| Schistosomiasis remains a serious parasitic disease. Crude adult worm antigens or crude egg antigens used for antibody detection are limited in material, and they can't differentiate infection time and need increase in specificity. Vaccine for schistosomiasis has been studied for a long time, but has not breakthrough. Schistosome is covered by a living syncytium, called tegument, which plays important roles in nutrient uptake and immune evasion and of which there may be some putative diagnosis, drug and vaccine targets. The tegumental proteome of Schistosoma japonicum identified 373 proteins, from which we chose 4 for further studies. They are Sjannexin (AY813612, AAW25344),Sj22 (AY815492, AAW27224),Sjinnexin (AY815599, AAW27331) and SjMRLC (AY815219, AAW26951). 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 immunolocalization, the evaluation of diagnosis value and animal protective experiment of recombinant proteins.Sjannexin is one relatively conserved protein with the high sequence similarity to the homogenous sequences of the hosts. Sjannexin contains four annexin repeat domains. We constructed the recombinant plasmid of pET-28a/Sjannexin. The rSjannexin is soluble and purified with high concentration and purity. The rSjannexin showed high immunogenicity by Western blot assay. The titre of rabbit antisera against rSjannexin was 1: 256, 000. Sjannexin showed higher transcription level in schistosomulae, female worms, male worms and paired adults and lower transcription level in sporocysts, miracidia, eggs and cercariae, while its encoding protein was mainly expressed in cercariae, schistosomulae, female worms, male worms and paired adults, but not found in eggs. Sjannexin was located in the tegument of male worms. The rSjannexin was used for BAS-ELISA analysis, showing 80% and 90% of sensitivity and specificity, respectively. The animal protective experiment of rSjannexin showed 3.82% in worm reduction, 21.13% in liver egg reduction, 31.44% and 24.17% in fecal egg reduction during 40d～42d and 43d～45d post infection, but there were not statistical significance (p>0.05). Sj22 is S. japonicum specific protein and contains a signal peptide. We constructed the recombinant plasmid of pET-28a/Sj22. The rSj22 was insoluble and purified with high concentration and purity. Western blot analysis demonstrated rSj22 had immunogenicity. The titre of rabbit antisera againat rSj22 was 1: 256, 000. Sj22 showed higher transcription level in female worms, male worms and paired adults, lower level in sporocyss, miracidia, cercariae and schistosomulae, but not in eggs, while its encoding protein was mainly expressed in male worms and paired adults. Sj22 was located in the gut of male worms, which is consistent with that Sj22 has a signal peptide. The rSj22 was used for BAS-ELISA analysis, showing 36.7% and 80% of sensitivity and specificity, respectively. Mouse immunized with rSj22 showed 17.28% in worms reduction , 9.03% in liver egg reduction and 22.53% in fecal egg reduction during 43d～45d post infection, but there were not statistical significance (p>0.05).The similarity of Sjinnexin with the homogenous sequences from the hosts of S. japonicum is lower than e-15. We constructed the recombinant plasmid of pET-28a/Sjinnexin which contained the second extracellular loop of Sjinnexin. The rSjinnexin was insoluble and purified with high concentration and purity. The rSjinnexin showed high immunogenicity by Western blot assay. Sj22 had similar transcription level in all developmental stages including sporocysts, miracidia, eggs, cercariae, schistosomulae, female worms, male worms and paired adults. The animal protective experiments of rSjinnexin showed 12.21% in worm reduction (p>0.05), 32.98% in liver egg reduction (p>0.05), 61.32% (p<0.05) and 53.55%(p>0.05) in fecal egg reduction during 40d～42d and 43d～45d post infection, demonstrating the potential of rSjinnexin for anti-reproduction vaccine.The similarity of SjMRLC with the homogenous sequences from the hosts of S. japonicum is lower than e-50 and they belong to different phylogenic branches. SjMRLC has one EF-hand calcium-binding domain. We constructed the recombinant plasmid of pGEX-4T-3/SjMRLC and obtained soluble recombinant protein. SjMRLC showed similar transcription level in all stages including sporocysts, miracidia, eggs, cercariae, schistosomulae, female worms, male worms and paired adults. |
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