The Study Of SjCRHSP-24 Gene's Functions

Schistosomiasis is a serious zoonotic parasitic disease of wide distribution, which is caused by schistosome. Schistosome is a infrequent bisexualism in Trematoda, Pair- ing between male and female worm of schistosomes is playing a important part in its growth and development. However, successful development of female maturation is o- bbligato precondition for its oviposition, and GCP is important protein transferred by Pairing. Ac- cordingly, so many people pay close attention to GCP about its mechanism of action and relative signaling pathways.sjCRHSP-24 is a valuable protein sieved by our lab, which can interaction with G- CP.According to report, sjCRHSP-24 is a Ca2+-regulated heat-stable protein in schistos- oma japomicum, it similar with CG9705-PA,AAW27166 is its Registered number in N- CBI site. The length of gene encoding this protein is 553bp, its expression product have 117 Amino Acids, Molecular Weight of protein is 13109Da,and it have a CSD domain. Nevertheless, the functions of sjCRHSP-24 isn't report by anybody. So, some experim- ent about sjCRHSP-24 was done by us, include clone and expression of sjCRHSP-24 gene; the gene expression in different worm development stage; RNAi and immunolo- gy research of it and immunofluorescence in worm.1.After the screening of sjCRHSP-24 by yeast two-hybrid system, the complete gene sequence of it was acquired, designed its primer and got amplification product by PCR, which have BamHI and XholⅠincision enzyme sites.After PCR product treated by incision enzyme, made it linked with pET-28b vector by ligase. When got recomb- inant plasmid , then the recombinant plasmid sjCRHSP-24/pET28(b) was transformed into E.coli BL21(DE3) to expressed recombinant protein. Our works is the first time to construct the recombinant plasmid and successfully expressed the recombinant protein in Escherichia coli, then analyzed the antigenicity of the protein. We immunized reco- mbinant protein as immunogen then get the antibody, the expression sites of sjCRHS- P-24 in worms was also identified used immunofluorescence localization. The result r- evealed that sjCRHSP-24 was homogeneous distribution in whole worms, and it also d- istributed in pellicle of worms. That displayed the interaction between GCP and sjCRHS- P-24 is poss- ible at the same time.2.Our research also have analyzed the genetic transcription level of sjCRHSP-24 gene in different developmental stage of worm, the result indicated that its genetic tra- nscription level is highest in 14d schistosomula.After schistosome infected host 15d or 16d, Pairing between male and female worm began take place, and before Pairing, sch- istosome should prepare lots of materies for it. So the high expression of sjCRHSP-24 may prepare for expression regulation of this materies.3.RNAi experiment was carried out in vivo and vitro regarding sjCRHSP-24 gene. Moreover, effect of RNAi was researched by Real time PCR and Western blot skill, the result showed that this genetic transcription and expression level can be commendably inhibited no matter RNAi in vivo or in vitro. At the same time, used microscope to obs- erve the worms after RNAi in vitro, the death rate and malformation rate of worms bec- ame higher, that demonstrated this gene may concerned with growth and development of schistosoma japomicum.4. After analyzed the immune type of activated mice, the consequence was that the content of four cytokines producted by splenocyte of mice immunized with recombinant protein including IL-4,IL-10,TNF-α,IFN-γhad apparently increase compared with untreated group after activated by recombinanted sjCRHSP-24. It's demonstrated that Th1 and Th2 cellular immunity may play a part in immunoprotection of mice and the former was predominant, but the existence of IL-10 can inhibit excessive Th1 immune reaction.