Identification And Preliminary Analysis Of Schistosome Japonicum Wnt Signalling Pathway Receptors And Ligands

Schistosomiasis caused by schistosome, is a serious hazard to human health of zoonotic parasitic diseases. A long time for the prevention and control of schistosomiasis has mainly taken the drug treatment (praziquantel), but the long-term and repeated medication will lead to schistosomiasis resistance, now the primay task is development a new anti-schistosomiasis therapy. In order to make the new drugs of schistosomiasis achieve a breakthrough progress, the researchers need to fully understand the schistosome growth and development process and its associated regulatory mechanism.Wnt proteins, receptor proteins of Frizzled family together with their associated signal molecules form a set of key signal pathways. The wnt signaling pathway is important role in a development processes including cell growth, cell differentiation. Our group has obtained singal proteins SjWnt4and SjWnt2, three potential receptors, and some other related molecules of S.japonicum wnt signaling. We aslo deduced the existence of wnt signaling pathway in Schistosoma japonicum through doing, expression and biological function analysis. However, the function and molecular mechanisms of the wnt signaling pathway in schistosome growth and development need to be further investigated.The method of this study were as follows:according to the known Wnt and Frizzled sequence of S. japonicum and S. mansoni, choose conservative amino acid sequence design primer, touch-up PCR amplification new genes EST of wnt signaling pathway in S. japonicum, and then used RACE procedure and electronic assembling methods to obtain full sequence; analysis the Wnt2gene structure of S. japonicum use GenomeWalkerTM Universal Kit. we recombined SjWnt2-(60aa-368aa) with pET28a and expression the recombinants in prokaryotic system which was identified by Western-blot assay, we prepared polyclonal anti-serum against SjWnt2-(60aa-368aa) by immunizing mice with the expressed proteins. Western-blot detection the anti-serum. We studied the tissue distribution of SjWnt2-(60aa-368aa) and SjFz5protein by means of immunohistochemical(IHC) staining.The main results were as follows:(1) In this study, we obtained four novel genes of Wnt signaling pathway from S. japonicum, one was (Frizzled8) Frizzled receptor gene, the others (Sjwnt5α, Sjwnt1, Sjwnt11) were the Wnt family genes; analysis the Wnt2gene structure we found that SjWnt2gene is consist of4introns, the size of intron1,3and4are unknown while intron2is653bp.(2) We successfully recombined SjWnt2-(60aa-368aa) with pET28a and expression the recombinants in prokaryotic system which was identified by Western-blot assay shows specific antigenicity, we prepared polyclonal anti-serum against SjWnt2-(60aa-368aa) by immunizing mice with the expressed proteins. Western-blot detection indicated that the anti-serum could recognize natural SjWnt2gene protein from23day schistosomula. Immunohistochemical (IHC) results show that SjWnt2-(60aa-368aa) protein was mainly localized in the subtegumental musculature and acetabulum musculature of schistosomulum and adult worms. Furthermore, SjWnt2-(60aa-368aa) was also found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female.(3) The immunohistochemical localization pattern of the SjFz5protein showed a broad tissue distribution in the musculature and acetabulum of schistosomulum and adult worms. Furthermore, SjFz5was found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female.In conclusion, we successfully obtained the related ligand receptor SjwntSa,Sjwntl, Sjwntll and Frizzled8gene of the wnt signaling pathway in S. japonicum. In order to explore the receptor ligand interaction of wnt signaling pathway in Schistosoma japonicum provides experimental material. And we studied the tissue distribution of SjWnt2and SjFz5proteins by means of immunohistochemical staining. The results showed that SjWnt2and SjFz5protein were mainly expressed in in the testes of the male and the ovary of the female. Our data primary suggest that Sj Wnt2and SjFz5may be participate in the development of the reproductive organs of both sexes in adult worm. All the data could provide basis for further studying on the ligand receptor interaction of S.japonicum wnt signalling. We will pay more attention to the growth and development of the wnt signaling pathway in S. japonicum, it also could provide the foundation for looking the key singaling molecules and finding the new drug target.