Studies On The Functions Of Exosomes Related Molecules In Schistosoma Japonicum

Schistosoma cause schistosomiasis,which is a major healthproblem in developing countries.In China Schistosoma japonicum is still a serious public health problem.Lots of studies show that SjEV plays an important role in pathogen-host interations.According to the previous research results in our programme,The presence of CD63 in SjEVs suggests that it is a potential diagnostic marker for the detection of schistosomiasis.And we want to explore a new way to treat schistosomiasis.1.Validation Schistosoma japonicum miR-125 b targeting murine CD276,and analyses the expression of CD276 in different tissues of mice infected with S.japonicum.In our previous study,we predicted that Schistosoma japonicum miR-125 b may target murine CD276.Here,we used PCR to amplify the putatively interacted regions of murine CD276 and cloned the amplified fragment into the downstream of a Gaussia luciferase vector(pGluc-Basic).Then,the recombinant plasmids,miR-125 b mimics,anti-sense miR-125 b were transfected into HEK293 cells and the luciferase activities were measured by using a dual luciferase reporter system.In addition,the expressions of CD276 in in vitro cultured murine macrophages were also determined by quantitative real-time PCR upon miR-125 b mimics transfection.Finally,the expressions of CD276 in different tissues of mice infected with Schistosoma japonicum were also determined by quantitative real-time PCR.The putatively interacted region of CD276 was obtained and the corresponding recombinant plasmid was successfully constructed.A significant downregulation of luciferase reporter activity was observed upon transfection of miR-125 b mimics into HEK293 T cells(P < 0.05).Transfection of miR-125 b mimics into murine macrophages led to the down-regulation of CD276.In addition,the expressions of CD276 in lymphocyte,liver,and spleen of mice infected with S.japonicum were also shown to be down-regulated(P < 0.01).Our results indicated that Schistosoma japonicum miR-125 b may directly target murine CD276 during schistosome infection,suggesting that miR-125 b may play an importantly regulatory role in parasite-host interactions.2.CD63 is a member of the tetraspanin protein family,which is widely expressed among eukaryotes.Previously,we identified a CD63 homolog from extracellular vesicles isolated from Schistosoma japonicum.In this study,we characterized this CD63 homolog using a molecular approach and evaluated the potential of its recombinant protein for the diagnosis of schistosomiasis.A sequence alignment indicated that S.japonicum CD63(SjCD63)has sequence identities of 76% and 28% with S.mansoni and human CD63,respectively.A phylogenetic analysis displayed that S.japonicum CD63 is related to S.mansoni and Opisthorchis viverrini CD63.The c DNA of SjCD63 was 240 bp long with an expected protein size of 12 kDa.A qRT-PCR analysis revealed significantly higher expression of SjCD63 m RNA in adult worms on days 21,28,and 35 than in 7-day schistosomula.Similarly,recombinant CD63 protein detected by ELISA revealed significantly higher optical density values compared to that of the negative control in both S.japonicum-infected mouse and rabbit sera,providing preliminary evidence for its diagnostic potential.Overall,these results provide insight into the molecular properties of SjCD63,its expression profiles,and its preliminary diagnostic potential.