A Preliminary Study Of Schistosoma Japonicum Vasa3 Gene Localization And Function

Objective: Gene mapping the whole worm of Schistosoma japonicum different developmental stages by using in situ hybridization, preliminary judgment of effect of Vasa3 in the development of S.japonicum reproductive system; Vasa3 gene function has revealed in the development and reproductive of S.japonicum through RNA interference; through this research we expects to find can interfere with and inhibit the key target of S.japonicum reproductive development, to provide theoretical and experimental basis for research and development of drugs to inhibit the development and activity of S.japonicum eggs or preparation, exploration of schistosomiasis control and reduce the harm to the human body new ways of S.japonicum.Methods: By using the BLAST, screening of specific sequences as in situ hybridization of the Vasa3 gene fragment with RNA probe, transcription in vitro synthesis of digoxin antisense RNA probe. After bleaching, hybriding, coloring and other processing, S.japonicum 24, 36 and 42 worms can by observation. In order to Screening a best RNA interference, three different target regions for Vasa3 siRNA by synthesis, then, construct plasmid vector expressing shRNA VSV-G retrovirus packaging, transfection to GP2-293 cells lines, virus can be harvest in the cell supernatant, concentrated by ultra centrifugation, and Retro-X qRT-PCR Titration Kit for virus titration. 18 days worm infected in retrovirus, The variation tendency of gene and protein expression level can be measurement by RT-PCR and western blot at 7 days later. Confocal microscopy to observe the morphological changes of the reproductive system by 14 days later.Results:Through the whole mount in situ hybridization, strong positive hybridization signal is visible in ovary and vitelline gland of 24, 36, 42 days female worms, Thers is no obvious positive signal in the male worms, this results indicate that SjVasa3 gene is highly expressed in ovary and vitelline gland of female. V2 target sequence(5’-cuaaacgcggagcugauautt-3’) has the best jamming effect in three siRNA, this sequence containing human U6 promoter was successfully constructed retroviral expression vector PLNHX_HsU6_Vasa3. With the PVSVG plasmid were transfected into GP2-293 cells, retrovirus titer can reach up 5×107cfu/ml. RT-PCR showed that the SjVasa3 gene level was decreased by 69%. Confocal observation of reproductive system morphology showed that the experimental group of testes size grow down, envelope is not structured well, testicular cell messy morphology, unclear boundaries. In ovary cells also decreased, cell boundary is not clear.Conclusion: Preliminary confirmed that SjVasa3 may be involved in the reproductive organs development of female worms and it can be used as female reproductive system of molecular markers S. japonicum Vasa3 gene can be effectively interfernce by retroviral expression system Through the study, contributes to the exploration of schistosomiasis control and reduce schistosomiasis on new ways. Provide a reliable method for long term inhibition of S.japonicum other gene expression.