 30.77% and 28.85% separately, which were tested by animal experiment. Meanwhile, caspase activity experiment showed that the caspase activity of interference group reduced by 89.73%, compared to control group. The result showed that Sjcaspase7 might have an important effect on worm growth and eggs spawn in S. japonicum. The study provides the basis for proceeding further study on the biological function of Sjcaspase7 and better understand the apoptosis mechanism of S. japonicum. 3. Cloning, eukaryotic expressing and function analyzing of S. japonicum apoptosis gene Sjcaspase3The cDNA encoding Sjcaspase3 of S. 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 33.5KDa 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 21 d 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 study provides the basis for proceeding further study on the biological function of Sjcaspase3 and better understand the apoptosis mechanism of S. japonicum.