| Schistosomiasis is a worldwide Parasitic zoonosis which seriously hazards the health of human and domestic animal. Schistosoma japonicum (S. japonicum ) is one of five schistosome species that infects humans. It is prevalent in China and other Eastern countries, and so far it still is an important public health problem. Microtus Fortis (M. fortis )is a non-suitable host of S. japonicum, with the capacity of natural resistance to S. japonicum. Characterizing the target molecules of S. japonicum related to the schistosoma resistance of M. fortis might aid in identifying potential chemotherapeutic targets and/or vaccine candidates against schistosome infection. Several positive clones were obtained in the previous work through screening a phage display cDNA library of S. japonicum with fresh sera or extracts from the liver and lungs of M. fortis. In this Paper, The immuno- protections of four positive clones obtained by screening with extracts from the liver of M. fortis were compared, and then, cloning, expression, vaccination and RNA-interference of SJCHGC06868 protein coding gene were carried out because the phage-displayed SJCHGC06868 could induce a high level of protection.1,Four Phage clones (Clone1, 2, 4 and 5) which were obtained by screening a phage display cDNA library of S. japonicum with extracts from the liver of M. fortis, were used to vaccinate mice. Both clone 1 and clone 4 induced significant (P <0.01) protection. Compared with control group of the negative phage, reductions of 28.23% (clone 1) and 24.28%(clone 4) for worm burden, and 51.73% (clone 1) and 45.52% (clone 4) for liver egg count, were obtained respectively in mice vaccinated with clone 1 and clone 4. The EST in clone1 is a partial sequence of S. japonicum protein SJCHGC06713 coding gene reported in GeneBank. BLASTX results revealed that the amino acid sequence of this protein had high similarity with many eukaryotic pyrophosphatase / phosphodiesterase protein. The EST in clone.4 is a partial sequence of S. japonicum SJCHGC06868 reported in GeneBank.2,According to the sequence of SJCHGC06868 encoding logged in GeneBank (accession number: AY809313), Two primers were designed and a 492bp (54-546) cDNA fragment was amplified by PCR using cDNAs prepared from schistosmula (7d or 14d) of S. japonicum as a template. Then the cDNA fragment was subcloned into the plasmid pET-32(a) to constract recombinant prokaryotic expression Plasmid. After transformation into E.coli rosetta (DE3), the gene was succesfully expressed when induced with IPTG. The recombinant protein was a 36KD soluble fusion protein which could be purified with 6×His chromatography column. The yield of purified protein was about 58.3mg / L E. coli culture.In the vaccine test trial, 31.63% and 29.19% worm reduction rates, 55.63% and 56.27% liver egg reduction rates were obtained from mice immunized with the recombiant antigen, compared respectively with the adjuvant control group and the PBS control group. ELISA test showed that a significant ( P < 0.05) increase of antigen-specific antibody IgG were induced in immunized group.3,The 492bp cDNA fragment was also subcloned into the eukaryotic plasmid pVAX1 to constract recombinant eukaryotic plasmid pVAX1-SJ06868.The recombinant eukaryotic plasmid pVAX1-SJ06868 was used to immunize BalB/c mice to assess the protective effect .The result of westernblotting analysis showed the SJCHGC06868 protein was expressed in mouse muscle injection site. The molecular weight of the expressed product is about18 KD. In infection experiment, worm reduction rates of 19.67% and 21.96%, liver egg reduction rates of 58.39% and 58.76% were obtained in pVAX1-SJ06868 injected group, compared respectively with the empty plasmid group and blank control group. The mechanism analysis showed that immunized mice produced a significant higher specific antibody level, CD4/CD8 ratio reduction, and significant increase of IL-2, IL-4 and other cytokines. These results indicated that injection of mice with recombinant plasmid pVAX1-SJ06868 induced an effective CTL response.4,The mRNA expression levels of this gene in various development stages of worm were analyzed with real-time fluorescence quantitative PCR. The results showed that gene expressed in all development stages in final host, and the higher expression was in 14dschistosomula.Three modified small interfering (si)RNA targeting the SJCHGC06868 protein coding gene were designed, synthesized and used to silence the the target gene expression in schistosomula cultured in vivo. Real-time fluorescence quantitative analysis showed that the expression at the transcript level was suppressed 32.6%, 37.2% and 44.9%, in siRNA1-4, siRNA2-244and siRNA-2-327 treated groups, respectively. Microscopic observation showed worms from interfered groups have low activity. After 6 days interference, worm survival rates in groups of siRNA1-4, siRNA2-244and siRNA-2-327 were 61%, 54% and 51%, respectively, which significantly lower than those of the control groups.In this paper, we assessed the protective effects of phage-displayed antigen, recombinant antigen and DNA vaccine of the unknown protein SJCHGC06868 of Schistosoma japonicum, confirmed that the gene was highly expressed in schistosomula and expression silence could cause schistosomula death. The data presented here indicated that the protein and its encoding gene has great value in anti-schistosomiasis vaccine and drug therapy research...
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