The Preliminary Study On Schistosoma Japonicum Tegumental Protein Dysferlin And Myoferlin

Schistosomes are parasitic blood helminths that infect millions of people in tropical and subtropicalcountries. Approximately779million people are at risk of being infected in76endemic countries and anestimated280,000deaths are directly or indirectly attributable to the disease annually. Besides humans,>40types of livestock and wild animals are reservoir hosts for Schistosoma japonicum in China. Currently,schistosomiasis control strategy is mainly based on treatment of infected individuals with praziquantel(PZQ). PZQ has been proved not to be sufficient to control disease transmission and prevent reinfection. Aneffective vaccine against schistosomiasis would be essential to the current control strategy, mainly becauseit would provide long-lasting immunity against infection. The ability of schistosomes to survive in theinhospitable environment of the mammalian bloodstream and avoid host immune responses can beattributed in part to their tegument. Schistosomal proteins on the surface of the tegument that are exposed tothe host may be ideal molecules for the discovery of vaccine candidates and drug targets. Based on thestudy of tegument surface proteins at different development stages of Schistosoma japonicum by proteomictechniques, myoferlin and dysferlin, which belong to protein of ferlin family,were found at differentstages.They may play important roles in Schistosoma japonicum’s life cycle. In this study, dysferlin andmyoferlin of S. japonicum were cloned, expressed and characterized, the potential of SjDF and SjMFrecombinant protein as a vaccine candidate were evaluated.1. Characterization Analysis of Schistosoma japonicum tegumental protein MyoferlinIn this study, myoferlin of S. japonicum (SjMF) was cloned, expressed and characterized. Thetranscription level of SjMF was up-regulated at42days’ worms by Real-time PCR analysis, and it wassignificantly higher in42-day-old female worms than that in male worms. Immunofluorescence showed thatthis protein was mainly distributed on the surface of worms at different stages. Western-blot revealed thatrSjMF showed strong immunogenicity. Vaccination of rSjMF with ISA206adjuvants induced a higher levelof specific IgG. Furthermore, results showed that mice in the vaccination group presented statisticallysignifcantly higher concentrations of IL-2, IL-4, IL-10, IL-12p70and IFN-g compared with the controlgroup. Purified rSjMF emulsified with ISA206adjuvant significantly reduced worm burden from21.8%to23.21%and liver egg number from42.58%to28.35%. Besides, SjMF transcription was downregulatedwhen worms were exposed to low-dose praziquantel (PZQ) and upregulated when PZQ was degraded,accompanied by recovery of damaged tegument. When worms were exposed to high-dose PZQ, SjMFtranscription was downregulated all the time and the damaged tegument did not recover. Besides, threesmall interfering (si)RNA duplexes targeting the SjMF gene were designed, and the silencing effects wereevaluated in vitro. Our results show that there were approximately91.18%、53.8%and70.5%reduction inthe transcript level as determined by RT-PCR analysis. These findings indicated that SjMF is a potential vaccine against S. japonicum and provides the basis for further investigations into the biological function ofSjMF.2. Cloning, expression and preliminary characterization of Schistosoma japonicum tegumental proteindysferlinTo evaluate the immunogenicity of Schistosoma japonicum dysferlin recombinant protein (rSjDF),SjDF was firstly subcloned into plasmid pET-28a(+) and successfully expressed in E coli. The purifiedrecombinant protein was used to inject BALB/c mice emulsified with ISA206adjuvant three times toproduce polyclonal antibodies. Real-time RT-PCR analysis showed that SjDF was upregulated mainly inadult worms, and the transcription level in42-day-old female worms was significantly higher than that inmales. Immuno-fluorescent analysis reveal that SjDF is mainly distributed on the tegument surfacemembrane. Western-blot analysis revealed that recombinant SjDF (rSjDF) had good immunogenicity. Highlevel of specific IgG antibodies as well as IL-2, IL-10, IL-12p70, IFN-g cytokines are detected after thevaccination of rSjDF. The results suggest that the recombinant protein can induce stronger immunologicresponse in mice. When the mice were treated with praziquantel35days post-infection, we noted that thedamage of the tegument and subtegument became more severe and could not be recovered36hpost-treatment in the high-dose group, accompanied by downregulation of SjDF at the transcript level,while the damage was reduced and recovered at this time in the low-dose group, accompanied byupregulation of SjDF. The study provid the basis for screening the vaccine candidate for schistosomiasis.These findings indicated that SjDF and SjMF are potential vaccines against S. japonicum and providesthe basis for further investigations into the biological function of SjDF and SjMF.