Proteomics Analysis Of 3D Schistosoma Japonicum And Characterization Of SjPHB

Schistosomiasis japonicum is a widespread and seriously harmful zoonotic parasitic disease.Lung-stage schistosomula are small in size and simple in structure.They are not only the early stages of schistosome development in definitivehost,but also the most important stage of the reductionin worm burden during migration.Therefore,Lung-stageschistosomula are regarded as the best targets for host immune system attack.1.Proteomic analysis of 3d lung-stage schistosomulaThe non-labeled quantitative proteomics analysis was used to analyze the protein composition and quantity differences between 3d larvae and 42 d adult worm.The results showed that there were 194 up-regulated differentially expressed proteins and 435 down-regulated differences.19 differentially expressed proteins including C1LVH1 and Q86E32 were selected for PRM verification.The results were consistent with the results of non-labeled quantitative proteomics.GO analysis showed that differentially expressed proteins were involved in biological processes such as macromolecular complex assembly,ATP-dependent microtubule motor regulation,ATP activity positive regulation,and ATP-dependent microtubule positive regulation.2.Cloning and expression of Schistosoma japonicum Prohibitin 1(SjPHB1)and Prohibitin 2(SjPHB2)and evaluation of immune protection effect in mouseThe gene fragments of SjPHB1 and SjPHB2 were amplified by PCR and RACE,respectively.Sequence analysis showed that SjPHB1 contains an ORF of 825 bp,encoding 274 amino acids,without signal peptide and transmembrane region;SjPHB2 contains an ORF of 900 bp,encoding 299 amino acids,without signal peptide,and has a transmembrane structure region.The pET-32a(+)-SjPHB1 and pGEX-4T-1-SjPHB2 recombinant expression plasmids were successfully constructed and expressed,both in the form of inclusion bodies.Under denaturing conditions,rSjPHB1 and rSjPHB2 fusion proteins were purified,with molecular weights of approximately 47 kDa and 59 kDa.Western blotting results showed that rSjPHB1 and rSjPHB2 can be probed by immune serum respectively,indicating that they have good antigenicity.The BALB/c mice were immunized with rSjPHB1 or rSjPHB2 combined with ISA206 adjuvant to conduct immune protection test,and the results showed that the recombinant protein failed to induce a significant protection effect.3.SjPHB1,SjPHB2 of expression,localization and RNA interference studiesUsing qPCR to analyze the expression of SjPHB1 and SjPHB2 in various stages of schistosoma japonicum development,the results showed that SjPHB1 and SjPHB2 were expressed at all stages,and their levels are quite different in schistosomula,adult worms,eggs,miracidium and cercariae.The expression of SjPHB2 in 3d and 7d schistosomula and eggs were significantly higher than that of SjPHB1 and that in other stages,but SjPHB1 was higher than SjPHB2 in miracidium.The transcription levels of SjPHB1 and SjPHB2 in females both ars higher than that in males,but their separate expression levels are opposite between males and females.The results of immunofluorescence histochemistry showed that both SjPHB1 and SjPHB2 were expressed in the cell membrane and cytoplasm,they also overlappedpartly in the membrane.In cultured worm,siRNA727 can interfere SjPHB1 transcription level with the reduction of 4.6% ~ 22.67%,siRNA796 and siRNA325 can interfere SjPHB2 with the reduction of 5.8% ~ 19.7%.Theeffect of siRNA727 and siRNA796 combined is better,which can induce SjPHB1 and SjPHB2 transcribed down by 37.39%~68.58% and 34% ~ 68.31%respectively,and make the worms black and thickened,with sectionly broken surface.In worm from mice treated with siRNA727/796 combined,the transcription levels of SjPHB1 and SjPHB2 decreased by 31.20% and 17.33%,respectively.Compared with the NC siRNA,the siRNA727/796 combination induced worm reduction of 31.35%in BALB/c mouse.Scanning electron microscope observation showed that siRNA727/796 treatment can cause the destruction of male and female body surface.