[Cloning, expression and characterization of a gene encoding signal transduction protein Wnt4 of Schistosoma japonicum]
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Published on: December 14, 2015
Li-Hong Tao1, Li-Xiao Yao, Zhi-Qiang Fu
1Shanghai Veterinary Research Institute of CAAS, National Laboratory of Animal Schistosomiasis Control, Key Laboratory of Animal Parasitology Ministry of Agriculture, Shanghai 200232, China.
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, 31 days 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.
Insights
Researchers identified and cloned the novel SjWnt4 gene in Schistosoma japonicum. This gene is crucial for development and shows differential expression across various life stages and genders, providing insights into Wnt signaling.
Area of Science:
- Molecular Biology
- Developmental Biology
- Parasitology
Background:
- Wnt signaling pathways regulate crucial developmental processes like cell growth, differentiation, and apoptosis.
- Wnt4 is a key regulator of gonadal differentiation in mammals.
- Schistosoma japonicum is a parasitic flatworm causing schistosomiasis.
Purpose of the Study:
- To clone and characterize a novel Wnt4 gene from Schistosoma japonicum (SjWnt4).
- To investigate the expression patterns of SjWnt4 during different developmental stages and genders of S. japonicum.
- To provide a basis for understanding Wnt signaling in S. japonicum development, particularly gonadal differentiation.
Main Methods:
- Rapid Amplification of Complementary DNA Ends (RACE) technique to clone the SjWnt4 gene.
- Sequence analysis to determine gene characteristics and protein properties.
- Real-time PCR to analyze SjWnt4 mRNA expression levels.
- Recombinant protein expression in E. coli and Western blotting for protein validation.
Main Results:
- A novel gene, SjWnt4, was successfully cloned with a complete open reading frame, sharing sequence similarity with other Wnt4 proteins.
- SjWnt4 exhibits stage- and gender-specific expression patterns, with highest mRNA levels observed in 19-day schistosomula.
- Recombinant SjWnt4 protein was expressed and detected using Western blotting, confirming its identity.
Conclusions:
- The identification and characterization of SjWnt4 provide a new molecular tool for studying Wnt signaling in S. japonicum.
- The differential expression of SjWnt4 suggests its critical role in the development and differentiation processes of the parasite.
- This research lays the groundwork for future investigations into the regulatory mechanisms of Wnt signaling in Schistosoma japonicum, especially concerning gonadal development.
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