| Schistosomiasis is a kind of serious zoonosis,which severely impact humanhealth, social and economic development. An effective and safe anti-schistosomiasisvaccine plays a key role in controlling and eliminating Schistosomiasis.Radiation-attenuated cercariae or schistosomula(RAV) can drive high level ofprotective effect, but they are not suitable for direct application. Therefore, it might bea practical strategy to make a simulation about the protective mechanisms ofRadiation-attenuated(RA) schistosoma japonicum for the development of safe andreliable vaccines against of Schistosomiasis.As a result, exploring the high protectivemechanisms of RAV, especially in the area of molecular and cellular mechanisms ofRAV’s immunogenicity, becomes an important topic for researchers.It has been suggested that higher anti-infective effect can be induced in variety ofanimal models vaccinated with RAV and the host present dominant Th1response,while normal S.japonicum cercariaes or schistosomula can’t induce the sameeffect. The host immune response pattern may be related to the expression of antigendriving from S.japonicum and innate and adaptive immunity. HMGB1is primarily anendogenous nuclear protein, highly conserved across several species, and it may makea translocation from nucleus to extracellular environment to play its biologic roleunder the condition of certain stimulus. Studies indicated that HMGB1may associateswith the host inflammatory immune responses triggered by schistosome and inducesthe maturation of dendritic cells (DCs), and stimulate Th1-biased response. Therefore,we speculate that there is a kind of molecules which driving from RA schistosomacercariae or schistosomula, such as SjHMGB1probably plays a critical function ininducing the host to produce high protective effect. Thus, we observed the location ofSjHMGB1in different developmental stages of S.japonicum, analyzed its functionalcharacteristics in stimulating the dendritic cells driving from hosts preliminarily, anddetected primary conditions of apoptosis and necrosis about different stages ofschistosomula which was transformed by syringe passage in vitro.These studies willprovide a foundation for further research on the immunobiological functions andmechanisms of SjHMGB1in RAV.Firstly, SjHMGB1was cloned into pET28a(+) expression vector by themolecular cloning technology to generate recombinant proteins in prokaryotic expression system. Mice were immunized with the fusion SjHMGB1protein toachieve antiserum. We successfully obtained the location of SjHMGB1in differentdevelopmental stages of the S.japonicum by immunofluorescence.The results showedthat SjHMGB1is expressed in various life cycle stages including7d,10d,14d,23d,32d and42d post-infection. Additonally, Significant levels of SmHMGB1werepresented in schistosomulum stages and testicles of adult males.Secondly, with the purpose of avoiding the interference from lipopolysaccharidein prokaryotic expression proteins, we choosed Bac-to-Bac baculovirus expressionsystem to obtain SjHMGB1proteins. Sf9cells which were transfected recombinantshuttle plasmid were detected by indirect immunoflurescence. The purified SjHMGB1was verified with Western Blot and Mass spectrometry (MS) to analysis itsimmunogenicity and protein spectrum. The results suggested that the SjHMGB1wastransformed into Sf9cells successfully and had excellent immunogenicity.Thirdly, in order to verify whether SjHMGB1could activate host DCs andparticipate in host inflammatory and immunological responses, Mice bonemarrow-derived dendritic cells(BMDCs) were isolated in aseptic conditions andincubated with recombinant SjHMGB1from eukaryon expression. The expression ofDC surface antigen molecule MHCⅡ, CD40, CD80, CD86were detected andanalyzed by flow cytometry(FCM). The results showed that SjHMGB1protein canincrease the expression of CD40, CD80, CD86and MHCⅡ, which preliminarilyindicated that SjHMGB1can promote phenotypic maturation of DCs and laid afoundation for subsequent function experiments.Finally, in order to study the relationship between high level of protection forceinduced by RAV and apoptosis and necrosis in schistosoma following RA, we havemade a preliminary analysis of apoptosis and necrosis in different stages ofS.japonicum schistosomula which was transformed from cercariae in vitro. Firstly, wecollected normal and RA schistosoma and transform to schistosomula by syringepassage in vitro, then cultured till4d,7d,10d and14d respectively. At last, weobserved ultrastructure changes by transmission electro-microscope(TEM) and madequantitative detection of schistosomula cells by flow cytometry(FCM). TEM resultsshowed that there was no significant change in4d normal and RA schistosomula cellsstructure; When the schistosomula cultured from7d to14d, the normalschistosomula presented apoptotic morphological and necrosis changesgradually,while the RA schistosomula showed vigorously typical apoptoticmorphological changes. FCM results revealed that the percentage of apoptosis onnormal schistosomula-derived cells was higher than those derived from RAschistosomula; When schistosomula cultured experience7d,and from10d to14d, theratio of RA schistosomula-derived cells apoptosis increased rapidly, while the rate ofapoptotic on the normal schistosomula-derived cells growed gradually. In other word,the normal and RA schistosomula can present different apoptotic/necrotic patterns.These studies established a foundation for further research of cellular mechanisms ofRAV model, which induced high protective effect on host.In conclusion, fusion protein SjHMGB1were obtained both in prokaryotic andeukaryon expression system, SjHMGB1was expressed in S.japonicum at7d,10d,14 d,23d,32d and42d post-infection, and preliminarily demonstrated that the fusionSjHMGB1protein can stimulate phenotypic maturation of BMDCs.Apoptotic/necrotic conditions were detected in terms of morphology and cellular level.The study will provide a basis for further clarifying the immunobiology functions andmechanisms of SjHMGB1as well as necrotic and apoptotic cells in RAV schistosomavaccine model. |
