Study Of Two Tyrosinases From The Trematode Schistosoma Japonicum

Schistosomiasis is a kind of parasitic zoonoses which is prevalent widely in76countries and territories. In China, Schistosomiasis is mainly caused by Schistosoma japonicum. The combination of drugs and vaccines is considered as a long-term effective strategy to control schistosomiasis by World Health Organization. So far, the drugs for treatment of schistosomiasis mainly depend on praziquantel and artemisinin. They are single variety and the active mechanism is not clearly. It can inevitably develop drug resistance for long term use. Besides, there is no effective vaccine for schistosomiasis. Consequently, it is urgent to develop new vaccines and drugs to prevent and treat schistosomiasis. It is of important application value for finding new vaccines and drug targets.Tyrosinase (TYR) is thought to play a critical role during trematode egg production. In this study, we analyzed two genes (S/TYR1and SjTYR2), derived from Schistosoma japonicum genome databases, which encode proteins of two tyrosinases. The bioinformatics analysis showed that the tyrosinases exhibited the typical TYR topology, including two copper-binding domains and a highly conserved cysteine-rich domain. Multiple sequence alignment with other species showed that the identity of tyrosinases form Schistosoma japonicum and human is low(only24%). Therefore it may be a potential drug target. Transcription levels of the two tyrosinases were determined via RT-PCR method, the transcription levels of the two tyrosinases in male worms was close to zero in different stages. In female worms, the transcription levels of the two tyrosinases rised sharply on the24th day and the maximal value occurred on the28th day. We expressed the recombiant proteins based on the whole ORF of two tyrosinases. The whole coding sequence of each tyrosinase was cloned and inserted into a pSJ2prokaryotic expression vector in Escherichia coli Rosetta Gami strains to express the recombiant proteins. The recomiant proteins we gained were insoluble. There was an additional protein with lower molecular weight besides the full length expression. It was truncated protein expression after identification by mass spectrometry. The recombiant proteins dissolved in8M urea after purification. The recombinant proteins SjTYR1and SjTYR2were used to produce their specific antibodies by immunizing the rabbits. Both the antibody titre of the rSjTYRl and rSjTYR2immunized rabbit serum were1:512000by indirect ELISA. Western blot experiments showed that the recombiant proteins SjTYR1and SjTYR2had good immunogenicity. And we also detected cross-reactive immunoresponse between the recombinant proteins SjTYR1and SjTYR2. The recombinant protein SjTYR1can be identified by the antibody of rSjTYR2. The full length protein expression of recombinant protein SjTYR2can be identified by the antibody of rSjTYR1while the truncated protein can’t. The recombinant SjTYR1and SjTYR2-specific rabbit antiserum were also used to identify native tyrosinases (TYRs) in S.japonicum, there was a fragment which molecular mass was approximately100KDa was identified in western blotting using SjTYR1-specific rabbit antiserum. This implies that the native tyrosinase in S.japonicum may be present as a homodimer. The recombiant proteins SjTYR1and SjTYR2were uesd to immune the mouse to evaluted the protective efficiency against the infection of S.japonicum. The results showed that SjTYR1immunized mouse induced a reduction rate of31.4%in worms and52.1%in eggs. The SjTYR2immunized mouse induced a reduction rate of29.5%in worms and8.4%in eggs.RNA interference was used to study the function of SjTYR1and SjTYR2in Schistosoma japonicum. After the worms were immersed in the medium with siRNA, the mRNA level of SjTYR1and SjTYR2were detected by RT-PCR. The results suggested that the interference effect of siTyrl-1or siTyr2-1was more obvious. The most obvious interference effect is realized when the siTyrl-1and siTyr2-1were uesd at the same time. No morphological change was observed.