Identifying The Function Of The Protein Coded By Gene Tsunagi In Schistosoma Japonicum

Schistosomiasis is still a serious infectious disease which is harmful to people's health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.According to in vitro dsRNA synthesis kit demand, we amplified Tsunagi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Tsunagi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjTsunagi mRNA levels were decreased by16%,57%,75% in test group,respectively,at day 1,3,5 after electroporation.The SjTsunagi protein expression levels were decreased by15%,38%,52% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of three to four worms of eight male in SjTsunagi group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjTsunagi dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Tsunagi is the gene associated with reproduction in schistosomiasis japonicum.