A Preliminary Study On SjTNFR Of Schistosoma Japonicum

Schistosomiasis is one of the world’s major neglected tropical diseases.The development of new drugs is increasingly important in the face of the potential for resistance to currently available treatments,and the lack of an effective vaccine.Apoptosis is a major way of programmed cell death.Gene expression and functional data indicate that apoptosis is active throughout the lifecycle.Moreover,drugs that activate apoptosis in human cells kill schistosome cells,raising the prospect of developing new treatments against schistosomiasis of humans.In this study,through the bioinformatics,cloning and expression of prokaryotic and eukaryotic analysis of Sj.TNFR gene from Schistosoma japonicum,providing a good foundation for further study on the mechanism of Schistosoma japonicum apoptosis1 Cloning and expression analysis of a functional fragment of associate genes Sj.TNFR(tumor necrosis factor receptor)in Schistosoma japonicumThe full length sequence of SjTNFR was cloned by PCR technique,the recombinant plasmid containing SjTNFR was constructed.The expression of SjTNFR in worms of different development stage of S.japonicum from non-susceptible host rat and susceptible host mouse were analyzed and compared using Real-time PCR technique.The gene encoding SjTNFR of S japonicum was cloned.The ORF of the gene contains 603 bp encoding 200 amino acid with the theoretical molecular weight of 22.3kDa,the theoretical isoelectric point of the molecule is 7.6.This gene does not contain signal peptide and transmembrane region sequences by bioinformatics analysis.Analysis also revealed that the molecule possess belong to the member of supter TNFR family.Real-time PCR analysis showed that the gene expressed in all of the worms from four testing development stages,the expression level in worms from rats was higher than those from mice after infected 14 d,and significant expression difference was observed in 14 d worms from two different hosts(P<0.05).2 The expression analysis of recombinant eukaryotic plasmid pxj-40-SjTNFR in293 T cellThis experiment applies PCR technique to amplify the functional fragment of SjTNFR.The fragment of SjTNFR was subcloned into a pxj-40 vector and the recombinant plasmid was transfected into 293 T cell by liposome transfection methodfor producing recombinant protein.Western blotting showed that the recombinant plasmid was successfully expressed in 293 T cells,and the recombinant protein molecular weights about 26 kDa.FITC-Annexin-V and PI were used to stain unicellular sample of different apoptosis stages.Flow cytometry results revealed that the proportion of early apoptotic cells of experimental group was 25.3%,the proportion of late apoptotic cells was 2.73%,and the proportion of early apoptotic cells of negative control group was 11.2%,the proportion of late apoptotic cells was2.73%.The proportion of apoptotic cells of experimental group was higher than that of negative control group.Cell immunofluorescence assay showed that 293 cells which were transfected with pxj-40-TNFR plasmid expressed protein of interest and no protein expression in control group.Together all date,presumably,Schistosoma japonnicum apoptosis-inducing factor(SjTNFR)has a certain effect on apoptosis-promoting.This article provides a basis for further studying of SjTNFR in some respects,for instance biological function of SjTNFR and screening for drug targets.