Funciton Studies On Lethal Giant Larvae Gene Of Schistosoma Japonicum

Schistosomiasis is the second most common globally parasitic disease of human and animals after malaria and mainly occurs in developing countries. Despite decades of control, there are still millions of people at risk of contracting this infection, the best long-term strategy to control schistosomiasis is immunization with an antischistosomiasis vaccine combined with drug treatment. To date, candidate antigens suggested by the World Health Organization (WHO) and reported by researchers do not provide a sufficiently protective effect to be used in the clinic; therefore, it is necessary to search for alternative highly protective vaccine candidates.Lethal giant larvae (Lg1) was the first neoplastic tumor suppressor gene (nTSG) described in Drosophila melanogaster. The zygotic mutant does not pupate and continues to grow, expressing the giant larva phenotype. Many studies showed that Lgl function is essential for the development of polarized epithelia, localization of cell fate, determinant Numb in Drosophila neuroblasts and association with the cytoskeletal complex. Our lab has preliminary researched gene Lgl, and to further study the function and evaluate Lglas a candidate vaccine, we researched from the following aspects:1. Alternative splicing Analysis of SjLgl gene.In our previous study, we found two transcritors of SjLgl. In this study, we tested the SjLGL protein by western blot with rat anti SjLGL protein serum, and recovered the reactive band. Then using ESI mass spectrum sequencing corresponding protein, the result shows that all of these strips include the peptide fragment of SjLGL protein. Nevertheless, the multiple protein protect could resulted in post-translational modification, so further study is needed.2. The effect of RNAi SjLgl gene in S. japonicum egg hatchingIn our study the RNAi were used to in interfere the expression of SjLgl gene. The hatching rates were reduced to55.98%in the long term RNAi. The RNAi in different development stage of S.japonicum were proceeded. The hatching rates were reduced to63.07%and42.24%in the early RNAi, and47.08%and37.89%in the late stage RNAi in two independed experiments. The result of real time RT-PCR shows that the transcript level of SjLgl gene were reduced more significant in early stage RNAi than in late stage RNAi, which is corresponding to the results in egg hatching rates. The results testified the impact of RNAi in early stage is more significant than in late stage.3. The immunoprotective effect of SjLGLIn this study we usd rSjLGL protein and montanide ISA206combined immuned mouse, and achieved certain liver egg burden reduction, every female laying egg reduction and stable and efficiency hatching rate reduction. The IgG level raised slowy after the first immunization and quickly got up in the second immunization. The IgE level also raised during this experiment. In combined immuned experiments, the eukaryon expression plasmid pcDNA-SjLgl was used for the first immunization and recombinant protein rSjLGLwere used for second and third immunization. In all the immunoprotective experiments certain worm and egg reduction, and stable and efficiency hatching rate reduction were achieved. But the combined experiments was not more effective than protain immunization. The IgG level of combined immunization didn’t raise after first DNA immunization, but raised quickly after second protein immunization.4. The immunoprotective effect of rSjGALEThis study used rSjGALE protein and montanide ISA206combined for mouse immunization.In three independent experiment, we achieved liver egg burden reduction of51.52%, every female laying egg reduction of13.7%, hatching rate reduction of49.01%. The result of ELISA test showed that the IgG level raised slowy after the first immunization and quickly after second immunization. The IgE keep a slow rising trend after first immunization. The immunoprotective effect was not so well as combined with Freund adjuvant, but still offers certain Immunoprotection.This study preliminary analysed the alternative splicing of SjLgl gene, studied its function in embryo development through RNAi, estimated its immunoprotective effect as immunogen, we first proposed the concept of egg hatching rate reduction in schistosomiasis immunoprotection, provideing a foundation for screening candidate molecules of Schistosoma.