Preliminary Characterization Of EsRRBL1and PGMRC2, The Sex Hormone Receptors Of Schistosoma Japonicum

In China, schistosomiasis, which caused by infection with Schistosoma japonicum, affects about362thousand individuals, with more than68.5million people at risk of infection in2010, as reported bythe China Health Statistics2011. In China, more than40species of mammals have been reported to benaturally infected with S. japonicum. Microtus fortis, a species of vole, is the only mammal in which theschistosome is found to be incompletely developed and unable to complete its life cycle. Differentialgene expression profiles between10-days-old (10d) schistosomula from infected BALB/c mice, rats andM. fortis were analyzed by oligonucleotide microarray. Several genes with specific biological functionswere identified as differentially expressed in the10d schistosomula from the susceptible BALB/c mousehost and the non-permissive M. fortis host. Some of the down-regulated genes in the schistosomulaobtained from M. fortis were known to contribute to developmental, metabolic, signal transduction andgrowth processes. Transcriptomic and proteomic profiling has revealed that, schistosomes encodereceptors for steroid hormones, implying that schistosomes can accept host hormone signals forcellproliferation, development, mating, and reproduction. In the previous microarray comparativeanalysis of10d schistosomula from BALB/c mice and M. fortis, two of the novel differentiallyexpressed genes encode the estrogen-related receptor beta like1(SjEsRRBL1) protein andmembrane-associated progesterone receptor component2(SjPGMRC2). In this study, SjEsRRBL1andSjPGMRC2was cloned and characterized.1. The development of Schistosoma japonicum induce by host sex40cercaria from the same batch of snails were injected in the peritoneum of the same old female ormale mice at the same conditions. At42days post infection, mice were sacrificed and worms werecounted and collected by perfusion from the hepatic portal system. The results show that thedevelopment rate of worms from the female mice (56.5%;64.1%) is higher than these from the malemice (40.8%;47%). The width of the female or male worms from female mice was biger than thesefrom male worms, but the difference was not significant.2. Characterization of Schistosoma japonicum estrogen-related receptor beta like1andimmunogenicity analysis of the recombinant proteinHere, the EsRRBL1gene from Schistosoma japonicum (SjEsRRBL1, JQ612589) was cloned on anORF of1374bp and expressed as a recombinant protein of72kDa. Quantitative real time PCR（qPCR）and Western blot analysis revealed that SjEsRRBL1was highly expressed in14-,18-,23-and28-days-old schistosomes at the transcriptional and protein levels. qPCR also showed thatschistosomula isolated from a S. japonicum-susceptible mouse host had3-to4-fold higher expressionof SjEsRRBL1than that from the S. japonicum non-permissive Microtus fortis host or the rat host.Moreover, SjEsRRBL1expression was2-fold higher in schistosomula from female mice than that frommale mice. Western blotting analysis showed that rSjEsRRBL1had good antigenicity. Afterimmunization of BALB/c mice with rSjEsRRBL1, partial and significantly protective efficacy was observed in two independent trials (30.84%and30.70%worm reduction;35.39%and35.61%livereggs reduction), as compared with the blank control group. An enzyme-linked immunosorbent assay(ELISA) showed that mice vaccinated with rSjEsRRBL1produced increased levels of specific IgG,IFN-γ and IL-4, but a reduced IgG1/IgG2a ratio, as compared to the adjuvant control group and theblank control group, suggesting that rSjEsRRBL1vaccination could induce a mixed Th1/Th2response.The results suggested that SjEsRRBL1might be a critical regulator of schistosome development andrepresent a promising vaccine target for schistosomiasis.2. Cloning, expression and characterization of Schistosoma japonicum membrane-associatedprogesterone receptor component2PGMRC2and the closely-related PGMRC1belong to the membrane-associated progesteronereceptor (MAPR) family. Progesterone membrane receptor component1is a protein with a singletransmembrane domain and a potential internal cytochrome b5binding sequence. The PGMRC1proteindisplays moderately high binding affinity for progesterone and also can bind to other moleculessuch astestosterone, glucocorticoids, heme, cholesterol metabolites and proteins.In this study we describe the cloning，expression and characterization of the SjPGMRC2,(FN317689.1). qPCR revealed that SjEsRRBL1was highly expressed in18-and23-days-oldschistosomes at the transcriptional levels. qPCR also showed that schistosomula isolated from a S.japonicum-susceptible mouse host was higher expression of SjPGMRC2than that from the S.japonicum non-permissive Microtus fortis host or the rat host. Moreover, SjPGMRC2expression washigher in schistosomula from female mice than that from male mice. Western blotting analysis showedthat rSjPGMRC2had good antigenicity. After immunization of BALB/c mice with rSjPGMRC2, partialprotective efficacy was observed in two independent trials (22.08%and22.097%worm reduction), ascompared with the blank control group. ELISA showed that mice vaccinated with rSjPGMRC2produced increased levels of specific IgG, IFN-γ and IL-4, but a reduced IgG1/IgG2a ratio, as comparedto the adjuvant control group and the blank control group, suggesting that rSjPGMRC2vaccinationcould induce a mixed Th1/Th2response. The RNAi specifically for SjPGMRC2induced24%wormreduction in BABL/c mice. The results suggested that SjPGMRC2might be a critical regulator ofschistosome development and represent a promising vaccine target for schistosomiasis.In conclusion, the differences of the development of schistosomiasis from different hosts show thatthe development of schistosomiasis is closely related to different environment of host. In this study,SjEsRRBL1and SjPGMRC2, which are two of the down-regulated genes in the schistosomula obtainedfrom M. fortis and both sex hormone receptors, were firstly cloned and expressed. Meantime theimmunogenicity of recombant protection was assessed by immunization with mice. This result will helpus speculate the mechanism of growth and development and discover new vaccines and drug targets.