The Study Of Schistosoma Japonicum Tollip And SSX As Candidates For Diagnosis And Vaccine

Schistosomiasis caused by Schistosome japonicum is one of the immune diseases. Sex differentiation, reproduction and oviposition are all have important influence on the survival and spread of Schistosome japonicum. Dozens of proteins which are likely to be associated to immune, sex and reproduction of Schistosome japonicum have been screened and of which there may be some putative diagnosis, drug and vaccine targets, from which we chose 2 for further studies. They are SjTollip(AY814053, AAW25785.1) and SjSSX(BU793503, CAX76185.1). Studies included bioinformatics analysis, the cloning of target genes, the expression and purification of recombinant proteins, the immunogenicity analysis of recombinant proteins, the preparation and titer evaluation of polyantibodies against the recombinant protein, the stage-specific transcription profile, the stage-specific expression profile, the evaluation of diagnosis value and animal protective experiment of recombinant proteins.Tollip is one of negative regulation factors in TLRs signaling pathway, which can inhibit the excessive and prolonged immune response. SjTollip is one relatively conserved protein. There are many homologous sequences in other species. We constructed the recombinant plasmid of pET-28a/SjTollip. The rSjTollip is insoluble and purified with high concentration and purity. Western blot analysis demonstrated rSjTollip had immunogenicity. The titre of rabbit antisera against rSjTollip was 1:256,000. rSjTollip showed higher transcription level in sporocysts, miracidia, eggs, schistosomulae, female wormsand paired adults and lower transcription level in male worms and cercariae, while its encoding protein was mainly expressed in schistosomulae and female worms. The rSjTollip was used for ELISA analysis, showing 70% and 80% of sensitivity and specificity, respectively. The animal protective experiment of rSjTollip showed 58.05% in worm reduction,59.84% and 78.68% in fecal egg reduction during 40d～42d and 43d～45d post infection, demonstrating the potential of rSjTollip for vaccine.SSX protein belongs to CT antigens family. The isotopic expression of CT antigens can lead to immune response, which can be used to build and develop immune therapies, so they will be targets for immunotherapy. We constructed the recombinant plasmid of pET-28a/SjSSX. The rSjSSX is insoluble and purified with high concentration and purity. Western blot analysis demonstrated rSjSSX had immunogenicity. The titre of rabbit antisera against rSjSSX was 1:256,000. rSjSSX showed higher transcription level in schistosomulae, female worms and paired adults and lower transcription level in sporocysts, eggs and lowest transcription level in miracidia, cercariae and male worms, while its encoding protein was expressed in all six stages. The rSjSSX was used for ELISA analysis, showing 70% and 80% of sensitivity and specificity, respectively. The animal protective experiment of rSjSSX showed 28.16% in worm reduction,4.25% in fecal egg reduction during 43d～45d post infection.