Cloning, Expression And Biological Function Analysis Of Signal Conduct Protein Wnt4 And Wnt10a Encoding Genes Of Schistosoma Japonicum

Schistosomiasis caused by schistosome, is a wide spread parasitic zoonosis that causes serious healthy problem to both human and animals. Schistosome presents differential gene expression pattern at each developmental stage of its life cycle, which results in dramatic changes in its biology and morphology and special metabolism and development. Wnt proteins together with their downstream effectors form a set of key signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Base on the studies on the stage and gender differential proteome on Schistosoma japonicum, two new genes encoding signal conduct proteins Wnt have been cloned.Two ESTs (GenBank accession NO.AAM89872,AY811118) were searched in the schistosoma EST bank by using a peptide sequence obtained from two-dimentional electrophoresis conbimed with peptide mass fingerprinting and sequencing as query. With RACE technique, two signal conduct protein encoding genes Sjwnt4 (GenBank accession NO.DQ643829) and Sjwnt10a(GenBank accession NO. DQ643830) were cloned.Bioinformatic analysis showed that the two genes had typical characteristics of Wnt family proteins: there were more than 100 conserved sites, containing a signal sequence followed by a highly conserved distribution of cycteines and three or four glycosylation sites. Sequence analysis showed that SjWnt4 shared 43% similarity to Dugesia Japonica and 37% to human Wnt4. The ORF of Sjwnt4 contained 1311 nucleotides, encoding 436 amino acid with a molecular weight of 49.6 kD. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 was highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential expression. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E.coli BL21(DE3) cells, and the production of recombinant Sjwnt4 protein was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. Sjwnt10a shared 26% similarity to mouse and human Wnt10a and had a complete ORF containing 1 896 nucleotides, encoding 631 amino acid with a molecular weight of 73.3 kD. Real-time PCR revealed that the mRNA level of Sjwnt10a was highest in the 19 days schistosomula, lower in 44 days male worms and 14 days schistosomula with only 8.8% and 5% of that of 19 days schistosomula respectively, and no expression was observed in 31 days adult worms and 44 days female worms.To analyse the function of Sjwnt4 gene, four siRNAs were designed, one of which duduced 86.4% reduction of mRNA expression in Schistosoma japonicum interfered for 7 days with a concentration of 100nM.The purified rSjwnt4 protein induced 19.90% worm reduction and 20.58% liver egg reduction. We obtained two signal conduct protein Wnt encoding genes for the first time, which are stage-and-gender differentially expressed and may play an important role in the development of schistosomula. Further studies like RNAi will assist us to understand more about the new knowledge on the regulation mechanism of the Wnt signaling pathway during the development of Schistosoma japonicum.