Cloning, Expression and Characterization of a Gene Encoding Signal Transduction Protein Wnt4 of Schistosoma japonicum

Abstract:

Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4（GenBank accession No. DQ643829）. Sequence analyses showed that Sjwnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4.The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real_time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31days adult worms and 44 days male worms, suggesting a stage_and_gender differential express. 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 fused to a GST tag 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. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.