Analysis Of Differentially Expressed Proteins Between Mature Female Worms And Single-sex Infected Female Worms Of Schistosoma Japonicum

Schistosomiasis,a zoonotic parasitic disease caused by schistosoma,can induce serious pathological damage to the host.The Schistosoma japonicum cercariae infects the host and enters into the host to develop into an adult worm.After couple with male worms,the female worms can be mature.The eggs produced by mature female worms,accumulating in the hepatic portal vein and mesenteric vein,will cause the main pathological damage to the host and the dissemination of schistosomiasis.Unpaired female worms will remain in a status of sexual immaturity and small physique;they do not spawn normally or cause pathological damage to the host and spread disease.In this study,iTRAQ-coupled LC-MS/MS was used to explore the whole proteome of single-sex infected female worms(SF)and bisexual infected mature female worms(MF).A total of 19,423 peptides and 3,953 proteins were identified,and 632 differentially expressed proteins(DEPs)were found in 18 d,589 DEPs were found in 21d,705 DEPs were found in 23d and 909 DEPs were found in 25d between SF and MF.MRM was used to analyze the expression of partial DEPs,it was found that MRM analysis was consistent with iTRAQ analysis.GO function annotation and KEGG pathway analysis showed,more proteins involved in synthesis and metabolism,translational modification,protein folding and metabolic processes in MF;more proteins involved in organism movement and cell cycle,redox reaction,energy metabolism in SF.As the development of female worms,the proteins involved in the reproductive process was up-regulated in MF,and the proteins involved in detoxification were only down-regulated in the 18d MF and 21d MF,the number of proteins involved in detoxification increased in 23d MF and 25d MF.The DEPs involved in binding and catalytic activity,translational regulation activity and antioxidant activity were up-regulated in MF.The DEPs took part in the synthetic and metabolic pathways were up-regulated in MF.Subcellular localization analysis revealed that the DEPs were located in the cytoplasm,extracellular matrix,nucleus and mitochondria.In this study,SjLwr Gene(Gene Bank login number: CAX77005.1)was cloned,fused and expressed.The SjLwr gene sequence was 501 bp,encoding 137 amino acids.The product of fusion protein was 24.5 kda,and Weston blot showed that the protein had good reactivity.Recombinant protein was purified by His nickel column affinity chromatography,and polyclonal antibodies were prepared for immunizing mice.Immunohistochemistry showed that the protein was localized on the body surface and in vivo.qRT-PCR analysis showed SjLwr was expressed at all examined developmental time points and exhibited the highest transcription level at 14d,which gradually decreased to the lowest level at 21d and was relatively stable thereafter.SjLwr expression was relatively more stable in male worms than in female worms,and higher in SF than in MF.S2 siRNA was successfully screened by in vivo RNAi assay,and S2 siRNA was used for long-term in vivo interference experiments.qRT-PCR was used to detecte the interference effect and found that RNA interference significantly inhibited the expression of SjLwr gene.The SjLwr gene silencing negatively affected the parasite growth and development in mice.51.37% of S.japonicum in hosts did not survive to adulthood,as compared with the NC groups.In addition,gene knockdown inhibited the reproductive capacity of the surviving adult female worms,as fertility decreased by 15.37%,as compared to worms in the NC groups.The loss of reproductive capacity led to a lower liver egg burden.The liver egg burden of the S2 siRNA group was 43.98%,as compared with NC groups.Moreover,64.95% of liver eggs failed to hatch into miracidia.Electron microscopy showed that compared with the NC group,there are white spotted protrusions and vesicular protrusions appeared on the body of the S2 group,the tegument was arranged irregularly.The lipid droplets are reduced,the cells are not saturated,and the cell gap was increased.In this study,iTRAQ-coupled LC-MS/MS was used to explore the whole proteome of SF and MF,DEPs were involved in the different reproduction and development processes of female worms;preliminary organisms research on SjLwr also provides a theoretical basis for further study on the role of this protein in the reproductive development of schistosoma japonicum females.