关键词: 日本血吸虫, 胰凝乳蛋白酶样蛋白, 原位杂交, RNA干扰, 免疫保护

Abstract:

 Objective  To investigate the gene expression, localization and potential functions of Schistosoma japonicum chymotrypsin-like protease（SjCTRL） in the host, and evaluate its potential immune-protection efficacy against S. japonicum infection in mice.  Methods  The physicochemical properties of SjCTRL and its phylogenetic relationship with homologous genes from other species were analyzed with bioinformatic software. The distribution of SjCTRL transcripts in 26-day-old worms was investigated using whole-mount in situ hybridization. The transcriptional levels of SjCTRL in male and female worms at four development stages（14, 18, 22, and 26 days after infection） were measured with quantitative real-time PCR. The SjCTRL-dsRNA was prepared and used to induce RNA interference（RNAi） in 26-day-old worms via soaking in vitro, and confocal microscopy was used to observe the morphological changes of worms after RNAi. Primers were designed to amplify the encoding sequence（excluding the transmembrane region） from the S. japonicum cDNA. The truncated gene was subcloned into the pET-28a plasmid, transformed into E. coli BL21（DE3） for expression. Mice were immunized with the purified recombinant protein and challenged with cercariae. The worms and mouse liver were collected on day 35 after the challenge, and the worm-reduction rate and egg-reduction rate were calculated.  Results  The in situ hybridization results showed that SjCTRL mRNA was located in the posterior segment of intestinal tract of female worms, and having abundence only in 26-day-old female worms. After RNAi with SjCTRL-dsRNA, the mRNA expression was reduced to 25.7% （P<0.05）, without significant morphological changes. Using the recombinant plasmid pET-28a/SjCTRL, expression of insoluble SjCTRL protein was induced. Mice immunized with this protein gained a worm-reduction rate of 25.4% and an egg-reduction rate of 80.5% in liver after being challenged with cercariae.  Conclusion  This study proves a high transcriptional level of SjCTRL in the posterior segment of intestinal tract in 26-day-old female worms, which can be reduced by RNAi treatment in vitro. Immunization with the SjCTRL protein can reduce adult worms and liver eggs.

Key words: Schistosoma japonicum, Chymotrypsin-like protease, Whole-mount in situ hybridization, RNAi, Immune protection